RU2811467C2 - Modified mitochondria and their use - Google Patents

Abstract

FIELD: molecular biology; biotechnology; medicine.

SUBSTANCE: invention relates to mitochondria modified by means of a fusion protein, and a pharmaceutical composition containing them as an active ingredient. The mitochondria of the invention can be efficiently delivered to a target, into a cell where they exhibit various effects, or into the body. Modified mitochondria can be used as an antitumor agent in the treatment of various diseases. In addition, a fusion protein containing the protein of interest and a fusion protein containing the targeting protein can be used to modify mitochondria.

EFFECT: invention provides an effective protein delivery system using mitochondria as a carrier for the treatment of diseases.

30 cl, 83 dwg, 12 tbl, 26 ex

Description

Technical field

The present invention relates to a fusion protein by which mitochondria can be modified, mitochondria modified by the fusion protein, and a pharmaceutical composition containing them as an active ingredient.

Prior Art

Mitochondria are cellular organelles of eukaryotic cells involved in the synthesis and regulation of adenosine triphosphate (ATP), an intracellular energy source. Mitochondria are associated with various metabolic pathways in vivo, such as cell signal transduction, cell differentiation, cell death, and control of the cell cycle and cell growth. Mitochondria have their own genomes and are organelles that play a key role in the energy metabolism of cells. Mitochondria produce energy through electron transport and oxidative phosphorylation and play an important role in apoptotic signal transduction pathways.

Decreased energy production due to decreased mitochondrial function has been described to cause various diseases. When the functioning of the electron transport chain decreases in accordance with changes in the genome and proteome of mitochondria, there is a decrease in ATP production, excess production of reactive oxygen species, a decrease in the function of calcium regulation, etc. In this case, a change in the permeability of the mitochondrial membrane occurs, the apoptosis function can be performed with deviations, which leads to the development of malignant neoplasms and incurable diseases.

In this regard, human diseases that have been described as resulting from mitochondrial dysfunction include mitochondrial-related genetic diseases (Wallace DC, 1999), diabetes mellitus (Maechler P, 2001), heart disease (Sorescu D, 2002), aging dementia such as Parkinson's disease or Alzheimer's disease (Lin MT, 2006), various malignant neoplasms (Petros JA, 2005), metastasis of malignant neoplasms (Ishikawa K, 2008), etc. In addition, features frequently found in more than 200 types of different malignancies consist of impaired apoptosis, increased inflammatory response, and increased abnormal metabolism. All of these processes are closely related to mitochondrial function, and the correlation between malignancies and mitochondria has received increasing attention.

On the other hand, it is known that normal cells produce 36 ATP per glucose molecule through the electron transport system, but malignant cells, unlike normal cells, produce 2 ATP per glucose molecule through glycolysis under sufficient oxygen conditions (aerobic glycolysis). In this regard, it is known that malignant cells, unlike normal cells, use the inefficient process of glycolysis in terms of energy to produce amino acids, lipids, nucleic acids, etc., necessary for rapid cell proliferation. Because of this, it is known that malignant cells require less oxygen and produce more lactic acid than normal cells.

Thus, changes in the composition of the tumor microenvironment due to abnormal metabolism occurring in malignant cells, inhibition of apoptosis caused by dysfunctional mitochondria, increased inflammatory response and abnormal metabolic response in malignant cells play a very important role in the proliferation of malignancy. Thus, the development of metabolism-related anticancer agents using these features may be a good way to solve the side effects and economic problems of conventional anticancer agents.

Mitochondria are known to enter cells when mitochondria present in cells are isolated and the cells are treated with them in vitro, or mitochondria are injected into the body. Using this phenomenon, it is possible to inject normal mitochondria isolated from cells into the body to treat diseases caused by mitochondrial dysfunction or, in particular, to treat diseases by efficiently delivering a specific protein into cells using mitochondria as a carrier, but this has not yet been described.

Technical problem

The purpose of the present invention is to provide an effective protein delivery system by demonstrating that mitochondria can be used as vehicles for the effective delivery of proteins capable of exerting various pharmacological effects in cells. It is further an object of the present invention to provide a recombinant protein for effective drug delivery and for producing modified mitochondria produced therewith. It is further an object of the present invention to provide a pharmaceutical composition containing modified mitochondria as an active ingredient.

Solving a technical problem

With regard to solving the above problems, the present invention relates to modified mitochondria in which a foreign protein is associated with the outer membrane of the mitochondria. Moreover, with respect to the production of modified mitochondria, the present invention provides a fusion protein comprising a mitochondrial outer membrane anchoring peptide and a desired pharmacological protein. Additionally, the present invention provides a fusion protein comprising an antibody or fragment thereof and a mitochondrial outer membrane anchoring peptide.

Invention effect

A foreign protein can be effectively delivered into a cell if the mitochondria to which the foreign protein is bound are introduced into the human body. In addition, with the help of a pharmacologically active protein delivered into the cell, it is possible to restore impaired cell function. In addition, if mitochondria to which a foreign protein containing a pharmacologically active protein is bound is delivered into a cell, the pharmacologically active protein dissociates from the mitochondria in the cell and can be expected to serve a beneficial role. In addition, modified mitochondria containing an antibody fragment can be efficiently delivered to target cells. In particular, if an antibody fragment against a protein present on the surface of malignant tissue is bound to the surface of mitochondria, the modified mitochondria can be efficiently delivered to the malignant cells. Thus, by introducing modified mitochondria, not only can the damaged electron transport system of cells be restored, but also various diseases can be prevented or treated using the pharmacologically active protein associated with the modified mitochondria.

Brief description of drawings

Figure 1 is a diagram of the method for producing pTA-p53.

Figure 2 is a diagram of the method for producing the pET15b-UB-p53 vector.

Figure 3 shows the expression of UB-p53 protein in E. coli.

Figure 4 is a diagram of the method for producing the pET11C-TOM70-UB-p53 vector.

Figure 5 shows the expression of TOM70-UB-p53 protein in E. coli.

Figure 6 is a diagram of the method for producing the pET11C-TOM70-(GGGGS)3-UB-p53 vector.

Figure 7 shows the protein expression of TOM70-(GGGGS)3-UB-p53 in E. coli.

Figure 8 shows the method for producing the pET11C-TOM70-(GGGGS)3-p53 vector.

Figure 9 shows the expression of TOM70-(GGGGS)3-p53 protein in E. coli.

Figure 10 shows the method for producing the pET15b-UB-p53-TOM7 vector.

Figure 11 shows the expression of UB-p53-TOM7 protein in E. coli.

Figure 12 shows the method for producing the pCMV-p53-myc/His vector.

Figure 13 shows p53-myc/His protein expression in transformed CHO.

Figure 14 shows the results of purification of the TOM70-(GGGGS)3-p53 protein and then its identification.

Figure 15 shows the purified TOM70-(GGGGS)3-p53 protein.

Figure 16 shows the results of purification of the TOM70-(GGGGS)3-UB-p53 protein and then its identification.

Figure 17 shows the purified TOM70-(GGGGS)3-UB-p53 protein.

Figure 18 shows the results of purification of the UB-p53 protein and then its identification.

Figure 19 shows the purified UB-p53 protein.

Figure 20 shows the results of purification of the UB-p53-TOM7 protein and then its identification.

Figure 21 shows the purified UB-p53-TOM7 protein.

Figure 22 shows the method for preparing the pTA-granzyme B vector.

Figure 23 shows the method for producing the pET11C-TOM70-(GGGGS)3-UB-granzyme B vector.

Figure 24 shows the expression of TOM70-(GGGGS)3-UB granzyme B protein in E. coli.

Figure 25 shows the method for producing the pET15b-UB-granzyme B-TOM7 vector.

Figure 26 shows the expression of the UB granzyme protein B-TOM7 in E. coli.

Figure 27 shows the purification results of the TOM70-(GGGGS)3-UB-granzyme B protein.

Figure 28 shows the purified TOM70-(GGGGS)3-UB granzyme B protein.

Figure 29 shows a method for producing the pTA-RKIP vector.

Figure 30 shows the method for producing the pET11C-TOM70-(GGGGS)3-UB-RKIP vector.

Figure 31 shows the expression of TOM70-(GGGGS)3-UB-RKIP protein in E. coli.

Figure 32 shows the purification results of the TOM70-(GGGGS)3-UB-RKIP protein.

Figure 33 shows the purified TOM70-(GGGGS)3-UB-RKIP protein.

Figure 34 shows a method for producing the pTA-PTEN vector.

Figure 35 shows the method for producing the pET11C-TOM70-(GGGGS)3-UB-PTEN vector.

Figure 36 shows the expression of TOM70-(GGGGS)3-UB-PTE protein in E. coli.

Figure 37 shows the purification results of the TOM70-(GGGGS)3-UB-PTEN protein.

Figure 38 shows the purified TOM70-(GGGGS)3-UB-PTEN protein.

Figure 39 shows the results of purification of the UB-GFP-TOM7 protein and then its identification.

Figure 40 shows the purified UB-GFP-TOM7 protein.

Figure 41 shows the results of purification of the TOM70-(GGGGS)3-UB-GFP protein and then its identification.

Figure 42 shows the purified TOM70-(GGGGS)3-UB-GFP protein.

Figure 43 shows the method for producing the vector pET15b-UB-scFvHER2-TOM7.

Figure 44 shows the protein expression of UB-scFvHER2-TOM7 in E. coli.

Figure 45 shows the method for producing the pCMV-scFvHER2-TOM7-myc/His vector.

Figure 46 shows the protein expression of scFvHER2-TOM7-myc/His in transformed CHO.

Figure 47 shows the purification results of the UB-ScFvHER2-TOM7 protein.

Figure 48 shows the purified UB-ScFvHER2-TOM7 protein.

Figure 49 shows the method for producing the vector pET15b-UB-scFvMEL-TOM7.

Figure 50 shows the expression of UB-scFvMEL-TOM7 protein in E. coli.

Figure 51 shows the method for producing the vector pCMV-scFvMEL-TOM7-myc/His.

Figure 52 shows the protein expression of scFvMEL-TOM7-myc/His in transformed CHO.

Figure 53 shows a method for producing the vector pCMV-scFvPD-L1-TOM7-myc/His.

Figure 54 shows the protein expression of scFvPD-L1-TOM7-myc/His in transformed CHO.

Figure 55 is an image that confirms that the fluorescent protein is associated with the outer membrane of mitochondria. In this case, mitochondria are stained using MitoTracker CMXRos to demonstrate red staining and TOM70-UB-GFP to demonstrate green staining. The area where the two parts overlap shows yellow coloration. In this case, the magnification at 55a is 200x, and the magnification at 55b is 600x.

Figure 56 shows the identification of recombinant protein TOM70-(GGGGS)3-UB-p53 and UB-p53-TOM7 associated with the outer membrane of foreign mitochondria by Western blot analysis.

Figure 57 shows the results of observing the degree of intracellular injection according to the concentration of mitochondria using a fluorescence microscope after isolating foreign mitochondria and then injecting mitochondria into cells.

Figure 58 is an image that confirms the influence of normal mitochondria on the proliferation of malignant skin cells.

Figure 59 is an image that confirms the effect of normal mitochondria in inhibiting the production of reactive oxygen species (ROS) in malignant skin cells.

Figure 60 is an image that confirms the influence of normal mitochondria on drug resistance.

Figure 61 is an image that confirms the influence of normal mitochondria on the expression of antioxidant genes in cells.

Figure 62 is an image showing the influence of normal mitochondria on the expression of genes involved in the metastasis of malignant cells.

Figure 63 is a diagram of a method for confirming loading of recombinant p53 protein onto the outer membrane of foreign mitochondria and injection of recombinant p53 protein into a cell.

Figure 64 is an image confirming that recombinant p53 protein is loaded onto the outer membrane of foreign mitochondria and that p53 is injected into the cell. In this case the magnification is 200x.

Figure 65 is an image in which it is confirmed that the recombinant p53 protein is loaded onto the outer membrane of foreign mitochondria and that p53 is injected into the cell. In this case, the magnification is 600x.

Figure 66 is a diagram of a method for confirming the apoptotic ability of modified mitochondria loaded with p53 injected into cells using a gastric cancer cell line.

Figure 67a is an image confirming the apoptotic ability of modified p53-loaded mitochondria injected into gastric cancer cells by TUNEL assay. In this case, the magnification is 600x.

Figure 67b is an image confirming the apoptotic ability of modified p53-loaded mitochondria injected into gastric cancer cells by fluorescence measurement.

Figure 68 is an image confirming the effect of inhibiting cancer cell metastasis by modified RKIP-loaded mitochondria in MDA-MB-231 cells.

Figure 69 is an image in which it is confirmed that a single chain variable fragment (ScFv) of an antibody for targeting malignant cells is expressed in cells.

Figure 70 is an image in which the single chain variable fragment (ScFv) of an antibody for targeting malignant cells is confirmed to be expressed and associated with mitochondria present in the cell using an experimental immunocytochemistry (ICC) method. In this case the magnification is 200x.

Figure 71 is an image in which the single chain variable fragment (ScFv) of an antibody for targeting malignant cells is confirmed to be expressed and associated with mitochondria present in the cell using an experimental immunocytochemistry (ICC) method. In this case, the magnification is 600x.

Figure 72 is an image comparing the effect of injecting mitochondria to which a single chain variable fragment of a cancer cell targeting antibody is bound into a gastric cancer cell line.

Figure 73 is a diagram of an animal experiment using modified mitochondria.

Figure 74 is a photograph showing an increase in tumor tissue.

Figure 75 is an image that confirms the change in body weight of mice after the introduction of mitochondria and modified mitochondria.

Figure 76 is an image that confirms the size of the tumor after the introduction of mitochondria and modified mitochondria.

Figure 77 is an image confirming that modified mitochondria loaded with TOM-UB-p53 protein are effective in inhibiting the proliferation of A431 cells.

Figure 78 is an image that confirms the function of isolated mitochondria in terms of ATP content.

Figure 79 is an image that confirms the function of isolated mitochondria in relation to membrane potential.

Figure 80 is an image that confirms the extent of damage to isolated mitochondria by measuring mitochondrial ROS (mROS production).

Figure 81a is an image showing the structure of a protein present in the outer membrane of mitochondria and the amino acid sequence of the N-terminal region of TOM70, TOM20 or OM45.

Figure 81b is an image showing the amino acid sequence of the C-terminal region of TOM5, TOM7, Fis1, VAMP1B, Cytb5, BCL-2 or BCL-X.

Figure 82 is an image confirming that the desired protein dissociates depending on the presence or absence of a linker between the outer membrane anchoring peptide and ubiquitin.

Figure 83 is an image that confirms that the desired protein associated with the modified mitochondria is separated from the mitochondria in the cell.

The best way to carry out the invention

Hereinafter, the present invention will be described in more detail.

In one aspect, the present invention relates to modified mitochondria in which a foreign protein is associated with the outer membrane of the mitochondria.

Mitochondria can be obtained from mammals, in particular they can be obtained from humans. In particular, mitochondria can be isolated from cells or tissues. For example, mitochondria can be obtained from somatic cells, germ cells or stem cells. In addition, the mitochondria may be normal mitochondria obtained from cells in which the biological activity of the mitochondria is normal. In addition, mitochondria can be cultured in vitro.

In addition, mitochondria can be obtained from an autologous, allogeneic or xenogeneic individual. In particular, the term "autologous mitochondria" refers to mitochondria obtained from tissues or cells of the same individual. In addition, the term "allogeneic mitochondria" refers to mitochondria obtained from an individual belonging to the same biological species as the individual and having different allele genotypes. In addition, the term "xenogeneic mitochondria" refers to mitochondria obtained from an individual belonging to a species other than the individual.

In particular, somatic cells may be muscle cells, hepatocytes, nerve cells, fibroblasts, epithelial cells, adipocytes, osteocytes, leukocytes, lymphocytes, platelets or mucosal cells. In addition, germ cells are cells that undergo meiosis and mitosis and can be sperm or eggs. In addition, the stem cells may be selected from the group consisting of mesenchymal stem cells, adult stem cells, induced pluripotent stem cells, embryonic stem cells, bone marrow stem cells, neural stem cells, limbal stem cells, and tissue stem cells. In this case, the mesenchymal stem cells can be selected from the group consisting of umbilical cord, umbilical cord blood, bone marrow, fat, muscle, nerves, skin, amniotic membrane and placenta.

On the other hand, if mitochondria are isolated from specific cells, mitochondria can be isolated by various known methods, for example, using a specific buffer solution or using a potential difference and a magnetic field, and the like.

As used herein, the term “foreign protein” refers to a protein, including a desired protein, capable of functioning inside and outside a cell. In this case, the foreign protein is a protein that does not exist in mitochondria and may be a recombinant protein. In particular, the foreign protein may contain a mitochondrial anchoring peptide and a desired protein. In addition, the foreign protein may be a recombinant fusion protein comprising a mitochondrial anchoring peptide and a desired protein. In this case, the foreign protein may contain a peptide that anchors in the mitochondria. Preferably, the mitochondrial anchoring peptide may be a peptide that can localize to the mitochondrial outer membrane. Thus, the foreign protein can be associated with the outer mitochondrial membrane via a mitochondrial anchoring peptide. The mitochondrial anchoring peptide may be a peptide containing the N-terminal region or C-terminal region of a protein present in a mitochondrial membrane protein and the N-terminal region or C-terminal region of a protein present in the outer membrane of mitochondria, the protein may be localized to outer membrane of mitochondria. In this case, the anchoring peptide may further comprise a mitochondrial signal sequence.

One embodiment of the protein present in the mitochondrial membrane may be selected from the group consisting of TOM20, TOM70, OM45, TOM5, TOM6, TOM7, TOM22, Fis1, Bcl-2, Bcl-x and VAMP1B. In particular, if the mitochondrial anchoring peptide is derived from any peptide selected from the group consisting of TOM20, TOM70 and OM45, it may contain the N-terminal region of TOM20, TOM70 or OM45. One embodiment of the mitochondrial anchoring peptide may be TOM70 derived from yeast and set forth in SEQ ID NO: 75, or TOM70 derived from human and set forth in SEQ ID NO: 76. Another embodiment may be TOM20 derived from yeast and set forth in SEQ ID NO: 77, or human-derived TOM20 set forth in SEQ ID NO: 78. Another embodiment may be yeast-derived OM45 set forth in SEQ ID NO: 79.

In addition, if the mitochondrial anchoring peptide is derived from any peptide selected from the group consisting of TOM5, TOM6, TOM7, TOM22, Fis1, Bcl-2, Bcl-x and VAMP1B, it may contain the C-terminal region of any peptide , selected from the group consisting of TOM5, TOM6, TOM7, TOM22, Fis1, Bcl-2, Bcl-x and VAMP1B. One embodiment of the mitochondrial anchoring peptide may be TOM5 derived from yeast, set forth in SEQ ID NO: 80, or TOM5, derived from human, set forth in SEQ ID NO: 81. Another embodiment may be TOM7, derived from yeast and set forth in SEQ ID NO: 82, or human-derived TOM7 set forth in SEQ ID NO: 83. Another embodiment may be yeast-derived TOM22 set forth in SEQ ID NO: 84, or human-derived TOM22 and set forth in SEQ ID NO: 85. Another embodiment may be Fis1 derived from yeast and set forth in SEQ ID NO: 86, or Fis1 derived from human and set forth in SEQ ID NO: 87. Another embodiment may be Bcl- 2 alpha, derived from human and set forth in SEQ ID NO: 88. Another embodiment may be VAMP1 derived from yeast, set forth in SEQ ID NO: 89, or VAMP1, derived from human, set forth in SEQ ID NO: 90.

In this case, the desired protein capable of functioning inside and outside the cell and incorporated into the foreign protein may be any protein selected from the group consisting of an active protein exhibiting activity in the cell, a protein present in the cell, and a protein having the ability to bind to a ligand or receptor present in the cell membrane.

An embodiment of the active protein or the protein present in the cell may be any protein selected from the group consisting of p53, granzyme B, Bax, Bak, PDCD5, E2F, AP-1 (Jun/Fos), EGR-1, retinoblastoma protein (RB), phosphatase and tensin homologue (PTEN), E-cadherin, neurofibromin-2 (NF-2), poly-ADP-ribose synthase 1 (PARP-1), BRCA-1, BRCA-2, adenomatous polyposis protein colon (APC), tumor necrosis factor receptor-associated factor (TRAF), RAF kinase inhibitory protein (RKIP), p16, KLF-10, LKB1, LHX6, C-RASSF, DKK-3PD1, Oct3/4, Sox2 , Klf4 and c-Myc. If the desired protein is selected from the above group, it can be coupled to an anchoring peptide containing the N-terminal region of TOM20, TOM70 or OM45.

Such a fusion protein can be linked in the following order:

N-terminal-anchoring peptide containing the N-terminal region of TOM20, TOM70 or OM45-desired protein-C-terminus.

In addition, the foreign protein may further comprise an amino acid sequence recognized by a proteolytic enzyme in eukaryotic cells, or ubiquitin or a fragment thereof between the mitochondrial anchoring peptide and the desired protein. The term "proteolytic enzyme" in eukaryotic cells refers to an enzyme that leads to the degradation of protein present in eukaryotic cells. In this case, because the foreign protein contains an amino acid sequence recognized by an enzyme leading to protein degradation, the foreign protein bound to the mitochondrial outer membrane can be separated in the cell into an anchoring peptide and the desired protein.

In this case, the ubiquitin fragment may contain the C-terminal amino acid sequence Gly-Gly SEQ ID NO: 71 and may contain from 3 to 75 amino acids located sequentially from the C-terminus. In addition, the foreign protein may further comprise a linker between the desired protein and ubiquitin or a fragment thereof. In this case, the linker may consist of, but is not limited to, 1 to 150 amino acids, or 10 to 100 amino acids, or 20 to 50 amino acids. The linker may consist of amino acids suitably selected from the 20 amino acids, preferably may consist of glycine and/or serine. One embodiment of the linker may consist of from 5 to 50 amino acids consisting of glycine and serine. One embodiment of the linker may be (G4S)n, wherein n is an integer from 1 to 10, and n may be 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

In addition, the protein having the ability to bind to a ligand or receptor present in the cell membrane may be a ligand or receptor present on the surface of the tumor cell. In this case, the ligand or receptor present on the surface of the tumor cell may be, by way of non-limiting examples, any ligand or receptor selected from the group consisting of CD19, CD20, melanoma-associated antigen E(MAGE), NY-ESO-1 , carcinoembryonic antigen (CEA), cell surface associated mucin 1 (MUC-1), prostatic acid phosphatase (PAP), prostate-specific antigen (PSA), survivin, tyrosinase-associated protein 1 (tyrp1), tyrosinase-related protein 1 (tyrp2), brachyury gene, mesothelin, epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (HER-2), ERBB2, Wilms tumor protein (WT1), FAP, EpCAM, PD-L1, ACPP, CPT1A , IFNΓ, CD274, FOLR1, EPCAM, ICAM2, NCAM1, LRRC4, UNC5H2 LILRB2, CEACAM, nectin-3 and combinations thereof.

In addition, the protein having the ability to bind to a ligand or receptor present in the cell membrane may be an antibody or a fragment thereof that binds to any protein selected from the above group. In particular, the term "antibody fragment" refers to a fragment having the same complementarity determining region (CDR) as the antibody. In particular, it may be Fab, scFv, F(ab')2 or a combination thereof.

In this case, the desired protein can be linked to an anchoring peptide containing the C-terminal region of any protein selected from the group consisting of TOM5, TOM6, TOM7, TOM22, Fis1, Bcl-2, Bcl-x and VAMP1B, and the foreign protein can be connected in the following order:

N-terminal is a desired protein-anchoring peptide containing the C-terminal region of any protein selected from the group consisting of TOM5, TOM6, TOM7, TOM22, Fis1, Bcl-2, Bcl-x and VAMP1B-C terminus.

In addition, the foreign protein may further comprise a linker between the desired protein and the C-terminal region of any protein selected from the group consisting of TOM5, TOM6, TOM7, TOM22, Fis1, Bcl-2, Bcl-x and VAMP1B. In this case, the linker is as described above. In this case, the desired protein, the active protein, the protein present in the cell, and the protein having the ability to bind to a ligand or receptor present in the cell membrane and the like are as described above.

In one embodiment of the desired protein, the antibody or fragment thereof against a particular cell may be in the form linked to an anchoring peptide containing the C-terminal region of any protein selected from the group consisting of TOM5, TOM6, TOM7, TOM22, Fis1, Bcl- 2, Bcl-x and VAMP1B. Modified mitochondria to which the desired protein is bound can easily be introduced into a specific target such that the mitochondria can effectively enter a specific cell.

One embodiment of the modified mitochondria may be in a form to which one or more desired proteins are associated. In particular, they may be in a form to which a desired p53-containing protein and a desired anti-HER-2 antibody-containing protein or fragment thereof are associated. Such modified mitochondria can be efficiently delivered to malignant cells expressing HER-2. In addition, malignant cells can be effectively killed by p53 associated with modified mitochondria.

Depending on the purpose of the modified mitochondria, the desired protein can be engineered to contain one or more active proteins and allowed to bind to the mitochondria. In addition, the desired cell-targeting protein can be engineered in a variety of ways depending on the target cell.

In another aspect, the present invention relates to a pharmaceutical composition containing the above-described modified mitochondria as an active ingredient. In this case, the pharmaceutical composition can be used for the prevention or treatment of malignancy. In this case, the malignancy may be a malignancy selected from the group consisting of stomach cancer, liver cancer, lung cancer, colorectal cancer, breast cancer, prostate cancer, ovarian cancer, pancreatic cancer, cervical cancer, thyroid cancer , laryngeal cancer, acute myeloid leukemia, brain tumor, neuroblastoma, retinoblastoma, head and neck cancer, salivary gland cancer and lymphoma.

In particular, if the active protein kills tumor cells like p53, or if the proliferation inhibitory protein is associated with mitochondria, modified mitochondria to which p53 is associated can be used as an antitumor agent. In addition, if a protein, such as RKIP, capable of inhibiting cancer cell metastasis is associated with mitochondria, the modified mitochondria to which RKIP is associated can be used as an inhibitor of tumor metastasis. If any protein selected from the group consisting of granzyme B, Bax, Bak, PDCD5, E2F, AP-1 (Jun/Fos), EGR-1, retinoblastoma protein (RB), phosphatase and tensin homolog (PTEN) is associated with mitochondria , E-cadherin, neurofibromin-2(NF-2), poly-ADP-ribose synthase 1 (PARP-1), BRCA-1, BRCA-2, adenomatous polyposis coli protein (APC), receptor-associated factor tumor necrosis factor (TRAF), p16, KLF-10, LKB1, LHX6, C-RASSF, DKK-3PD1 and combinations thereof, which are proteins that inhibit the proliferation of malignant cells, or control the phosphorylation reaction in malignant cells, or inhibit the metastasis of malignant cells, modified mitochondria, to which the active protein is bound, can be used as an antitumor agent.

Additionally, in the case of a pharmaceutical composition, mitochondria may be included in concentrations of, but not limited to, 0.1 μg/ml to 500 μg/ml, 0.2 μg/ml to 450 μg/ml, or 0.5 μg/ml. ml up to 400 µg/ml. Inclusion of mitochondria within this range may facilitate adjustment of the dose of mitochondria after administration and may increase the degree of improvement in the patient's disease symptoms. In this case, the mitochondrial dose can be determined by quantitative mitochondrial analysis by counting the membrane protein in the isolated mitochondria. In particular, isolated mitochondria can be quantitatively analyzed using the Bradford protein assay (James D. McCully (J Vis Exp. 2014;(91):51682)).

Additionally, in the case of a pharmaceutical composition, the active mitochondrial binding protein may be included at a concentration of, but not limited to, 0.1 μg/ml to 500 μg/ml, 0.2 μg/ml to 450 μg/ml, or from 0.5 µg/ml to 400 µg/ml. Inclusion of the active protein within the above range may facilitate adjustment of the dose of the active protein after administration and may increase the degree of improvement in the patient's symptoms.

Additionally, in the case of a pharmaceutical composition, a targeting protein capable of targeting mitochondria to a specific cell may be included in a concentration of, but not limited to, 0.1 μg/ml to 500 μg/ml, 0.2 μg/ml to 450 µg/ml, from 0.5 µg/ml to 400 µg/ml. Inclusion of the targeting protein within the above range may facilitate adjustment of the dose of the targeting protein after administration and may increase the degree of improvement in the patient's disease symptoms.

In particular, the pharmaceutical composition of the present invention can be administered to mitochondria in an amount of, but not limited to, 0.01-5 mg/kg, 0.1-4 mg/kg, or 0.25-2.5 mg/kg per dose. times based on the body weight of the individual to whom they will be administered. Those. It is most preferred in terms of cell activity to administer the pharmaceutical composition such that the number of modified mitochondria falls within the above range based on the body weight of the individual having the cancerous tissue. In addition, the pharmaceutical composition can be administered 1-10 times, 3-8 times or 5-6 times, and preferably 5 times. In this case, the interval between administrations may be 1-7 days or 2-5 days and preferably 3 days.

In addition, the pharmaceutical composition of the present invention can be administered to a human or other mammal prone to developing or suffering from cancer. In addition, the pharmaceutical composition may be an injectable drug that can be administered intravenously or an injectable drug that can be administered topically, and preferably may be an injectable drug.

Thus, the pharmaceutical composition of the present invention can be prepared as a physically or chemically highly stable drug by adjusting the pH of the composition with a buffer solution such as an aqueous acid or phosphate that can be used in the injectable drug to ensure the stability of the product during distribution of the injectable drugs .

In particular, the pharmaceutical composition of the present invention may contain water for injection. Water for injection is distilled water prepared to dissolve a solid injectable drug or dilute a water-soluble injectable drug, and may be glucose injection, xylitol injection, D-mannitol injection, fructose injection, saline, Dextran 40 injection, dextran 70 for injection, amino acid injection, Ringer's solution, Ringer's lactate, phosphate buffer solution having a pH of 3.5 to 7.5, sodium dihydrogen phosphate citrate buffer solution or the like.

In addition, the pharmaceutical composition of the present invention may include a stabilizer or solubilizer. For example, the stabilizer may be sodium pyrosulfite or ethylenediaminetetraacetic acid, and the solubilizer may be hydrochloric acid, acetic acid, sodium hydroxide, sodium hydrogen carbonate, sodium carbonate or potassium hydroxide.

In addition, the present invention may relate to a method of preventing or treating malignancy, comprising administering to an individual the above pharmaceutical composition. As used herein, the individual may be a mammal, and preferably a human.

In one aspect, the present invention relates to a method for producing modified mitochondria, comprising the step of mixing the isolated mitochondria with a desired protein containing an active protein and/or a desired protein containing a targeting protein.

In this case, the desired protein and mitochondria can be mixed in a suitable ratio. For example, the desired protein:mitochondria can be mixed in a ratio of 1:100 to 100:1 on a weight basis. In particular, they can be mixed in a ratio of 1:10, 1:5, 1:4, 1:3, 1:2 or 1:1. In addition, the ratio can be 10:1, 5:1, 4:1, 3:1 or 2:1.

In another aspect, the present invention relates to a method for producing modified mitochondria from transformed cells by injecting a polynucleotide encoding the desired protein described above into eukaryotic cells. In particular, the present invention relates to a method for producing the above-described fusion protein, comprising the step of transforming prokaryotic cells or eukaryotic cells using the above-described polynucleotide without a ubiquitin degradation enzyme or a proteolytic enzyme in eukaryotic cells; and the step of producing the fusion protein. This production method is suitable if the desired protein does not contain an amino acid sequence recognized by a proteolytic enzyme in eukaryotic cells, or ubiquitin or a fragment thereof.

In another aspect of the present invention, the desired protein can be obtained using a prokaryotic cell or an extract of prokaryotic cells. Moreover, the present invention relates to a method for producing modified mitochondria using eukaryotic cells without a ubiquitin degradation enzyme or proteolytic enzyme or eukaryotic cell extract.

In another aspect, the present invention relates to the use of mitochondria as vehicles for the delivery of foreign protein. In particular, modified mitochondria can be used as a means of intracellular and extracellular delivery of a foreign protein containing a desired protein that can function inside and outside the cell. Mitochondria can be efficiently incorporated into cells, in which case the foreign protein that is desired to be delivered into the cells can be efficiently delivered into the cells. In this case, mitochondria can be used as an effective protein delivery system. The desired protein is as described above.

Another aspect of the present invention relates to a fusion protein comprising a mitochondrial outer membrane anchoring peptide and a desired protein. In this case, the desired protein is as described above.

As used herein, the term "mitochondrial outer membrane-anchoring peptide" may mean the N-terminus or C-terminus of a protein present in the outer membrane of mitochondria. The mitochondrial outer membrane anchoring peptide may have an amino acid sequence specifically located in the mitochondrial outer membrane. In this case, the mitochondrial outer membrane anchoring peptide allows the fusion protein described herein to be attached to the outer membrane of the mitochondria. In this case, a mitochondrial outer membrane anchoring peptide can be used in the same sense as a mitochondrial outer membrane targeting peptide.

In addition, the mitochondrial outer membrane anchoring peptide prevents the fusion protein provided herein from entering the mitochondria. The TOM (translocase outer membrane) complex present in the mitochondrial outer membrane has a mitochondrial target sequence and a separate outer membrane anchoring domain at the amino terminus, and most of the carboxy terminus may have a structure exposed towards the cytoplasm (Figure 81a). The TOM (translocase outer membrane) complex present in the mitochondrial outer membrane has a mitochondrial target sequence and a separate outer membrane anchoring domain at the carboxy terminus, and most of the amino terminus may also have a structure exposed towards the cytoplasm (Figure 81b) . In addition, the protein present in the outer membrane of mitochondria can be selected from proteins present in mitochondria found in a eukaryotic cell. For example, it may be selected from proteins present in the mitochondrial outer membrane found in yeast, animal cells or human cells.

In this case, the embodiment of the protein present in the mitochondrial outer membrane may be any protein selected from the group consisting of TOM20, TOM70, OM45, TOM5, TOM6, TOM7, TOM22, Fis1, Bcl-2, Bcl-x and VAMP1B or its fragment. In this case, the mitochondrial outer membrane anchoring peptide may be a fragment of any protein selected from the group consisting of TOM20, TOM70, OM45, TOM5, TOM6, TOM7, TOM22, Fis1, Bcl-2, Bcl-x and VAMP1B. In this case, the peptide anchoring in the mitochondrial outer membrane may be the C-terminal or N-terminal polypeptide TOM20, TOM70, OM45, TOM5, TOM6, TOM7, TOM22, Fis1, Bcl-2, Bcl-x and VAMP1B, localized in the mitochondrial outer membrane.

In particular, if the mitochondrial outer membrane anchoring peptide is fused to the N-terminus of a desired protein, the mitochondrial outer membrane anchoring peptide may comprise the terminal sequence of a protein selected from the group consisting of TOM20, TOM70 and OM45. Preferably, it may be the N-terminal sequence of a protein selected from the group consisting of TOM20, TOM70 and OM45. An embodiment of the mitochondrial outer membrane anchoring peptide is as described above.

Additionally, if the mitochondrial outer membrane-anchoring peptide is fused to the C-terminus of the desired protein, the outer membrane targeting protein may comprise the terminal sequence of a protein selected from the group consisting of TOM5, TOM6, TOM7, TOM22, Fis1, Bcl-2 , Bcl-X and VAMP1B. Preferably, it may be the C-terminal sequence of a protein selected from the group consisting of TOM5, TOM6, TOM7, TOM22, Fis1, Bcl-2, Bcl-X and VAMP1B. An embodiment of the mitochondrial outer membrane anchoring peptide is as described above.

Within the scope of the invention, the term "active protein" may be a protein exhibiting physiological activity. One embodiment of such an active protein may be a reduced function protein or a modified protein present in damaged cancer cells. One embodiment of the active protein may be a protein that enhances cell activity. An embodiment of such an active protein is as described above.

The fusion protein may be a protein to which the mitochondrial outer membrane targeting protein and the desired protein are linked, from the N-terminus to the C-terminus. In this case, it may further contain ubiquitin or a fragment thereof having a specific ubiquitin protease (glycine-glycine) cleavage site between the mitochondrial outer membrane targeting protein and the desired protein. In this case, to facilitate degradation by a ubiquitin protease, it may further comprise a linker containing the hydrophilic and polar amino acids serine, glycine and threonine between the mitochondrial outer membrane targeting protein and the ubiquitin protein.

As used herein, the term "ubiquitin" refers to a protein involved in the proteolytic process, also referred to as UB. One embodiment of ubiquitin may be ubiquitin found in humans or ubiquitin found in yeast. Ubiquitin, present in the human body, consists of 76 amino acids. In this case, ubiquitin can be used in its mature form. As used herein, the term "mature form" may refer to the protein in a form from which the signal peptide has been removed.

Additionally, an enzyme designated as ubiquitin protease or UBP (ubiquitin-specific protease) is naturally present in eukaryotic cells and can induce natural dissociation of the desired protein by cleaving the C-terminal amino acid glycine-glycine region of ubiquitin in the cell.

In this case, the ubiquitin fragment may contain the amino acids Gly-Gly of the C-terminus of ubiquitin and may contain from 3 to 75 consecutive amino acids from the C-terminus. In particular, an embodiment of the ubiquitin moiety may be Arg-Gly-Gly, Leu-Arg-Gly-Gly, Arg-Leu-Arg-Gly-Gly, or Leu-Arg-Leu-Arg-Gly-Gly. In addition, the ubiquitin moiety may have the amino acid sequence of SEQ ID NO: 71.

A fusion protein comprising a mitochondrial outer membrane targeting protein and a desired protein can be referred to as a mitochondrial activity-modifying fusion protein. Such a fusion protein may have any of the following structures:

<Structural formula 1>

N-terminus - mitochondrial outer membrane-anchoring peptide - desired protein - C-terminus

<Structural formula 2>

N-terminus - mitochondrial outer membrane-anchoring peptide - ubiquitin or its fragment - desired protein - C-terminus

<Structural formula 3>

N-terminus-mitochondrial outer membrane targeting protein-linker 1-ubiquitin or its fragment-desired protein-C-terminus

<Structural formula 4>

N-terminus - peptide anchoring in the mitochondrial outer membrane - ubiquitin or its fragment - linker 2 - desired protein - C-terminus

<Structural formula 5>

N-terminus-peptide anchoring in the mitochondrial outer membrane-linker 1-ubiquitin or its fragment-linker 2-desired protein-C-terminus

In the above structural formulas 1 to 5, the outer membrane anchoring peptide may be a terminal sequence of a protein selected from the group consisting of TOM20, TOM70 and OM45, and the desired protein may be any protein selected from the group consisting of p53, granzyme B, Bax, Bak, PDCD5, E2F, AP-1 (Jun/Fos), EGR-1, retinoblastoma protein (RB), phosphatase and tensin homologue (PTEN), E-cadherin, neurofibromin-2 (NF-2) , poly-ADP-ribose synthase 1 (PARP-1), BRCA-1, BRCA-2, adenomatous polyposis coli protein (APC), tumor necrosis factor receptor-associated factor (TRAF), RAF kinase inhibitory protein ( RKIP), p16, KLF-10, LKB1, LHX6, C-RASSF and DKK-3PD1.

In this case, linker 1 or 2 may be a polypeptide of 1 to 100, 1 to 80, 1 to 50, or 1 to 30 amino acids, respectively, and preferably may be a polypeptide of 1 to 30 amino acids consisting of serine, glycine or threonine, alone or in combination. In addition, linker 1 or 2 may be a polypeptide consisting of 5 to 15 amino acids, respectively, and preferably may be a polypeptide consisting of 5 to 15 amino acids consisting of serine, glycine or threonine alone or in combination. One embodiment of the linker may be (GGGGS) ₃ (SEQ ID NO: 70).

<Structural formula 6>

N-terminus - desired protein-peptide anchored in the mitochondrial outer membrane - C-terminus

<Structural formula 7>

N-terminus - desired protein - ubiquitin or its fragment - peptide anchored in the mitochondrial outer membrane - C-terminus

<Structural formula 8>

N-terminus - desired linker protein 1-ubiquitin or its fragment - peptide anchoring in the mitochondrial outer membrane - C-terminus

<Structural formula 9>

N-terminus - desired protein - ubiquitin or its fragment - linker 2 - peptide anchoring in the mitochondrial outer membrane - C-terminus

<Structural formula 10>

N-terminus-desired linker protein 1-ubiquitin or its fragment-linker 2-peptide for mitochondrial outer membrane targeting-C-terminus

In the above structural formulas 6-10, the outer membrane anchoring peptide may be the terminal sequence of a protein selected from the group consisting of TOM5, TOM6, TOM7, TOM22, Fis1, Bcl-2, Bcl-X and VAMP1B, and the desired protein may be any protein selected from the group consisting of p53, granzyme B, Bax, Bak, PDCD5, E2F, AP-1 (Jun/Fos), EGR-1, retinoblastoma protein (RB), phosphatase and tensin homolog (PTEN) , E-cadherin, neurofibromin-2(NF-2), poly-ADP-ribose synthase 1 (PARP-1), BRCA-1, BRCA-2, adenomatous polyposis coli protein (APC), receptor-associated factor tumor necrosis factor (TRAF), RAF kinase inhibitory protein (RKIP), p16, KLF-10, LKB1, LHX6, C-RASSF, DKK-3PD1, Oct3/4, Sox2, Klf4 and c-Myc. In this case, linker 1 or 2 is as described above.

In one aspect, the present invention provides a polynucleotide encoding a fusion protein comprising a mitochondrial outer membrane anchoring peptide and a desired protein.

Additionally, in one aspect, the present invention provides a vector loaded with a polynucleotide encoding a fusion protein containing a desired protein.

Moreover, in one aspect, the present invention relates to a host cell into which a vector loaded with a polynucleotide encoding a fusion protein containing the desired protein is inserted.

In one aspect, the present invention provides a fusion protein comprising a target targeting protein and a mitochondrial outer membrane targeting protein.

In this case, the target targeting protein and the mitochondrial outer membrane anchoring peptide can be linked from the N-terminus to the C-terminus. In this case, the mitochondrial outer membrane anchoring peptide may be any peptide selected from the group consisting of TOM20, TOM70, OM45, TOM5, TOM6, TOM7, TOM22, Fis1, Bcl-2, Bcl-x and VAMP1B.

Within the scope of the invention, the term "target" refers to the location to which the modified mitochondria are to be delivered. One embodiment of the target may be a malignant cell. In particular, one embodiment of the target may be a biomarker located on the surface of malignant cells. In particular, the target may be a tumor-specific antigen (TAA). In this case, the tumor-specific antigen may be any antigen selected from the group consisting of CD19, CD20, melanoma-associated antigen E (MAGE), NY-ESO-1, carcinoembryonic antigen (CEA), mucin 1 associated with cell surface (MUC -1), prostatic acid phosphatase (PAP), prostate-specific antigen (PSA), survivin, tyrosinase-related protein 1 (tyrp1), tyrosinase-related protein 1 (tyrp2), brachyury gene, mesothelin, epidermal growth factor receptor (EGFR) ), human epidermal growth factor receptor 2 (HER-2), ERBB2, Wilms tumor protein (WT1), FAP, EpCAM, PD-L1, ACPP, CPT1A, IFNΓ, CD274, FOLR1, EPCAM, ICAM2, NCAM1, LRRC4, UNC5H2 LILRB2, CEACAM, nectin-3 and combinations thereof.

Within the scope of the invention, the term “target targeting protein” may mean a protein sequence capable of binding to the target described above. In this case, one embodiment of the target targeting protein may be a protein that binds to a biomarker located on the surface of cancer cells. In this case, an embodiment of the biomarker located on the surface of the malignant cells may be, but are not limited to, ICAM2, NCAM1, LRRC4, UNC5H2, LILRB2, CEACAM, or nectin-3. In this case, the target targeting protein can be included in the foreign protein described above.

One embodiment of the target targeting protein may be an antibody or fragment thereof. In particular, it may be an antibody or a fragment thereof that specifically binds to a tumor-specific antigen. In addition, the antibody fragment may be any fragment selected from the group consisting of Fab, Fab', scFv and F(ab)2.

One embodiment of the target targeting protein may be a scFvHER capable of binding to the epidermal growth factor receptor. Another embodiment may be scFvMEL, which can be used to target melanoma. Another embodiment may be scFvPD-L1 capable of binding to PD-L1 overexpressed on the surface of cancer cells. Another embodiment may be PD-1 capable of binding to PDL-1 overexpressed on the surface of cancer cells.

One aspect of the present invention may further comprise ubiquitin or a fragment thereof between the target targeting protein and the mitochondrial outer membrane targeting protein. A fusion protein comprising a mitochondrial target targeting protein and a desired protein can be referred to as a mitochondrial activity-modifying fusion protein. Such a fusion protein may have any of the following structures:

<Structural formula 11>

N-terminus-target targeting protein-mitochondrial outer membrane anchoring peptide-C-terminus

<Structural formula 12>

N-terminus - target targeting protein - ubiquitin or its fragment - peptide anchoring in the mitochondrial outer membrane - C-terminus

<Structural formula 13>

N-terminus-target targeting protein-linker 1-ubiquitin or its fragment-peptide anchoring in the mitochondrial outer membrane-C-terminus

<Structural formula 14>

N-terminus-target targeting protein-ubiquitin or its fragment-linker 2-peptide anchoring in the mitochondrial outer membrane-C-terminus

<Structural formula 15>

N-terminus-target targeting protein-linker 1-ubiquitin or its fragment-linker 2-peptide anchoring in the mitochondrial outer membrane-C-terminus

In the above structural formulas 11-15, the outer membrane anchoring peptide may be a terminal sequence of a protein selected from the group consisting of TOM5, TOM6, TOM7, TOM22, Fis1, Bcl-2, Bcl-X and VAMP1B, and a protein for The target targeting protein may be any protein selected from the group consisting of tumor-associated antigens, CD19, CD20, melanoma-associated antigen E (MAGE), NY-ESO-1, carcinoembryonic antigen (CEA), cell surface associated mucin 1 (MUC -1), prostatic acid phosphatase (PAP), prostate-specific antigen (PSA), survivin, tyrosinase-related protein 1 (tyrp1), tyrosinase-related protein 1 (tyrp2), brachyury gene, mesothelin, epidermal growth factor receptor (EGFR ), human epidermal growth factor receptor 2 (HER-2), ERBB2, Wilms tumor protein (WT1), FAP, EpCAM, PD-L1, ACPP, CPT1A, IFNΓ, CD274, FOLR1, EPCAM, ICAM2, NCAM1, LRRC4, UNC5H2 LILRB2, CEACAM, nectin-3 and combinations thereof. In addition, the target targeting protein may be an antibody that specifically binds to a tumor-associated antigen or a fragment thereof. In this case, the linker 1 or 2 and the amino acid sequence recognized by the proteolytic enzyme are as described above.

<Structural formula 16>

N-terminus-mitochondrial outer membrane-anchoring peptide-target targeting protein-C-terminus

<Structural formula 17>

N-terminus - mitochondrial outer membrane anchoring peptide - ubiquitin or its fragment - target targeting protein - C-terminus

<Structural formula 18>

N-terminus-mitochondrial outer membrane anchoring peptide-linker 1-ubiquitin or its fragment-target targeting protein-C-terminus

<Structural formula 19>

N-terminus - mitochondrial outer membrane anchoring peptide - ubiquitin or its fragment - linker 2 - target targeting protein - C-terminus

<Structural formula 20>

N-terminus-mitochondrial outer membrane anchoring peptide-linker 1-ubiquitin or its fragment-linker 2-target targeting protein-C-terminus

In the above structural formulas 16-20, the outer membrane anchoring peptide may be any peptide selected from the group consisting of TOM20, TOM70 and OM45. In addition, the target targeting protein, ubiquitin or a fragment thereof, and linker 1 or 2 are as described above.

In one aspect, the present invention provides a polynucleotide encoding a fusion protein comprising a protein for targeting a target.

Additionally, in one aspect, the present invention provides a vector loaded with a polynucleotide encoding a fusion protein comprising a target targeting protein.

Additionally, in one aspect, the present invention relates to a host cell into which a vector loaded with a polynucleotide encoding a fusion protein comprising a target targeting protein is inserted. The host cell may be a prokaryotic cell or a eukaryotic cell. In this case, preferably, the eukaryotic cell may be a strain from which the ubiquitin degrading enzyme has been removed.

Additionally, in one aspect, the present invention provides a method for producing modified mitochondria from transformed cells by injecting a polynucleotide encoding a fusion protein into eukaryotic cells.

METHOD FOR IMPLEMENTING THE INVENTION

Hereinafter, in order to facilitate understanding of the present invention, a preferred embodiment will be presented in the present description. However, the following examples are presented solely to facilitate understanding of the present invention, and the present invention is not limited to them.

I. Preparation of a fusion protein containing a mitochondrial outer membrane anchoring peptide, a linker, ubiquitin and the desired protein

Example 1: Preparation of a Fusion Protein Containing p53

Example 1.1. p53 gene amplification

To express human p53 in a recombinant protein, total RNA was isolated from human epithelial cells and cDNA was synthesized from it. Specifically, human dermal fibroblasts were cultured in 10% serum, 5% carbon dioxide, and 37°C (1 x 10 ⁶ cells). The culture solution was then removed, the cells were washed twice by adding PBS buffer, and 0.5 mL of RNA extract (TRIzol reagent, Thermo Fisher Scientific) was added directly. The mixture to which the RNA extract was added was left at ambient temperature for 10 minutes, and then 0.1 ml of chloroform was added and mixed for 15 seconds, and then centrifuged at approximately 12,000 x g for 10 minutes. The separated supernatant was then collected and the same volume of isopropyl alcohol was added and centrifuged again at 12,000×g for 10 minutes. The liquid was then removed and washed once with 75% ethanol, and the RNA was dried at ambient temperature.

Approximately 50 μl of RNase-free purified distilled water was added and the quantity and purity of RNA was measured using a spectrophotometer. For cDNA synthesis, 2 μg of purified total RNA was subjected to an oligo-dT coupling reaction at 70°C for 5 min. Then, 10× reverse transcription reaction buffer, 10 mM dNTP, RNase and M-MLV reverse transcriptase inhibitor (Enzynomics, Korea) were added, and the cDNA synthesis reaction was carried out at 42°C for 60 minutes. Next, reverse transcriptase was inactivated by heating at 72°C for 5 minutes, and then RNase H was added to remove single-stranded RNA, which was used as a template for polymerase chain reaction of the p53 gene.

To obtain the p53 gene from human dermal fibroblasts, from which the signal peptide sequence was removed, a primer (T2p53) encoding from the amino-terminal glutamic acid and a primer (Xp53) encoding from the carboxy-terminus were synthesized, and then PCR was carried out using cDNA, obtained as described above as a matrix. The sequence of each primer is presented in Table 1 below.

[Table 1]

Primer Subsequence SEQ ID NO. T2p53 5'-AAA AAA CCG CGG TGG TGA GGA GCC GCA GTC AGA TCC TAG-3' SEQ ID NO: 1 Xp53 5'- AAA AAA CTC GAG TGA GTC TGA GTC AGG CCC TTC TG-3' SEQ ID NO: 2

0.2 pmol of T2p53 primer and 0.2 pmol of Xp53 primer were mixed with 0.2 nM dNTP, 1× AccuPrime Taq DNA polymerase reaction buffer (Invitrogen, USA) and 1 unit. AccuPrime Taq DNA polymerases. Amplification reactions were then carried out in the polymerase chain reaction apparatus at 95°C for 40 seconds, 58°C for 30 seconds, 72°C for 1 minute for 40 cycles. After the reaction, the amplified DNA fragment of approximately 1.2 kb. isolated by electrophoresis on a 1% agarose gel and then inserted into the pGEM-T easy vector (Promega, USA) using T4 DNA ligase. By sequencing the DNA thus obtained, the cDNA encoding the human p53 protein was confirmed. The resulting p53 gene was designated pTA-p53, and its base sequence is the same as that of SEQ ID NO: 3 (FIG. 1).

Example 1.2. Preparation of the E. coli expression vector for p53

Example 1.2.1. Preparation of plasmid pET15b-UB-p53

To obtain the p53 protein in its ubiquitin-fused form, the following expression vector was prepared. To obtain the ubiquitin gene, primer NdeUB and primer T2UB were prepared. The sequence of each primer is shown in Table 2 below.

[Table 2]

Primer Subsequence SEQ ID NO. NdeUB 5'-GGA TTC CAT ATG CAA CTT TTC GTC AAA ACT CTA AC-3' SEQ ID NO: 4 T2UB 5'-ATG ACC ACC GCG GAG TCT CAA CAC CAA-3' SEQ ID NO: 5

0.2 pmol of NdeUB primer and 0.2 pmol of T2UB primer were mixed with 0.2 nM dNTP, 1× AccuPrime Taq DNA polymerase reaction buffer (Invitrogen, USA) and 1 unit. AccuPrime Taq DNA polymerases. Amplification reactions were then carried out in a polymerase chain reaction apparatus at 95°C for 40 seconds, 58°C for 30 seconds, 72°C for 1 minute for 25 cycles to obtain the ubiquitin (UB) gene. The amplified ubiquitin gene was digested with restriction enzymes NdeI and SacII, and plasmid pTA-p53 was digested with restriction enzymes SacII and XhoI. DNA fragments of approximately 210 bp are then and 1200 bp prepared by 2% agarose gel electrophoresis, respectively, and then inserted into the pET15b vector digested with NdeI and XhoI restriction enzymes using T4 DNA ligase to obtain plasmid pET15b-UB-p53 (Figure 2). In this case, UB-p53 is represented by the base sequence SEQ ID NO: 6.

E. coli strain BL21 (DE3) was transformed using plasmid pET15b-UB-p53. The transformed strain was then cultured in solid Luria-Bertani (LB) medium to which the antibiotic ampicillin was added, and the resulting colonies were then cultured in liquid LB medium at 37°C. Then, when the cell density reached an optical density of approximately 0.2 at OD600, IPTG was added to achieve a final concentration of 1 mM, followed by shaking culture for approximately 4 more hours.

Some E. coli cells were obtained by centrifugation, and then the cells were destroyed and electrophoresis was performed on SDS-PAGE. As shown in Figure 3, expression of the ubiquitin fusion protein p53, which is approximately 60 kDa in size, was confirmed. In this case, lane M in Figure 3 corresponds to the protein molecular weight marker, and lane 1 corresponds to the precipitate centrifuged after E. coli disruption 4 hours after the addition of IPTG, and lane 2 corresponds to the supernatant centrifuged after E. coli disruption.

Example 1.2.2. Preparation of plasmid pET11C-TOM70-UB-p53

To produce p53 protein in a form fused to TOM70, which binds to the mitochondrial outer membrane, and ubiquitin, an expression vector capable of expressing p53 in a form fused to TOM70 and ubiquitin was prepared. To obtain TOM70 and ubiquitin genes, primer NdeTOM70, primer TOM70-AS, primer TOM70UB-S and primer T2UB-AS were prepared. The sequence of each primer is shown in Table 3 below.

[Table 3]

Primer Subsequence SEQ ID NO. NdeTOM70 5'-GAA TTC CAT ATG AAA AGT TTT ATA ACT CGG AAT AAA ACT GCA ATT TTC GCA ACT GTT GC -3' SEQ ID NO: 7 TOM70-AS 5'- GGT GCA TAC TAC TAT TAT CAAA CTT TTC GTC AAA ACT C-3' SEQ ID NO: 8 TOM70UB-S 5'- GGC TAC GT ATT TAT TTC CAA CTT TTC GTC AAA ACT C-3' SEQ ID NO: 9 T2UB-AS 5'- GGC ACC ACC GCG GAG TCT CAA CAC 3' SEQ ID NO: 10

To obtain the TOM70 gene, 0.2 pmol of the NdeTOM70 primer and 0.2 pmol of the TOM70-AS primer were mixed with 0.2 nM dNTP, 1x reaction buffer solution for AccuPrime Taq DNA polymerase (Invitrogen, USA) and 1 unit. AccuPrime Taq DNA polymerases. Then, to obtain the TOM70 gene, amplification reactions were carried out in a polymerase chain reaction apparatus at 95°C for 40 seconds, 58°C for 30 seconds, 72°C for 1 minute for 25 cycles. The amplified DNA fragment was designated N-TOM70. Plasmid pET15b-UB-p53 obtained in example 1.2.1. above, used as a template, added 0.2 pmol of primer TOM70UB-S and 0.2 pmol of primer T2UB-AS and mixed with 0.2 nM dNTP, 1x reaction buffer solution for AccuPrime Taq DNA polymerase (Invitrogen, USA ) and 1 unit. AccuPrime Taq DNA polymerases. Then, to obtain the UB gene, amplification reactions were carried out in a polymerase chain reaction apparatus at 95°C for 40 seconds, 58°C for 30 seconds, 72°C for 1 minute for 25 cycles. The amplified DNA fragment was designated C-UB.

The amplified DNA of N-TOM70 and C-UB was used as a template, and 0.2 pmol NdeTOM70 primer, 0.2 pmol T2UB-AS primer was mixed with 0.2 nM dNTP, 1× AccuPrime Taq DNA polymerase reaction buffer (Invitrogen, USA) and 1 unit. AccuPrime Taq DNA polymerases. Then, to obtain the TOM70-UB ubiquitin gene to which the amplified TOM70 is fused, amplification reactions were carried out in a polymerase chain reaction apparatus at 95°C for 40 seconds, 58°C for 30 seconds, 72°C for 1 minute for 25 cycles.

The amplified TOM70-UB gene was digested with restriction enzymes NdeI and SacII, plasmid pTA-p53 was digested with SacII and XhoI to obtain 330 bp DNA fragments. and 1500 bp by electrophoresis in 2% agarose gel, respectively. They were then inserted into the NdeI and SalI restriction enzyme-digested vector pET11c using T4 DNA ligase to produce plasmid pET11c-TOM70-UB-p53 (Figure 4). In this case, TOM70-UB-p53 is represented by the base sequence SEQ ID NO: 11.

E. coli strain BL21 (DE3) was transformed using plasmid pET11c-TOM70-UB-p53. The transformed strain was then cultured in solid Luria-Bertani (LB) medium to which the antibiotic ampicillin was added, and then the resulting colonies were cultured in liquid LB medium in a shaking incubator at 37°C. Then, when the cell density reached an optical density of approximately 0.2 at OD600, IPTG was added to achieve a final concentration of 1 mM, followed by shaking culture for approximately 4 more hours.

Some E. coli cells were obtained by centrifugation, and then the cells were destroyed and electrophoresis was performed on SDS-PAGE. As shown in Figure 5, p53 protein, which is approximately 62 kDa in size, was confirmed to be expressed in a form to which TOM70 and ubiquitin are fused. In this case, lane M corresponds to the protein molecular weight marker, and lane 1 corresponds to the supernatant centrifuged after E. coli disruption 4 hours after the addition of IPTG.

Example 1.2.3. Preparation of plasmid pET11c-TOM70-(GGGGS)3-UB-p53

To obtain the p53 protein in the form to which TOM70, the mitochondrial outer membrane binding, the linker (GGGGSGGGGSGGGGS (SEQ ID NO: 70)) and ubiquitin are fused, an expression vector capable of expressing the p53 protein in the form to which the TOM70, the linker and ubiquitin. To obtain the linker gene associated with TOM70, primer TOM70(G)3-AS, primer (G)3UB-S, and primer Xp53 (noT) were prepared. The sequence of each primer is shown in Table 4 below.

[Table 4]

Primer Subsequence SEQ ID NO. TOM70(G) ₃ -AS 5'-GCC CCC GGA TCC TCC ACC CCC GCT TCC GCC ACC TCC ATA ATA GT AGT ATG CAC CAA TAG -3' SEQ ID NO: 12 (G) ₃ UB-S 5'-GGT GGA GGA TCC GGG GGC GC GGA AGC CAA ATC-3' SEQ ID NO: 13 Xp53(noT) 5'- AAA AAA CTC GAG GTC TGA GTC AGG CCC TTC TG-3' SEQ ID NO: 14

Plasmid pET11c-TOM70-UB-p53 obtained in example 1.2.2. above, was used as a template, and added 0.2 pmol of NdeTOM70 primer and 0.2 pmol of TOM70(G)3-AS primer, and mixed with 0.2 nM dNTP, 1x AccuPrime Taq DNA polymerase reaction buffer (Invitrogen, USA) and 1 unit. AccuPrime Taq DNA polymerases. Then, to obtain the TOM70-G3 gene in which the TOM70 gene and the linker are linked, amplification reactions were carried out in a polymerase chain reaction apparatus at 95°C for 40 seconds, 58°C for 30 seconds, 72°C for 1 minute at for 25 cycles. In addition, the plasmid pET15b-UB-p53 obtained in example 1.2.1. above, was used as a template and mixed 0.2 pmol primer (G)3UB-S and 0.2 pmol primer Xp53 (noT) with 0.2 nM dNTPs, 1× AccuPrime Taq DNA polymerase reaction buffer (Invitrogen , USA) and 1 unit. AccuPrime Taq DNA polymerases.

Then, to obtain UB-p53, p53 fused to the ubiquitin gene, amplification reactions were carried out in a polymerase chain reaction apparatus at 95°C for 40 seconds, 58°C for 30 seconds, 72°C for 1 minute for 25 cycles. The amplified TOM70-G3 gene was digested with restriction enzymes NdeI and BamHI, the amplified UB-p53 gene was digested with BamHI and XhoI, and 100 bp DNA fragments were digested. and 1500 bp obtained by electrophoresis in 2% agarose gel, respectively. They were then inserted into the vector pET11c, digested with NdeI and SalI restriction enzymes using T4 DNA ligase, to obtain the plasmid pET11c-TOM70-(GGGGS)3-UB-p53 (Figure 6). In this case, TOM70-(GGGGS)3-UB-p53 is represented by the base sequence SEQ ID NO: 15.

E. coli strain BL21 (DE3) was transformed using the plasmid pET11c-TOM70-(GGGGS)3-UB-p53. The transformed strain was then cultured in solid Luria-Bertani (LB) medium to which the antibiotic ampicillin was added, and the resulting colonies were then cultured in liquid LB medium at 37°C. Then, when the cell density reached an optical density of approximately 0.2 at OD600, IPTG was added to achieve a final concentration of 1 mM, followed by shaking culture for approximately 4 more hours.

Some E. coli cells were obtained by centrifugation, and then the cells were destroyed and electrophoresis was performed on SDS-PAGE. As shown in Figure 7, it was confirmed that p53 protein, having a size of approximately 62 kDa, was expressed in a form to which TOM70, linker and ubiquitin were fused. In this case, lane M corresponds to the protein molecular weight marker, lane 1 corresponds to the precipitate centrifuged after E. coli disruption 4 hours after the addition of IPTG, and lane 2 corresponds to the supernatant centrifuged after E. coli disruption.

Example 1.2.4. Preparation of plasmid pET11c-TOM70-(GGGGS)3-p53

To obtain the p53 protein in the form to which TOM70, which binds to the mitochondrial outer membrane, and the linker are fused (GGGGSGGGGSGGGGS), an expression vector capable of expressing the p53 protein in the form to which TOM70 and the linker are fused was prepared. To obtain the p53 gene, to which TOM70 and the linker are fused, a primer (B(G)3p53) was obtained. The sequence of each primer is shown in Table 5 below.

[Table 5]

Primer Subsequence SEQ ID NO. B(G) ₃ p53 5'-GGT GGA GGA TCC GGG GGC GGC GGA AGC GAG GAG CCG CAG TCA GAT CCT AGC-3' SEQ ID NO: 16

Plasmid pET11c-TOM70-UB-p53 obtained in example 1.2.2. above, used as a template, added 0.2 pmol NdeTOM70 primer and 0.2 pmol TOM70(G)3-AS primer and mixed with 0.2 nM dNTP, 1× AccuPrime Taq DNA polymerase reaction buffer (Invitrogen , USA) and 1 unit. AccuPrime Taq DNA polymerases. Then, to obtain the TOM70 gene, amplification reactions were carried out in a polymerase chain reaction apparatus at 95°C for 40 seconds, 58°C for 30 seconds, 72°C for 1 minute for 25 cycles. The amplified DNA fragment was designated TOM70-G3.

Plasmid pET15b-UB-p53 obtained in example 1.2.1. above, was used as a template, and 0.2 pmol of B(G)3p53 primer and 0.2 pmol of Xp53 (noT) primer were mixed with 0.2 nM dNTPs, 1× AccuPrime Taq DNA polymerase reaction buffer (Invitrogen , USA) and 1 unit. AccuPrime Taq DNA polymerases. Amplification reactions were then carried out in the polymerase chain reaction apparatus at 95°C for 40 seconds, 58°C for 30 seconds, 72°C for 1 minute for 25 cycles. The amplified DNA fragment was designated G3-p53.

The amplified DNA fragment, TOM70-G3, was digested with NdeI and BamHI, and the DNA fragment G3-53 was digested with restriction enzymes BamHI and XhoI. Then DNA fragments of approximately 150 bp in size. and 1300 bp obtained by electrophoresis in 2% agarose gel, respectively, and then inserted into the vector pET11c, digested with restriction enzymes NdeI and SalI using T4 DNA ligase to obtain plasmid pET11c-TOM70-(GGGGS)3-p53 (Figure 8). In this case, TOM70-(GGGGS)3-p53 is represented by the base sequence SEQ ID NO: 17.

E. coli strain BL21(DE3) was transformed using the plasmid pET11c-TOM70-(GGGGS)3-p53. The transformed strain was then cultured in solid Luria-Bertani (LB) medium to which the antibiotic ampicillin was added, and the resulting colonies were cultured in liquid LB medium in a shaking incubator at 37°C. Then, when the cell density reached an optical density of approximately 0.2 at OD600, IPTG was added to achieve a final concentration of 1 mM, followed by shaking culture for approximately 4 more hours.

Some E. coli cells were obtained by centrifugation, and then the cells were destroyed and electrophoresis was performed on SDS-PAGE. As shown in Figure 9, expression of the approximately 60 kDa p53 protein in the TOM70 fused form was confirmed. In this case, lane M corresponds to the protein molecular weight marker, lane 1 corresponds to the precipitate centrifuged after E. coli disruption 4 hours after the addition of IPTG, and lane 2 corresponds to the supernatant centrifuged after E. coli disruption.

Example 1.2.5. pET15b-UB-p53-TOM7

To produce p53 protein in a ubiquitin-TOM7 fused form that binds to the mitochondrial outer membrane, an expression vector capable of expressing p53 in a ubiquitin-p53-TOM fused form was prepared. To obtain the p53 gene to which TOM7 and ubiquitin are fused, primer Xp53(noT), primer XTOM7 and primer LTOM7 were prepared. The sequence of each primer is shown in Table 6 below.

[Table 6]

Primer Subsequence SEQ ID NO. Xp53(noT) 5'-AAA AAA CTC GAG GTC TGA GTC AGG CCC TTC TG -3' SEQ ID NO: 18 XTOM7 5'-AAA AAA CTC GAG ttt gcc att cgc tgg ggc ttt atc-3' SEQ ID NO: 19 LTOM7 5'-AAA AAA GTC GAC TTA TCC CCA AAG TAG GCT CAA AAC AG-3' SEQ ID NO: 20

Plasmid pET15b-UB-p53 obtained in example 1.2.1. above, used as a template, added 0.2 pmol of NdeUB primer and 0.2 pmol of Xp53(noT) primer and mixed with 0.2 nM dNTP, 1x reaction buffer solution for AccuPrime Taq DNA polymerase (Invitrogen, USA) and 1 unit. AccuPrime Taq DNA polymerases. Then, to obtain the UB-p53 gene, amplification reactions were carried out in a polymerase chain reaction apparatus at 95°C for 40 seconds, 58°C for 30 seconds, 72°C for 1 minute for 25 cycles. In addition, the cDNA obtained as described above was used as a template, and 0.2 pmol of XTOM7 primer and 0.2 pmol of LTOM7 primer were mixed with 0.2 nM dNTP, 1× AccuPrime Taq DNA polymerase reaction buffer ( Invitrogen, USA) and 1 unit. AccuPrime Taq DNA polymerases.

Then, to obtain the TOM7 gene, amplification reactions were carried out in a polymerase chain reaction apparatus at 95°C for 40 seconds, 58°C for 30 seconds, 72°C for 1 minute for 40 cycles. The amplified DNA fragment, UB-p53, was digested with restriction enzymes NdeI and XhoI, and the amplified TOM7 gene was digested with restriction enzymes XhoI and SalI. DNA fragments approximately 1500 bp in size. and 150 bp obtained by electrophoresis in 2% agarose gel, respectively, and then inserted into the vector pET15b, digested with restriction enzymes NdeI and XhoI, using T4 DNA ligase to obtain plasmid pET15b-UB-p53-TOM7 (Figure 10). In this case, UB-p53-TOM7 is represented by the base sequence SEQ ID NO: 21.

E. coli strain BL21 (DE3) was transformed using plasmid pET15b-UB-p53-TOM7. The transformed strain was then cultured in solid Luria-Bertani (LB) medium to which the antibiotic ampicillin was added, and the resulting colonies were then cultured in liquid LB medium at 37°C. Then, when the cell density reached an optical density of approximately 0.2 at OD600, IPTG was added to achieve a final concentration of 1 mM, followed by shaking culture for approximately 4 more hours.

Some E. coli cells were obtained by centrifugation, and then the cells were destroyed and electrophoresis was performed on SDS-PAGE. As shown in Figure 11, expression of the approximately 60 kDa p53 protein was confirmed in a ubiquitin-TOM7 fused form. In this case, lane M corresponds to the protein molecular weight marker, lane 1 corresponds to the precipitate centrifuged after E. coli disruption 4 hours after the addition of IPTG, and lane 2 corresponds to the supernatant centrifuged after E. coli disruption.

Example 1.2.6. Construction of the mammalian expression vector pCMV-p53-myc/His

An animal cell expression vector capable of expressing p53 was prepared. To obtain the p53 gene, primer Rp53 was prepared. The sequence of each primer is shown in Table 7 below.

[Table 7]

Primer Subsequence SEQ ID NO. Rp53 5'-AAA AAA GAA TTC ATG GTC TGA GTC AGG CCC TTC TG -3' SEQ ID NO: 23

Plasmid pET-UB-p53 obtained in example 1.2.1. above, was used as a template and mixed 0.2 pmol of Rp53 primer and 0.2 pmol of Xp53(noT) primer with 0.2 nM dNTP, 1x AccuPrime Taq DNA polymerase reaction buffer (Invitrogen, USA) and 1 units AccuPrime Taq DNA polymerases. Amplification reactions were then carried out in a polymerase chain reaction apparatus at 95°C for 40 seconds, 58°C for 30 seconds, 72°C for 1 minute for 25 cycles to obtain the p53 gene.

The amplified p53 gene was digested with restriction enzymes EcoRI and XhoI, and a DNA fragment of approximately 1300 bp was obtained. was prepared by 2% agarose gel electrophoresis and then inserted into the pcDNA3.1-myc/His A vector digested with EcoRI and XhoI using T4 DNA ligase to obtain plasmid pCMV-p53-myc/His ( figure 12). In this case, p53-myc/His is represented by the base sequence SEQ ID NO: 23.

CHO animal cells were transfected with the pCMV-p53-myc/His plasmid, the cells were disrupted, and then subjected to SDS-PAGE and analyzed by Western blotting using an anti-c-myc antibody. As shown in Figure 13, expression of the p53 protein, which is approximately 55 kDa in size, was confirmed. In this case, lane M corresponds to the protein molecular weight marker, and lane 1 shows that CHO animal cells were transfected and the cells were disrupted and then subjected to SDS-PAGE and then confirmed by Western blotting using anti-c- myc.

Example 1.3. Isolation and purification of a fusion protein containing p53

Example 1.3.1. Isolation and purification of recombinant protein TOM70-(GGGGS)3-p53 obtained from E. coli

The production E. coli strain BL21(DE3), expressing the recombinant protein TOM70-(GGGGS)3-p53, was inoculated into liquid LB medium and cultured at 37°C. Then, when the optical density reached 0.4 at OD600, 0.5 mM IPTG was added, and shaking culture was carried out for another 4 hours to express TOM70-(GGGGS)3-p53 protein.

After completion of culture, the cells were isolated by centrifugation and the isolated cells were washed once using PBS, and then the cells were suspended using a PBS solution and the suspended cells were disrupted using an ultrasonic homogenizer. The disrupted cells were centrifuged using a high-speed centrifuge, and then the insoluble fractions were isolated and the isolated insoluble fractions were washed three times using 50 mM Tris, 100 mM ethylenediaminetetraacetic acid (EDTA), pH 8.0. They were then dissolved in a solution of 6 M guanidine, 100 mM sodium phosphate, 10 mM Tris, pH 8.0, and filtered using a 0.45 μm filter, and then loaded onto a packed nickel chromatography column to perform primary purification.

A solution containing the TOM70-(GGGGS)3-p53 protein was loaded and then a wash solution of 8 M urea, 50 mM sodium phosphate, 500 mM NaCl, 10 mM imidazole, pH 8.0 was passed through until no unbound impurities will be undetectable, and the protein was eluted using 8 M urea, 50 mM sodium phosphate, 500 mM NaCl, 500 mM imidazole, pH 8.0, varying the imidazole concentration to 50 mM, 100 mM, 250 mM, 500 mM (Figure 14). In this case, lane M in Figure 14 corresponds to the protein molecular weight marker, and lane 1 corresponds to the loaded sample for nickel affinity chromatography. Lane 2 shows that it did not bind to the nickel affinity resin. Lanes 3-4 correspond to the elution results using a solution of 8 M urea/50 mM Na phosphate/500 mM NaCl/50 mM imidazole. Lanes 5-7 correspond to the elution results using a solution of 8 M urea/50 mM Na phosphate/500 mM NaCl/100 mM imidazole. Lanes 8-9 correspond to the elution results using a solution of 8 M urea/50 mM Na phosphate/500 mM NaCl/250 mM imidazole. Lanes 10-11 correspond to the elution results using a solution of 8 M urea/50 mM Na phosphate/500 mM NaCl/500 mM imidazole.

The eluted solution isolated by nickel chromatography was subjected to buffer exchange with PBS using the principle of osmotic pressure. After completion of the buffer exchange, the eluted solution was centrifuged to recover the supernatant, and the amount of protein in the recovered eluted solution was measured by protein quantitation method and confirmed using SDS-PAGE. As shown in Figure 15, after confirmation, the TOM70-(GGGGS)3-p53 protein was rapidly cooled with liquid nitrogen and stored in a cryogenic freezer at -80°C. In this case, lane M corresponds to the protein molecular weight marker, and lane 1 corresponds to the TOM70-(GGGGS)3-p53 protein obtained after dialysis in PBS buffer.

Example 1.3.2. Isolation and purification of recombinant protein TOM70-(GGGGS)3-UB-p53 obtained from E. coli

E. coli expressing the recombinant TOM70-(GGGGS)3-UB-p53 protein was used to isolate and purify the TOM70-(GGGGS)3-UB-p53 protein in the same manner as in Example 1.3.1. As a result, the TOM70-(GGGGS)3-UB-p53 protein was eluted (Figure 16). In this case, lane M in Figure 16 corresponds to the protein molecular weight marker, and lane 1 corresponds to the loaded sample for nickel affinity chromatography. In track 2 you can see that it is not bound to the nickel affinity resin. Lane 3 corresponds to the elution results using 8 M urea/50 mM Na phosphate/500 mM NaCl/50 mM imidazole. Lanes 4-7 correspond to the elution results using a solution of 8 M urea/50 mM Na phosphate/500 mM NaCl/100 mM imidazole. Lanes 8-11 correspond to the elution results using a solution of 8 M urea/50 mM Na phosphate/500 mM NaCl/250 mM imidazole.

The amount of protein in the recovered eluted solution was measured by protein quantitation and confirmed using SDS-PAGE. As shown in Figure 17, after confirmation, the TOM70-(GGGGS)3-UB-p53 protein was rapidly cooled with liquid nitrogen and stored in a cryogenic freezer at -80°C. In this case, lane M in Figure 17 corresponds to the protein molecular weight marker, and lane 1 shows the TOM70-(GGGGS)3-UB-p53 protein obtained after dialysis in PBS buffer.

Example 1.3.3. Isolation and purification of recombinant UB-p53 protein derived from E. coli

The production strain BL21 (DE3), expressing the ubiquitin-fused mature form of UB-p53 protein, was inoculated into liquid LB medium and cultured in a shaking incubator at 37°C. When the optical density reached 0.4 at OD600, 0.5 mM IPTG was added and shaking culture was carried out for another 4 hours to express the ubiquitin-fused mature form of UB-p53 protein.

The UB-p53 protein was then isolated and purified using the same method as in Example 1.3.1. As a result, the UB-p53 protein was eluted (Figure 18). In this case, lane M in Figure 18 corresponds to the protein molecular weight marker, and lane 1 corresponds to the loaded sample for nickel affinity chromatography. In track 2 you can see that it is not bound to the nickel affinity resin. Lane 3 corresponds to the elution results using 8 M urea/50 mM Na phosphate/500 mM NaCl/50 mM imidazole. Lanes 4-6 correspond to the elution results using a solution of 8 M urea/50 mM Na phosphate/500 mM NaCl/100 mM imidazole. Lanes 7-9 correspond to the elution results using a solution of 8 M urea/50 mM Na phosphate/500 mM NaCl/250 mM imidazole. Lanes 10-11 correspond to the elution results using a solution of 8 M urea/50 mM Na phosphate/500 mM NaCl/500 mM imidazole.

The amount of protein in the recovered eluted solution was measured by protein quantitation and confirmed using SDS-PAGE. As shown in Figure 19, after confirmation, the UB-p53 protein was rapidly cooled with liquid nitrogen and stored in a cryogenic freezer at -80°C. In this case, lane M in Figure 19 corresponds to the molecular weight marker of the proteins, and lane 1 shows the UB-p53 protein obtained after dialysis in PBS buffer.

Example 1.3.4. Isolation and purification of recombinant protein UB-p53-TOM7 obtained from E. coli

Production strain E. coli BL21 (DE3), expressing the UB-p53-TOM7 protein in a mature form to which ubiquitin is fused, was inoculated into liquid LB medium and cultured at 37°C. When the optical density reached 0.4 at OD600, 0.5 mM IPTG was added and shaking culture was carried out for another 4 hours to express the UB-p53-TOM7 protein in the mature ubiquitin-fused form.

The UB-p53-TOM7 protein was then isolated and purified using the same method as in Example 1.3.1. As a result, the UB-p53-TOM7 protein was eluted (Figure 20). In this case, lane M in Figure 20 corresponds to the protein molecular weight marker, and lane 1 corresponds to the loaded sample for nickel affinity chromatography. In track 2 you can see that it is not bound to the nickel affinity resin. Lane 3 corresponds to the elution results using 8 M urea/50 mM Na phosphate/500 mM NaCl/10 mM imidazole. Lane 4 corresponds to the elution results using 8 M urea/50 mM Na phosphate/500 mM NaCl/50 mM imidazole. Lanes 5-7 correspond to the elution results using a solution of 8 M urea/50 mM Na phosphate/500 mM NaCl/100 mM imidazole. Lanes 8-9 correspond to the elution results using a solution of 8 M urea/50 mM Na phosphate/500 mM NaCl/250 mM imidazole. Lanes 10-11 correspond to the elution results using a solution of 8 M urea/50 mM Na phosphate/500 mM NaCl/500 mM imidazole.

The amount of protein in the recovered eluted solution was measured by protein quantitation and confirmed using SDS-PAGE. As shown in Figure 21, after confirmation, the UB-p53 protein was rapidly cooled with liquid nitrogen and stored in a cryogenic freezer at -80°C. In this case, lane M in Figure 21 corresponds to the protein molecular weight marker, and lane 1 shows the UB-p53-TOM7 protein obtained after dialysis in PBS buffer.

Example 2: Preparation of a fusion protein containing granzyme B

Example 2.1. Granzyme B gene amplification

To express human granzyme B in a recombinant protein, total RNA was isolated from human natural killer cells and cDNA was synthesized from it. In particular, human natural killer cells were cultured in a medium with 10% serum under conditions of 5% carbon dioxide and 37°C (1×10 ⁶ cells). RNA was then obtained in the same way as in example 1.1, and then it was used as a template for the polymerase chain reaction of the granzyme B gene.

To obtain the granzyme B gene, from which the signal peptide sequence has been removed in human natural killer cells, primer T2GZMB, encoding from the amino-terminal isoleucine, and primer XGZMB(noT), encoding from the carboxy-terminus, were synthesized, and then PCR was carried out using the cDNA obtained , as described above, as a matrix. The sequence of each primer is shown in Table 8 below.

[Table 8]

Primer Subsequence SEQ ID NO. T2GZMB 5'-AAA AAA CCG CGG TGG TAT CAT CGG GGG ACA TGA GGC ACA TGA GGC CAA GCC -3' SEQ ID NO: 24 XGZMB(noT) 5'-AAA AAA CTC GAG GTA GCG TTT CAT GGT TTT CTT TAT CC-3' SEQ ID NO: 25

The cDNA prepared as described above was used as a template and 0.2 pmol of T2GZMB primer and 0.2 pmol of XGZMB(noT) primer were mixed with 0.2 nM dNTPs, 1× AccuPrime Taq DNA polymerase reaction buffer ( Invitrogen, USA) and 1 unit. AccuPrime Taq DNA polymerases. Amplification reactions were then carried out in the polymerase chain reaction apparatus at 95°C for 40 seconds, 58°C for 30 seconds, 72°C for 1 minute for 40 cycles. After the reaction, an amplified DNA fragment of approximately 700 bp in size. isolated by 1% agarose gel electrophoresis and then inserted into the pGEM-T easy vector (Promega, USA) using T4 DNA ligase. By sequencing the DNA thus obtained, the cDNA encoding the human granzyme B protein was confirmed. The resulting granzyme B gene was designated as pTA-granzyme B, and the granzyme B gene is represented by the base sequence of SEQ ID NO: 26 (FIG. 22).

Example 2.2. Preparation of E. coli expression vector for granzyme B protein

Example 2.2.1. Preparation of plasmid pET11c-TOM70-(GGGGS)3-UB-granzyme B

To produce granzyme B protein in the form to which TOM70, mitochondrial outer membrane binding, linker (GGGGSGGGGSGGGGS) and ubiquitin are fused, an expression vector capable of expressing granzyme B in the form to which TOM70, linker and ubiquitin is fused was prepared.

Plasmid pTA-granzyme B obtained in example 2.1. above, was digested with the restriction enzymes SacII and XhoI, and a DNA fragment of approximately 700 bp in size. obtained by electrophoresis in 2% agarose gel. It was then inserted into the vector pET11c-TOM70-(GGGGS)3-UB-(p53), digested with the restriction enzymes SacII and XhoI, using T4 DNA ligase to obtain the plasmid pET11c-TOM70-(GGGGS)3-UB-granzyme B (SEQ ID NO: 27) (FIG. 23).

E. coli strain BL21 (DE3) was transformed using the plasmid pET11c-TOM70-(GGGGS)3-UB-granzyme B. The transformed strain was then cultured in solid Luria-Bertani (LB) medium, to which the antibiotic ampicillin was added, and then the resulting colonies were cultured in liquid LB medium in a shaking incubator at 37°C. Then, when the cell density reached an optical density of approximately 0.2 at OD600, IPTG was added to achieve a final concentration of 0.5 mM, followed by shaking culture for approximately 4 more hours.

Some E. coli cells were obtained by centrifugation, and then the cells were destroyed and electrophoresis was performed on SDS-PAGE. As shown in Figure 24, it was confirmed that granzyme B protein, having a size of approximately 35 kDa, was expressed in a form to which TOM70, linker and ubiquitin were fused. In this case, lane M in Figure 24 corresponds to the protein molecular weight marker, lane 1 corresponds to the precipitate centrifuged after E. coli disruption 4 hours after the addition of IPTG, and lane 2 corresponds to the supernatant centrifuged after E. coli disruption.

Example 2.2.2. Preparation of plasmid pET15b-UB-granzyme B-TOM7

To produce granzyme B protein in a ubiquitin-TOM7 fused form that binds to the mitochondrial outer membrane, an expression vector capable of expressing granzyme B protein in a ubiquitin-granzyme B-TOM7 fused form was prepared.

Plasmid pTA-granzyme B obtained in example 2.1. above, was digested with the restriction enzymes SacII and XhoI, and a DNA fragment of approximately 700 bp in size. obtained by electrophoresis in 2% agarose gel. It was then inserted into the SacII and XhoI digested vector pET15b-UB-(p53)-TOM7 using T4 DNA ligase to produce the plasmid pET15b-UB-granzyme B-TOM7 (Figure 25). In this case, the UB granzyme B-TOM7 is represented by the base sequence SEQ ID NO: 28.

E. coli strain BL21 (DE3) was transformed using the plasmid pET15b-UB-granzyme B-TOM7. The transformed strain was then cultured in solid Luria-Bertani (LB) medium to which the antibiotic ampicillin was added, and then the resulting colonies were cultured in liquid LB medium at 37°C. Then, when the cell density reached an optical density of approximately 0.2 at OD600, IPTG was added to achieve a final concentration of 0.5 mM, followed by shaking culture for approximately 4 more hours.

Some E. coli cells were obtained by centrifugation, and then the cells were destroyed and electrophoresis was performed on SDS-PAGE. As shown in Figure 26, it was confirmed that granzyme B protein, having a size of approximately 35 kDa, was expressed in a form to which ubiquitin and TOM70 were fused. In this case, lane M in Figure 26 corresponds to the protein molecular weight marker, lane 1 corresponds to the precipitate centrifuged after E. coli disruption 4 hours after the addition of IPTG, and lane 2 corresponds to the supernatant centrifuged after E. coli disruption.

Example 2.3. Isolation and purification of recombinant protein TOM70-(GGGGS)3-UB-granzyme B obtained from E. coli

The TOM70-(GGGGS)3-UB-granzyme B protein was isolated and purified using the same method as in Example 1.3.1. As a result, the TOM70-(GGGGS)3-UB-granzyme B protein was eluted (Figure 27). In this case, lane M in Figure 27 corresponds to the protein molecular weight marker, and lane 1 corresponds to the loaded sample for nickel affinity chromatography. In track 2 you can see that it is not bound to the nickel affinity resin. Lanes 3 and 4 correspond to elution results using 8 M urea/50 mM Na phosphate/500 mM NaCl/50 mM imidazole. Lanes 5-7 correspond to the elution results using a solution of 8 M urea/50 mM Na phosphate/500 mM NaCl/100 mM imidazole. Lanes 8-9 correspond to the elution results using a solution of 8 M urea/50 mM Na phosphate/500 mM NaCl/250 mM imidazole.

The amount of protein in the recovered eluted solution was measured by protein quantitation and confirmed using SDS-PAGE. As shown in Figure 28, after confirmation, the TOM70-(GGGGS)3-UB-granzyme B protein was rapidly cooled with liquid nitrogen and stored in a cryogenic freezer at -80°C. In this case, lane M in Figure 28 corresponds to the protein molecular weight marker, and lane 1 shows the TOM70-(GGGGS)3-UB-granzyme B protein obtained after dialysis in PBS buffer.

Example 3: Preparation of a Fusion Protein Containing RKIP

Example 3.1. RKIP gene amplification

To express the gene for the recombinant human RKIP protein (Raf kinase inhibitory protein), total RNA was isolated from human epithelial cells and cDNA was synthesized from it. Human dermal fibroblasts were cultured in 10% serum, 5% carbon dioxide and 37°C (1 x 10 ⁶ cells). RNA was then obtained in the same way as in example 1.1, and then used as a template for the polymerase chain reaction of the RKIP gene.

To obtain the RKIP gene from human dermal fibroblasts, from which the signal peptide sequence was removed, primer T2RKIP, encoding from the amino-terminal proline, and primer XRKIP(noT), encoding from the carboxy-terminus, were synthesized, and then PCR was carried out using the cDNA obtained as above, as a matrix. The sequence of each primer is shown in Table 9 below.

[Table 9]

Primer Subsequence SEQ ID NO. T2RKIP 5'-AAA AAA CCG CGG TGG Tcc ggt gga cct cag caa gtg gtc-3' SEQ ID NO: 29 XRKIP(noT) 5'- AAA AAA CTC GAG CTT CCC AGA CAG CTG CTC GTA CAG TTT GG-3' SEQ ID NO: 30

The cDNA obtained as described above was used as a template, and 0.2 pmol of T2RKIP primer and 0.2 pmol of XRKIP(noT) primer were mixed with 0.2 nM dNTPs, 1× AccuPrime Taq DNA polymerase reaction buffer (Invitrogen, USA) and 1 unit. AccuPrime Taq DNA polymerases. Amplification reactions were then carried out in the polymerase chain reaction apparatus at 95°C for 40 seconds, 58°C for 30 seconds, 72°C for 1 minute for 40 cycles. After the reaction, an amplified DNA fragment of approximately 560 bp was obtained. isolated by 1% agarose gel electrophoresis and then inserted into the pGEM-T easy vector (Promega, USA) using T4 DNA ligase. By sequencing the DNA thus obtained, the cDNA encoding the human RKIP protein was confirmed. The resulting RKIP gene was designated pTA-RKIP (Figure 29), and the base sequence of the RKIP gene is represented by the base sequence of SEQ ID NO: 31.

Example 3.2. Preparation of an E. coli expression vector for the RKIP protein

Example 3.2.1. Preparation of plasmid pET11c-TOM70-(GGGGS)3-UB-RKIP

To produce the RKIP protein in the form to which TOM70, the mitochondrial outer membrane binding, linker (GGGGSGGGGSGGGGS) and ubiquitin are fused, an expression vector capable of expressing RKIP in the form to which TOM70, the linker and ubiquitin is fused was prepared.

Plasmid pTA-RKIP obtained in Example 3.1 was digested with restriction enzymes SacII and XhoI and a DNA fragment of approximately 560 bp was digested. was obtained by electrophoresis in 2% agarose gel, and then it was inserted into the vector pET11c-TOM70-(GGGGS)3-UB-(p53), digested with restriction enzymes SacII and XhoI, using T4 DNA ligase to obtain plasmid pET11- TOM70-(GGGGS)3-UB-RKIP (Figure 30). In this case, TOM70-(GGGGS)3-UB-RKIP is represented by the base sequence SEQ ID NO: 32.

E. coli strain BL21(DE3) was transformed using the plasmid pET11c-TOM70-(GGGGS)3-UB-RKIP. The transformed strain was then cultured in solid Luria-Bertani (LB) medium to which the antibiotic ampicillin was added, and the resulting colonies were cultured in liquid LB medium in a shaking incubator at 37°C. Then, when the cell density reached an optical density of approximately 0.2 at OD600, IPTG was added to achieve a final concentration of 0.5 mM, followed by shaking culture for approximately 4 more hours.

Some E. coli cells were obtained by centrifugation, and then the cells were destroyed and electrophoresis was performed on SDS-PAGE. As shown in Figure 31, it was confirmed that the RKIP protein, having a size of approximately 33 kDa, was expressed in a form to which TOM70, linker and ubiquitin were fused. In this case, lane M in Figure 31 corresponds to the protein molecular weight marker, lane 1 corresponds to the precipitate centrifuged after E. coli disruption 4 hours after the addition of IPTG, and lane 2 corresponds to the supernatant centrifuged after E. coli disruption.

Example 3.3. Isolation and purification of recombinant protein TOM70-(GGGGS)3-UB-RKIP obtained from E. coli

Production strain E. coli BL21 (DE3), expressing recombinant TOM70-(GGGGS)3-UB-RKIP, was inoculated into liquid LB medium and cultured at 37°C. When the optical density reached 0.3 at OD600, the culture was placed in a refrigerator to reduce the temperature of the culture solution, and the incubator temperature was changed to 18°C, and then 0.5 mM IPTG was added and shaking culture was carried out for another 1 day for expression protein TOM70-(GGGGS)3-UB-RKIP.

The TOM70-(GGGGS)3-UB-RKIP protein was then isolated and purified using the same method as in Example 1.3.1. As a result, the TOM70-(GGGGS)3-UB-RKIP protein was eluted (Figure 32). In this case, lane M in Figure 32 corresponds to the protein molecular weight marker, and lane 1 corresponds to the loaded sample for nickel affinity chromatography. In track 2 you can see that it is not bound to the nickel affinity resin. Lane 3 corresponds to the elution results using 50 mM Na phosphate/500 mM NaCl/10 mM imidazole. Lanes 4-6 correspond to elution results using 50 mM Na phosphate/500 mM NaCl/50 mM imidazole. Lanes 7-8 correspond to elution results using 50 mM Na phosphate/500 mM NaCl/100 mM imidazole. Lanes 9-10 correspond to elution results using 50 mM Na phosphate/500 mM NaCl/175 mM imidazole. Lanes 11-13 correspond to elution results using 50 mM Na phosphate/500 mM NaCl/250 mM imidazole. Lanes 14-16 correspond to elution results using 50 mM Na phosphate/500 mM NaCl/500 mM imidazole.

The amount of protein in the recovered eluted solution was measured by protein quantitation and confirmed using SDS-PAGE. As shown in Figure 33, after confirmation, the TOM70-(GGGGS)3-UB-RKIP protein was rapidly cooled with liquid nitrogen and stored in a cryogenic freezer at -80°C. In this case, lane M in Figure 33 corresponds to the protein molecular weight marker, and lane 1 shows the TOM70-(GGGGS)3-UB-RKIP protein obtained after dialysis in PBS buffer.

Example 4: Preparation of a fusion protein containing PTEN

Example 4.1. PTEN gene amplification

To express human PTEN (a homologue of phosphatase and tensin) in a recombinant protein, total RNA was isolated from human epithelial cells and cDNA was synthesized from it. Fibroblasts (human dermal fibroblasts) were cultured in 10% serum, 5% carbon dioxide and 37°C (1 x 10 ⁶ cells). The RNA was then obtained in the same way as in example 1.1, and then it was used as a template for the polymerase chain reaction of the PTEN gene.

To obtain the PTEN gene from human dermal fibroblasts, from which the signal peptide sequence was removed, primer T2PTEN, encoding from the amino-terminal threonine, and primer XPTEN(noT), encoding from the carboxy-terminus, were synthesized, and then PCR was carried out using the cDNA obtained as described above, as a matrix. The sequence of each primer is shown in Table 10 below.

[Table 10]

Primer Subsequence SEQ ID NO. T2PTEN 5'-AAA AAA CCG CGG TGG Tac agc CAT CAT caa aga gat cgt tag -3' SEQ ID NO: 33 XPTEN(noT) 5'- AAA AAA CTC GAG GAC TTT TGT AAT TTG TGT ATG CTG -3' SEQ ID NO: 34

The cDNA prepared as described above was used as a template, and 0.2 pmol T2PTEN primer and 0.2 pmol XPTEN(noT) primer were mixed with 0.2 nM dNTP, 1× AccuPrime Taq DNA polymerase reaction buffer (Invitrogen, USA) and 1 unit. AccuPrime Taq DNA polymerases. Amplification reactions were then carried out in the polymerase chain reaction apparatus at 95°C for 40 seconds, 58°C for 30 seconds, 72°C for 1 minute for 40 cycles. After the reaction, an amplified DNA fragment of approximately 1200 bp in size. isolated by 1% agarose gel electrophoresis and then inserted into the pGEM-T easy vector (Promega, USA) using T4 DNA ligase. By sequencing the DNA thus obtained, the cDNA encoding the human RKIP protein was confirmed. The resulting PTEN gene was designated pTA-PTEN (Figure 34), and the base sequence of PTEN is represented by the base sequence of SEQ ID NO: 35.

Example 4.2. Preparation of an E. coli expression vector for the PTEN protein

Example 4.2.1. Preparation of plasmid pET11c-TOM70-(GGGGS)3-UB-PTEN

To obtain the PTEN protein in the form to which TOM70, the mitochondrial outer membrane binding, linker (GGGGSGGGGSGGGGS) and ubiquitin are fused, an expression vector capable of expressing the PTEN gene in the form to which TOM70, linker and ubiquitin is fused was prepared.

Plasmid pTA-PTEN obtained in example 4.1. above, was digested with the restriction enzymes SacII and XhoI to obtain a DNA fragment of approximately 1200 bp. by electrophoresis in 2% agarose gel. It was then inserted into the vector pET11c-TOM70-(GGGGS)3-UB-(p53), digested with the restriction enzymes SacII and XhoI, using T4 DNA ligase to obtain the plasmid pET11c-TOM70-(GGGGS)3-UB-PTEN (figure 35). In this case, TOM70-(GGGGS)3-UB-PTEN is represented by the base sequence SEQ ID NO: 36.

E. coli strain BL21(DE3) was transformed using the plasmid pET11c-TOM70-(GGGGS)3-UB-PTEN. The transformed strain was then cultured in solid Luria-Bertani (LB) medium to which the antibiotic ampicillin was added, and the resulting colonies were then cultured in liquid LB medium at 37°C. Then, when the cell density reached an optical density of approximately 0.2 at OD600, IPTG was added to achieve a final concentration of 0.5 mM, followed by shaking culture for approximately 4 more hours.

Some E. coli cells were obtained by centrifugation, and then the cells were destroyed and electrophoresis was performed on SDS-PAGE. As shown in Figure 36, expression of the approximately 73 kDa PTEN protein was confirmed in the form to which TOM70, linker and ubiquitin are fused. In this case, lane M in Figure 36 corresponds to the protein molecular weight marker, lane 1 corresponds to the precipitate centrifuged after E. coli disruption 4 hours after the addition of IPTG, and lane 2 corresponds to the supernatant centrifuged after E. coli disruption.

Example 4.3 Isolation and purification of recombinant protein TOM70-(GGGGS)3-UB-PTEN obtained from E. coli

The TOM70-(GGGGS)3-UB-PTEN protein was isolated and purified using the same method as in example 1.3.1. As a result, the TOM70-(GGGGS)3-UB-PTEN protein was eluted (Figure 37). In this case, lane M in Figure 37 corresponds to the protein molecular weight marker, and lane 1 corresponds to the loaded sample for nickel affinity chromatography. In track 2 you can see that it is not bound to the nickel affinity resin. Lane 3 corresponds to the elution results using 8 M urea/50 mM Na phosphate/500 mM NaCl/10 mM imidazole. Lane 4 corresponds to the elution results using 8 M urea/50 mM Na phosphate/500 mM NaCl/50 mM imidazole. Lanes 5-8 correspond to the elution results using a solution of 8 M urea/50 mM Na phosphate/500 mM NaCl/100 mM imidazole. Lanes 9-10 correspond to the elution results using a solution of 8 M urea/50 mM Na phosphate/500 mM NaCl/250 mM imidazole. Lane 11 corresponds to the elution results using 8 M urea/50 mM Na phosphate/500 mM NaCl/500 mM imidazole.

The amount of protein in the recovered eluted solution was measured by protein quantitation and confirmed using SDS-PAGE. As shown in Figure 38, after confirmation, the TOM70-(GGGGS)3-UB-PTEN protein was rapidly cooled with liquid nitrogen and stored in a cryogenic freezer at -80°C. In this case, lane M in Figure 38 corresponds to the protein molecular weight marker, and lane 1 shows the TOM70-(GGGGS)3-UB-PTEN protein obtained after dialysis in PBS buffer.

Example 5: Preparation of a Fusion Protein Containing Mitochondrial Outer Membrane Protein, Ubiquitin and GFP

Example 5.1. Isolation and purification of recombinant UB-GFP-TOM7 protein derived from E. coli

Production strain E. coli BL21 (DE3), expressing the UB-GFP-TOM7 protein in a mature form to which ubiquitin is fused, was inoculated into liquid LB medium and cultured at 37°C. When the optical density reached 0.3 at OD600, the culture was placed in a refrigerator to reduce the temperature of the culture solution, and the incubator temperature was changed to 18°C, and then 0.5 mM IPTG was added and shaking culture was carried out for another 1 day for expression the GFP-TOM7 protein in its mature form, to which ubiquitin is fused.

After completion of culture, the cells were isolated by centrifugation, and the isolated cells were washed once using PBS, and then the cells were suspended using a solution of 50 mM sodium phosphate, 500 mM NaCl, 10 mM imidazole, pH 8.0, and the suspended cells were disrupted using an ultrasonic homogenizer. . The disrupted cells were centrifuged using a high-speed centrifuge, and then the supernatant was isolated, and the isolated supernatant was filtered using a 0.45 μm filter and then loaded onto a packed nickel chromatography column to perform primary purification.

Load a cell disruption solution containing the UB-GFP-TOM7 protein in its mature form to which ubiquitin is fused, and then run a wash solution of 50 mM sodium phosphate, 500 mM NaCl, 20 mM imidazole, pH 8.0 until no unbound impurities are present. undetectable, and the protein was eluted according to a concentration gradient using a solution of 50 mM sodium phosphate, 500 mM NaCl, 500 mM imidazole, pH 8.0 (Figure 39). In this case, lane M in Figure 39 corresponds to the protein molecular weight marker, and lane 1 corresponds to the loaded sample for nickel affinity chromatography. In track 2 you can see that it is not bound to the nickel affinity resin. Lane 3 corresponds to the elution results using 50 mM Na phosphate/500 mM NaCl/20 mM imidazole. Lane 4 corresponds to the elution results using 50 mM Na phosphate/500 mM NaCl/55 mM imidazole. Lane 5 corresponds to the elution results using 50 mM Na phosphate/500 mM NaCl/60 mM imidazole. Lane 6 corresponds to the elution results using 50 mM Na phosphate/500 mM NaCl/65 mM imidazole. Lane 7 corresponds to the elution results using 50 mM Na phosphate/500 mM NaCl/70 mM imidazole. Lane 8 corresponds to the elution results using 50 mM Na phosphate/500 mM NaCl/75 mM imidazole. Lane 9 corresponds to the elution results using 50 mM Na phosphate/500 mM NaCl/80 mM imidazole. Lane 10 corresponds to the elution results using 50 mM Na phosphate/500 mM NaCl/85 mM imidazole. Lane 11 corresponds to the elution results using 50 mM Na phosphate/500 mM NaCl/90 mM imidazole. Lane 12 corresponds to the elution results using 50 mM Na phosphate/500 mM NaCl/95 mM imidazole. Lane 13 corresponds to the elution results using 50 mM Na phosphate/500 mM NaCl/100 mM imidazole. Lane 14 corresponds to the elution results using 50 mM Na phosphate/500 mM NaCl/105 mM imidazole.

To remove imidazole from the eluting solution, dialysis was carried out using the principle of osmotic pressure with a solution of 50 mM sodium phosphate, 500 mM NaCl, pH 8.0 (Figure 40). The identified final UB-GFP-TOM7 was rapidly cooled with liquid nitrogen and stored in a cryogenic freezer at -80°C. In this case, lane M in Figure 40 corresponds to the molecular weight marker of the proteins, and lane 1 corresponds to the protein obtained after dialyzing with 50 mM Na phosphate/500 mM NaCl after mixing with the fusion protein fraction.

Example 5.2. Isolation and purification of recombinant protein TOM70-(GGGGS)3-UB-GFP obtained from E. coli

Production strain E. coli BL21 (DE3), expressing the recombinant protein TOM70-(GGGGS)3-UB-GFP, was inoculated into liquid LB medium and cultured at 37°C. When the optical density reached 0.3 at OD600, the culture was placed in a refrigerator to reduce the temperature of the culture solution and the incubator temperature was changed to 18°C, and then 0.5 mM IPTG was added and shaking culture was carried out for another 1 day to express the recombinant TOM70-(GGGGS)3-UB-GFP protein.

After completion of culture, the cells were isolated by centrifugation and the isolated cells were washed once using PBS, and then the cells were suspended using a solution of 50 mM sodium phosphate, 500 mM NaCl, 10 mM imidazole, pH 8.0, and the suspended cells were disrupted using an ultrasonic homogenizer. The disrupted cells were centrifuged using a high speed centrifuge and then the supernatant was isolated and the recovered supernatant was filtered using a 0.45 μm filter and then loaded onto a packed nickel chromatography column to perform primary purification.

The cell disruption solution containing the recombinant TOM70-(GGGGS)3-UB-GFP protein was loaded onto a column containing nickel resins, and then a wash solution of 50 mM sodium phosphate, 500 mM NaCl, 20 was passed until unbound impurities were undetectable. mM imidazole, pH 8.0. The protein was then eluted using a solution of 50 mM sodium phosphate, 500 mM NaCl, 500 mM imidazole, pH 8.0, changing the imidazole concentration to 50 mM, 100 mM, 250 mM, 500 mM (Figure 41). In this case, lane M in Figure 41 corresponds to the protein molecular weight marker, and lane 1 corresponds to the loaded sample for nickel affinity chromatography. In track 2 you can see that it is not bound to the nickel affinity resin. Lane 3 corresponds to the elution results using 50 mM Na phosphate/500 mM NaCl/20 mM imidazole. Lane 4 corresponds to the elution results using 50 mM Na phosphate/500 mM NaCl/50 mM imidazole. Lanes 5-8 correspond to elution results using 50 mM Na phosphate/500 mM NaCl/100 mM imidazole. Lanes 9-11 correspond to elution results using 50 mM Na phosphate/500 mM NaCl/250 mM imidazole. Lane 12 corresponds to the elution results using 50 mM Na phosphate/500 mM NaCl/500 mM imidazole.

The eluted solution obtained by nickel chromatography was subjected to buffer exchange with PBS buffer using the principle of osmotic pressure. After buffer exchange was completed, the isolated final TOM70-(GGGGS)3-UB-GFP protein was identified using quantitative analysis and SDS-PAGE. As shown in Figure 42, after identification was completed, the TOM70-(GGGGS)3-UB-GFP protein was rapidly cooled with liquid nitrogen and stored in a cryogenic freezer at -80°C. In this case, lane M in Figure 42 corresponds to the molecular weight marker of the proteins, and lane 1 corresponds to the TOM70-(GGGGS)3-UB-GFP protein obtained after dialysis carried out in PBS buffer solution.

II. Preparation of a fusion protein containing a mitochondrial outer membrane targeting protein and a target targeting protein

Example 6: Preparation of a fusion protein containing scFvHER2

Example 6.1. Synthesis of the scFvHER2 gene

In the case of expression of human scFvHER2 in a recombinant protein, the scFvHER2 gene obtained through custom gene synthesis at Bionics Co., Ltd. was designated pUC57-scFvHER2, and the base sequence of scFvHER2 was the same as that of SEQ ID NO: 37.

Example 6.2. Preparation of an expression vector for the scFvHER2 protein

Example 6.2.1. pET15b-UB-scFvHER2-TOM7

To produce the scFvHER2 protein in a ubiquitin-TOM7 fused form that binds to the mitochondrial outer membrane, an expression vector capable of expressing the scFvHER2 gene in a ubiquitin-TOM7 fused form was prepared.

Plasmid pUC57-scFvHER2 obtained in Example 6.1 was digested with restriction enzymes SacII and XhoI to obtain a DNA fragment of approximately 750 bp. by electrophoresis in a 2% agarose gel, and then it was inserted into the vector pET15b-UB-(p53)-TOM7, digested with restriction enzymes SacII and XhoI, using T4 DNA ligase to obtain the plasmid pET15b-UB-scFvHER2-TOM7 ( figure 39). In this case, UB-scFvHER2-TOM7 is represented by the base sequence SEQ ID NO: 38.

E. coli strain BL21(DE3) was transformed using the plasmid pET15b-UB-scFvHER2-TOM7. The transformed strain was then cultured in solid Luria-Bertani (LB) medium to which the antibiotic ampicillin was added, and the resulting colonies were then cultured in liquid LB medium at 37°C. Then, when the cell density reached an optical density of approximately 0.2 at OD600, IPTG was added to achieve a final concentration of 1 mM, followed by shaking culture for approximately 4 more hours.

Some E. coli cells were obtained by centrifugation, and then the cells were destroyed and electrophoresis was performed on SDS-PAGE. As shown in Figure 44, the scFvHER2 protein, which is approximately 35 kDa in size, was confirmed to be expressed in a ubiquitin-TOM7 fused form. In this case, lane M in Figure 44 corresponds to the protein molecular weight marker, lane 1 corresponds to the precipitate centrifuged after E. coli disruption 4 hours after the addition of IPTG, and lane 2 corresponds to the supernatant centrifuged after E. coli disruption.

Example 6.2.2. Generation of pCMV-scFvHER2-TOM7-myc/His

To produce scFvHER2 protein in the TOM7 fused form that binds to the mitochondrial outer membrane, an animal cell expression vector capable of expressing scFvHER2 in the TOM7 fused form was prepared. To obtain the TOM7 and scFvHER2 genes, primer RscFvHER2 and primer XTOM7(noT) were obtained. The sequence of each primer is shown in Table 11 below.

[Table 11]

Primer Subsequence SEQ ID NO. RscFvHER2 5'-AAA AAA GAA TTC ATG GAA GTG CAA CTT GTT GAG AGT GG -3' SEQ ID NO: 39 XTOM7(noT) 5'- AAA AAA CTC GAG TCC CCA AAG TAG GCT CAA AAC AG-3' SEQ ID NO: 40

Plasmid pET15b-UB-scFvHER2-TOM7, obtained in example 6.2.1., was used as a template and mixed 0.2 pmol of primer (RscFvHER2) and 0.2 pmol of primer (XTOM7(noT)) with 0.2 nM dNTP, 1x reaction buffer solution for DNA polymerase AccuPrime Taq (Invitrogen, USA) and 1 unit. AccuPrime Taq DNA polymerases. Then, to obtain the scFvHER2-TOM7 gene, amplification reactions were carried out in a polymerase chain reaction apparatus at 95°C for 40 seconds, 58°C for 30 seconds, 72°C for 1 minute for 25 cycles. The amplified scFvHER2-TOM7 gene was digested with restriction enzymes EcoRI and XhoI to obtain approximately 850 bp DNA fragments, respectively, by 1% agarose gel electrophoresis and then inserted into the pcDNA3.1-myc/His A vector. digested with restriction enzymes EcoRI and XhoI using T4 DNA ligase to obtain plasmid pCMV-scFvHER2-TOM7-myc/His (Figure 45).

In this case, scFvHER2-TOM7-myc/His is represented by the base sequence SEQ ID NO: 41. It was used to transfect CHO animal cells using the plasmid pCMV-scFvHER2-TOM7-myc/His, the cells were destroyed, and then SDS PAGE was performed. and analyzed by Western blotting using anti-c-myc antibody. As shown in Figure 46, expression of the approximately 35 kDa scFvHER2 protein in the TOM7 fused form was confirmed. In this case, lane M in Figure 46 corresponds to the protein molecular weight marker, and lane 1 shows that it was used to transfect CHO animal cells, disrupt the cells, and then perform SDS-PAGE and then confirm by Western blotting using antibodies against c-myc.

Example 6.3. Isolation and purification of recombinant protein UB-ScFvHER2-TOM7 obtained from E. coli

The UB-ScFvHER2-TOM7 protein was isolated and purified using the same method as in example 1.3.1. As a result, the UB-ScFvHER2-TOM7 protein was eluted (Figure 47). In this case, lane M in Figure 47 corresponds to the protein molecular weight marker, and lane 1 corresponds to the loaded sample for nickel affinity chromatography. In track 2 you can see that it is not bound to the nickel affinity resin. Lane 3 corresponds to the elution results using 8 M urea/50 mM Na phosphate/500 mM NaCl/10 mM imidazole. Lanes 4-5 correspond to the elution results using 8 M urea/50 mM Na phosphate/500 mM NaCl/50 mM imidazole. Lanes 6-8 correspond to elution results using 8 M urea/50 mM Na phosphate/500 mM NaCl/100 mM imidazole. Lanes 9-10 correspond to the elution results using 8 M urea/50 mM Na phosphate/500 mM NaCl/250 mM imidazole. Lane 11 corresponds to the elution results using 8 M urea/50 mM Na phosphate/500 mM NaCl/500 mM imidazole.

The amount of protein in the recovered eluted solution was measured by protein quantitation and confirmed using SDS-PAGE. As shown in Figure 48, after confirmation, the UB-ScFvHER2-TOM7 protein was rapidly cooled with liquid nitrogen and stored in a cryogenic freezer at -80°C. In this case, lane M in Figure 48 corresponds to the protein molecular weight marker, and lane 1 corresponds to the UB-ScFvHER2-TOM7 protein obtained after dialysis in PBS buffer.

Example 7: Preparation of a Fusion Protein Containing scFvMEL

Example 7.1. Synthesis of the scFvMEL gene

To express human scFvMEL in a recombinant protein as an anti-melanoma antibody fragment, the scFvMEL gene obtained through custom gene synthesis at Bionics Co., Ltd. was designated pUC57-scFvMEL, and the base sequence of scFvMEL was the same as the base sequence of SEQ ID NO: 42.

Example 7.2. Preparation of an expression vector for the scFvMEL protein

Example 7.2.1. Preparation of pET15b-UB-scFvMEL-TOM7

To produce scFvMEL protein in a ubiquitin-TOM7 fused form that binds to the mitochondrial outer membrane, an expression vector capable of expressing scFvMEL in a ubiquitin-TOM7 fused form was prepared.

Plasmid pUC57-scFvMEL obtained in Example 7.1 was digested with restriction enzymes SacII and XhoI to obtain a DNA fragment of approximately 750 bp. by electrophoresis in a 2% agarose gel, and then inserted into the vector pET15b-UB-(p53)-TOM7, digested with the restriction enzymes SacII and XhoI, using T4 DNA ligase to obtain the plasmid pET15b-UB-scFvMEL-TOM7 (Figure 49). In this case, UB-scFvMEL-TOM7 is represented by the base sequence SEQ ID NO: 43.

E. coli strain BL21(DE3) was transformed using plasmid pET15b-UB-scFvMEL-TOM7. The transformed strain was then cultured in solid Luria-Bertani (LB) medium to which the antibiotic ampicillin was added, and the resulting colonies were cultured in liquid LB medium in a shaking incubator at 37°C. Then, when the cell density reached an optical density of approximately 0.2 at OD600, IPTG was added to achieve a final concentration of 1 mM, followed by shaking culture for approximately 4 more hours.

Some E. coli cells were obtained by centrifugation, and then the cells were destroyed and electrophoresis was performed on SDS-PAGE. As shown in Figure 50, scFvMEL protein having a size of approximately 35 kDa was confirmed to be expressed in a form with ubiquitin and TOM7 fused. In this case, lane M in Figure 50 corresponds to the protein molecular weight marker, lane 1 corresponds to the precipitate centrifuged after E. coli disruption 4 hours after the addition of IPTG, and lane 2 corresponds to the supernatant centrifuged after E. coli disruption.

Example 7.2.2. Preparation of pCMV-scFvMEL-TOM7-myc/His

To produce scFvMEL protein in the TOM7 fused form that binds to the mitochondrial outer membrane, an animal cell expression vector capable of expressing scFvMEL in the TOM7 fused form was prepared. A primer (RscFvMEL) was prepared to obtain the TOM7 and scFvMEL genes. The sequence of each primer is shown in Table 12 below.

[Table 12]

Primer Subsequence SEQ ID NO. RscFvMEL 5'- AAA AAA GAA TTC ATG AAA ACA AGT AAC CCA GGA GTG-3' SEQ ID NO: 44

Plasmid pET15b-UB-scFvMEL-TOM7, obtained in example 6.2.1., was used as a template, and 0.2 pmol of RscFvMEL primer and 0.2 pmol of XTOM7(noT) primer were mixed with 0.2 nM dNTP, 1× reaction buffer solution for DNA polymerase AccuPrime Taq (Invitrogen, USA) and 1 unit. AccuPrime Taq DNA polymerases. Amplification reactions were then performed in a polymerase chain reaction apparatus at 95°C for 40 seconds, 58°C for 30 seconds, 72°C for 1 minute for 25 cycles to obtain scFvMEL-TOM7. The amplified scFvMEL-TOM7 gene was digested with EcoRI and XhoI restriction enzymes to obtain a DNA fragment of approximately 850 bp. by electrophoresis in 1% agarose gel. It was then inserted into the EcoRI and XhoI digested vector pcDNA3.1-myc/His A using T4 DNA ligase to produce the plasmid pCMV-scFvMEL-TOM7-myc/His (Figure 51). In this case, scFvMEL-TOM7-myc/His is represented by the base sequence SEQ ID NO: 45.

It was used to transfect CHO animal cells using the plasmid pCMV-scFvMEL-TOM7-myc/His, the cells were disrupted, and then subjected to SDS-PAGE and analyzed by Western blotting using an anti-c-myc antibody. As shown in Figure 52, expression of the approximately 35 kDa scFvMEL protein in the TOM7 fused form was confirmed. In this case, lane M in Figure 52 corresponds to a protein molecular weight marker, and lane 1 shows that CHO animal cells were transfected with it and the cells were disrupted and then subjected to SDS-PAGE and then confirmed by Western blotting using antibodies against c-myc.

Example 8: Preparation of a Fusion Protein Containing scFvPD-L1

Example 8.1. Synthesis of the scFvPD-L1 gene

In the case of expression of human scFvPD-L1 in a recombinant protein, the scFvPD-L1 gene obtained through custom gene synthesis at Bionics Co., Ltd. was designated pUC57-scFvPD-L1, the base sequence of which was the same as the base sequence of SEQ ID NO: 46.

Example 8.2. Preparation of an expression vector for the scFvPD-L1 protein

Example 8.2.1. Preparation of pCMV-scFvPD-L1-TOM7-myc/His

To produce scFvPD-L1 protein in a TOM7-fused form that binds to the mitochondrial outer membrane, an animal cell expression vector capable of expressing scFvPD-L1 in a ubiquitin-TOM7 fused form was prepared.

Plasmid pUC57-scFvPD-L1 was digested with restriction enzymes EcoRI and XhoI to obtain a DNA fragment of approximately 760 bp. by electrophoresis in 1% agarose gel. It was then inserted into the EcoRI and XhoI digested vector pCMV-(scFvMEL)-TOM7-myc/His using T4 DNA ligase to obtain plasmid pCMV-scFvPD-L1-TOM7-myc/His (Figure 53) . In this case, scFvPD-L1-TOM7-myc/His is represented by the base sequence SEQ ID NO: 47.

It was used to transfect CHO animal cells using the plasmid pCMV-scFvPD-L1-TOM7-myc/His, and the cells were disrupted, then subjected to SDS-PAGE and analyzed by Western blotting using an anti-c-myc antibody. As shown in Figure 54, expression of the approximately 35 kDa scFvPD-L1 protein in the TOM7 fused form was confirmed. In this case, lane M in Figure 54 corresponds to the protein molecular weight marker, and lane 1 shows that it was transfected into CHO animal cells, the cells were disrupted, and then subjected to SDS-PAGE and then confirmed by Western blotting using the antibody against c-myc.

III. Preparation of modified mitochondria to which the fusion protein is associated

Example 9. Preparation of modified mitochondria

The following experiment was performed to confirm whether a fluorescent protein fused to the mitochondrial outer membrane binding site binds to the mitochondrial outer membrane. First, mitochondria were isolated from umbilical cord-derived mesenchymal stem cells (UC-MSC) by centrifugation. They were then stained with MitoTracker CMXRos Red. They were mixed with recombinant TOM70-(GGGGS)3-UB-GFP protein isolated from E. coli as described above and incubated at ambient temperature for approximately 30 minutes.

Unreacted protein was then removed by centrifugation and washed twice with PBS buffer. The fluorescent protein in mitochondria-associated form was then observed using a fluorescence microscope. Purified GFP protein lacking the mitochondrial outer membrane binding site was used as a control group. The results confirmed that the fluorescent protein fused to the mitochondrial outer membrane binding site (TOM70-(GGGGS)3-UB-GFP) was located in the same location as mitochondria from umbilical cord-derived mesenchymal stem cells (UC-MSCs) (Figure 55a, Figure 55b).

Example 10: Confirmation of the ability of recombinant p53 protein to bind to the foreign mitochondrial outer membrane

Mitochondria isolated from umbilical cord-derived mesenchymal stem cells by centrifugation were mixed with purified recombinant TOM70-(GGGGS)3-UB-p53 or UB-p53-TOM7 protein and allowed to bind in a 1:1 ratio under reaction conditions at 4 °C for 1 hour. Mitochondria not mixed with protein were used as a control group. The possibility of binding mitochondria and p53 was confirmed by Western blotting (Figure 56).

First, mitochondria and p53 protein were bound, and then centrifugation was performed at 13,000 rpm. for 10 minutes to obtain the mitochondria or mitochondria to which p53 is bound as a pellet. Protein unbound to mitochondria was removed by washing twice with PBS, and the washed pellet was subjected to protein electrophoresis (SDS-PAGE) and then Western blotting. Rabbit anti-p53 antibody was used as the primary antibody and HRP-conjugated anti-rabbit IgG antibody was used as the secondary antibody. The presence of bands at the same position was confirmed, corresponding to the size of 60 kDa, the molecular weight expected in the experimental group for mitochondria bound to TOM70-(GGGGS)3-UB-p53 or UB-p53-TOM7, compared to the control group of mitochondria alone , not bound to the protein (Figure 56).

IV. Confirmation of the activity of modified mitochondria to which the active protein is bound

Example 11. Isolation and intracellular injection of foreign mitochondria

Mitochondria were isolated from umbilical cord-derived mesenchymal stem cells (UC-MSC) by centrifugation. Isolated mitochondria were stained using Mitotracker CMX Ros dye and the concentration and total number of isolated mitochondria were confirmed by BCA quantitation method, and 0 μg, 1 μg, 5 μg, 10 μg, 50 μg, 100 μg mitochondria were injected into SNU-484 cells, a cell line stomach cancer, by centrifugation. As a result of the fluorescence microscope experiment, it was confirmed that the number of mitochondria injected into the cells depended on the mitochondrial concentration (Figure 57).

Example 12. Confirmation of the influence of normal mitochondria on malignant cells

The following experiment was performed to investigate how mitochondria derived from normal cells affect malignant cell proliferation and ROS production. First, liver cells (WRL-68), fibroblasts, and umbilical cord-derived mesenchymal stem cells (UC-MSC) were selected as mitochondrial donor cells. Mitochondria were isolated from cells by centrifugation fractionation method, respectively. The cancer cell used as the mitochondrial recipient cell was an epidermal skin cancer cell line, A431. In this case, mitochondria are delivered to epidermal skin cancer cells using centrifugal force according to concentration (see Korean Patent Application No. 10-2017-0151526).

Proliferation of epidermal malignant skin cells and production of reactive oxygen species (ROS) were observed at 24, 48 and 72 hours after administration. As a result, it was confirmed that when mitochondria obtained from normal cells of various origins were injected into malignant cells, a concentration-dependent effect of inhibiting the proliferation of malignant cells was observed. In addition, it was confirmed that ROS production in malignant cells was inhibited depending on the concentration of normal mitochondria (Figures 58 and 59).

Example 13: Confirmation of the influence of normal mitochondria on drug resistance

The present inventors investigated the effect on drug resistance, antioxidant gene expression, cancer metastasis, which are hallmarks of malignant cells, when mitochondria derived from normal cells were injected into malignant cells by the following methods. First, normal liver cells (WRL-68) were used as mitochondrial donor cells, mitochondria were isolated from the cells by centrifugation fractionation method, and the mitochondria were used. HepG2 cells, a malignant liver cell line, were used as malignant mitochondrial recipient cells. Mitochondria were delivered to cancerous liver cells using centrifugal force according to concentration, and it was then confirmed that when resistance to the antitumor drug doxorubicin was observed, cancer cell lines injected with mitochondria showed higher drug sensitivity (Figure 60).

Example 14 Confirmation of the influence of normal mitochondria on the antioxidant effect

Because Mitochondria isolated from normal cells were injected into HepG2 cells, a liver malignant cell line, according to the concentration, increased gene expression of catalase enzyme, antioxidant protein, and SOD-2 (superoxide dismutase-2) in malignant cells was confirmed (Figure 61).

Example 15. Confirmation of the influence of normal mitochondria on metastasis of malignant cells

Regarding metastasis, the presence of α-smooth muscle actin (α-SMA) gene expression, one of the genes involved in EMT (epithelial-mesenchymal transition), was confirmed. In this case, it was found that in the case of malignant liver cells that were injected with mitochondria, α-SMA protein expression was significantly reduced depending on the concentration of mitochondria compared to malignant liver cells that were not injected with mitochondria. In contrast, E-cadherin protein, one of the cell adhesion proteins, was found to increase as a function of mitochondrial concentration (Figure 62). It was confirmed that changes in proteins known to be involved in cancer metastasis occurred under the influence of normal mitochondria injected into malignant cells and thus also affected cancer cell metastasis.

Example 16 Confirmation of recombinant p53 protein loading on foreign mitochondrial outer membrane and injection into cells

Mitochondria were isolated from mesenchymal stem cells obtained from umbilical cord by centrifugation, and then stained using Mitotracker CMX Ros dye, mixed with purified recombinant protein TOM70-(GGGGS)3-UB-p53 or UB-p53-TOM7 and incubated in a ratio of 1 :1 under reaction conditions at 4°C for 1 hour, and then centrifuged to remove unreacted proteins, and then washed twice with PBS buffer, and then mitochondria in the form to which the p53 protein is bound were injected into SNU-484 cells, line gastric cancer cells by centrifugation method (Figure 63). In this case, the control group was a group that did not use mitochondria and a group that used mitochondria alone. After one day of culture, the p53 protein loaded on foreign mitochondria injected into the cells was analyzed by a fluorescence microscope using immunocytochemistry (ICC).

Rabbit anti-p53 antibody was used as the primary antibody and Alexa Fluor 488-conjugated goat anti-rabbit IgG antibody was used as the secondary antibody. The results confirmed that the protein TOM70-(GGGGS)3-UB-p53 (colored in green) or UB-p53-TOM7 (colored in green) loaded on foreign mitochondria (colored in red) was located in the cytoplasm in cells injected with foreign mitochondria. mitochondria (Figure 64, 200x magnification, and Figure 65, 400x magnification). As a result, it was found that the recombinant protein was easily injected into the cell using mitochondria.

Example 17 Confirmation of p53-loaded mitochondrial activity in a malignant cell line

Example 17.1. Confirmation of the apoptotic capacity of p53-loaded foreign mitochondria injected into cells using a gastric cancer cell line

Mitochondria isolated from umbilical cord-derived mesenchymal stem cells by centrifugation were mixed with recombinant TOM70-(GGGGS)3-UB-p53 or UB-p53-TOM7 protein isolated from E. coli and allowed to bind in a ratio of 1: 1 under reaction conditions at 4°C for 1 hour. UB-p53 protein, which does not contain TOM70, and TOM70-(GGGGS)3-p53, which does not contain ubiquitin, were used as a control group. Unbound proteins were removed by centrifugation and washing with PBS, and mitochondria to which the proteins were bound were injected into the gastric cancer cell line SNU-484, which lacks p53 capabilities due to a change in the p53 gene, by centrifugation (Figure 66). After one day of culture, the cells were fixed with 4% paraformaldehyde for 1 hour and then permeabilized using a permeabilization solution (0.1% sodium citrate buffer containing 0.1% Triton-X-100, pH 7.4) and reacted with TUNEL solution (in situ cell death detection kit, TMR RED, Roche) at 37°C for 1 hour.

In the TUNEL assay, the part where nucleic acid fragmentation occurs (DNA fragmentation) is stained red, indicating that apoptosis is occurring. Cells injected with mitochondria to which TOM70-(GGGGS)3-ub-p53 or p53-TOM7 is associated showed significant red staining, in contrast to the control group, indicating that apoptosis occurs under the influence of mitochondria to which associated TOM70-(GGGGS)3-UB-p53 or UB-p53-TOM7. In particular, it was confirmed that apoptosis occurred to a greater extent in the case of mitochondria to which the protein in the form of TOM70-(GGGGS)3-UB-p53 was associated (Figure 67a).

Example 17.2. Confirmation of the apoptotic ability of foreign p53-loaded mitochondria bound to luciferase

To confirm whether the biological activity of the introduced TOM70-(GGGGS)3-UB-p53 protein was maintained in the recipient cell after delivery of the TOM70-(GGGGS)3-UB-p53 protein in the form associated with mitochondria obtained in example 5.2. higher into recipient cells, cellular analysis was performed using a reporter gene. Because the p53 protein is a transcription factor, a gene was synthesized in which the base sequence RRRCWWGYYY (where R is G or A, W is A or T, and Y is C or T) is repeated 6 times, which the p53 transcription factor can bind to, with the following sequence. The base sequence of P53-promter-S is (5'-GGG CAT GCT CGG GCA TGC CCG GGC ATG CTC GGG CAT GCC CGG GCA TGC TCG GGC ATG CCC-3') (SEQ ID NO: 91), and the base sequence of P53- promter-AS is as follows (5'-GGG CAT GCC CGA GCA TGC CCG GGC ATG CCC GAG CAT GCC CGG GCA TGC CCG AGC ATG CCC-3') (SEQ ID NO: 92).

5 μg of the synthesized P53-promter-S gene and 5 μg of the synthesized P53-promter-AS gene were incubated at 70°C for 20 minutes to promote the synthesis of the double helix of the gene, and then a phosphorylation reaction was induced using the polynucleotide T4 kinase enzyme. The double helix of the gene, in which phosphorylation was induced, was inserted into the pGL3 vector, digested with the restriction enzyme Sma I, and the gene, in which the 6-fold repeated base sequence (RRRCWWGYYY), to which the p53 transcription factor can bind, was allowed to bind to luciferase, a reporter gene, to obtain plasmid p6xp53-Luc. Using plasmid p6xp53-Luc and plasmid pRSVb-gal, a beta-galactosidase expression vector, HEK293 cells, human kidney cells, were transformed by the Lipofectamine method.

Then, after 6 hours, HEK293 cells were treated with a combination in which 10 μg of mitochondria and 5 μg, 10 μg, and 20 μg of TOM70-(GGGGS)3-UB-p53 protein coupled, respectively. In this case, as a control group, cells were treated with 10 μg of mitochondria bound to PBS or p53 protein, respectively. Treated cells were cultured for 18 hours and then luciferase activity was measured and analyzed. In this case, to correct for transformation efficiency, the corrected luciferase value was determined to be the luciferase value divided by the value obtained by measuring beta-galactosidase activity.

It was confirmed that the luciferase value was increased in cells treated with a combination in which 10 μg of mitochondria and 5 μg, 10 μg, and 20 μg of TOM70-(GGGGS)3-UB-p53 protein were bound, respectively. Thus, it was confirmed that the p53 protein entered the cells and was active (Figure 67b).

Example 18: Confirmation of the ability of foreign RKIP-loaded mitochondria injected into cells to reduce metastasis of malignant cell lines

Mitochondria isolated from umbilical cord-derived mesenchymal stem cells by centrifugation were mixed with purified recombinant TOM70-(GGGGS)3-UB-RKIP protein and allowed to bind in a 1:1 ratio under reaction conditions at 4°C for 1 hour . Mitochondria to which the protein is bound were injected by centrifugation into the MDA-MB-231 breast cancer cell line, which is known to have an increased ability to metastasize due to a decrease in the RKIP protein.

To confirm the ability of malignant cells to metastasize, a cell invasion assay was performed using a Transwell plate. The upper chamber of the Transwell, which has a pore size of 8 μm, was coated with Matrigel for 30 minutes at 37°C. The test group was MDA-MB-231 cells injected with mitochondria alone and MDA-MB-231 cells injected with mitochondria bound to the RKIP protein. Each cell type, 1 x 10 ⁵ cells, was placed in the upper chamber of a Transwell containing serum-free medium, and the lower chamber was placed in medium containing 10% bovine serum. After culture at 37°C for 12 hours, fixation with 4% paraformaldehyde was carried out for 1 hour, and then cells passing through Matrigel were stained with 1% crystal violet.

As a result of microscopic observation, violet-stained cells were identified in the membrane below the upper chamber, and this could be said to represent the process by which cell metastasis occurs. It was confirmed that the number of violet-stained cells was reduced in the experimental group treated with mitochondria alone and the experimental group treated with mitochondria to which RKIP is associated, compared with the control group which was not treated with anything. Four parts were randomly selected, and then the number of stained cells was measured and plotted (Figure 68).

IV. Confirmation of the delivery rate of modified mitochondria to which the target targeting protein is bound

Example 19 Confirmation of intracellular expression of a single chain variable fragment (ScFv) antibody for targeting malignant cells and confirmation of binding to mitochondria in cells

To express pCMV-ScFv-HER2-TOM7 or pCMV-ScFv-MEL-TOM7 or pCMV-ScFv-PD-L1-TOM7 in animal cells, CHO cells were transfected with DNA using Lipofectamine LTX and PLUS or Lipofectamine 2000. As a control groups used GFP-TOM7 DNA. To confirm that they were expressed in a cell and bound to mitochondria in the same cell, cytosol and mitochondria were isolated from transfected cells by centrifugation and corrected for the same protein using a BCA assay, followed by PAGE and the results obtained. by Western blotting. Anti-c-myc monoclonal antibody was used as the primary antibody, and anti-mouse IgG HRP-conjugated antibody was used as the secondary antibody.

ScFv-HER2-TOM7 or ScFv-MEL-TOM7 protein bands were identified in the expected 35 kDa region. Given that they were all identified in the mitochondrial layer, it can be expected that the transfected and expressed proteins are associated with mitochondria in cells through TOM7 (Figure 69).

Then, to confirm the binding of a target protein expressed in a cell to mitochondria in the same cell, the ScFv-HER2-TOM7, ScFv-MEL-TOM7, or ScFv-PD-L1-TOM7 protein expressed in the cell was observed using a fluorescence microscope in the following manner: immunocytochemistry (ICC). Anti-c-myc monoclonal antibody was used as the primary antibody and Alexa Fluor 488-conjugated goat anti-mouse IgG antibody was used as the secondary antibody. Mitochondria in the cell were stained using Mitotracker CMX Ros dye. As a result, it was confirmed that the expressed proteins ScFv-HER2-TOM7, ScFv-MEL-TOM7 or ScFv-PD-L1-TOM7 colocalized with mitochondria and were associated with mitochondria in the cell (Figures 70 and 71).

Example 20 Isolation of mitochondria to which a single chain variable fragment of an antibody is bound for targeting malignant cells and comparison of injection of mitochondria into gastric cancer cell lines

Mitochondria were isolated from CHO cells that were transfected with pCMV-ScFv-HER2-TOM7 or pCMV-ScFv-PD-L1-TOM7. Mitochondria from CHO cells that were not transformed were isolated and used as a control group. Mitochondria isolated from each cell were stained using Mitotracker CMX Ros. SNU-484, a gastric cancer cell line, was treated with the same amount of mitochondria and the next day the number of mitochondria injected into the cells was compared and confirmed using a fluorescence microscope. It was confirmed that, compared with the control group, mitochondria to which ScFv-HER2-TOM7 or ScFv-PD-L1-TOM7 was associated were injected into malignant cells in greater quantities than mitochondria obtained from the control group (Figure 72). Thus, it was found that mitochondria to which the target protein was bound were more easily injected into malignant cells than when using mitochondria alone.

VI. Confirmation of in vivo activity of modified mitochondria to which the active protein is bound

Example 21 Construction of a xenograft model (SNU-484) and administration of test substance

Example 21.1. Obtaining malignant cells

On the day of the experiment, the SNU-484 cell line, a gastric cancer cell line, was prepared at a rate of 5×10 ⁶ cells per mouse. The cell culture medium was removed and then PBS was added to wash the cells. The cells were dissociated using trypsin-EDTA solution, and then the cells were placed in a 50 ml tube and washed twice with PBS buffer solution, and then 20 ml PBS was added and the cell number and viability were measured. Based on the measured number of cells, the number of cells was adjusted to 5×10 ⁶ cells per mouse, and the cells were prepared by dividing them into groups. The volume to be transplanted per mouse was adjusted to the same amount of 100 μl. A group of 100 μL of malignant cells individually was prepared as a control group.

Example 21.2. Obtaining the test substance

Mitochondria isolated from umbilical cord blood mesenchymal stem cells as described above were prepared for transplantation in an amount of 50 μg per mouse, taking into account the protein concentration. In the case of the group in which mitochondria were administered alone, mitochondria were prepared by thoroughly mixing with 100 μL of PBS in which the malignant cells were mixed. For the modified mitochondria group, TOM70-(GGGGS)3-UB-p53 protein was mixed at a 1:1 concentration ratio with the amount of mitochondria prepared in an Eppendorf tube before mixing with malignant cells and left at ambient temperature for 1 hour. At the end of the reaction time, the supernatant was removed after centrifugation at 20,000×g for 10 minutes, and the mitochondria (MT+TOM70-(GGGGS)3-UB-p53) with which the protein was bound was precipitated. It was washed twice with PBS buffer, and then the mitochondria (MT+TOM70-(GGGGS)3-UB-p53) to which the p53 protein is bound were prepared by thoroughly mixing with 100 μL of PBS in which the malignant cells were mixed.

Example 21.3. Obtaining an experimental animal and transplanting the test substance

For transplantation, the same amount of Matrigel (BD) as PBS was added to the sample prepared for the groups and mixed gently with the cells to obtain 200 μl of test substance per mouse. In this case, all actions were carried out on ice. To construct the model, Balb/c nude mice (female 7 weeks old) were purchased from RAONBIO and anesthetized via isoflurane inhalation for malignant cell transplantation, and then the right posterior region (per animal) was sterilized using an alcohol swab. Then, 200 μl was injected subcutaneously into the right posterior region of the experimental animal using a 1 ml syringe containing the injection solution. After administration, the animal's weight and tumor size were measured twice a week and the results were analyzed at a follow-up of up to 3 weeks (FIG. 73).

Example 21.4. Confirmation of tumor formation

Tumor volume was calculated by measuring the long axis size and short axis size of the tumor and using the following equation.

<Math Equation 1>

Long axis × short axis × short axis × 0.5 = tumor volume (mm ³ )

Example 21.5. Observation of physiological and morphological changes

To observe physiological and morphological changes in mice upon administration of candidate antitumor agents, changes in body weight and tumor size were measured twice per week from the time of administration of malignant cells and test substances (FIG. 74).

Mouse weight was measured using a scale and group changes were analyzed using values measured twice per week (Figure 75). It was confirmed that there were no significant differences in changes in body weight over 3 weeks between the group in which no mitochondria were injected, the group in which mitochondria were injected alone, and the group in which modified mitochondria were injected. Tumor size was calculated by measuring the long axis (length) and short axis (width) size of the tumor using a caliper and then using them in the above mathematical equation 1. Changes in the group were analyzed using values measured twice a week (Figure 76) . It was found that tumor size increased significantly over time in the group that did not receive mitochondria, while in the case of mice that received mitochondria, the increase in tumor size slowed down over time. It was further confirmed that the increase in tumor size was significantly reduced in the p53 protein-loaded mitochondrial group as compared to the mitochondrial alone group (FIG. 76).

Example 22 Confirmation of the effect of modified mitochondria in inhibiting the proliferation of malignant skin cells

Mitochondria obtained as described above, to which p53 is associated, were introduced into A431 cells, which are malignant skin cells, by a centrifugation method, and then the proliferation of A431 cells was observed. In this case, saline was used as a control group and an equivalent number of mitochondria to which the p53 protein was not fused was used as a test control group. It was confirmed that mitochondria loaded with p53 protein, an apoptosis-inducing protein, could significantly inhibit the proliferation of A431 cells compared with the control group and the mitochondria-only group (Figure 76).

V. Confirmation of the activity of isolated mitochondria

Example 23. Confirmation of the function of isolated mitochondria: ATP content

To isolate intracellular mitochondria from umbilical cord-derived mesenchymal stem cells (UC-MSC), homogenization was performed using a syringe to disrupt the cells, and then continuous centrifugation was performed to obtain mitochondria. To confirm the function of the isolated mitochondria, the concentration of mitochondrial proteins in the isolated mitochondria was quantitatively analyzed by BCA assay, yielding 5 μg of mitochondria. The amount of ATP in mitochondria was confirmed using a CellTiter-Glo luminescence assay kit (Promega, Madison, WI).

The prepared mitochondria were mixed in 100 μl of PBS and then prepared in a 96-well plate and compared with 100 μl of mitochondria-free PBS as a control group. In the same way, add 100 μL of the test solution included in the kit, react and mix thoroughly for 2 minutes using a stirrer, and then react at ambient temperature for 10 minutes, and then measure the amount of ATP using a fluorescence reading spectrophotometer microplates. It was confirmed that ATP was increased by mitochondrial inclusion compared with the control group and confirmed the function of mitochondria (Figure 78).

Example 24: Confirmation of Isolated Mitochondria Function: Membrane Potential

JC-1 dye (Molecular Probes, cat. no. 1743159) was used to confirm the membrane potential of isolated mitochondria. The resulting mitochondria were mixed in 50 μl of PBS and then prepared in a 96-well plate. A mitochondria-free PBS group (50 μL) was obtained as a control group and a CCCP treatment group (R&D systems, CAS 555-60-2). CCCP, a mitochondrial ionophore, inhibits mitochondrial function by depolarizing the mitochondrial membrane potential. The CCCP group reacted with isolated mitochondria at 50 μM for 10 min at room temperature.

Then, the dye JC-1 (2 μM) was reacted in the same manner, and then the absorbance was measured using the property that it has a different spectrum according to the concentration generated by changing the membrane potential. At low concentrations it exists as a monomer and exhibits green fluorescence and at high concentrations the dye aggregates (J-aggregate) exhibiting red fluorescence. Mitochondrial membrane potential was analyzed by calculating the ratio of green fluorescence to red fluorescence. After completion of the reaction, mitochondrial membrane potential was measured using a fluorescence microplate reading spectrophotometer (monomer: excitation 485/emission 530, J-aggregate: excitation 535/emission 590). The results are shown in Figure 79.

Example 25 Confirmation of the degree of damage to isolated mitochondria by confirming mROS production

To confirm whether the 5 μg of mitochondria obtained as described above were damaged, the red indicator dye MitoSOX (Invitrogen, cat. no. M36008) was used, which can be used to analyze mitochondrial reactive oxygen species in the isolated mitochondria. The resulting mitochondria were mixed in 50 μl of PBS and then prepared in a 96-well plate and compared with 50 μl of mitochondria-free PBS as a control group. MitoSOX red dye was mixed in 50 µl PBS to a concentration of 10 µM and placed in a 96-well plate (final concentration 5 µM) and then reacted in a 37°C CO ₂ incubator for 20 minutes. After completion of the reaction, the amount of ROS in the mitochondria was measured using a microplate reader (excitation 510/emission 580). The results are shown in Figure 80.

VI. Confirmation of dissociation of desired protein bound to mitochondrial outer membrane protein outside and inside cells

Example 26: Confirmation of Dissociation of Desired Protein Associated with Mitochondrial Outer Membrane Protein Outside the Cell

To obtain the desired protein in free form, when the active protein associated with mitochondria was injected into the cell, a fusion protein (TOM70-UB-p53 or TOM-UB-GFP) was prepared from E. coli in a form in which the ubiquitin protein is inserted between the protein mitochondrial outer membrane and the desired protein. To confirm whether the ubiquitin sequence was cleaved by UBP1, a ubiquitin-degrading enzyme, the recombinant TOM70-UB-p53 fusion protein was reacted with the UBP1 enzyme at 37°C for 1 hour.

It was then confirmed by SDS-PAGE analysis that dissociation of the ubiquitin protein from the fusion protein did not occur by UBP1. This was considered to be an interference phenomenon of the mitochondrial outer membrane protein structure, and thus a linker protein consisting of the amino acids glycine and serine was inserted between the mitochondrial outer membrane protein and the ubiquitin protein to obtain a new fusion protein (TOM70-(GGGGS)3-UB-p53 or TOM70-(GGGGS)3-UB-GFP) by purification from E. coli, and then reacted with the UBP1 enzyme at 37°C for 1 hour as described above. As a result, it was confirmed by SDS-PAGE that the 3' end of ubiquitin was cleaved by the UBP1 enzyme, and only the p53 protein was dissociated, as expected (Figure 82).

Example 26: Confirmation of Dissociation of Desired Protein Associated with Mitochondrial Outer Membrane Protein Within Cells

If the fusion protein (TOM70-(GGGGS)3-UB-p53 or TOM70-(GGGGS)3-UB-GFP) obtained in the example described above enters the cell in the mitochondria-associated state, it is determined whether the active protein has dissociated under by the action of a ubiquitin-cleaving enzyme present in the cell. First, mitochondria derived from umbilical cord blood mesenchymal cells and the TOM70-(GGGGS)3-UB-GFP fusion protein were reacted for 1 hour in a microtube to allow them to bind, and then the unbound fusion protein was removed by centrifugation and then washed twice PBS buffer solution. In this case, a fusion protein (TOM70-(GGGGS)3-GFP) from which ubiquitin was removed was used as a control group.

The mitochondria-associated protein was then injected into MDA-MB-231 cells, a breast cancer cell line, by centrifugation. After one day, MDA-MB-231 cells were disrupted and fractionated into mitochondrial portion and cytosolic portion, respectively, using differential centrifugation. As a result of analysis by SDS-PAGE and Western blot analysis, it was found that in the case of the fusion protein that included ubiquitin, the GFP proteins dissociated from the mitochondrial outer membrane protein, the linker protein and ubiquitin were mainly detected in the cytosolic part. and found that in the case of the fusion protein from which ubiquitin was removed, GFP proteins in the form to which the mitochondrial outer membrane protein and linker protein are bound were mainly detected in the mitochondrial part (Figure 83).

As a result, it was found that when the mitochondrial outer membrane protein-linker-ubiquitin-active protein associated with mitochondria was injected into cells, the junction site of ubiquitin and the active protein was cleaved, and the dissociated active protein was released into the cytoplasm, and it was found that, thus , mitochondria can be used as a delivery vehicle as one way to efficiently deliver beneficial protein into cells.

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LIST OF SEQUENCES

<110> Paean Biotechnology Inc.

<120> MODIFIED MITOCHONDRIA AND THEIR APPLICATION

<130>PCB903024PBT/PCT

<150> KR 10-2018-0048486

<151> 2018-04-26

<160> 92

<170> KoPatentIn 3.0

<210> 1

<211> 39

<212> DNA

<213> Artificial sequence

<220>

<223> T2p53 primer

<400> 1

aaaaaaccgc ggtggtgagg agccgcagtc agatcctag

39

<210> 2

<211> 35

<212> DNA

<213> Artificial sequence

<220>

<223> Primer Xp53

<400> 2

aaaaaactcg agtgagtctg agtcaggccc ttctg

35

<210> 3

<211> 1186

<212> DNA

<213> Artificial sequence

<220>

<223> Nucleotide sequence encoding p53

<400> 3

ccgcggtggt gaggagccgc agtcagatcc tagcgtcgag ccccctctga gtcaggaaac

60

attttcagac ctgtggaaac tacttcctga aaacaacgtt ctgtccccct tgccgtccca

120

agcaatggat gatttgatgc tgtccccgga cgatattgaa caatggttca ctgaagaccc

180

aggtccagat gaagctccca gaatgccaga ggctgctccc cgcgtggccc ctgcaccagc

240

agctcctaca ccggcggccc ctgcaccagc cccctcctgg cccctgtcat cttctgtccc

300

ttcccagaaa acctaccagg gcagctacgg tttccgtctg ggcttcttgc attctgggac

360

agccaagtct gtgacttgca cgtactcccc tgccctcaac aagatgtttt gccaactggc

420

caagacctgc cctgtgcagc tgtgggttga ttccacaccc ccgcccggca cccgcgtccg

480

cgccatggcc atctacaagc agtcacagca catgacggag gttgtgaggc gctgccccca

540

ccatgagcgc tgctcagata gcgatggtct ggcccctcct cagcatctta tccgagtgga

600

aggaaatttg cgtgtggagt atttggatga cagaaacact tttcgacata gtgtggtggt

660

gccctatgag ccgcctgagg ttggctctga ctgtaccacc atccactaca actacatgtg

720

taacagttcc tgcatgggcg gcatgaaccg gaggcccatc ctcaccatca tcacactgga

780

agactccagt ggtaatctac tgggacggaa cagctttgag gtgcgtgttt gtgcctgtcc

840

tgggagagac cggcgcacag aggaagagaa tctccgcaag aaaggggagc ctcaccacga

900

gctgccccca gggagcacta agcgagcact gcccaacaac accagctcct ctccccagcc

960

aaagaagaaa ccactggatg gagaatattt cacccttcag atccgtgggc gtgagcgctt

1020

cgagatgttc cgagagctga atgaggcctt ggaactcaag gatgcccagg ctgggaagga

1080

gccagggggg agcagggctc actccagcca cctgaagtcc aaaaagggtc agtctacctc

1140

ccgccataaa aaactcatgt tcaagacaga agggcctgac tcagac

1186

<210> 4

<211> 35

<212> DNA

<213> Artificial sequence

<220>

<223> Primer NdeUB

<400> 4

ggattccata tgcaactttt cgtcaaaact ctaac

35

<210> 5

<211> 27

<212> DNA

<213> Artificial sequence

<220>

<223> Primer T2UB

<400> 5

atgaccaccg cggagtctca acaccaa

27

<210> 6

<211> 1460

<212> DNA

<213> Artificial sequence

<220>

<223> Nucleotide sequence encoding UB-p53

<400> 6

atgggcagca gccatcatca tcatcatcac agcagcggcc tggtgccgcg cggcagccat

60

atgcaaatct tcgtcaaaac tctaacaggg aagactataa ccctagaggt tgaaccatcc

120

gacactattg aaaacgtcaa agctaaaatt caagataaag aaggtatccc tccggatcag

180

cagagattga tttttgctgg taagcaacta gaagatggta gaaccttgtc tgactacaac

240

atccaaaagg aatctactct tcacttggtg ttgagactcc gcggtggtga ggagccgcag

300

tcagatccta gcgtcgagcc ccctctgagt caggaaacat tttcagacct gtggaaacta

360

cttcctgaaa acaacgttct gtcccccttg ccgtcccaag caatggatga tttgatgctg

420

tccccggacg atattgaaca atggttcact gaagacccag gtccagatga agctcccaga

480

atgccagagg ctgctccccg cgtggcccct gcaccagcag ctcctacacc ggcggcccct

540

gcaccagccc cctcctggcc cctgtcatct tctgtccctt cccagaaaac ctaccagggc

600

agctacggtt tccgtctggg cttcttgcat tctgggacag ccaagtctgt gacttgcacg

660

tactcccctg ccctcaacaa gatgttttgc caactggcca agacctgccc tgtgcagctg

720

tgggttgatt ccacaccccc gcccggcacc cgcgtccgcg ccatggccat ctacaagcag

780

tcacagcaca tgacggaggt tgtgaggcgc tgcccccacc atgagcgctg ctcagatagc

840

gatggtctgg cccctcctca gcatcttatc cgagtggaag gaaatttgcg tgtggagtat

900

ttggatgaca gaaacacttt tcgacatagt gtggtggtgc cctatgagcc gcctgaggtt

960

ggctctgact gtaccaccat cactacaac tacatgtgta acagttcctg catgggcggc

1020

atgaaccgga ggcccatcct caccatcatc acactggaag actccagtgg taatctactg

1080

ggacggaaca gctttgaggt gcgtgtttgt gcctgtcctg gggagagaccg gcgcacagag

1140

gaagagaatc tccgcaagaa aggggagcct caccacgagc tgcccccagg gagcactaag

1200

cgagcactgc ccaacaacac cagctcctct ccccagccaa agaagaaacc actggatgga

1260

gaatatttca cccttcagat ccgtgggcgt gagcgcttcg agatgttccg agagctgaat

1320

gaggccttgg aactcaagga tgcccaggct gggaaggagc caggggggag cagggctcac

1380

tccagccacc tgaagtccaa aaagggtcag tctacctccc gccataaaaa actcatgttc

1440

aagacagaag ggcctgatag

1460

<210> 7

<211> 59

<212> DNA

<213> Artificial sequence

<220>

<223> Primer NdeTOM70

<400> 7

gaattccata tgaaaagttt tataactcgg aataaaactg caattttcgc aactgttgc

59

<210> 8

<211> 38

<212> DNA

<213> Artificial sequence

<220>

<223> Primer TOM70-AS

<400> 8

ggtgcatact actattatca aacttttcgt caaaactc

38

<210> 9

<211> 36

<212> DNA

<213> Artificial sequence

<220>

<223> Primer TOM70UB-S

<400> 9

ggctacgtat ttatttccaa cttttcgtca aaactc

36

<210> 10

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> Primer T2UB-AS

<400> 10

ggcaccaccg cggagtctca acac

24

<210> 11

<211> 1515

<212> DNA

<213> Artificial sequence

<220>

<223> Nucleotide sequence encoding TOM70-UB-p53

<400> 11

atgaaaagtt ttataactcg gaataaaact gcaattttcg caactgttgc tgctacggga

60

accgctattg gtgcatacta ctattatcaa atcttcgtca aaactctaac agggaagact

120

ataaccctag aggttgaacc atccgacact attgaaaacg tcaaagctaa aattcaagat

180

aaagaaggta tccctccgga tcagcagaga ttgatttttg ctggtaagca actagaagat

240

ggtagaacct tgtctgacta caacatccaa aaggaatcta ctcttcactt ggtgttgaga

300

ctccgcggtg gtgaggagcc gcagtcagat cctagcgtcg agccccctct gagtcaggaa

360

acattttcag acctgtggaa actacttcct gaaaacaacg ttctgtcccc cttgccgtcc

420

caagcaatgg atgatttgat gctgtccccg gacgatattg aacaatggtt cactgaagac

480

ccaggtccag atgaagctcc cagaatgcca gaggctgctc cccgcgtggc ccctgcacca

540

gcagctccta caccggcggc ccctgcacca gccccctcct ggcccctgtc atcttctgtc

600

ccttcccaga aaacctacca gggcagctac ggtttccgtc tgggcttctt gcattctggg

660

acagccaagt ctgtgacttg cacgtactcc cctgccctca acaagatgtt ttgccaactg

720

gccaagacct gccctgtgca gctgtgggtt gattccacac ccccgcccgg cacccgcgtc

780

cgcgccatgg ccatctacaa gcagtcacag cacatgacgg aggttgtgag gcgctgcccc

840

caccatgagc gctgctcaga tagcgatggt ctggcccctc ctcagcatct tatccgagtg

900

gaaggaaatt tgcgtgtgga gtatttggat gacagaaaca cttttcgaca tagtgtggtg

960

gtgccctatg agccgcctga ggttggctct gactgtacca ccatccacta caactacatg

1020

tgtaacagtt cctgcatggg cggcatgaac cggaggccca tcctcaccat catcacactg

1080

gaagactcca gtggtaatct actgggacgg aacagctttg aggtgcgtgt ttgtgcctgt

1140

cctggggagag accggcgcac agaggaagag aatctccgca agaaagggga gcctcaccac

1200

gagctgcccc cagggagcac taagcgagca ctgcccaaca acaccagctc ctctccccag

1260

ccaaagaaga aaccactgga tggagaatat ttcacccttc agatccgtgg gcgtgagcgc

1320

ttcgagatgt tccgagagct gaatgaggcc ttggaactca aggatgccca ggctgggaag

1380

gagccagggg ggagcagggc tcactccagc cacctgaagt ccaaaaaggg tcagtctacc

1440

tcccgccata aaaaactcat gttcaagaca gaagggcctg actcagacct cgagcaccac

1500

caccaccacc actag

1515

<210> 12

<211> 59

<212> DNA

<213> Artificial sequence

<220>

<223> Primer TOM70(G)3-AS

<400> 12

gcccccggat cctccacccc cgcttccgcc acctccataa tagtagtatg caccaatag

59

<210> 13

<211> 32

<212> DNA

<213> Artificial sequence

<220>

<223> Primer (G)3UB-S

<400> 13

ggtggaggat ccggggggcgc ggaagccaaa tc

32

<210> 14

<211> 32

<212> DNA

<213> Artificial sequence

<220>

<223> Primer Xp53(noT)

<400> 14

aaaaaactcg aggtctgagt caggcccttc tg

32

<210> 15

<211> 1560

<212> DNA

<213> Artificial sequence

<220>

<223> Nucleotide sequence encoding

TOM70-(GGGGS)3-UB-p53

<400> 15

atgaaaagtt ttataactcg gaataaaact gcaattttcg caactgttgc tgctacggga

60

accgctattg gtgcatacta ctattatgga ggtggcggaa gcggggggtgg aggatccggg

120

ggcggcggaa gccaaatctt cgtcaaaact ctaacaggga agactataac cctagaggtt

180

gaaccatccg acactattga aaacgtcaaa gctaaaattc aagataaaga aggtatccct

240

ccggatcagc agagattgat ttttgctggt aagcaactag aagatggtag aaccttgtct

300

gactacaaca tccaaaagga atctactctt cacttggtgt tgagactccg cggtggtgag

360

gagccgcagt cagatcctag cgtcgagccc cctctgagtc aggaaacatt ttcagacctg

420

tggaaactac ttcctgaaaa caacgttctg tcccccttgc cgtcccaagc aatggatgat

480

ttgatgctgt ccccggacga tattgaacaa tggttcactg aagacccagg tccagatgaa

540

gctcccagaa tgccagaggc tgctccccgc gtggcccctg caccagcagc tcctacaccg

600

gcggcccctg caccagcccc ctcctggccc ctgtcatctt ctgtcccttc ccagaaaacc

660

taccagggca gctacggttt ccgtctgggc ttcttgcatt ctgggacagc caagtctgtg

720

acttgcacgt actcccctgc cctcaacaag atgttttgcc aactggccaa gacctgccct

780

gtgcagctgt gggttgattc cacacccccg cccggcaccc gcgtccgcgc catggccatc

840

tacaagcagt cacagcacat gacggaggtt gtgaggcgct gcccccacca tgagcgctgc

900

tcagatagcg atggtctggc ccctcctcag catcttatcc gagtggaagg aaatttgcgt

960

gtggagtatt tggatgacag aaacactttt cgacatagtg tggtggtgcc ctatgagccg

1020

cctgaggttg gctctgactg taccaccatc cactacaact acatgtgtaa cagttcctgc

1080

atgggcggca tgaaccggag gcccatcctc accatcatca cactggaaga ctccagtggt

1140

aatctactgg gacggaacag ctttgaggtg cgtgtttgtg cctgtcctgg gagagaccgg

1200

cgcacagagg aagagaatct ccgcaagaaa ggggagcctc accacgagct gcccccaggg

1260

agcactaagc gagcactgcc caacaacacc agctcctctc cccagccaaa gaagaaacca

1320

ctggatggag aatatttcac ccttcagatc cgtgggcgtg agcgcttcga gatgttccga

1380

gagctgaatg aggccttgga actcaaggat gcccaggctg ggaaggagcc aggggggagc

1440

agggctcact ccagccacct gaagtccaaa aagggtcagt ctacctcccg ccataaaaaa

1500

ctcatgttca agacagaagg gcctgactca gacctcgagc accaccacca ccaccactag

1560

1560

<210> 16

<211> 51

<212> DNA

<213> Artificial sequence

<220>

<223> B(G)3p53

<400> 16

ggtggaggat ccggggggcgg cggaagcgag gagccgcagt cagatcctag c

51

<210> 17

<211> 1335

<212> DNA

<213> Artificial sequence

<220>

<223> Nucleotide sequence encoding

TOM70-(GGGGS)3-p53

<400> 17

atgaaaagtt ttataactcg gaataaaact gcaattttcg caactgttgc tgctacggga

60

accgctattg gtgcatacta ctattatgga ggtggcggaa gcggggggtgg aggatccggg

120

ggcggcggaa gcgaggagcc gcagtcagat cctagcgtcg agccccctct gagtcaggaa

180

acattttcag acctgtggaa actacttcct gaaaacaacg ttctgtcccc cttgccgtcc

240

caagcaatgg atgatttgat gctgtccccg gacgatattg aacaatggtt cactgaagac

300

ccaggtccag atgaagctcc cagaatgcca gaggctgctc cccgcgtggc ccctgcacca

360

gcagctccta caccggcggc ccctgcacca gccccctcct ggcccctgtc atcttctgtc

420

ccttcccaga aaacctacca gggcagctac ggtttccgtc tgggcttctt gcattctggg

480

acagccaagt ctgtgacttg cacgtactcc cctgccctca acaagatgtt ttgccaactg

540

gccaagacct gccctgtgca gctgtgggtt gattccacac ccccgcccgg cacccgcgtc

600

cgcgccatgg ccatctacaa gcagtcacag cacatgacgg aggttgtgag gcgctgcccc

660

caccatgagc gctgctcaga tagcgatggt ctggcccctc ctcagcatct tatccgagtg

720

gaaggaaatt tgcgtgtgga gtatttggat gacagaaaca cttttcgaca tagtgtggtg

780

gtgccctatg agccgcctga ggttggctct gactgtacca ccatccacta caactacatg

840

tgtaacagtt cctgcatggg cggcatgaac cggaggccca tcctcaccat catcacactg

900

gaagactcca gtggtaatct actgggacgg aacagctttg aggtgcgtgt ttgtgcctgt

960

cctggggagag accggcgcac agaggaagag aatctccgca agaaagggga gcctcaccac

1020

gagctgcccc cagggagcac taagcgagca ctgcccaaca acaccagctc ctctccccag

1080

ccaaagaaga aaccactgga tggagaatat ttcacccttc agatccgtgg gcgtgagcgc

1140

ttcgagatgt tccgagagct gaatgaggcc ttggaactca aggatgccca ggctgggaag

1200

gagccagggg ggagcagggc tcactccagc cacctgaagt ccaaaaaggg tcagtctacc

1260

tcccgccata aaaaactcat gttcaagaca gaagggcctg actcagacct cgagcaccac

1320

caccaccacc actag

1335

<210> 18

<211> 32

<212> DNA

<213> Artificial sequence

<220>

<223> Primer Xp53(noT)

<400> 18

aaaaaactcg aggtctgagt caggcccttc tg

32

<210> 19

<211> 36

<212> DNA

<213> Artificial sequence

<220>

<223> Primer XTOM7

<400> 19

aaaaaactcg agtttgccat tcgctggggc tttatc

36

<210> 20

<211> 38

<212> DNA

<213> Artificial sequence

<220>

<223> Primer LTOM7

<400> 20

aaaaaagtcg acttatcccc aaagtaggct caaaacag

38

<210> 21

<211> 1578

<212> DNA

<213> Artificial sequence

<220>

<223> Nucleotide sequence encoding UB-p53-TOM7

<400> 21

atgggcagca gccatcatca tcatcatcac agcagcggcc tggtgccgcg cggcagccat

60

atgcaaatct tcgtcaaaac tctaacaggg aagactataa ccctagaggt tgaaccatcc

120

gacactattg aaaacgtcaa agctaaaatt caagataaag aaggtatccc tccggatcag

180

cagagattga tttttgctgg taagcaacta gaagatggta gaaccttgtc tgactacaac

240

atccaaaagg aatctactct tcacttggtg ttgagactcc gcggtggtga ggagccgcag

300

tcagatccta gcgtcgagcc ccctctgagt caggaaacat tttcagacct atggaaacta

360

cttcctgaaa acaacgttct gtcccccttg ccgtcccaag caatggatga tttgatgctg

420

tccccggacg atattgaaca atggttcact gaagacccag gtccagatga agctcccaga

480

atgccagagg ctgctccccg cgtggcccct gcaccagcag ctcctacacc ggcggcccct

540

gcaccagccc cctcctggcc cctgtcatct tctgtccctt cccagaaaac ctaccagggc

600

agctacggtt tccgtctggg cttcttgcat tctgggacag ccaagtctgt gacttgcacg

660

tactcccctg ccctcaacaa gatgttttgc caactggcca agacctgccc tgtgcagctg

720

tgggttgatt ccacaccccc gcccggcacc cgcgtccgcg ccatggccat ctacaagcag

780

tcacagcaca tgacggaggt tgtgaggcgc tgcccccacc atgagcgctg ctcagatagc

840

gatggtctgg cccctcctca gcatcttatc cgagtggaag gaaatttgcg tgtggagtat

900

ttggatgaca gaaacacttt tcgacatagt gtggtggtgc cctatgagcc gcctgaggtt

960

ggctctgact gtaccaccat cactacaac tacatgtgta acagttcctg catgggcggc

1020

atgaaccgga ggcccatcct caccatcatc acactggaag actccagtgg taatctactg

1080

ggacggaaca gctttgaggt gcatgtttgt gcctgtcctg gggagagaccg gcgcacagag

1140

gaagagaatc tccgcaagaa aggggagcct caccacgagc tgcccccagg gagcactaag

1200

cgagcactgt ccaacaacac cagctcctct ccccagccaa agaagaaacc actggatgga

1260

gaatatttca cccttcagat ccgtgggcgt gagcgcttcg agatgttccg agagctgaat

1320

gaggccttgg aactcaagga tgcccaggct gggaaggagc caggggggag cagggctcac

1380

tccagccacc tgaagtccaa aaagggtcag tctacctccc gccataaaaa actcatgttc

1440

aagacagaag ggcctgactc agacctcgag tttgccattc gctggggctt tatccctctt

1500

gtgatttacc tgggatttaa gaggggtgca gatcccggaa tgcctgaacc aactgttttg

1560

agcctacttt ggggatag

1578

<210> 22

<211> 35

<212> DNA

<213> Artificial sequence

<220>

<223> Rp53 primer

<400> 22

aaaaaagaat tcatggtctg agtcaggccc ttctg

35

<210> 23

<211> 1266

<212> DNA

<213> Artificial sequence

<220>

<223> Nucleotide sequence encoding p53-myc/His

<400> 23

atggagggagc cgcagtcaga tcctagcgtc gagccccctc tgagtcagga aacattttca

60

gacctgtgga aactacttcc tgaaaacaac gttctgtccc ccttgccgtc ccaagcaatg

120

gatgatttga tgctgtcccc ggacgatatt gaacaatggt tcactgaaga cccaggtcca

180

gatgaagctc ccagaatgcc agaggctgct ccccgcgtgg cccctgcacc agcagctcct

240

acaccggcgg cccctgcacc agccccctcc tggcccctgt catcttctgt cccttcccag

300

aaaacctacc agggcagcta cggtttccgt ctgggcttct tgcattctgg gacagccaag

360

tctgtgactt gcacgtactc ccctgccctc aacaagatgt tttgccaact ggccaagacc

420

tgccctgtgc agctgtgggt tgattccaca cccccgcccg gcacccgcgt ccgcgccatg

480

gccatctaca agcagtcaca gcacatgacg gaggttgtga ggcgctgccc cccacatgag

540

cgctgctcag atagcgatgg tctggcccct cctcagcatc ttatccgagt ggaaggaaat

600

ttgcgtgtgg agtatttgga tgacagaaac acttttcgac atagtgtggt ggtgccctat

660

gagccgcctg aggttggctc tgactgtacc accatccact acaactacat gtgtaacagt

720

tcctgcatgg gcggcatgaa ccggaggccc atcctcacca tcatcacact ggaagactcc

780

agtggtaatc tactgggacg gaacagcttt gaggtgcgtg tttgtgcctg tcctgggaga

840

gaccggcgca cagaggaaga gaatctccgc aagaaagggg agcctcacca cgagctgccc

900

ccagggagca ctaagcgagc actgcccaac aacaccagct cctctcccca gccaaagaag

960

aaaccactgg atggagaata tttcaccctt cagatccgtg ggcgtgagcg cttcgagatg

1020

ttccgagagc tgaatgaggc cttggaactc aaggatgccc aggctgggaa ggagccaggg

1080

ggggagcaggg ctcactccag ccacctgaag tccaaaaagg gtcagtctac ctcccgccat

1140

aaaaaactca tgttcaagac agaagggcct gactcagacc tcgagtctag agggcccttc

1200

gaacaaaaac tcatctcaga agaggatctg aatatgcata ccggtcatca tcaccatcac

1260

cattga

1266

<210> 24

<211> 51

<212> DNA

<213> Artificial sequence

<220>

<223> Primer T2GZMB

<400> 24

aaaaaaccgc ggtggtatca tcgggggaca tgaggcacat gaggccaagc c

51

<210> 25

<211> 38

<212> DNA

<213> Artificial sequence

<220>

<223> Primer XGZMB(noT)

<400> 25

aaaaaactcg aggtagcgtt tcatggtttt ctttatcc

38

<210> 26

<211> 691

<212> DNA

<213> Artificial sequence

<220>

<223> Nucleotide sequence encoding GranzymeB

<400> 26

ccgcggtggt atcatcgggg gacatgaggc caagccccac tcccgcccct acatggctta

60

tcttatgatc tgggatcaga agtctctgaa gaggtgcggt ggcttcctga tacaagacga

120

cttcgtgctg acagctgctc actgttgggg aagctccata aatgtcacct tggggggccca

180

caatatcaaa gaacaggac cgacccagca gtttatccct gtgaaaagac ccatccccca

240

tccagcctat aatcctaaga acttctccaa cgacatcatg ctactgcagc tggagagaaa

300

ggccaagcgg accagagctg tgcagcccct caggctacct agcaacaagg cccaggtgaa

360

gccagggcag acatgcagtg tggccggctg ggggcagacg gcccccctgg gaaaacactc

420

acacacacta caagaggtga agatgacagt gcaggaagat cgaaagtgcg aatctgactt

480

acgccattat tacgacagta ccattgagtt gtgcgtgggg gacccagaga ttaaaaagac

540

ttcctttaag ggggactctg gaggccctct tgtgtgtaac aaggtggccc agggcattgt

600

ctcctatgga cgaaacaatg gcatgcctcc acgagcctgc accaaagtct caagctttgt

660

acactggata aagaaaacca tgaaacgcta c

691

<210> 27

<211> 1065

<212> DNA

<213> Artificial sequence

<220>

<223> Nucleotide sequence encoding

TOM70-(GGGGS)3-UB-granzyme B

<400> 27

atgaaaagtt ttataactcg gaataaaact gcaattttcg caactgttgc tgctacggga

60

accgctattg gtgcatacta ctattatgga ggtggcggaa gcggggggtgg aggatccggg

120

ggcggcggaa gccaaatctt cgtcaaaact ctaacaggga agactataac cctagaggtt

180

gaaccatccg acactattga aaacgtcaaa gctaaaattc aagataaaga aggtatccct

240

ccggatcagc agagattgat ttttgctggt aagcaactag aagatggtag aaccttgtct

300

gactacaaca tccaaaagga atctactctt cacttggtgt tgagactccg cggtggtatc

360

atcgggggac atgaggccaa gccccactcc cgcccctaca tggcttatct tatgatctgg

420

gatcagaagt ctctgaagag gtgcggtggc ttcctgatac gagacgactt cgtgctgaca

480

gctgctcact gttggggaag ctccataaat gtcaccttgg gggcccacaa tatcaaagaa

540

caggagccga cccagcagtt tatccctgtg aaaagaccca tcccccatcc agcctataat

600

cctaagaact tctccaacga catcatgcta ctgcagctgg agagaaaggc caagcggacc

660

agagctgtgc agcccctcag gctacctagc aacaaggccc aggtgaagcc agggcagaca

720

tgcagtgtgg ccggctgggg gcagacggcc cccctgggaa aacactcaca cacactacaa

780

gaggtgaaga tgacagtgca ggaagatcga aagtgcgaat ctgacttacg ccattattac

840

gacagtacca ttgagttgtg cgtgggggac ccagagatta aaaagacttc ctttaagggg

900

gactctggag gccctcttgt gtgtaacaag gtggcccagg gcattgtctc ctatggacga

960

aacaatggca tgcctccacg agcctgcacc aaagtctcaa gctttgtaca ctggataaag

1020

aaaaccatga aacgctacct cgagcaccac caccaccacc actag

1065

<210> 28

<211> 1083

<212> DNA

<213> Artificial sequence

<220>

<223> Nucleotide sequence encoding UB granzyme

B-TOM7

<400> 28

atgggcagca gccatcatca tcatcatcac agcagcggcc tggtgccgcg cggcagccat

60

atgcaaatct tcgtcaaaac tctaacaggg aagactataa ccctagaggt tgaaccatcc

120

gacactattg aaaacgtcaa agctaaaatt caagataaag aaggtatccc tccggatcag

180

cagagattga tttttgctgg taagcaacta gaagatggta gaaccttgtc tgactacaac

240

atccaaaagg aatctactct tcacttggtg ttgagactcc gcggtggtat catcggggga

300

catgaggcca agccccactc ccgcccctac atggcttatc ttatgatctg ggatcagaag

360

tctctgaaga ggtgcggtgg cttcctgata cgagacgact tcgtgctgac agctgctcac

420

tgttggggaa gctccataaa tgtcaccttg ggggcccaca atatcaaaga acaggagccg

480

acccagcagt ttatccctgt gaaaagaccc atcccccatc cagcctataa tcctaagaac

540

ttctccaacg acatcatgct actgcagctg gagagaaagg ccaagcggac cagagctgtg

600

cagcccctca ggctacctag caacaaggcc caggtgaagc cagggcagac atgcagtgtg

660

gccggctggg ggcagacggc ccccctggga aaacactcac acacactaca agaggtgaag

720

atgacagtgc aggaagatcg aaagtgcgaa tctgacttac gccattatta cgacagtacc

780

attgagttgt gcgtggggga cccagagatt aaaaagactt cctttaaggg ggactctgga

840

ggccctcttg tgtgtaacaa ggtggcccag ggcattgtct cctatggacg aaacaatggc

900

atgcctccac gagcctgcac caaagtctca agctttgtac actggataaa gaaaaccatg

960

aaacgctacc tcgagtttgc cattcgctgg ggctttatcc ctcttgtgat ttacctggga

1020

tttaagagg gtgcagatcc cggaatgcct gaaccaactg ttttgagcct actttgggga

1080

tag

1083

<210> 29

<211> 39

<212> DNA

<213> Artificial sequence

<220>

<223> Primer T2RKIP

<400> 29

aaaaaaccgc ggtggtccgg tggacctcag caagtggtc

39

<210> 30

<211> 41

<212> DNA

<213> Artificial sequence

<220>

<223> Primer XRKIP(noT)

<400> 30

aaaaaactcg agcttcccag acagctgctc gtacagtttg g

41

<210> 31

<211> 568

<212> DNA

<213> Artificial sequence

<220>

<223> Nucleotide sequence encoding RKIP

<400> 31

ccgcggtggt ccggtggacc tcagcaagtg gtccgggccc ttgagcctgc aagaagtgga

60

cgagcagccg cagcacccac tgcatgtcac ctacgccggg gcggcggtgg acgagctggg

120

caaagtgctg acgcccaccc aggttaagaa tagacccacc agcatttcgt gggatggtct

180

tgattcaggg aagctctaca ccttggtcct gacagacccg gatgctccca gcaggaagga

240

tcccaaatac agagaatggc atcatttcct ggtggtcaac atgaagggca atgacatcag

300

cagtggcaca gtcctctccg attatgtggg ctcggggcct cccaagggca caggcctcca

360

ccgctatgtc tggctggttt acgagcagga caggccgcta aagtgtgacg agcccatcct

420

cagcaaccga tctggagacc accgtggcaa attcaaggtg gcgtccttcc gtaaaaagta

480

tgagctcagg gccccggtgg ctggcacgtg ttaccaggcc gagtgggatg actatgtgcc

540

caaactgtac gagcagctgt ctgggaag

568

<210> 32

<211> 942

<212> DNA

<213> Artificial sequence

<220>

<223> Nucleotide sequence encoding

TOM70-(GGGGS)3-UB-RKIP

<400> 32

atgaaaagtt ttataactcg gaataaaact gcaattttcg caactgttgc tgctacggga

60

accgctattg gtgcatacta ctattatgga ggtggcggaa gcggggggtgg aggatccggg

120

ggcggcggaa gccaaatctt cgtcaaaact ctaacaggga agactataac cctagaggtt

180

gaaccatccg acactattga aaacgtcaaa gctaaaattc aagataaaga aggtatccct

240

ccggatcagc agagattgat ttttgctggt aagcaactag aagatggtag aaccttgtct

300

gactacaaca tccaaaagga atctactctt cacttggtgt tgagactccg cggtggtccg

360

gtggacctca gcaagtggtc cgggcccttg agcctgcaag aagtggacga gcagccgcag

420

cacccactgc atgtcaccta cgccggggcg gcggtggacg agctgggcaa agtgctgacg

480

cccacccagg ttaagaatag acccaccagc atttcgtggg atggtcttga ttcagggaag

540

ctctacacct tggtcctgac agacccggat gctcccagca ggaaggatcc caaatacaga

600

gaatggcatc atttcctggt ggtcaacatg aagggcaatg acatcagcag tggcacagtc

660

ctctccgatt atgtgggctc ggggcctccc aagggcacag gcctccaccg ctatgtctgg

720

ctggtttacg agcaggacag gccgctaaag tgtgacgagc ccatcctcag caaccgatct

780

ggagaccacc gtggcaaatt caaggtggcg tccttccgta aaaagtatga gctcagggcc

840

ccggtggctg gcacgtgtta ccaggccgag tgggatgact atgtgcccaa actgtacgag

900

cagctgtctg ggaagctcga gcaccaccac caccaccact ag

942

<210> 33

<211> 42

<212> DNA

<213> Artificial sequence

<220>

<223> Primer T2PTEN

<400> 33

aaaaaaccgc ggtggtacag ccatcatcaa agagatcgtt ag

42

<210> 34

<211> 36

<212> DNA

<213> Artificial sequence

<220>

<223> Primer XPTEN(noT)

<400> 34

aaaaaactcg aggacttttg taatttgtgt atgctg

36

<210> 35

<211> 1216

<212> DNA

<213> Artificial sequence

<220>

<223> Nucleotide sequence encoding PTEN

<400> 35

ccgcggtggt acagccatca tcaaagagat cgttagcaga aacaaaagga gatatcaaga

60

ggatggattc gacttagact tgacctatat ttatccaaac attattgcta tgggatttcc

120

tgcagaaaga cttgaaggcg tatacaggaa caatattgat gatgtagtaa ggtttttgga

180

ttcaaagcat aaaaaccatt acaagatata caatctttgt gctgaaagac attatgacac

240

cgccaaattt aattgcagag ttgcacaata tccttttgaa gaccataacc caccacagct

300

agaacttatc aaaccctttt gtgaagatct tgaccaatgg ctaagtgaag atgacaatca

360

tgttgcagca attcactgta aagctggaaa gggacgaact ggtgtaatga tatgtgcata

420

tttattacat cggggcaaat ttttaaaggc acaagaggcc ctagatttct atggggaagt

480

aaggaccaga gacaaaaagg gagtaactat tcccagtcag aggcgctatg tgtattatta

540

tagctacctg ttaaagaatc atctggatta tagaccagtg gcactgttgt ttcacaagat

600

gatgtttgaa actattccaa tgttcagtgg cggaacttgc aatcctcagt ttgtggtctg

660

ccagctaaag gtgaagatat attcctccaa ttcaggaccc acacgacggg aagacaagtt

720

catgtacttt gagttccctc agccgttacc tgtgtgtggt gatatcaaag tagagttctt

780

ccacaaacag aacaagatgc taaaaaagga caaaatgttt cacttttggg taaatacatt

840

cttcatacca ggaccagagg aaacctcaga aaaagtagaa aatggaagtc tatgtgatca

900

agaaatcgat agcatttgca gtatagagcg tgcagataat gacaaggaat atctagtact

960

tactttaaca aaaaatgatc ttgacaaagc aaataaagac aaagccaacc gatacttttc

1020

tccaaatttt aaggtgaagc tgtacttcac aaaaacagta gaggagccgt caaatccaga

1080

ggctagcagt tcaacttctg taacaccaga tgttagtgac aatgaacctg atcattatag

1140

atattctgac accactgact ctgatccaga gaatgaacct tttgatgaag atcagcatac

1200

aaaattaca aaagtc

1216

<210> 36

<211> 1590

<212> DNA

<213> Artificial sequence

<220>

<223> Nucleotide sequence encoding

TOM70-(GGGGS)3-UB-PTEN

<400> 36

atgaaaagtt ttataactcg gaataaaact gcaattttcg caactgttgc tgctacggga

60

accgctattg gtgcatacta ctattatgga ggtggcggaa gcggggggtgg aggatccggg

120

ggcggcggaa gccaaatctt cgtcaaaact ctaacaggga agactataac cctagaggtt

180

gaaccatccg acactattga aaacgtcaaa gctaaaattc aagataaaga aggtatccct

240

ccggatcagc agagattgat ttttgctggt aagcaactag aagatggtag aaccttgtct

300

gactacaaca tccaaaagga atctactctt cacttggtgt tgagactccg cggtggtaca

360

gccatcatca aagagatcgt tagcagaaac aaaaggagat atcaagagga tggattcgac

420

ttagacttga cctatattta tccaaacatt attgctatgg gatttcctgc agaaagactt

480

gaaggcgtat acaggaacaa tattgatgat gtagtaaggt ttttggattc aaagcataaa

540

aaccattaca agatatacaa tctttgtgct gaaagacatt atgacaccgc caaatttaat

600

tgcagagttg cacaatatcc ttttgaagac cataacccac cacagctaga acttatcaaa

660

cccttttgtg aagatcttga ccaatggcta agtgaagatg acaatcatgt tgcagcaatt

720

cactgtaaag ctggaaaggg acgaactggt gtaatgatat gtgcatattt attacatcgg

780

ggcaaatttt taaaggcaca agaggcccta gatttctatg gggaagtaag gaccagagac

840

aaaaagggag taactattcc cagtcagagg cgctatgtgt attattatag ctacctgtta

900

aagaatcatc tggattatag accagtggca ctgttgtttc acaagatgat gtttgaaact

960

attccaatgt tcagtggcgg aacttgcaat cctcagtttg tggtctgcca gctaaaggtg

1020

aagatatatt cctccaattc aggacccaca cgacgggaag acaagttcat gtactttgag

1080

ttccctcagc cgttacctgt gtgtggtgat atcaaagtag agttcttcca caaacagaac

1140

aagatgctaa aaaaggacaa aatgtttcac ttttgggtaa atacattctt cataccagga

1200

ccagaggaaa cctcagaaaa agtagaaaat ggaagtctat gtgatcaaga aatcgatagc

1260

atttgcagta tagagcgtgc agataatgac aaggaatatc tagtacttac tttaacaaaa

1320

aatgatcttg acaaagcaaa taaagacaaa gccaaccgat acttttctcc aaattttaag

1380

gtgaagctgt acttcacaaa aacagtagag gagccgtcaa atccagaggc tagcagttca

1440

acttctgtaa caccagatgt tagtgacaat gaacctgatc attatagata ttctgacacc

1500

actgactctg atccagagaa tgaacctttt gatgaagatc agcatacaca aattacaaaa

1560

gtcctcgagc accaccacca ccaccactag

1590

<210> 37

<211> 739

<212> DNA

<213> Artificial sequence

<220>

<223> Nucleotide sequence encoding scFvHER2

<400> 37

ccgcggtggt gaagtgcaac ttgttgagag tggcggagga ctggtccaac cgggcggttc

60

acttaggctc tcatgtgcag cttcagggtt cacgttcacg gactatacaa tggactgggt

120

gaggcaagcc cctggtaagg gactggagtg ggttgctgac gttaacccta attccggtgg

180

gtccatctac aaccagcgat tcaagggacg atttactctt tcagtcgaca gaagcaaaaa

240

caccctctac ctccagatga actccttgcg ggcagaggat acagcggtct actattgcgc

300

gagaaacttg ggaccaagct tctacttcga ctactggggg caaggaacgc ttgttacggt

360

ttcaagcgga ggtggaggaa gtggaggtgg cggttccggc ggtggcggtt cagatataca

420

gatgacccaa tcacccagtt ctcttagcgc gtctgtaggc gacagggtaa ccataacctg

480

caaggcgtcc caggacgtgt caattggagt tgcctggtat cagcaaaaac ctgggaaagc

540

tccgaagctc ctgatttaca gcgcatctta ccgatatact ggtgtccctt caaggttcag

600

tggcagtgga tctgggacag actttacgct tactatcagc agtctgcaac ctgaggattt

660

cgcgacctac tactgtcagc agtattacat ctatccgtac acgttcggtc aaggtacaaa

720

ggtagaaata aaacgcact

739

<210> 38

<211> 1125

<212> DNA

<213> Artificial sequence

<220>

<223> Nucleotide sequence encoding UB-scFvHER2-TOM7

<400> 38

atgggcagca gccatcatca tcatcatcac agcagcggcc tggtgccgcg cggcagccat

60

atgcaaatct tcgtcaaaac tctaacaggg aagactataa ccctagaggt tgaaccatcc

120

gacactattg aaaacgtcaa agctaaaatt caagataaag aaggtatccc tccggatcag

180

cagagattga tttttgctgg taagcaacta gaagatggta gaaccttgtc tgactacaac

240

atccaaaagg aatctactct tcacttggtg ttgagactcc gcggtggtga agtgcaactt

300

gttgagagtg gcggaggact ggtccaaccg ggcggttcac ttaggctctc atgtgcagct

360

tcagggttca cgttcacgga ctatacaatg gactgggtga ggcaagcccc tggtaaggga

420

ctggagtggg ttgctgacgt taaccctaat tccggtgggt ccatctacaa ccagcgattc

480

aagggacgat ttactctttc agtcgacaga agcaaaaaca ccctctacct cgagatgaac

540

tccttgcggg cagaggatac agcggtctac tattgcgcga gaaacttggg accaagcttc

600

tacttcgact actggggggca aggaacgctt gttacggttt caagcggagg tggaggaagt

660

ggaggtggcg gttccggcgg tggcggttca gatatacaga tgacccaatc acccagttct

720

cttagcgcgt ctgtaggcga cagggtaacc ataacctgca aggcgtccca ggacgtgtca

780

attggagttg cctggtatca gcaaaaacct gggaaagctc cgaagctcct gatttacagc

840

gcatcttacc gatatactgg tgtcccttca aggttcagtg gcagtggatc tgggacagac

900

tttacgctta ctatcagcag tctgcaacct gaggatttcg cgacctacta ctgtcagcag

960

tattacatct atccgtacac gttcggtcaa ggtacaaagg tagaaataaa acgcactttt

1020

gccattcgct ggggctttat ccctcttgtg atttacctgg gatttaagag gggtgcagat

1080

cccggaatgc ctgaaccaac tgttttgagc ctactttggg gatag

1125

<210> 39

<211> 38

<212> DNA

<213> Artificial sequence

<220>

<223> RscFvHER2 primer

<400> 39

aaaaaagaat tcatggaagt gcaacttgtt gagagtgg

38

<210> 40

<211> 35

<212> DNA

<213> Artificial sequence

<220>

<223> Primer XTOM7(noT)

<400> 40

aaaaaactcg agtccccaaa gtaggctcaa aacag

35

<210> 41

<211> 924

<212> DNA

<213> Artificial sequence

<220>

<223> Nucleotide sequence encoding

scFvHER2-TOM7-myc/His

<400> 41

atggaagtgc aacttgttga gagtggcgga ggactggtcc aaccgggcgg ttcacttagg

60

ctctcatgtg cagcttcagg gttcacgttc acggactata caatggactg ggtgaggcaa

120

gcccctggta agggactgga gtgggttgct gacgttaacc ctaattccgg tgggtccatc

180

tacaaccagc gattcaaggg acgatttact ctttcagtcg acagaagcaa aaacaccctc

240

tacctccaga tgaactcctt gcgggcagag gatacagcgg tctactattg cgcgagaaac

300

ttgggaccaa gcttctactt cgactactgg gggcaaggaa cgcttgttac ggtttcaagc

360

ggaggtggag gaagtggagg tggcggttcc ggcggtggcg gttcagatat acagatgacc

420

caatcaccca gttctcttag cgcgtctgta ggcgacaggg taaccataac ctgcaaggcg

480

tcccaggacg tgtcaattgg agttgcctgg tatcagcaaa aacctgggaa agctccgaag

540

ctcctgattt acagcgcatc ttaccgatat actggtgtcc cttcaaggtt cagtggcagt

600

ggatctggga cagactttac gcttactatc agcagtctgc aacctgagga tttcgcgacc

660

tactactgtc agcagtatta catctatccg tacacgttcg gtcaaggtac aaaggtagaa

720

ataaaacgca cttttgccat tcgctggggc tttatccctc ttgtgattta cctgggattt

780

aagaggggtg cagatcccgg aatgcctgaa ccaactgttt tgagcctact ttggggactc

840

gagtctagag ggcccttcga acaaaaactc atctcagaag aggatctgaa tatgcatacc

900

ggtcatcatc accatcacca ttga

924

<210> 42

<211> 760

<212> DNA

<213> Artificial sequence

<220>

<223> Nucleotide sequence encoding scFvMEL

<400> 42

ccgcggtggt acggacattg tgatgaccca gtctcaaaaa ttcatgtcca catcagtagg

60

agacaggtc agcgtcacct gcaaggccag tcagaatgtg gatactaatg tagcctggta

120

tcaacaaaaa ccagggcaat ctcctgaacc actgcttttc tcggcatcct accgttacac

180

tggagtccct gatcgcttca caggcagtgg atctgggaca gatttcactc tcaccatcag

240

caatgtgcag tctgaagact tggcagagta tttctgtcag caatataaca gctatcctct

300

gacgttcggt ggaggcacca agctggagat caaaggctcc accagcggca gcggtaagcc

360

aggctccggc gaaggcagca ccaaaggcga agtgaaggtt gaggagtctg gaggaggctt

420

ggtgcaacct ggaggatcca tgaaactctc ctgtgttgtc tctggattca ctttcggtaa

480

ttactggatg aactgggtcc gccagtctcc agagaagggg cttgagtgga ttgcagaaat

540

tagattgaaa tccaataatt ttgcaagata ttatgcggag tctgtgaaag ggaggttcac

600

catctcaaga gatgattcca aaagtagtgt ctacctgcaa atgatcaacc taagagctga

660

agatactggc atttattact gtaccagtta tggtaactac gttgggcact attttgacca

720

ctggggccaa ggcaccactc tcaccgtctc cctttgggga

760

<210> 43

<211> 1137

<212> DNA

<213> Artificial sequence

<220>

<223> Nucleotide sequence encoding UB-scFvMEL-TOM7

<400> 43

atgggcagca gccatcatca tcatcatcac agcagcggcc tggtgccgcg cggcagccat

60

atgcaaatct tcgtcaaaac tctaacaggg aagactataa ccctagaggt tgaaccatcc

120

gacactattg aaaacgtcaa agctaaaatt caagataaag aaggtatccc tccggatcag

180

cagagattga tttttgctgg taagcaacta gaagatggta gaaccttgtc tgactacaac

240

atccaaaagg aatctactct tcacttggtg ttgagactcc gcggtggtac ggacattgtg

300

atgacccagt ctcaaaaatt catgtccaca tcagtaggag acagggtcag cgtcacctgc

360

aaggccagtc agaatgtgga tactaatgta gcctggtatc aacaaaaacc agggcaatct

420

cctgaaccac tgcttttctc ggcatcctac cgttacactg gagtccctga tcgcttcaca

480

ggcagtggat ctgggacaga tttcactctc accatcagca atgtgcagtc tgaagacttg

540

gcagagtatt tctgtcagca atataacagc tatcctctga cgttcggtgg aggcaccaag

600

ctggagatca aaggctccac cagcggcagc ggtaagccag gctccggcga aggcagcacc

660

aaaggcgaag tgaaggttga ggagtctgga ggaggcttgg tgcaacctgg aggatccatg

720

aaactctcct gtgttgtctc tggattcact ttcggtaatt actggatgaa ctgggtccgc

780

cagtctccag agaaggggct tgagtggatt gcagaaatta gattgaaatc caataatttt

840

gcaagatatt atgcggagtc tgtgaaaggg aggttcacca tctcaagaga tgattccaaa

900

agtagtgtct acctgcaaat gatcaaccta agagctgaag atactggcat ttattactgt

960

accagttatg gtaactacgt tgggcactat tttgaccact ggggccaagg caccactctc

1020

accgtctcct catttgccat tcgctggggc tttatccctc ttgtgattta cctgggattt

1080

aagaggggtg cagatcccgg aatgcctgaa ccaactgttt tgagcctact ttgggga

1137

<210> 44

<211> 36

<212> DNA

<213> Artificial sequence

<220>

<223> RscFvMEL primer

<400> 44

aaaaaagaat tcatgaaaac aagtaaccca ggagtg

36

<210> 45

<211> 939

<212> DNA

<213> Artificial sequence

<220>

<223> Nucleotide sequence encoding

scFvMEL-TOM7-myc/His

<400> 45

atgacggaca ttgtgatgac ccagtctcaa aaattcatgt ccacatcagt aggagacagg

60

gtcagcgtca cctgcaaggc cagtcagaat gtggatacta atgtagcctg gtatcaacaa

120

aaaccagggc aatctcctga accactgctt ttctcggcat cctaccgtta cactggagtc

180

cctgatcgct tcacaggcag tggatctggg acagatttca ctctcaccat cagcaatgtg

240

cagtctgaag acttggcaga gtatttctgt cagcaatata acagctatcc tctgacgttc

300

ggtggaggca ccaagctgga gatcaaaggc tccaccagcg gcagcggtaa gccaggctcc

360

ggcgaaggca gcaccaaagg cgaagtgaag gttgaggagt ctggaggagg cttggtgcaa

420

cctggaggat ccatgaaact ctcctgtgtt gtctctggat tcactttcgg taattactgg

480

atgaactggg tccgccagtc tccagagaag gggcttgagt ggattgcaga aattagattg

540

aaatccaata attttgcaag atattatgcg gagtctgtga aagggaggtt caccatctca

600

agagatgatt ccaaaagtag tgtctacctg caaatgatca acctaagagc tgaagatact

660

ggcatttatt actgtaccag ttatggtaac tacgttgggc actattttga ccactggggc

720

caaggcacca ctctcaccgt ctcctcattt gccattcgct ggggctttat ccctcttgtg

780

atttacctgg gatttaagag gggtgcagat cccggaatgc ctgaaccaac tgttttgagc

840

ctactttggg gactcgagtc tagagggccc ttcgaacaaa aactcatctc agaagaggat

900

ctgaatatgc ataccggtca tcatcaccat caccattga

939

<210> 46

<211> 750

<212> DNA

<213> Artificial sequence

<220>

<223> Nucleotide sequence encoding scFvPD-L1

<400> 46

atggacatcg tgatgagcca gtctcccagc agcctggctg tgtctgctgg ggagaaggtg

60

accatgtcct gcaagagctc ccagtccctg ctgaacagcc gcaccaggaa gaactacctg

120

gcctggtacc agcagaagcc aggccagagc cccaagctcc tcatctactg ggccagcacc

180

cggggagagcg gggtgcctga ccgcttcact ggaagtggca gcggcacaga cttcaccctg

240

accatcagct ctgtgcaggc cgaggacctg gcagtgtact actgccagca aagctatgat

300

gtggtgacat ttggagctgg caccaagctg gagctgaagg gaggtggcgg aagcgggggt

360

ggaggatccg ggggcggcgg aagccaggtc caggtgcagc agagcggggc tgagctggcc

420

gagcccgggg cctctgtgaa gatgagctgc aaggcttctg gctacatctt caccagctac

480

tggatgcact ggctcaagca gaggcctggg caggggctgg agtggatcgg ctatatcaac

540

cccagcagtg actacaatga atattctgag aagttcatgg acaaagccac cctgactgct

600

gacaaggcca gcaccaccgc ctacatgcag ctgatcagcc tgacctcaga ggacagcgct

660

gtgtactact gtgcccggag cggctggctg gtgcacgggg actattattt tgattattgg

720

ggccagggca ccacactgac agtgagcagc

750

<210> 47

<211> 948

<212> DNA

<213> Artificial sequence

<220>

<223> Nucleotide sequence encoding

scFvPD-L1-TOM7-myc/His

<400> 47

atggacatcg tgatgagcca gtctcccagc agcctggctg tgtctgctgg ggagaaggtg

60

accatgtcct gcaagagctc ccagtccctg ctgaacagcc gcaccaggaa gaactacctg

120

gcctggtacc agcagaagcc aggccagagc cccaagctcc tcatctactg ggccagcacc

180

cggggagagcg gggtgcctga ccgcttcact ggaagtggca gcggcacaga cttcaccctg

240

accatcagct ctgtgcaggc cgaggacctg gcagtgtact actgccagca aagctatgat

300

gtggtgacat ttggagctgg caccaagctg gagctgaagg gaggtggcgg aagcgggggt

360

ggaggatccg ggggcggcgg aagccaggtc caggtgcagc agagcggggc tgagctggcc

420

gagcccgggg cctctgtgaa gatgagctgc aaggcttctg gctacatctt caccagctac

480

tggatgcact ggctcaagca gaggcctggg caggggctgg agtggatcgg ctatatcaac

540

cccagcagtg actacaatga atattctgag aagttcatgg acaaagccac cctgactgct

600

gacaaggcca gcaccaccgc ctacatgcag ctgatcagcc tgacctcaga ggacagcgct

660

gtgtactact gtgcccggag cggctggctg gtgcacgggg actattattt tgattattgg

720

ggccagggca ccacactgac agtgagcagc ctcgagtttg ccattcgctg gggctttatc

780

cctcttgtga tttacctggg atttaagagg ggtgcagatc ccggaatgcc tgaaccaact

840

gttttgagcc tactttgggg actcgagtct agaggggccct tcgaacaaaa actcatctca

900

gaagaggatc tgaatatgca taccggtcat catcaccatc accattga

948

<210> 48

<211> 395

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Amino acid sequence of p53

<400> 48

Arg Gly Gly Glu Glu Pro Gln Ser Asp Pro Ser Val Glu Pro Pro Leu

1 5 10 15

Ser Gln Glu Thr Phe Ser Asp Leu Trp Lys Leu Leu Pro Glu Asn Asn

20 25 30

Val Leu Ser Pro Leu Pro Ser Gln Ala Met Asp Asp Leu Met Leu Ser

35 40 45

Pro Asp Asp Ile Glu Gln Trp Phe Thr Glu Asp Pro Gly Pro Asp Glu

50 55 60

Ala Pro Arg Met Pro Glu Ala Ala Pro Arg Val Ala Pro Ala Pro Ala

65 70 75 80

Ala Pro Thr Pro Ala Ala Pro Ala Pro Ala Pro Ser Trp Pro Leu Ser

85 90 95

Ser Ser Val Pro Ser Gln Lys Thr Tyr Gln Gly Ser Tyr Gly Phe Arg

100 105 110

Leu Gly Phe Leu His Ser Gly Thr Ala Lys Ser Val Thr Cys Thr Tyr

115 120 125

Ser Pro Ala Leu Asn Lys Met Phe Cys Gln Leu Ala Lys Thr Cys Pro

130 135 140

Val Gln Leu Trp Val Asp Ser Thr Pro Pro Pro Gly Thr Arg Val Arg

145 150 155 160

Ala Met Ala Ile Tyr Lys Gln Ser Gln His Met Thr Glu Val Val Arg

165 170 175

Arg Cys Pro His His Glu Arg Cys Ser Asp Ser Asp Gly Leu Ala Pro

180 185 190

Pro Gln His Leu Ile Arg Val Glu Gly Asn Leu Arg Val Glu Tyr Leu

195 200 205

Asp Asp Arg Asn Thr Phe Arg His Ser Val Val Val Pro Tyr Glu Pro

210 215 220

Pro Glu Val Gly Ser Asp Cys Thr Thr Ile His Tyr Asn Tyr Met Cys

225 230 235 240

Asn Ser Ser Cys Met Gly Gly Met Asn Arg Arg Pro Ile Leu Thr Ile

245 250 255

Ile Thr Leu Glu Asp Ser Ser Gly Asn Leu Leu Gly Arg Asn Ser Phe

260 265 270

Glu Val Arg Val Cys Ala Cys Pro Gly Arg Asp Arg Arg Thr Glu Glu

275 280 285

Glu Asn Leu Arg Lys Lys Gly Glu Pro His His Glu Leu Pro Pro Gly

290 295 300

Ser Thr Lys Arg Ala Leu Pro Asn Asn Thr Ser Ser Ser Pro Gln Pro

305 310 315 320

Lys Lys Lys Pro Leu Asp Gly Glu Tyr Phe Thr Leu Gln Ile Arg Gly

325 330 335

Arg Glu Arg Phe Glu Met Phe Arg Glu Leu Asn Glu Ala Leu Glu Leu

340 345 350

Lys Asp Ala Gln Ala Gly Lys Glu Pro Gly Gly Ser Arg Ala His Ser

355 360 365

Ser His Leu Lys Ser Lys Lys Gly Gln Ser Thr Ser Arg His Lys Lys

370 375 380

Leu Met Phe Lys Thr Glu Gly Pro Asp Ser Asp

385 390 395

<210> 49

<211> 488

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Amino acid sequence of UB-p53

<400> 49

Met Gly Ser Ser His His His His His Ser Ser Gly Leu Val Pro

1 5 10 15

Arg Gly Ser His Met Gln Ile Phe Val Lys Thr Leu Thr Gly Lys Thr

20 25 30

Ile Thr Leu Glu Val Glu Pro Ser Asp Thr Ile Glu Asn Val Lys Ala

35 40 45

Lys Ile Gln Asp Lys Glu Gly Ile Pro Pro Asp Gln Gln Arg Leu Ile

50 55 60

Phe Ala Gly Lys Gln Leu Glu Asp Gly Arg Thr Leu Ser Asp Tyr Asn

65 70 75 80

Ile Gln Lys Glu Ser Thr Leu His Leu Val Leu Arg Leu Arg Gly Gly

85 90 95

Glu Glu Pro Gln Ser Asp Pro Ser Val Glu Pro Pro Leu Ser Gln Glu

100 105 110

Thr Phe Ser Asp Leu Trp Lys Leu Leu Pro Glu Asn Asn Val Leu Ser

115 120 125

Pro Leu Pro Ser Gln Ala Met Asp Asp Leu Met Leu Ser Pro Asp Asp

130 135 140

Ile Glu Gln Trp Phe Thr Glu Asp Pro Gly Pro Asp Glu Ala Pro Arg

145 150 155 160

Met Pro Glu Ala Ala Pro Arg Val Ala Pro Ala Pro Ala Ala Pro Thr

165 170 175

Pro Ala Ala Pro Ala Pro Ala Pro Ser Trp Pro Leu Ser Ser Ser Val

180 185 190

Pro Ser Gln Lys Thr Tyr Gln Gly Ser Tyr Gly Phe Arg Leu Gly Phe

195 200 205

Leu His Ser Gly Thr Ala Lys Ser Val Thr Cys Thr Tyr Ser Pro Ala

210 215 220

Leu Asn Lys Met Phe Cys Gln Leu Ala Lys Thr Cys Pro Val Gln Leu

225 230 235 240

Trp Val Asp Ser Thr Pro Pro Pro Gly Thr Arg Val Arg Ala Met Ala

245 250 255

Ile Tyr Lys Gln Ser Gln His Met Thr Glu Val Val Arg Arg Cys Pro

260 265 270

His His Glu Arg Cys Ser Asp Ser Asp Gly Leu Ala Pro Pro Gln His

275 280 285

Leu Ile Arg Val Glu Gly Asn Leu Arg Val Glu Tyr Leu Asp Asp Arg

290 295 300

Asn Thr Phe Arg His Ser Val Val Val Pro Tyr Glu Pro Pro Glu Val

305 310 315 320

Gly Ser Asp Cys Thr Thr Ile His Tyr Asn Tyr Met Cys Asn Ser Ser

325 330 335

Cys Met Gly Gly Met Asn Arg Arg Pro Ile Leu Thr Ile Ile Thr Leu

340 345 350

Glu Asp Ser Ser Gly Asn Leu Leu Gly Arg Asn Ser Phe Glu Val Arg

355 360 365

Val Cys Ala Cys Pro Gly Arg Asp Arg Arg Thr Glu Glu Glu Asn Leu

370 375 380

Arg Lys Lys Gly Glu Pro His His Glu Leu Pro Pro Gly Ser Thr Lys

385 390 395 400

Arg Ala Leu Pro Asn Asn Thr Ser Ser Ser Pro Gln Pro Lys Lys Lys

405 410 415

Pro Leu Asp Gly Glu Tyr Phe Thr Leu Gln Ile Arg Gly Arg Glu Arg

420 425 430

Phe Glu Met Phe Arg Glu Leu Asn Glu Ala Leu Glu Leu Lys Asp Ala

435 440 445

Gln Ala Gly Lys Glu Pro Gly Gly Ser Arg Ala His Ser Ser His Leu

450 455 460

Lys Ser Lys Lys Gly Gln Ser Thr Ser Arg His Lys Lys Leu Met Phe

465 470 475 480

Lys Thr Glu Gly Pro Asp Ser Asp

485

<210> 50

<211> 504

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Amino acid sequence of TOM70-UB-p53

<400> 50

Met Lys Ser Phe Ile Thr Arg Asn Lys Thr Ala Ile Phe Ala Thr Val

1 5 10 15

Ala Ala Thr Gly Thr Ala Ile Gly Ala Tyr Tyr Tyr Tyr Gln Ile Phe

20 25 30

Val Lys Thr Leu Thr Gly Lys Thr Ile Thr Leu Glu Val Glu Pro Ser

35 40 45

Asp Thr Ile Glu Asn Val Lys Ala Lys Ile Gln Asp Lys Glu Gly Ile

50 55 60

Pro Pro Asp Gln Gln Arg Leu Ile Phe Ala Gly Lys Gln Leu Glu Asp

65 70 75 80

Gly Arg Thr Leu Ser Asp Tyr Asn Ile Gln Lys Glu Ser Thr Leu His

85 90 95

Leu Val Leu Arg Leu Arg Gly Gly Glu Glu Pro Gln Ser Asp Pro Ser

100 105 110

Val Glu Pro Pro Leu Ser Gln Glu Thr Phe Ser Asp Leu Trp Lys Leu

115 120 125

Leu Pro Glu Asn Asn Val Leu Ser Pro Leu Pro Ser Gln Ala Met Asp

130 135 140

Asp Leu Met Leu Ser Pro Asp Asp Ile Glu Gln Trp Phe Thr Glu Asp

145 150 155 160

Pro Gly Pro Asp Glu Ala Pro Arg Met Pro Glu Ala Ala Pro Arg Val

165 170 175

Ala Pro Ala Pro Ala Ala Pro Thr Pro Ala Ala Pro Ala Pro Ala Pro

180 185 190

Ser Trp Pro Leu Ser Ser Ser Val Pro Ser Gln Lys Thr Tyr Gln Gly

195 200 205

Ser Tyr Gly Phe Arg Leu Gly Phe Leu His Ser Gly Thr Ala Lys Ser

210 215 220

Val Thr Cys Thr Tyr Ser Pro Ala Leu Asn Lys Met Phe Cys Gln Leu

225 230 235 240

Ala Lys Thr Cys Pro Val Gln Leu Trp Val Asp Ser Thr Pro Pro Pro

245 250 255

Gly Thr Arg Val Arg Ala Met Ala Ile Tyr Lys Gln Ser Gln His Met

260 265 270

Thr Glu Val Val Arg Arg Cys Pro His His Glu Arg Cys Ser Asp Ser

275 280 285

Asp Gly Leu Ala Pro Pro Gln His Leu Ile Arg Val Glu Gly Asn Leu

290 295 300

Arg Val Glu Tyr Leu Asp Asp Arg Asn Thr Phe Arg His Ser Val Val

305 310 315 320

Val Pro Tyr Glu Pro Pro Glu Val Gly Ser Asp Cys Thr Thr Ile His

325 330 335

Tyr Asn Tyr Met Cys Asn Ser Ser Cys Met Gly Gly Met Asn Arg Arg

340 345 350

Pro Ile Leu Thr Ile Ile Thr Leu Glu Asp Ser Ser Gly Asn Leu Leu

355 360 365

Gly Arg Asn Ser Phe Glu Val Arg Val Cys Ala Cys Pro Gly Arg Asp

370 375 380

Arg Arg Thr Glu Glu Glu Asn Leu Arg Lys Lys Gly Glu Pro His His

385 390 395 400

Glu Leu Pro Pro Gly Ser Thr Lys Arg Ala Leu Pro Asn Asn Thr Ser

405 410 415

Ser Ser Pro Gln Pro Lys Lys Lys Pro Leu Asp Gly Glu Tyr Phe Thr

420 425 430

Leu Gln Ile Arg Gly Arg Glu Arg Phe Glu Met Phe Arg Glu Leu Asn

435 440 445

Glu Ala Leu Glu Leu Lys Asp Ala Gln Ala Gly Lys Glu Pro Gly Gly

450 455 460

Ser Arg Ala His Ser Ser His Leu Lys Ser Lys Lys Gly Gln Ser Thr

465 470 475 480

Ser Arg His Lys Lys Leu Met Phe Lys Thr Glu Gly Pro Asp Ser Asp

485 490 495

Leu Glu His His His His His

500

<210> 51

<211> 519

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Amino acid sequence of TOM70-(GGGGS)3-UB-p53

<400> 51

Met Lys Ser Phe Ile Thr Arg Asn Lys Thr Ala Ile Phe Ala Thr Val

1 5 10 15

Ala Ala Thr Gly Thr Ala Ile Gly Ala Tyr Tyr Tyr Tyr Gly Gly Gly

20 25 30

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Ile Phe Val

35 40 45

Lys Thr Leu Thr Gly Lys Thr Ile Thr Leu Glu Val Glu Pro Ser Asp

50 55 60

Thr Ile Glu Asn Val Lys Ala Lys Ile Gln Asp Lys Glu Gly Ile Pro

65 70 75 80

Pro Asp Gln Gln Arg Leu Ile Phe Ala Gly Lys Gln Leu Glu Asp Gly

85 90 95

Arg Thr Leu Ser Asp Tyr Asn Ile Gln Lys Glu Ser Thr Leu His Leu

100 105 110

Val Leu Arg Leu Arg Gly Gly Glu Glu Pro Gln Ser Asp Pro Ser Val

115 120 125

Glu Pro Pro Leu Ser Gln Glu Thr Phe Ser Asp Leu Trp Lys Leu Leu

130 135 140

Pro Glu Asn Asn Val Leu Ser Pro Leu Pro Ser Gln Ala Met Asp Asp

145 150 155 160

Leu Met Leu Ser Pro Asp Asp Ile Glu Gln Trp Phe Thr Glu Asp Pro

165 170 175

Gly Pro Asp Glu Ala Pro Arg Met Pro Glu Ala Ala Pro Arg Val Ala

180 185 190

Pro Ala Pro Ala Ala Pro Thr Pro Ala Ala Pro Ala Pro Ala Pro Ser

195 200 205

Trp Pro Leu Ser Ser Ser Val Pro Ser Gln Lys Thr Tyr Gln Gly Ser

210 215 220

Tyr Gly Phe Arg Leu Gly Phe Leu His Ser Gly Thr Ala Lys Ser Val

225 230 235 240

Thr Cys Thr Tyr Ser Pro Ala Leu Asn Lys Met Phe Cys Gln Leu Ala

245 250 255

Lys Thr Cys Pro Val Gln Leu Trp Val Asp Ser Thr Pro Pro Pro Gly

260 265 270

Thr Arg Val Arg Ala Met Ala Ile Tyr Lys Gln Ser Gln His Met Thr

275 280 285

Glu Val Val Arg Arg Cys Pro His His Glu Arg Cys Ser Asp Ser Asp

290 295 300

Gly Leu Ala Pro Pro Gln His Leu Ile Arg Val Glu Gly Asn Leu Arg

305 310 315 320

Val Glu Tyr Leu Asp Asp Arg Asn Thr Phe Arg His Ser Val Val Val

325 330 335

Pro Tyr Glu Pro Pro Glu Val Gly Ser Asp Cys Thr Thr Ile His Tyr

340 345 350

Asn Tyr Met Cys Asn Ser Ser Cys Met Gly Gly Met Asn Arg Arg Pro

355 360 365

Ile Leu Thr Ile Ile Thr Leu Glu Asp Ser Ser Gly Asn Leu Leu Gly

370 375 380

Arg Asn Ser Phe Glu Val Arg Val Cys Ala Cys Pro Gly Arg Asp Arg

385 390 395 400

Arg Thr Glu Glu Glu Asn Leu Arg Lys Lys Gly Glu Pro His His Glu

405 410 415

Leu Pro Pro Gly Ser Thr Lys Arg Ala Leu Pro Asn Asn Thr Ser Ser

420 425 430

Ser Pro Gln Pro Lys Lys Lys Pro Leu Asp Gly Glu Tyr Phe Thr Leu

435 440 445

Gln Ile Arg Gly Arg Glu Arg Phe Glu Met Phe Arg Glu Leu Asn Glu

450 455 460

Ala Leu Glu Leu Lys Asp Ala Gln Ala Gly Lys Glu Pro Gly Gly Ser

465 470 475 480

Arg Ala His Ser Ser His Leu Lys Ser Lys Lys Gly Gln Ser Thr Ser

485 490 495

Arg His Lys Lys Leu Met Phe Lys Thr Glu Gly Pro Asp Ser Asp Leu

500 505 510

Glu His His His His His

515

<210> 52

<211> 444

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Amino acid sequence of TOM70-(GGGGS)3-p53

<400> 52

Met Lys Ser Phe Ile Thr Arg Asn Lys Thr Ala Ile Phe Ala Thr Val

1 5 10 15

Ala Ala Thr Gly Thr Ala Ile Gly Ala Tyr Tyr Tyr Tyr Gly Gly Gly

20 25 30

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Glu Pro Gln

35 40 45

Ser Asp Pro Ser Val Glu Pro Pro Leu Ser Gln Glu Thr Phe Ser Asp

50 55 60

Leu Trp Lys Leu Leu Pro Glu Asn Asn Val Leu Ser Pro Leu Pro Ser

65 70 75 80

Gln Ala Met Asp Asp Leu Met Leu Ser Pro Asp Asp Ile Glu Gln Trp

85 90 95

Phe Thr Glu Asp Pro Gly Pro Asp Glu Ala Pro Arg Met Pro Glu Ala

100 105 110

Ala Pro Arg Val Ala Pro Ala Pro Ala Ala Pro Thr Pro Ala Ala Pro

115 120 125

Ala Pro Ala Pro Ser Trp Pro Leu Ser Ser Ser Val Pro Ser Gln Lys

130 135 140

Thr Tyr Gln Gly Ser Tyr Gly Phe Arg Leu Gly Phe Leu His Ser Gly

145 150 155 160

Thr Ala Lys Ser Val Thr Cys Thr Tyr Ser Pro Ala Leu Asn Lys Met

165 170 175

Phe Cys Gln Leu Ala Lys Thr Cys Pro Val Gln Leu Trp Val Asp Ser

180 185 190

Thr Pro Pro Pro Gly Thr Arg Val Arg Ala Met Ala Ile Tyr Lys Gln

195 200 205

Ser Gln His Met Thr Glu Val Val Arg Arg Cys Pro His His Glu Arg

210 215 220

Cys Ser Asp Ser Asp Gly Leu Ala Pro Pro Gln His Leu Ile Arg Val

225 230 235 240

Glu Gly Asn Leu Arg Val Glu Tyr Leu Asp Asp Arg Asn Thr Phe Arg

245 250 255

His Ser Val Val Val Pro Tyr Glu Pro Pro Glu Val Gly Ser Asp Cys

260 265 270

Thr Thr Ile His Tyr Asn Tyr Met Cys Asn Ser Ser Cys Met Gly Gly

275 280 285

Met Asn Arg Arg Pro Ile Leu Thr Ile Ile Thr Leu Glu Asp Ser Ser

290 295 300

Gly Asn Leu Leu Gly Arg Asn Ser Phe Glu Val Arg Val Cys Ala Cys

305 310 315 320

Pro Gly Arg Asp Arg Arg Thr Glu Glu Glu Asn Leu Arg Lys Lys Gly

325 330 335

Glu Pro His His Glu Leu Pro Pro Gly Ser Thr Lys Arg Ala Leu Pro

340 345 350

Asn Asn Thr Ser Ser Ser Pro Gln Pro Lys Lys Lys Pro Leu Asp Gly

355 360 365

Glu Tyr Phe Thr Leu Gln Ile Arg Gly Arg Glu Arg Phe Glu Met Phe

370 375 380

Arg Glu Leu Asn Glu Ala Leu Glu Leu Lys Asp Ala Gln Ala Gly Lys

385 390 395 400

Glu Pro Gly Gly Ser Arg Ala His Ser Ser His Leu Lys Ser Lys Lys

405 410 415

Gly Gln Ser Thr Ser Arg His Lys Lys Leu Met Phe Lys Thr Glu Gly

420 425 430

Pro Asp Ser Asp Leu Glu His His His His His

435 440

<210> 53

<211> 525

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Amino acid sequence of UB-p53-TOM7

<400> 53

Met Gly Ser Ser His His His His His Ser Ser Gly Leu Val Pro

1 5 10 15

Arg Gly Ser His Met Gln Ile Phe Val Lys Thr Leu Thr Gly Lys Thr

20 25 30

Ile Thr Leu Glu Val Glu Pro Ser Asp Thr Ile Glu Asn Val Lys Ala

35 40 45

Lys Ile Gln Asp Lys Glu Gly Ile Pro Pro Asp Gln Gln Arg Leu Ile

50 55 60

Phe Ala Gly Lys Gln Leu Glu Asp Gly Arg Thr Leu Ser Asp Tyr Asn

65 70 75 80

Ile Gln Lys Glu Ser Thr Leu His Leu Val Leu Arg Leu Arg Gly Gly

85 90 95

Glu Glu Pro Gln Ser Asp Pro Ser Val Glu Pro Pro Leu Ser Gln Glu

100 105 110

Thr Phe Ser Asp Leu Trp Lys Leu Leu Pro Glu Asn Asn Val Leu Ser

115 120 125

Pro Leu Pro Ser Gln Ala Met Asp Asp Leu Met Leu Ser Pro Asp Asp

130 135 140

Ile Glu Gln Trp Phe Thr Glu Asp Pro Gly Pro Asp Glu Ala Pro Arg

145 150 155 160

Met Pro Glu Ala Ala Pro Arg Val Ala Pro Ala Pro Ala Ala Pro Thr

165 170 175

Pro Ala Ala Pro Ala Pro Ala Pro Ser Trp Pro Leu Ser Ser Ser Val

180 185 190

Pro Ser Gln Lys Thr Tyr Gln Gly Ser Tyr Gly Phe Arg Leu Gly Phe

195 200 205

Leu His Ser Gly Thr Ala Lys Ser Val Thr Cys Thr Tyr Ser Pro Ala

210 215 220

Leu Asn Lys Met Phe Cys Gln Leu Ala Lys Thr Cys Pro Val Gln Leu

225 230 235 240

Trp Val Asp Ser Thr Pro Pro Pro Gly Thr Arg Val Arg Ala Met Ala

245 250 255

Ile Tyr Lys Gln Ser Gln His Met Thr Glu Val Val Arg Arg Cys Pro

260 265 270

His His Glu Arg Cys Ser Asp Ser Asp Gly Leu Ala Pro Pro Gln His

275 280 285

Leu Ile Arg Val Glu Gly Asn Leu Arg Val Glu Tyr Leu Asp Asp Arg

290 295 300

Asn Thr Phe Arg His Ser Val Val Val Pro Tyr Glu Pro Pro Glu Val

305 310 315 320

Gly Ser Asp Cys Thr Thr Ile His Tyr Asn Tyr Met Cys Asn Ser Ser

325 330 335

Cys Met Gly Gly Met Asn Arg Arg Pro Ile Leu Thr Ile Ile Thr Leu

340 345 350

Glu Asp Ser Ser Gly Asn Leu Leu Gly Arg Asn Ser Phe Glu Val His

355 360 365

Val Cys Ala Cys Pro Gly Arg Asp Arg Arg Thr Glu Glu Glu Asn Leu

370 375 380

Arg Lys Lys Gly Glu Pro His His Glu Leu Pro Pro Gly Ser Thr Lys

385 390 395 400

Arg Ala Leu Ser Asn Asn Thr Ser Ser Ser Pro Gln Pro Lys Lys Lys

405 410 415

Pro Leu Asp Gly Glu Tyr Phe Thr Leu Gln Ile Arg Gly Arg Glu Arg

420 425 430

Phe Glu Met Phe Arg Glu Leu Asn Glu Ala Leu Glu Leu Lys Asp Ala

435 440 445

Gln Ala Gly Lys Glu Pro Gly Gly Ser Arg Ala His Ser Ser His Leu

450 455 460

Lys Ser Lys Lys Gly Gln Ser Thr Ser Arg His Lys Lys Leu Met Phe

465 470 475 480

Lys Thr Glu Gly Pro Asp Ser Asp Leu Glu Phe Ala Ile Arg Trp Gly

485 490 495

Phe Ile Pro Leu Val Ile Tyr Leu Gly Phe Lys Arg Gly Ala Asp Pro

500 505 510

Gly Met Pro Glu Pro Thr Val Leu Ser Leu Leu Trp Gly

515 520 525

<210> 54

<211> 421

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Amino acid sequence of p53-myc/His

<400> 54

Met Glu Glu Pro Gln Ser Asp Pro Ser Val Glu Pro Pro Leu Ser Gln

1 5 10 15

Glu Thr Phe Ser Asp Leu Trp Lys Leu Leu Pro Glu Asn Asn Val Leu

20 25 30

Ser Pro Leu Pro Ser Gln Ala Met Asp Asp Leu Met Leu Ser Pro Asp

35 40 45

Asp Ile Glu Gln Trp Phe Thr Glu Asp Pro Gly Pro Asp Glu Ala Pro

50 55 60

Arg Met Pro Glu Ala Ala Pro Arg Val Ala Pro Ala Pro Ala Ala Pro

65 70 75 80

Thr Pro Ala Ala Pro Ala Pro Ala Pro Ser Trp Pro Leu Ser Ser Ser

85 90 95

Val Pro Ser Gln Lys Thr Tyr Gln Gly Ser Tyr Gly Phe Arg Leu Gly

100 105 110

Phe Leu His Ser Gly Thr Ala Lys Ser Val Thr Cys Thr Tyr Ser Pro

115 120 125

Ala Leu Asn Lys Met Phe Cys Gln Leu Ala Lys Thr Cys Pro Val Gln

130 135 140

Leu Trp Val Asp Ser Thr Pro Pro Pro Gly Thr Arg Val Arg Ala Met

145 150 155 160

Ala Ile Tyr Lys Gln Ser Gln His Met Thr Glu Val Val Arg Arg Cys

165 170 175

Pro His His Glu Arg Cys Ser Asp Ser Asp Gly Leu Ala Pro Pro Gln

180 185 190

His Leu Ile Arg Val Glu Gly Asn Leu Arg Val Glu Tyr Leu Asp Asp

195 200 205

Arg Asn Thr Phe Arg His Ser Val Val Val Pro Tyr Glu Pro Pro Glu

210 215 220

Val Gly Ser Asp Cys Thr Thr Ile His Tyr Asn Tyr Met Cys Asn Ser

225 230 235 240

Ser Cys Met Gly Gly Met Asn Arg Arg Pro Ile Leu Thr Ile Ile Thr

245 250 255

Leu Glu Asp Ser Ser Gly Asn Leu Leu Gly Arg Asn Ser Phe Glu Val

260 265 270

Arg Val Cys Ala Cys Pro Gly Arg Asp Arg Arg Thr Glu Glu Glu Asn

275 280 285

Leu Arg Lys Lys Gly Glu Pro His His Glu Leu Pro Pro Gly Ser Thr

290 295 300

Lys Arg Ala Leu Pro Asn Asn Thr Ser Ser Ser Pro Gln Pro Lys Lys

305 310 315 320

Lys Pro Leu Asp Gly Glu Tyr Phe Thr Leu Gln Ile Arg Gly Arg Glu

325 330 335

Arg Phe Glu Met Phe Arg Glu Leu Asn Glu Ala Leu Glu Leu Lys Asp

340 345 350

Ala Gln Ala Gly Lys Glu Pro Gly Gly Ser Arg Ala His Ser Ser His

355 360 365

Leu Lys Ser Lys Lys Gly Gln Ser Thr Ser Arg His Lys Lys Leu Met

370 375 380

Phe Lys Thr Glu Gly Pro Asp Ser Asp Leu Glu Ser Arg Gly Pro Phe

385 390 395 400

Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu Asn Met His Thr Gly His

405 410 415

His His His His

420

<210> 55

<211> 230

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Amino acid sequence of granzyme B

<400> 55

Arg Gly Gly Ile Ile Gly Gly His Glu Ala Lys Pro His Ser Arg Pro

1 5 10 15

Tyr Met Ala Tyr Leu Met Ile Trp Asp Gln Lys Ser Leu Lys Arg Cys

20 25 30

Gly Gly Phe Leu Ile Gln Asp Asp Phe Val Leu Thr Ala Ala His Cys

35 40 45

Trp Gly Ser Ser Ile Asn Val Thr Leu Gly Ala His Asn Ile Lys Glu

50 55 60

Gln Glu Pro Thr Gln Gln Phe Ile Pro Val Lys Arg Pro Ile Pro His

65 70 75 80

Pro Ala Tyr Asn Pro Lys Asn Phe Ser Asn Asp Ile Met Leu Leu Gln

85 90 95

Leu Glu Arg Lys Ala Lys Arg Thr Arg Ala Val Gln Pro Leu Arg Leu

100 105 110

Pro Ser Asn Lys Ala Gln Val Lys Pro Gly Gln Thr Cys Ser Val Ala

115 120 125

Gly Trp Gly Gln Thr Ala Pro Leu Gly Lys His Ser His Thr Leu Gln

130 135 140

Glu Val Lys Met Thr Val Gln Glu Asp Arg Lys Cys Glu Ser Asp Leu

145 150 155 160

Arg His Tyr Tyr Asp Ser Thr Ile Glu Leu Cys Val Gly Asp Pro Glu

165 170 175

Ile Lys Lys Thr Ser Phe Lys Gly Asp Ser Gly Gly Pro Leu Val Cys

180 185 190

Asn Lys Val Ala Gln Gly Ile Val Ser Tyr Gly Arg Asn Asn Gly Met

195 200 205

Pro Pro Arg Ala Cys Thr Lys Val Ser Ser Phe Val His Trp Ile Lys

210 215 220

Lys Thr Met Lys Arg Tyr

225 230

<210> 56

<211> 354

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Amino acid sequence of TOM70-(GGGGS)3-UB-granzyme

B

<400> 56

Met Lys Ser Phe Ile Thr Arg Asn Lys Thr Ala Ile Phe Ala Thr Val

1 5 10 15

Ala Ala Thr Gly Thr Ala Ile Gly Ala Tyr Tyr Tyr Tyr Gly Gly Gly

20 25 30

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Ile Phe Val

35 40 45

Lys Thr Leu Thr Gly Lys Thr Ile Thr Leu Glu Val Glu Pro Ser Asp

50 55 60

Thr Ile Glu Asn Val Lys Ala Lys Ile Gln Asp Lys Glu Gly Ile Pro

65 70 75 80

Pro Asp Gln Gln Arg Leu Ile Phe Ala Gly Lys Gln Leu Glu Asp Gly

85 90 95

Arg Thr Leu Ser Asp Tyr Asn Ile Gln Lys Glu Ser Thr Leu His Leu

100 105 110

Val Leu Arg Leu Arg Gly Gly Ile Ile Gly Gly His Glu Ala Lys Pro

115 120 125

His Ser Arg Pro Tyr Met Ala Tyr Leu Met Ile Trp Asp Gln Lys Ser

130 135 140

Leu Lys Arg Cys Gly Gly Phe Leu Ile Arg Asp Asp Phe Val Leu Thr

145 150 155 160

Ala Ala His Cys Trp Gly Ser Ser Ile Asn Val Thr Leu Gly Ala His

165 170 175

Asn Ile Lys Glu Gln Glu Pro Thr Gln Gln Phe Ile Pro Val Lys Arg

180 185 190

Pro Ile Pro His Pro Ala Tyr Asn Pro Lys Asn Phe Ser Asn Asp Ile

195 200 205

Met Leu Leu Gln Leu Glu Arg Lys Ala Lys Arg Thr Arg Ala Val Gln

210 215 220

Pro Leu Arg Leu Pro Ser Asn Lys Ala Gln Val Lys Pro Gly Gln Thr

225 230 235 240

Cys Ser Val Ala Gly Trp Gly Gln Thr Ala Pro Leu Gly Lys His Ser

245 250 255

His Thr Leu Gln Glu Val Lys Met Thr Val Gln Glu Asp Arg Lys Cys

260 265 270

Glu Ser Asp Leu Arg His Tyr Tyr Asp Ser Thr Ile Glu Leu Cys Val

275 280 285

Gly Asp Pro Glu Ile Lys Lys Thr Ser Phe Lys Gly Asp Ser Gly Gly

290 295 300

Pro Leu Val Cys Asn Lys Val Ala Gln Gly Ile Val Ser Tyr Gly Arg

305 310 315 320

Asn Asn Gly Met Pro Pro Arg Ala Cys Thr Lys Val Ser Ser Phe Val

325 330 335

His Trp Ile Lys Lys Thr Met Lys Arg Tyr Leu Glu His His His His

340 345 350

His His

<210> 57

<211> 360

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Amino acid sequence of UB-granzyme B-TOM7

<400> 57

Met Gly Ser Ser His His His His His Ser Ser Gly Leu Val Pro

1 5 10 15

Arg Gly Ser His Met Gln Ile Phe Val Lys Thr Leu Thr Gly Lys Thr

20 25 30

Ile Thr Leu Glu Val Glu Pro Ser Asp Thr Ile Glu Asn Val Lys Ala

35 40 45

Lys Ile Gln Asp Lys Glu Gly Ile Pro Pro Asp Gln Gln Arg Leu Ile

50 55 60

Phe Ala Gly Lys Gln Leu Glu Asp Gly Arg Thr Leu Ser Asp Tyr Asn

65 70 75 80

Ile Gln Lys Glu Ser Thr Leu His Leu Val Leu Arg Leu Arg Gly Gly

85 90 95

Ile Ile Gly Gly His Glu Ala Lys Pro His Ser Arg Pro Tyr Met Ala

100 105 110

Tyr Leu Met Ile Trp Asp Gln Lys Ser Leu Lys Arg Cys Gly Gly Phe

115 120 125

Leu Ile Arg Asp Asp Phe Val Leu Thr Ala Ala His Cys Trp Gly Ser

130 135 140

Ser Ile Asn Val Thr Leu Gly Ala His Asn Ile Lys Glu Gln Glu Pro

145 150 155 160

Thr Gln Gln Phe Ile Pro Val Lys Arg Pro Ile Pro His Pro Ala Tyr

165 170 175

Asn Pro Lys Asn Phe Ser Asn Asp Ile Met Leu Leu Gln Leu Glu Arg

180 185 190

Lys Ala Lys Arg Thr Arg Ala Val Gln Pro Leu Arg Leu Pro Ser Asn

195 200 205

Lys Ala Gln Val Lys Pro Gly Gln Thr Cys Ser Val Ala Gly Trp Gly

210 215 220

Gln Thr Ala Pro Leu Gly Lys His Ser His Thr Leu Gln Glu Val Lys

225 230 235 240

Met Thr Val Gln Glu Asp Arg Lys Cys Glu Ser Asp Leu Arg His Tyr

245 250 255

Tyr Asp Ser Thr Ile Glu Leu Cys Val Gly Asp Pro Glu Ile Lys Lys

260 265 270

Thr Ser Phe Lys Gly Asp Ser Gly Gly Pro Leu Val Cys Asn Lys Val

275 280 285

Ala Gln Gly Ile Val Ser Tyr Gly Arg Asn Asn Gly Met Pro Pro Arg

290 295 300

Ala Cys Thr Lys Val Ser Ser Phe Val His Trp Ile Lys Lys Thr Met

305 310 315 320

Lys Arg Tyr Leu Glu Phe Ala Ile Arg Trp Gly Phe Ile Pro Leu Val

325 330 335

Ile Tyr Leu Gly Phe Lys Arg Gly Ala Asp Pro Gly Met Pro Glu Pro

340 345 350

Thr Val Leu Ser Leu Leu Trp Gly

355 360

<210> 58

<211> 189

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Amino acid sequence of RKIP

<400> 58

Arg Gly Gly Pro Val Asp Leu Ser Lys Trp Ser Gly Pro Leu Ser Leu

1 5 10 15

Gln Glu Val Asp Glu Gln Pro Gln His Pro Leu His Val Thr Tyr Ala

20 25 30

Gly Ala Ala Val Asp Glu Leu Gly Lys Val Leu Thr Pro Thr Gln Val

35 40 45

Lys Asn Arg Pro Thr Ser Ile Ser Trp Asp Gly Leu Asp Ser Gly Lys

50 55 60

Leu Tyr Thr Leu Val Leu Thr Asp Pro Asp Ala Pro Ser Arg Lys Asp

65 70 75 80

Pro Lys Tyr Arg Glu Trp His His Phe Leu Val Val Asn Met Lys Gly

85 90 95

Asn Asp Ile Ser Ser Gly Thr Val Leu Ser Asp Tyr Val Gly Ser Gly

100 105 110

Pro Pro Lys Gly Thr Gly Leu His Arg Tyr Val Trp Leu Val Tyr Glu

115 120 125

Gln Asp Arg Pro Leu Lys Cys Asp Glu Pro Ile Leu Ser Asn Arg Ser

130 135 140

Gly Asp His Arg Gly Lys Phe Lys Val Ala Ser Phe Arg Lys Lys Tyr

145 150 155 160

Glu Leu Arg Ala Pro Val Ala Gly Thr Cys Tyr Gln Ala Glu Trp Asp

165 170 175

Asp Tyr Val Pro Lys Leu Tyr Glu Gln Leu Ser Gly Lys

180 185

<210> 59

<211> 313

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Amino acid sequence TOM70-(GGGGS)3-UB-RKIP

<400> 59

Met Lys Ser Phe Ile Thr Arg Asn Lys Thr Ala Ile Phe Ala Thr Val

1 5 10 15

Ala Ala Thr Gly Thr Ala Ile Gly Ala Tyr Tyr Tyr Tyr Gly Gly Gly

20 25 30

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Ile Phe Val

35 40 45

Lys Thr Leu Thr Gly Lys Thr Ile Thr Leu Glu Val Glu Pro Ser Asp

50 55 60

Thr Ile Glu Asn Val Lys Ala Lys Ile Gln Asp Lys Glu Gly Ile Pro

65 70 75 80

Pro Asp Gln Gln Arg Leu Ile Phe Ala Gly Lys Gln Leu Glu Asp Gly

85 90 95

Arg Thr Leu Ser Asp Tyr Asn Ile Gln Lys Glu Ser Thr Leu His Leu

100 105 110

Val Leu Arg Leu Arg Gly Gly Pro Val Asp Leu Ser Lys Trp Ser Gly

115 120 125

Pro Leu Ser Leu Gln Glu Val Asp Glu Gln Pro Gln His Pro Leu His

130 135 140

Val Thr Tyr Ala Gly Ala Ala Val Asp Glu Leu Gly Lys Val Leu Thr

145 150 155 160

Pro Thr Gln Val Lys Asn Arg Pro Thr Ser Ile Ser Trp Asp Gly Leu

165 170 175

Asp Ser Gly Lys Leu Tyr Thr Leu Val Leu Thr Asp Pro Asp Ala Pro

180 185 190

Ser Arg Lys Asp Pro Lys Tyr Arg Glu Trp His His Phe Leu Val Val

195 200 205

Asn Met Lys Gly Asn Asp Ile Ser Ser Gly Thr Val Leu Ser Asp Tyr

210 215 220

Val Gly Ser Gly Pro Pro Lys Gly Thr Gly Leu His Arg Tyr Val Trp

225 230 235 240

Leu Val Tyr Glu Gln Asp Arg Pro Leu Lys Cys Asp Glu Pro Ile Leu

245 250 255

Ser Asn Arg Ser Gly Asp His Arg Gly Lys Phe Lys Val Ala Ser Phe

260 265 270

Arg Lys Lys Tyr Glu Leu Arg Ala Pro Val Ala Gly Thr Cys Tyr Gln

275 280 285

Ala Glu Trp Asp Asp Tyr Val Pro Lys Leu Tyr Glu Gln Leu Ser Gly

290 295 300

Lys Leu Glu His His His His His

305 310

<210> 60

<211> 405

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Amino acid sequence of PTEN

<400> 60

Arg Gly Gly Thr Ala Ile Ile Lys Glu Ile Val Ser Arg Asn Lys Arg

1 5 10 15

Arg Tyr Gln Glu Asp Gly Phe Asp Leu Asp Leu Thr Tyr Ile Tyr Pro

20 25 30

Asn Ile Ile Ala Met Gly Phe Pro Ala Glu Arg Leu Glu Gly Val Tyr

35 40 45

Arg Asn Asn Ile Asp Asp Val Val Arg Phe Leu Asp Ser Lys His Lys

50 55 60

Asn His Tyr Lys Ile Tyr Asn Leu Cys Ala Glu Arg His Tyr Asp Thr

65 70 75 80

Ala Lys Phe Asn Cys Arg Val Ala Gln Tyr Pro Phe Glu Asp His Asn

85 90 95

Pro Pro Gln Leu Glu Leu Ile Lys Pro Phe Cys Glu Asp Leu Asp Gln

100 105 110

Trp Leu Ser Glu Asp Asp Asn His Val Ala Ala Ile His Cys Lys Ala

115 120 125

Gly Lys Gly Arg Thr Gly Val Met Ile Cys Ala Tyr Leu Leu His Arg

130 135 140

Gly Lys Phe Leu Lys Ala Gln Glu Ala Leu Asp Phe Tyr Gly Glu Val

145 150 155 160

Arg Thr Arg Asp Lys Lys Gly Val Thr Ile Pro Ser Gln Arg Arg Tyr

165 170 175

Val Tyr Tyr Tyr Ser Tyr Leu Leu Lys Asn His Leu Asp Tyr Arg Pro

180 185 190

Val Ala Leu Leu Phe His Lys Met Met Phe Glu Thr Ile Pro Met Phe

195 200 205

Ser Gly Gly Thr Cys Asn Pro Gln Phe Val Val Cys Gln Leu Lys Val

210 215 220

Lys Ile Tyr Ser Ser Asn Ser Gly Pro Thr Arg Arg Glu Asp Lys Phe

225 230 235 240

Met Tyr Phe Glu Phe Pro Gln Pro Leu Pro Val Cys Gly Asp Ile Lys

245 250 255

Val Glu Phe Phe His Lys Gln Asn Lys Met Leu Lys Lys Asp Lys Met

260 265 270

Phe His Phe Trp Val Asn Thr Phe Phe Ile Pro Gly Pro Glu Glu Thr

275 280 285

Ser Glu Lys Val Glu Asn Gly Ser Leu Cys Asp Gln Glu Ile Asp Ser

290 295 300

Ile Cys Ser Ile Glu Arg Ala Asp Asn Asp Lys Glu Tyr Leu Val Leu

305 310 315 320

Thr Leu Thr Lys Asn Asp Leu Asp Lys Ala Asn Lys Asp Lys Ala Asn

325 330 335

Arg Tyr Phe Ser Pro Asn Phe Lys Val Lys Leu Tyr Phe Thr Lys Thr

340 345 350

Val Glu Glu Pro Ser Asn Pro Glu Ala Ser Ser Ser Thr Ser Val Thr

355 360 365

Pro Asp Val Ser Asp Asn Glu Pro Asp His Tyr Arg Tyr Ser Asp Thr

370 375 380

Thr Asp Ser Asp Pro Glu Asn Glu Pro Phe Asp Glu Asp Gln His Thr

385 390 395 400

Gln Ile Thr Lys Val

405

<210> 61

<211> 529

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Amino acid sequence TOM70-(GGGGS)3-UB-PTEN

<400> 61

Met Lys Ser Phe Ile Thr Arg Asn Lys Thr Ala Ile Phe Ala Thr Val

1 5 10 15

Ala Ala Thr Gly Thr Ala Ile Gly Ala Tyr Tyr Tyr Tyr Gly Gly Gly

20 25 30

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Ile Phe Val

35 40 45

Lys Thr Leu Thr Gly Lys Thr Ile Thr Leu Glu Val Glu Pro Ser Asp

50 55 60

Thr Ile Glu Asn Val Lys Ala Lys Ile Gln Asp Lys Glu Gly Ile Pro

65 70 75 80

Pro Asp Gln Gln Arg Leu Ile Phe Ala Gly Lys Gln Leu Glu Asp Gly

85 90 95

Arg Thr Leu Ser Asp Tyr Asn Ile Gln Lys Glu Ser Thr Leu His Leu

100 105 110

Val Leu Arg Leu Arg Gly Gly Thr Ala Ile Ile Lys Glu Ile Val Ser

115 120 125

Arg Asn Lys Arg Arg Tyr Gln Glu Asp Gly Phe Asp Leu Asp Leu Thr

130 135 140

Tyr Ile Tyr Pro Asn Ile Ile Ala Met Gly Phe Pro Ala Glu Arg Leu

145 150 155 160

Glu Gly Val Tyr Arg Asn Asn Ile Asp Asp Val Val Arg Phe Leu Asp

165 170 175

Ser Lys His Lys Asn His Tyr Lys Ile Tyr Asn Leu Cys Ala Glu Arg

180 185 190

His Tyr Asp Thr Ala Lys Phe Asn Cys Arg Val Ala Gln Tyr Pro Phe

195 200 205

Glu Asp His Asn Pro Pro Gln Leu Glu Leu Ile Lys Pro Phe Cys Glu

210 215 220

Asp Leu Asp Gln Trp Leu Ser Glu Asp Asp Asn His Val Ala Ala Ile

225 230 235 240

His Cys Lys Ala Gly Lys Gly Arg Thr Gly Val Met Ile Cys Ala Tyr

245 250 255

Leu Leu His Arg Gly Lys Phe Leu Lys Ala Gln Glu Ala Leu Asp Phe

260 265 270

Tyr Gly Glu Val Arg Thr Arg Asp Lys Lys Gly Val Thr Ile Pro Ser

275 280 285

Gln Arg Arg Tyr Val Tyr Tyr Tyr Ser Tyr Leu Leu Lys Asn His Leu

290 295 300

Asp Tyr Arg Pro Val Ala Leu Leu Phe His Lys Met Met Phe Glu Thr

305 310 315 320

Ile Pro Met Phe Ser Gly Gly Thr Cys Asn Pro Gln Phe Val Val Cys

325 330 335

Gln Leu Lys Val Lys Ile Tyr Ser Ser Asn Ser Gly Pro Thr Arg Arg

340 345 350

Glu Asp Lys Phe Met Tyr Phe Glu Phe Pro Gln Pro Leu Pro Val Cys

355 360 365

Gly Asp Ile Lys Val Glu Phe Phe His Lys Gln Asn Lys Met Leu Lys

370 375 380

Lys Asp Lys Met Phe His Phe Trp Val Asn Thr Phe Phe Ile Pro Gly

385 390 395 400

Pro Glu Glu Thr Ser Glu Lys Val Glu Asn Gly Ser Leu Cys Asp Gln

405 410 415

Glu Ile Asp Ser Ile Cys Ser Ile Glu Arg Ala Asp Asn Asp Lys Glu

420 425 430

Tyr Leu Val Leu Thr Leu Thr Lys Asn Asp Leu Asp Lys Ala Asn Lys

435 440 445

Asp Lys Ala Asn Arg Tyr Phe Ser Pro Asn Phe Lys Val Lys Leu Tyr

450 455 460

Phe Thr Lys Thr Val Glu Glu Pro Ser Asn Pro Glu Ala Ser Ser Ser

465 470 475 480

Thr Ser Val Thr Pro Asp Val Ser Asp Asn Glu Pro Asp His Tyr Arg

485 490 495

Tyr Ser Asp Thr Thr Asp Ser Asp Pro Glu Asn Glu Pro Phe Asp Glu

500 505 510

Asp Gln His Thr Gln Ile Thr Lys Val Leu Glu His His His His

515 520 525

His

<210> 62

<211> 246

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Amino acid sequence of scFvHER2

<400> 62

Arg Gly Gly Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln

1 5 10 15

Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe

20 25 30

Thr Asp Tyr Thr Met Asp Trp Val Arg Gln Ala Pro Gly Lys Gly Leu

35 40 45

Glu Trp Val Ala Asp Val Asn Pro Asn Ser Gly Gly Ser Ile Tyr Asn

50 55 60

Gln Arg Phe Lys Gly Arg Phe Thr Leu Ser Val Asp Arg Ser Lys Asn

65 70 75 80

Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val

85 90 95

Tyr Tyr Cys Ala Arg Asn Leu Gly Pro Ser Phe Tyr Phe Asp Tyr Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly

115 120 125

Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr Gln Ser

130 135 140

Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys

145 150 155 160

Lys Ala Ser Gln Asp Val Ser Ile Gly Val Ala Trp Tyr Gln Gln Lys

165 170 175

Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ser Ala Ser Tyr Arg Tyr

180 185 190

Thr Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe

195 200 205

Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr

210 215 220

Cys Gln Gln Tyr Tyr Ile Tyr Pro Tyr Thr Phe Gly Gln Gly Thr Lys

225 230 235 240

Val Glu Ile Lys Arg Thr

245

<210> 63

<211> 374

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Amino acid sequence of UB-scFvHER2-TOM7

<400> 63

Met Gly Ser Ser His His His His His Ser Ser Gly Leu Val Pro

1 5 10 15

Arg Gly Ser His Met Gln Ile Phe Val Lys Thr Leu Thr Gly Lys Thr

20 25 30

Ile Thr Leu Glu Val Glu Pro Ser Asp Thr Ile Glu Asn Val Lys Ala

35 40 45

Lys Ile Gln Asp Lys Glu Gly Ile Pro Pro Asp Gln Gln Arg Leu Ile

50 55 60

Phe Ala Gly Lys Gln Leu Glu Asp Gly Arg Thr Leu Ser Asp Tyr Asn

65 70 75 80

Ile Gln Lys Glu Ser Thr Leu His Leu Val Leu Arg Leu Arg Gly Gly

85 90 95

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

100 105 110

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Asp Tyr

115 120 125

Thr Met Asp Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

130 135 140

Ala Asp Val Asn Pro Asn Ser Gly Gly Ser Ile Tyr Asn Gln Arg Phe

145 150 155 160

Lys Gly Arg Phe Thr Leu Ser Val Asp Arg Ser Lys Asn Thr Leu Tyr

165 170 175

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

180 185 190

Ala Arg Asn Leu Gly Pro Ser Phe Tyr Phe Asp Tyr Trp Gly Gln Gly

195 200 205

Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

210 215 220

Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr Gln Ser Pro Ser Ser

225 230 235 240

Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Lys Ala Ser

245 250 255

Gln Asp Val Ser Ile Gly Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys

260 265 270

Ala Pro Lys Leu Leu Ile Tyr Ser Ala Ser Tyr Arg Tyr Thr Gly Val

275 280 285

Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

290 295 300

Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln

305 310 315 320

Tyr Tyr Ile Tyr Pro Tyr Thr Phe Gly Gln Gly Thr Lys Val Glu Ile

325 330 335

Lys Arg Thr Phe Ala Ile Arg Trp Gly Phe Ile Pro Leu Val Ile Tyr

340 345 350

Leu Gly Phe Lys Arg Gly Ala Asp Pro Gly Met Pro Glu Pro Thr Val

355 360 365

Leu Ser Leu Leu Trp Gly

370

<210> 64

<211> 307

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Amino acid sequence scFvHER2-TOM7-myc/His

<400> 64

Met Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly

1 5 10 15

Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Asp

20 25 30

Tyr Thr Met Asp Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp

35 40 45

Val Ala Asp Val Asn Pro Asn Ser Gly Gly Ser Ile Tyr Asn Gln Arg

50 55 60

Phe Lys Gly Arg Phe Thr Leu Ser Val Asp Arg Ser Lys Asn Thr Leu

65 70 75 80

Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr

85 90 95

Cys Ala Arg Asn Leu Gly Pro Ser Phe Tyr Phe Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly

115 120 125

Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr Gln Ser Pro Ser

130 135 140

Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Lys Ala

145 150 155 160

Ser Gln Asp Val Ser Ile Gly Val Ala Trp Tyr Gln Gln Lys Pro Gly

165 170 175

Lys Ala Pro Lys Leu Leu Ile Tyr Ser Ala Ser Tyr Arg Tyr Thr Gly

180 185 190

Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu

195 200 205

Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln

210 215 220

Gln Tyr Tyr Ile Tyr Pro Tyr Thr Phe Gly Gln Gly Thr Lys Val Glu

225 230 235 240

Ile Lys Arg Thr Phe Ala Ile Arg Trp Gly Phe Ile Pro Leu Val Ile

245 250 255

Tyr Leu Gly Phe Lys Arg Gly Ala Asp Pro Gly Met Pro Glu Pro Thr

260 265 270

Val Leu Ser Leu Leu Trp Gly Leu Glu Ser Arg Gly Pro Phe Glu Gln

275 280 285

Lys Leu Ile Ser Glu Glu Asp Leu Asn Met His Thr Gly His His His

290 295 300

His His His

305

<210> 65

<211> 251

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Amino acid sequence of scFvMEL

<400> 65

Arg Gly Gly Thr Asp Ile Val Met Thr Gln Ser Gln Lys Phe Met Ser

1 5 10 15

Thr Ser Val Gly Asp Arg Val Ser Val Thr Cys Lys Ala Ser Gln Asn

20 25 30

Val Asp Thr Asn Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro

35 40 45

Glu Pro Leu Leu Phe Ser Ala Ser Tyr Arg Tyr Thr Gly Val Pro Asp

50 55 60

Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser

65 70 75 80

Asn Val Gln Ser Glu Asp Leu Ala Glu Tyr Phe Cys Gln Gln Tyr Asn

85 90 95

Ser Tyr Pro Leu Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Gly

100 105 110

Ser Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser Thr Lys

115 120 125

Gly Glu Val Lys Val Glu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly

130 135 140

Gly Ser Met Lys Leu Ser Cys Val Val Ser Gly Phe Thr Phe Gly Asn

145 150 155 160

Tyr Trp Met Asn Trp Val Arg Gln Ser Pro Glu Lys Gly Leu Glu Trp

165 170 175

Ile Ala Glu Ile Arg Leu Lys Ser Asn Asn Phe Ala Arg Tyr Tyr Ala

180 185 190

Glu Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser

195 200 205

Ser Val Tyr Leu Gln Met Ile Asn Leu Arg Ala Glu Asp Thr Gly Ile

210 215 220

Tyr Tyr Cys Thr Ser Tyr Gly Asn Tyr Val Gly His Tyr Phe Asp His

225 230 235 240

Trp Gly Gln Gly Thr Thr Leu Thr Val Ser Ser

245 250

<210> 66

<211> 378

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Amino acid sequence of UB-scFvMEL-TOM7

<400> 66

Met Gly Ser Ser His His His His His Ser Ser Gly Leu Val Pro

1 5 10 15

Arg Gly Ser His Met Gln Ile Phe Val Lys Thr Leu Thr Gly Lys Thr

20 25 30

Ile Thr Leu Glu Val Glu Pro Ser Asp Thr Ile Glu Asn Val Lys Ala

35 40 45

Lys Ile Gln Asp Lys Glu Gly Ile Pro Pro Asp Gln Gln Arg Leu Ile

50 55 60

Phe Ala Gly Lys Gln Leu Glu Asp Gly Arg Thr Leu Ser Asp Tyr Asn

65 70 75 80

Ile Gln Lys Glu Ser Thr Leu His Leu Val Leu Arg Leu Arg Gly Gly

85 90 95

Thr Asp Ile Met Thr Gln Ser Gln Lys Phe Met Ser Thr Ser Val Gly

100 105 110

Asp Arg Val Ser Val Thr Cys Lys Ala Ser Gln Asn Val Asp Thr Asn

115 120 125

Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Glu Pro Leu Leu

130 135 140

Phe Ser Ala Ser Tyr Arg Tyr Thr Gly Val Pro Asp Arg Phe Thr Gly

145 150 155 160

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Asn Val Gln Ser

165 170 175

Glu Asp Leu Ala Glu Tyr Phe Cys Gln Gln Tyr Asn Ser Tyr Pro Leu

180 185 190

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Gly Ser Thr Ser Gly

195 200 205

Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser Thr Lys Gly Glu Val Lys

210 215 220

Val Glu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Met Lys

225 230 235 240

Leu Ser Cys Val Val Ser Gly Phe Thr Phe Gly Asn Tyr Trp Met Asn

245 250 255

Trp Val Arg Gln Ser Pro Glu Lys Gly Leu Glu Trp Ile Ala Glu Ile

260 265 270

Arg Leu Lys Ser Asn Asn Phe Ala Arg Tyr Tyr Ala Glu Ser Val Lys

275 280 285

Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Ser Val Tyr Leu

290 295 300

Gln Met Ile Asn Leu Arg Ala Glu Asp Thr Gly Ile Tyr Tyr Cys Thr

305 310 315 320

Ser Tyr Gly Asn Tyr Val Gly His Tyr Phe Asp His Trp Gly Gln Gly

325 330 335

Thr Thr Leu Thr Val Ser Ser Phe Ala Ile Arg Trp Gly Phe Ile Pro

340 345 350

Leu Val Ile Tyr Leu Gly Phe Lys Arg Gly Ala Asp Pro Gly Met Pro

355 360 365

Glu Pro Thr Val Leu Ser Leu Leu Trp Gly

370 375

<210> 67

<211> 312

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Amino acid sequence of scFvMEL-TOM7-myc/His

<400> 67

Met Thr Asp Ile Val Met Thr Gln Ser Gln Lys Phe Met Ser Thr Ser

1 5 10 15

Val Gly Asp Arg Val Ser Val Thr Cys Lys Ala Ser Gln Asn Val Asp

20 25 30

Thr Asn Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Glu Pro

35 40 45

Leu Leu Phe Ser Ala Ser Tyr Arg Tyr Thr Gly Val Pro Asp Arg Phe

50 55 60

Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Asn Val

65 70 75 80

Gln Ser Glu Asp Leu Ala Glu Tyr Phe Cys Gln Gln Tyr Asn Ser Tyr

85 90 95

Pro Leu Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Gly Ser Thr

100 105 110

Ser Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser Thr Lys Gly Glu

115 120 125

Val Lys Val Glu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser

130 135 140

Met Lys Leu Ser Cys Val Val Ser Gly Phe Thr Phe Gly Asn Tyr Trp

145 150 155 160

Met Asn Trp Val Arg Gln Ser Pro Glu Lys Gly Leu Glu Trp Ile Ala

165 170 175

Glu Ile Arg Leu Lys Ser Asn Asn Phe Ala Arg Tyr Tyr Ala Glu Ser

180 185 190

Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Ser Val

195 200 205

Tyr Leu Gln Met Ile Asn Leu Arg Ala Glu Asp Thr Gly Ile Tyr Tyr

210 215 220

Cys Thr Ser Tyr Gly Asn Tyr Val Gly His Tyr Phe Asp His Trp Gly

225 230 235 240

Gln Gly Thr Thr Leu Thr Val Ser Ser Phe Ala Ile Arg Trp Gly Phe

245 250 255

Ile Pro Leu Val Ile Tyr Leu Gly Phe Lys Arg Gly Ala Asp Pro Gly

260 265 270

Met Pro Glu Pro Thr Val Leu Ser Leu Leu Trp Gly Leu Glu Ser Arg

275 280 285

Gly Pro Phe Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu Asn Met His

290 295 300

Thr Gly His His His His His

305 310

<210> 68

<211> 250

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Amino acid sequence of scFvPD-L1

<400> 68

Met Asp Ile Val Met Ser Gln Ser Pro Ser Ser Leu Ala Val Ser Ala

1 5 10 15

Gly Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn

20 25 30

Ser Arg Thr Arg Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly

35 40 45

Gln Ser Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly

50 55 60

Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu

65 70 75 80

Thr Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Val Tyr Tyr Cys Gln

85 90 95

Gln Ser Tyr Asp Val Val Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu

100 105 110

Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

115 120 125

Gln Val Gln Val Gln Gln Ser Gly Ala Glu Leu Ala Glu Pro Gly Ala

130 135 140

Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Ile Phe Thr Ser Tyr

145 150 155 160

Trp Met His Trp Leu Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile

165 170 175

Gly Tyr Ile Asn Pro Ser Ser Asp Tyr Asn Glu Tyr Ser Glu Lys Phe

180 185 190

Met Asp Lys Ala Thr Leu Thr Ala Asp Lys Ala Ser Thr Thr Ala Tyr

195 200 205

Met Gln Leu Ile Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

210 215 220

Ala Arg Ser Gly Trp Leu Val His Gly Asp Tyr Tyr Phe Asp Tyr Trp

225 230 235 240

Gly Gln Gly Thr Thr Leu Thr Val Ser Ser

245 250

<210> 69

<211> 315

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Amino acid sequence of scFvPD-L1-TOM7-myc/His

<400> 69

Met Asp Ile Val Met Ser Gln Ser Pro Ser Ser Leu Ala Val Ser Ala

1 5 10 15

Gly Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn

20 25 30

Ser Arg Thr Arg Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly

35 40 45

Gln Ser Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly

50 55 60

Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu

65 70 75 80

Thr Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Val Tyr Tyr Cys Gln

85 90 95

Gln Ser Tyr Asp Val Val Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu

100 105 110

Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

115 120 125

Gln Val Gln Val Gln Gln Ser Gly Ala Glu Leu Ala Glu Pro Gly Ala

130 135 140

Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Ile Phe Thr Ser Tyr

145 150 155 160

Trp Met His Trp Leu Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile

165 170 175

Gly Tyr Ile Asn Pro Ser Ser Asp Tyr Asn Glu Tyr Ser Glu Lys Phe

180 185 190

Met Asp Lys Ala Thr Leu Thr Ala Asp Lys Ala Ser Thr Thr Ala Tyr

195 200 205

Met Gln Leu Ile Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

210 215 220

Ala Arg Ser Gly Trp Leu Val His Gly Asp Tyr Tyr Phe Asp Tyr Trp

225 230 235 240

Gly Gln Gly Thr Thr Leu Thr Val Ser Ser Leu Glu Phe Ala Ile Arg

245 250 255

Trp Gly Phe Ile Pro Leu Val Ile Tyr Leu Gly Phe Lys Arg Gly Ala

260 265 270

Asp Pro Gly Met Pro Glu Pro Thr Val Leu Ser Leu Leu Trp Gly Leu

275 280 285

Glu Ser Arg Gly Pro Phe Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu

290 295 300

Asn Met His Thr Gly His His His His His

305 310 315

<210> 70

<211> 15

<212> DNA

<213> Artificial sequence

<220>

<223> Linker

<400> 70

ggggsggggs ggggs

15

<210> 71

<211> 75

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Amino acid sequence of ubiquitin

<400> 71

Gln Leu Phe Val Lys Thr Leu Thr Gly Lys Thr Val Thr Leu Glu Val

1 5 10 15

Glu Ser Ser Asp Thr Ile Asp Asn Val Lys Ser Lys Ile Gln Asp Lys

20 25 30

Glu Gly Ile Pro Pro Asp Gln Gln Arg Leu Ile Phe Ala Gly Lys Gln

35 40 45

Leu Glu Asp Gly Arg Thr Leu Ser Asp Tyr Asn Ile Gln Lys Glu Ser

50 55 60

Thr Leu His Leu Val Leu Arg Leu Arg Gly Gly

65 70 75

<210> 72

<211> 225

<212> DNA

<213> Artificial sequence

<220>

<223> Nucleotide sequence encoding ubiquitin

<400> 72

caacttttcg tcaaaactct aacaggaag actgtaaccc tagaggttga atcttccgac

60

actattgaca acgtcaaaag taaaattcaa gataaagaag gtatccctcc ggatcagcag

120

agattgattt ttgctggtaa gcaactagaa gatggtagaa ccttgtctga ctacaacatc

180

caaaaggaat ctactcttca cttggtgttg agactccgcg gtggt

225

<210> 73

<211> 400

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Amino acid sequence of p53

<400> 73

Glu Glu Pro Gln Ser Asp Pro Ser Val Glu Pro Pro Leu Ser Gln Glu

1 5 10 15

Thr Phe Ser Asp Leu Trp Lys Leu Leu Pro Glu Asn Asn Val Leu Ser

20 25 30

Pro Leu Pro Ser Gln Ala Met Asp Asp Leu Met Leu Ser Pro Asp Asp

35 40 45

Ile Glu Gln Trp Phe Thr Glu Asp Pro Gly Pro Asp Glu Ala Pro Arg

50 55 60

Met Pro Glu Ala Ala Pro Arg Val Ala Pro Ala Pro Ala Ala Pro Thr

65 70 75 80

Pro Ala Ala Pro Ala Pro Ala Pro Ser Trp Pro Leu Ser Ser Ser Val

85 90 95

Pro Ser Gln Lys Thr Tyr Gln Gly Ser Tyr Gly Phe Arg Leu Gly Phe

100 105 110

Leu His Ser Gly Thr Ala Lys Ser Val Thr Cys Thr Tyr Ser Pro Ala

115 120 125

Leu Asn Lys Met Phe Cys Gln Leu Ala Lys Thr Cys Pro Val Gln Leu

130 135 140

Trp Val Asp Ser Thr Pro Pro Pro Gly Thr Arg Val Arg Ala Met Ala

145 150 155 160

Ile Tyr Lys Gln Ser Gln His Met Thr Glu Val Val Arg Arg Cys Pro

165 170 175

His His Glu Arg Cys Ser Asp Ser Asp Gly Leu Ala Pro Pro Gln His

180 185 190

Leu Ile Arg Val Glu Gly Asn Leu Arg Val Glu Tyr Leu Asp Asp Arg

195 200 205

Asn Thr Phe Arg His Ser Val Val Val Pro Tyr Glu Pro Pro Glu Val

210 215 220

Gly Ser Asp Cys Thr Thr Ile His Tyr Asn Tyr Met Cys Asn Ser Ser

225 230 235 240

Cys Met Gly Gly Met Asn Arg Arg Pro Ile Leu Thr Ile Ile Thr Leu

245 250 255

Glu Asp Ser Ser Gly Asn Leu Leu Gly Arg Asn Ser Phe Glu Val Arg

260 265 270

Val Cys Ala Cys Pro Gly Arg Asp Arg Arg Thr Glu Glu Glu Asn Leu

275 280 285

Arg Lys Lys Gly Glu Pro His His Glu Leu Pro Pro Gly Ser Thr Lys

290 295 300

Arg Ala Leu Pro Asn Asn Thr Ser Ser Ser Pro Gln Pro Lys Lys Lys

305 310 315 320

Pro Leu Asp Gly Glu Tyr Phe Thr Leu Gln Ile Arg Gly Arg Glu Arg

325 330 335

Phe Glu Met Phe Arg Glu Leu Asn Glu Ala Leu Glu Leu Lys Asp Ala

340 345 350

Gln Ala Gly Lys Glu Pro Gly Gly Ser Arg Ala His Ser Ser His Leu

355 360 365

Lys Ser Lys Lys Gly Gln Ser Thr Ser Arg His Lys Lys Leu Met Phe

370 375 380

Lys Thr Glu Gly Pro Asp Ser Asp Leu Glu His His His His His

385 390 395 400

<210> 74

<211> 1203

<212> DNA

<213> Artificial sequence

<220>

<223> Nucleotide sequence encoding p53

<400> 74

gaggagccgc agtcagatcc tagcgtcgag ccccctctga gtcaggaaac attttcagac

60

ctgtggaaac tacttcctga aaacaacgtt ctgtccccct tgccgtccca agcaatggat

120

gatttgatgc tgtccccgga cgatattgaa caatggttca ctgaagaccc aggtccagat

180

gaagctccca gaatgccaga ggctgctccc cgcgtggccc ctgcaccagc agctcctaca

240

ccggcggccc ctgcaccagc cccctcctgg cccctgtcat cttctgtccc ttcccagaaa

300

acctaccagg gcagctacgg tttccgtctg ggcttcttgc attctgggac agccaagtct

360

gtgacttgca cgtactcccc tgccctcaac aagatgtttt gccaactggc caagacctgc

420

cctgtgcagc tgtgggttga ttccacaccc ccgcccggca cccgcgtccg cgccatggcc

480

atctacaagc agtcacagca catgacggag gttgtgaggc gctgccccca ccatgagcgc

540

tgctcagata gcgatggtct ggcccctcct cagcatctta tccgagtgga aggaaatttg

600

cgtgtggagt atttggatga cagaaacact tttcgacata gtgtggtggt gccctatgag

660

ccgcctgagg ttggctctga ctgtaccacc atccactaca actacatgtg taacagttcc

720

tgcatgggcg gcatgaaccg gaggcccatc ctcaccatca tcacactgga agactccagt

780

ggtaatctac tgggacggaa cagctttgag gtgcgtgttt gtgcctgtcc tgggagagac

840

cggcgcacag aggaagagaa tctccgcaag aaaggggagc ctcaccacga gctgccccca

900

gggagcacta agcgagcact gcccaacaac accagctcct ctccccagcc aaagaagaaa

960

ccactggatg gagaatattt cacccttcag atccgtgggc gtgagcgctt cgagatgttc

1020

cgagagctga atgaggcctt ggaactcaag gatgcccagg ctgggaagga gccagggggg

1080

agcagggctc actccagcca cctgaagtcc aaaaagggtc agtctacctc ccgccataaa

1140

aaactcatgt tcaagacaga agggcctgac tcagacctcg agcaccacca ccaccaccac

1200

tag

1203

<210> 75

<211> 29

<212> PROTEIN

<213> Artificial sequence

<220>

<223> TOM70 S. cerevisiae

<400> 75

Met Lys Ser Phe Ile Thr Arg Asn Lys Thr Ala Ile Phe Ala Thr Val

1 5 10 15

Ala Ala Thr Gly Thr Ala Ile Gly Ala Tyr Tyr Tyr Tyr

20 25

<210> 76

<211> 60

<212> PROTEIN

<213> Artificial sequence

<220>

<223> TOM70 Homo sapiens

<400> 76

Met Ala Ala Ser Lys Pro Val Glu Ala Ala Val Val Ala Ala Ala Val

1 5 10 15

Pro Ser Ser Gly Ser Gly Val Gly Gly Gly Gly Thr Ala Gly Pro Gly

20 25 30

Thr Gly Gly Leu Pro Arg Trp Gln Leu Ala Leu Ala Val Gly Ala Pro

35 40 45

Leu Leu Leu Gly Ala Gly Ala Ile Tyr Leu Trp Ser

50 55 60

<210> 77

<211> 29

<212> PROTEIN

<213> Artificial sequence

<220>

<223> TOM20 S. cerevisiae

<400> 77

Met Ser Gln Ser Asn Pro Ile Leu Arg Gly Leu Ala Ile Thr Thr Ala

1 5 10 15

Ile Ala Ala Leu Ser Ala Thr Gly Tyr Ala Ile Tyr Phe

20 25

<210> 78

<211> 24

<212> PROTEIN

<213> Artificial sequence

<220>

<223> TOM20 Homo sapiens

<400> 78

Met Val Gly Arg Asn Ser Ala Ile Ala Ala Gly Val Cys Gly Ala Leu

1 5 10 15

Phe Ile Gly Tyr Cys Ile Tyr Phe

20

<210> 79

<211> 22

<212> PROTEIN

<213> Artificial sequence

<220>

<223> OM45 S. cerevisiae

<400> 79

Met Ser Ser Arg Ile Ile Val Gly Ser Ala Ala Leu Ala Ala Ala Ile

1 5 10 15

Thr Ala Ser Ile Met Val

20

<210> 80

<211> 23

<212> PROTEIN

<213> Artificial sequence

<220>

<223> TOM5 S. cerevisiae

<400> 80

Ala Ala Tyr Val Ala Ala Phe Leu Trp Val Ser Pro Met Ile Trp His

1 5 10 15

Leu Val Lys Lys Gln Trp Lys

20

<210> 81

<211> 24

<212> PROTEIN

<213> Artificial sequence

<220>

<223> TOM5 Homo sapiens

<400> 81

Ile Arg Asn Phe Leu Ile Tyr Val Ala Leu Leu Arg Val Thr Pro Phe

1 5 10 15

Ile Leu Lys Lys Leu Asp Ser Ile

20

<210> 82

<211> 41

<212> PROTEIN

<213> Artificial sequence

<220>

<223> TOM7 S. cerevisiae

<400> 82

Ile Leu Thr Leu Thr His Asn Val Ala His Tyr Gly Trp Ile Pro Phe

1 5 10 15

Val Leu Tyr Leu Gly Trp Ala His Thr Ser Asn Arg Pro Asn Phe Leu

20 25 30

Asn Leu Leu Ser Pro Leu Pro Ser Val

35 40

<210> 83

<211> 35

<212> PROTEIN

<213> Artificial sequence

<220>

<223> TOM7 Homo sapiens

<400> 83

Phe Ala Ile Arg Trp Gly Phe Ile Pro Leu Val Ile Tyr Leu Gly Phe

1 5 10 15

Lys Arg Gly Ala Asp Pro Gly Met Pro Glu Pro Thr Val Leu Ser Leu

20 25 30

Leu Trp Gly

35

<210> 84

<211> 55

<212> PROTEIN

<213> Artificial sequence

<220>

<223> TOM22 S. cerevisiae

<400> 84

Leu Ala Trp Thr Leu Thr Thr Thr Ala Leu Leu Leu Gly Val Pro Leu

1 5 10 15

Ser Leu Ser Ile Leu Ala Glu Gln Gln Leu Ile Glu Met Glu Lys Thr

20 25 30

Phe Asp Leu Gln Ser Asp Ala Asn Asn Ile Leu Ala Gln Gly Glu Lys

35 40 45

Asp Ala Ala Ala Thr Ala Asn

50 55

<210> 85

<211> 60

<212> PROTEIN

<213> Artificial sequence

<220>

<223> TOM22 Homo sapiens

<400> 85

Ala Ala Leu Trp Ile Gly Thr Thr Ser Phe Met Ile Leu Val Leu Pro

1 5 10 15

Val Val Phe Glu Thr Glu Lys Leu Gln Met Glu Gln Gln Gln Gln Leu

20 25 30

Gln Gln Arg Gln Ile Leu Leu Gly Pro Asn Thr Gly Leu Ser Gly Gly

35 40 45

Met Pro Gly Ala Leu Pro Ser Leu Pro Gly Lys Ile

50 55 60

<210> 86

<211> 25

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Fis1 S. cerevisiae

<400> 86

Gly Val Val Val Ala Gly Gly Val Leu Ala Gly Ala Val Ala Val Ala

1 5 10 15

Ser Phe Phe Leu Arg Asn Lys Arg Arg

20 25

<210> 87

<211> 31

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Fis1 Homo sapiens

<400> 87

Gly Leu Val Gly Met Ala Ile Val Gly Gly Met Ala Leu Gly Val Ala

1 5 10 15

Gly Leu Ala Gly Leu Ile Gly Leu Ala Val Ser Lys Ser Lys Ser

20 25 30

<210> 88

<211> 25

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Bcl-2 alpha Homo sapiens

<400> 88

Leu Ser Leu Lys Thr Leu Leu Ser Leu Ala Leu Val Gly Ala Cys Ile

1 5 10 15

Thr Leu Gly Ala Tyr Leu Gly His Lys

20 25

<210> 89

<211> 19

<212> PROTEIN

<213> Artificial sequence

<220>

<223> VAMP1 S. cerevisiae

<400> 89

Met Ile Met Leu Gly Ala Ile Cys Ala Ile Ile Val Val Val Ile Val

1 5 10 15

Arg Arg Asp

<210> 90

<211> 22

<212> PROTEIN

<213> Artificial sequence

<220>

<223> VAMP1 Homo sapiens

<400> 90

Met Met Ile Met Leu Gly Ala Ile Cys Ala Ile Ile Val Val Val Ile

1 5 10 15

Val Ile Tyr Phe Phe Thr

20

<210> 91

<211> 60

<212> DNA

<213> Artificial sequence

<220>

<223> P53-promter-S

<400> 91

gggcatgctc gggcatgccc gggcatgctc gggcatgccc gggcatgctc gggcatgccc

60

60

<210> 92

<211> 60

<212> DNA

<213> Artificial sequence

<220>

<223> P53-promter-AS

<400> 92

gggcatgccc gagcatgccc gggcatgccc gagcatgccc gggcatgccc gagcatgccc

60

60

<---

Claims (37)

1. A modified mitochondrion for delivering a foreign protein into cells, in which the foreign protein is associated with the outer membrane of mitochondria, where the foreign protein is a fusion protein containing a mitochondria-anchoring peptide and a desired protein capable of functioning inside and outside the cell, or fusion protein , containing a target targeting protein having the ability to bind to a ligand or receptor present in the cell membrane, and a peptide anchoring in the mitochondrial outer membrane, wherein the mitochondria-anchoring peptide contains the N-terminal region or the C-terminal region of a protein present in a mitochondrial membrane protein, and the protein present in a mitochondrial membrane protein is any protein selected from the group consisting of TOM20, TOM70, OM45, TOM5, TOM6, TOM7, TOM22, Fis1, Bcl-2, Bcl-x and VAMP1B. 2. Modified mitochondria according to claim 1, where the mitochondria is isolated from cells or tissues. 3. The modified mitochondrion of claim 2, wherein the cell is any cell selected from the group consisting of somatic cells, germ cells, stem cells, and combinations thereof. 4. The modified mitochondrion according to claim 1, wherein the foreign protein is bound to the outer membrane of the mitochondrion via a mitochondria-anchoring peptide. 5. The modified mitochondrion according to claim 1, characterized in that the N-terminal region or C-terminal region of the protein present in the mitochondrial membrane protein is located on the outer membrane of the mitochondria. 6. The modified mitochondrion according to claim 1, characterized in that the anchoring peptide contains the N-terminal region of any protein selected from the group consisting of TOM20, TOM70 and OM45. 7. A modified mitochondrion according to claim 1, characterized in that the mitochondria-anchoring peptide contains the C-terminal region of any protein selected from the group consisting of TOM5, TOM6, TOM7, TOM22, Fis1, Bcl-2, Bcl-x and VAMP1B. 8. The modified mitochondrion of claim 1, wherein the desired protein is any protein selected from the group consisting of an active protein exhibiting activity in a cell, a protein present in the cell, and a protein having the ability to bind to a ligand or receptor present in a membrane cells. 9. The modified mitochondrion of claim 8, wherein the desired protein is any protein selected from the group consisting of p53, granzyme B, Bax, Bak, PDCD5, E2F, AP-1 (Jun/Fos), EGR-1, retinoblastoma protein (RB), phosphatase and tensin homolog (PTEN), E-cadherin, neurofibromin-2(NF-2), poly-ADP-ribose synthase 1 (PARP-1), BRCA-1, BRCA-2, adenomatous polyposis protein colon (APC), tumor necrosis factor receptor-associated factor (TRAF), RAF kinase inhibitory protein (RKIP), p16, KLF-10, LKB1, LHX6, C-RASSF, DKK-3PD1, Oct3/4, Sox2 , Klf4 and c-Myc. 10. The modified mitochondrion according to claim 1, wherein the foreign protein is a desired protein associated with the N-terminal region of TOM20, TOM70 or OM45. 11. Modified mitochondria according to claim 10, where the foreign protein is bound in the following order: N-terminal-N-terminal region of TOM20, TOM70 or OM45-desired protein-C-terminal. 12. The modified mitochondrion according to claim 11, wherein the foreign protein further comprises an amino acid sequence recognized by a proteolytic enzyme in eukaryotic cells, or ubiquitin or a fragment thereof between the anchoring peptide and the desired protein. 13. The modified mitochondrion according to claim 12, wherein the ubiquitin fragment contains the C-terminal Gly-Gly amino acid sequence SEQ ID NO: 71 and contains from 3 to 75 consecutive amino acids from the C-terminus. 14. The modified mitochondrion according to claim 12, wherein the foreign protein further comprises a linker between the desired protein and ubiquitin or a fragment thereof. 15. The modified mitochondrion according to claim 14, where the linker is a polypeptide of SEQ ID NO: 70. 16. The modified mitochondrion according to claim 8, wherein the protein having the ability to bind to a ligand or receptor present in the cell membrane is a ligand or receptor present on the surface of the tumor cell. 17. The modified mitochondrion according to claim 16, wherein the ligand or receptor present on the surface of the tumor cell is any ligand or receptor selected from the group consisting of CD19, CD20, melanoma-associated antigen E (MAGE), NY-ESO-1 , carcinoembryonic antigen (CEA), cell surface associated mucin 1 (MUC-1), prostatic acid phosphatase (PAP), prostate-specific antigen (PSA), survivin, tyrosinase-related protein 1 (tyrp1), tyrosinase-related protein 1 (tyrp2), brachyury gene, mesothelin, epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (HER-2), ERBB2, Wilms tumor protein (WT1), FAP, EpCAM, PD-L1, ACPP, CPT1A , IFNG, CD274, FOLR1, EPCAM, ICAM2, NCAM1, LRRC4, UNC5H2 LILRB2, CEACAM, nectin-3 or combinations thereof. 18. The modified mitochondrion according to claim 1, wherein the foreign protein is associated with the C-terminal region of any protein selected from the group consisting of TOM5, TOM6, TOM7, TOM22, Fis1, Bcl-2, Bcl-x and VAMP1B. 19. Modified mitochondria according to claim 18, where the foreign protein is bound in the following order: N-terminal-desired protein-C-terminal region of any protein selected from the group consisting of TOM5, TOM6, TOM7, TOM22, Fis1, Bcl-2, Bcl-x and VAMP1B-C-terminus. 20. The modified mitochondrion of claim 19, wherein the foreign protein further comprises a linker between the desired protein and the C-terminal region of any protein selected from the group consisting of TOM5, TOM6, TOM7, TOM22, Fis1, Bcl-2, Bcl-x and VAMP1B. 21. The modified mitochondrion according to claim 20, where the linker is a polypeptide of SEQ ID NO: 70. 22. A pharmaceutical composition for the prevention or treatment of malignant neoplasms, containing a modified mitochondria according to any one of claims 1 to 21 as an active ingredient and a buffer solution, wherein the malignant neoplasm is any malignant neoplasm selected from the group consisting of gastric cancer, liver cancer, lung cancer, colorectal cancer, breast cancer, prostate cancer, ovarian cancer, pancreatic cancer, cervical cancer, thyroid cancer, cancer larynx, acute myeloid leukemia, brain tumor, neuroblastoma, retinoblastoma, head and neck cancer, salivary gland cancer and lymphoma. 23. Use of modified mitochondria according to paragraphs 1-21 for the prevention or treatment of malignant neoplasms, wherein the malignant neoplasm is any malignant neoplasm selected from the group consisting of gastric cancer, liver cancer, lung cancer, colorectal cancer, breast cancer, prostate cancer, ovarian cancer, pancreatic cancer, cervical cancer, thyroid cancer, cancer larynx, acute myeloid leukemia, brain tumor, neuroblastoma, retinoblastoma, head and neck cancer, salivary gland cancer and lymphoma. 24. Use of a modified mitochondria according to any one of paragraphs. 1-21 as a means of intracellular and extracellular delivery of a foreign protein containing the desired protein capable of functioning outside and inside the cell. 25. Use of a modified mitochondrion according to claim 24, wherein the foreign protein contains a mitochondrial outer membrane-anchoring peptide and is associated with the outer membrane of the mitochondria through the outer membrane-anchoring peptide and is delivered into and outside the cell. 26. The modified mitochondrion according to claim 1, wherein the target targeting protein having the ability to bind to a ligand or receptor present in the cell membrane and the mitochondrial outer membrane anchoring peptide are linked from the N-terminus to the C-terminus. 27. The modified mitochondrion according to claim 26, wherein the target targeting protein having the ability to bind to a ligand or receptor present in the cell membrane is an antibody or a fragment thereof. 28. The modified mitochondrion according to claim 27, wherein the antibody fragment is any fragment selected from the group consisting of Fab, Fab', scFv and F(ab)2. 29. The method of obtaining a modified mitochondrion according to claim 1, including the step of mixing the isolated mitochondria with a foreign protein, where the foreign protein is a fusion protein containing a peptide anchoring in mitochondria and a desired protein capable of functioning inside and outside the cell, or fusion protein, containing a target targeting protein having the ability to bind to a ligand or receptor present in the cell membrane, and a peptide anchoring in the mitochondrial outer membrane, wherein the mitochondria-anchoring peptide contains the N-terminal region or the C-terminal region of a protein present in a mitochondrial membrane protein, and the protein present in a mitochondrial membrane protein is any protein selected from the group consisting of TOM20, TOM70, OM45, TOM5, TOM6, TOM7, TOM22, Fis1, Bcl-2, Bcl-x and VAMP1B. 30. The method of obtaining a modified mitochondrion according to claim 1 from transformed cells by injecting a polynucleotide encoding a foreign protein, where the foreign protein is a fusion protein containing a peptide anchoring in mitochondria and a desired protein capable of functioning inside and outside the cell, or fusion protein , containing a target targeting protein having the ability to bind to a ligand or receptor present in the cell membrane, and a peptide anchoring in the mitochondrial outer membrane, wherein the mitochondria-anchoring peptide contains the N-terminal region or the C-terminal region of a protein present in a mitochondrial membrane protein, and the protein present in a mitochondrial membrane protein is any protein selected from the group consisting of TOM20, TOM70, OM45, TOM5, TOM6, TOM7, TOM22, Fis1, Bcl-2, Bcl-x and VAMP1B.