WO2006089455A1 - Targeting anti-tumour fusion protein containing adenovirus e4orf4 protein - Google Patents

Abstract

A recombination human epidermal growth factor -adenovirus E4orf4 fusion protein is disclosed by the present invention. A fusion gene coding the said fusion protein, a express vector containing this fusion gene and a method using methylotrophic yeast to express the secretory recombination fusion protein are disclosed too. In addition, this invention also relates to a medicine combination containing the said protein and the treatment application thereof.

Description

 Targeted anti-tumor fusion protein containing adenovirus E4orf4 protein

 The present invention relates to a recombinant human epidermal growth factor-adenovirus early transcribed region 4 fourth open reading frame protein product (E4orf4) fusion protein, a fusion gene encoding the fusion protein, an expression vector containing the fusion gene, and the use of methyl nutrition The yeast is a method for secretory expression of such a recombinant fusion protein, a pharmaceutical composition containing the same, and a therapeutic use thereof. Background technique

 The early transcribed region 4 (E4) transcription unit of the human adenovirus gene contains at least 7 open reading frames, and the respective products have no obvious similarity. The product encoded by the fourth open reading frame 4 (E4orf4) is a 14kDa small molecule protein consisting of 114 amino acids, and its amino acid sequence has no homology with any known protein. The E4orf4 protein has a highly conserved sequence, RXKRRXRRRR, which also has a proline-rich sequence at the amino terminus, potentially providing a potential Src homology region 3 (SH3) binding site. Deletion of any small portion of an amino acid in a polypeptide chain, including both the amino terminus and the carboxy terminus region, results in a non-functional and often unstable mutant. In the early stage of adenovirus infection, the E4orf4 gene is transcribed from the E4 promoter to produce i RNA. Although the transcription was stopped in the late E4 promoter, the E4orf4 protein in the cells remained at a stable level. This indicates that the E4orf4 protein is highly stable in cells (Branton PE and Roopc and DE. Oncogene, 2001; (20): 7855-7865; Marcellus RC, Chan H, Paquette D et al. J Virol, 2000; 74 (17 ): 7869-7877).

Many viruses can induce apoptosis and kill infected cells during their life cycle. Human adenovirus infection forms a complex process that induces and inhibits apoptosis. The adenovirus early region 1A (E1A) product leads to p53-dependent apoptosis by enhancing the activity and stability of the p53 gene. Early zone IB (E1B) encodes two eggs of 55kDa and 19kDa White, together with the E4orf6 protein, produces a large number of viruses by blocking the toxic effects of E1A and inhibiting apoptosis. Further experiments revealed that infection with adenovirus with p53-deficient cells and expression of intact E1A induced apoptosis, suggesting that this process is caused by other viral products expressed under the control of the E1A transactivation region. After a series of experiments, the cytotoxic function was finally localized to E4orf4 protein. In the absence of other adenoviral proteins, E4orf4 protein has high cytotoxicity, induces apoptosis independent of p53, and pcDNA plasmid expressing E4orf4 can induce tumor cells. With a mortality rate of 80-90%, it is the only E4 product that kills cells by itself. The adenovirus E4orf4 protein is a multifunctional viral regulatory protein that specifically induces apoptosis in transformed cells in vitro, but has no effect on normal cells; apoptosis induced by E4orf4 protein is p53-independent Its role in inducing apoptosis requires interaction with protein phosphatase 2A; E4orf4 regulates the activity of Src kinase and triggers cytoplasmic expression of apoptosis (Maecell s RC, Tendorn JG, Wu T et al. J Virol. 1996, 70:6207-6215; Marcellus RC, Lavoie JN, Boivin D et al. J. Virol. 1998, 72: 7144-7153; Shtricheman R, Sharf R, Barr H et al. Proc. Natl. Acad Sci. USA. 1999, 96: 10080-10085).

Numerous studies have confirmed that EGF receptors are overexpressed in most tumor cells such as breast, lung, brain, bladder, kidney, prostate, etc., and their cell lines are often inhibited by EGF after growth or transplantation in vitro. The results showed that chemically linked RNase 1 and recombinant EGF form a complex EGF-RNase 1, which has a dose-dependent cytotoxic effect on breast cancer and squamous cell carcinoma with high expression of EGF receptor, ie, the more EGF receptor is expressed The higher the cell line, the more obvious the cytotoxic effect. Recently, Dmitriev et al. expressed a CAR-EGF fusion protein (CAR, Coxsackie virus and adenovirus receptor) using a baculovirus expression system, and efficiently transported adenoviral vectors through the targeting of EGF binding to EGF receptors. Increased reporter gene release into tumor cells (Yang Yu'an, Huang Bingren, Cai Liangbiao, et al. Journal of Chinese Academy of Medical Sciences. 1996, 18(3): 171; Ueda M, Psarras K, Ji, H et al. Breast Cancer 1997, 4(4): 253-255; Dmitriev I, Kashentseva E, Rogers BE et al. J. Virol. 2000, 74(15): 6875-6884).

 Since a high proportion of p53 gene mutations in human tumors and the conventional prognosis of these tumors are often poor, it is a very interesting attempt to find a new method for treating p53 mutant tumors. Adenovirus is a promising gene therapy vector. However, while gene therapy for adenovirus provides many benefits, the technology is not yet fully mature, and there is a certain risk in the safety of treatment. Therefore, further exploration of methods and drugs for treatment with adenovirus remains a hot spot in the field of cancer therapy. Summary of invention

 The present invention relates to a recombinant human epidermal growth factor-adenovirus E4orf4 fusion protein, a pharmaceutical composition containing the same, and its use for targeted tumor therapy. In one embodiment, the fusion gene encoding the fusion protein of the present invention has the sequence of SEQ ID NO: 1, and the amino acid sequence of the fusion protein is as set forth in SEQ ID NO: 2.

 Another aspect of the invention relates to a fusion gene encoding the fusion protein, an expression vector containing the fusion gene, and an engineered strain. A genetically engineered strain according to the present invention, named Pichia pastoris GS115-3 EGF-E4orf4, deposited on July 15, 2002 at the General Microbiology Center (CGMCC) of the China Microbial Culture Collection Management Committee, The number is 0771. The deposit was transferred to the international deposit under the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Patent Procedure on February 22, 2005.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1: Overlapping PCR to construct the α-factor leader peptide-EGF-E4orf4 gene fragment; Figure 2: Recombinant plasmid pUC-EGF-E4orf4 digestion and identification electrophoresis map; Figure 3: Recombinant plasmid pUC-EGF-E4orf4 site-directed mutagenesis; Figure 4: Physical map of plasmid pA0815, pA0815 consists of 7709 nucleotides, wherein base 1 940 is a 5ΆΟΧ1 promoter fragment, bases 943-948 are EcoRJ sites, and bases 950-1277 are 3ΆΟΧ1 transcription terminators ( TT), base 4199-1665 is HISORF, bases 4554-5310 are 3ΆΟΧ1 fragments, bases 6394-5740 are starting with ColEl, and bases 7399-6539 are ampicillin resistance genes;

 Figure 5: Construction of recombinant plasmid pAO-EGF-E4orf4;

 Figure 6: Electrophoresis pattern of the recombinant plasmid pAO-EGF-E4orf4;

 Figure 7: Construction of recombinant plasmid pAO-3EGF-E4orf4;

 Figure 8: Electrophoresis pattern of the recombinant plasmid pAO-3EGF-E4orf4;

Figure 9: Comparison of Mut ⁺ and Mut ^s transformants;

 Figure 10: SDS-PAGE electrophoresis pattern of EGF-E4orf4 secreted by GS115-3EGF-E4orf4;

 Figure 11: Western Blot analysis of EG115-3EGF-E4orf4 secreting EGF-E4orf4.

 Figure 12: Results of recombinant EGF-E4orf4 fusion protein purified by cation exchange chromatography;

 Figure 13: MTT colorimetric assay for BT325 cell viability;

 Figure 14: MTT colorimetric assay for MDA-MB-231 cell viability;

 Figure 15: MDA-MB-231 cells were cultured for 72 hours, and apoptosis was analyzed by flow cytometry;

Figure 16: MDA-MB-231 cells were co-cultured with the fusion protein EGF-E4orf4 (10μ _§ /3χ10 ⁵ cells) for 72 hours, and apoptosis was analyzed by flow cytometry;

Figure 17: MDA-MB-231 cells were co-cultured with the fusion protein EGF_E4orf4 (15μ _§ /3χ10 ⁵ cells) for 72 hours, and apoptosis was analyzed by flow cytometry; Figure 18: MDA-MB-231 cells were co-cultured with the fusion protein EGF-E4orf4 (20μ _§ /3χ10 ⁵ cells) for 72 hours, and apoptosis was analyzed by flow cytometry;

Figure 19: MDA-MB-231 cells were co-cultured with the fusion protein EGF-E4orf4 (25μ _§ /3χ10 ⁵ cells) for 72 hours, and apoptosis was analyzed by flow cytometry. Detailed description of the invention

 According to an aspect of the present invention, a recombinant human epidermal growth factor (EGF)-adenovirus E4orf4 fusion protein comprising human epidermal growth factor, a linker, and an adenovirus E4orf4 protein from N-terminal to C-terminal is provided. In a preferred embodiment, the amino acid sequence of the fusion protein is set forth in SEQ ID NO: 2.

 According to another aspect of the present invention, the present invention provides a fusion gene encoding the fusion protein of the present invention.

 Preferably, the artificial fusion gene encoding the 51 amino acid EGF gene fragment is used in the fusion gene of the present invention, and the gene fragment is described in Chinese Patent Application No. 97115284.5. The E4orf4 protein gene sequence of the adenovirus in the fusion gene of the present invention is preferably a modified E4orf4 protein gene sequence encoding adenovirus A5, which is a site-directed mutation that mutates the 228th base from T to G, thereby The restriction endonuclease Bglll cleavage site was eliminated, but the amino acid sequence was not altered to facilitate the construction of the yeast multiple copy tandem expression vector. The linker between the two gene fragments of EGF and E4orf4 is preferably a gene sequence encoding 11 amino acids consisting of glycine and serine, so that it can form an arm to ensure that EGF and E4orf4 proteins can not interfere with each other, and each forms an independent Advanced structure, exercising normal biological activity. In a preferred embodiment, the gene sequence encoding the EGF-E4orf4 fusion protein of the present invention is represented by SEQ ID NO: 1.

According to still another aspect of the present invention, an expression vector comprising the fusion gene of the present invention and an engineered bacteria containing the expression vector are provided. Preferably, the expression vector of the present invention is An expression vector for secreted expression of an integrated single-copy or triple-copy EGF-E4orf4 fusion protein by fusing fusion of a DNA fragment encoding a yeast alpha-factor leader peptide with a gene sequence of an EGF-E4orf4 fusion protein, and The α-factor leader peptide was added to the Kozak sequence and cloned downstream of the alcohol oxidase promoter (AOX1). And a single copy of the present invention, for example, three copies of an expression vector pAO-EGF-E4orf4 and _{P AO-3EGF-E4orf4,} 5 and 7 shown in FIG. Preferably, the engineered bacteria of the present invention is a methylotrophic yeast (also known as Pichia pastoris) comprising the expression vector of the present invention, particularly Pichia pastoris GS115-3 EGF-E4orf4, which was in 2002. Deposited on July 15th at the General Microbiology Center of China Microbial Culture Collection Management Committee

(CGMCC) with a deposit number of 0771. The strain is an integrated Mut ⁺ genotype strain (GS115-3 EGF-E4orf4) screened by transforming the Pichia pastoris strain GS115 into a 3-copy vector. After high-density culture and induced expression, the strain can secrete EGF- E4orf4 fusion protein. The molecular weight of the secreted EGF-E4orf4 fusion protein was consistent with the predicted. Western Blot analysis was performed using rabbit anti-human EGF polyclonal antibody and rabbit anti-adenovirus E4orf4 protein antiserum, respectively, and it was confirmed that the specific expressed protein band was positively reacted with both antibodies, and it was a fusion protein composed of EGF and E4orf4. . The expressed protein is separated and purified by column chromatography to have a purity of 90% or higher.

 In addition, the present invention has two terminators TAA and TGA linked to the coding sequence of the 176th amino acid of the EGF-E4orf4 fusion protein. This design facilitates the isolation and purification of the gene expression product and the termination of gene transcription. The addition of the Kozak sequence provides conditions for efficient expression of the EGF-E4orf4 fusion protein.

The fusion protein of the invention has tumor targeting property and can specifically kill tumor cells, especially tumors deficient in p53 gene, and the targeting is to utilize the high expression of EGF receptor on the surface of most tumor cells, and the fusion protein contains EGF. E4orf4 protein can be introduced into tumor cells by binding EGF to EGF receptor. E4orf4 is a highly cytotoxic protein in adenovirus, which can induce tumor cell-independent p53. Death, thereby achieving targeted treatment of tumors. The activity of the fusion proteins of the invention is verified as described in the Examples section.

 Another aspect of the invention relates to a pharmaceutical composition comprising a pharmaceutically effective amount of an EGF-E4orf4 fusion protein as an active ingredient. Such compositions contain a pharmaceutically acceptable carrier, adjuvant, diluent or filler. The pharmaceutical composition can be administered by injection or by topical administration to a tumor, in order to be therapeutically suitable for clinical use in a clinical setting.

 The invention will be further described below with reference to the following examples. Example 1 : Overlapping PCR construction of α-factor leader peptide- EGF-E4orf4 gene fragment In this example, the following PCR primers were used:

Bow I 1 (31-mer):

 5'-GGAA TC ACC ATG AGA TTT CCT TCA ATT TTT-3'

 EcoRI Kozak

Primer 2 (45 -mer):

 5'-ACC ACC ACC GGA ACC ACC ACC ACC TTC CCA CCA CTT CAA GTC TCT-3'

Primer 3 (45-mer):

 5,-GGT TCC GGT GGT GGT GGT TCC TCC ATG GTT CTT CCA GCT CTT CCC-3'

Primer 4 (31 -mer):

5 ^? -GGAATTC TCA TTA CTG TAC GGA GTG CGC CGA-3' EcoRI Termination Password

Primer 1 contains an EcoRI cleavage site and a Kozak sequence, and is complementary to the 5' end of the α-factor leader peptide; ligate 2 is complementary to the 3' end of EGF and incorporates an amino acid partially encoded by glycine and serine. Sequence of arms; primer 3 and 5, end of E4orf4 Supplement, and added a sequence encoding an amino acid arm consisting of glycine and serine, this sequence is complementary to primer 3; primer 4 is complementary to the 3' end of E4orf4, and two terminators and an EcoRI cleavage site are added.

Using plasmid pYA41EGF as a template (Huang Bingren, Zhang Wei, Chi Lai Shun, et al. Journal of Chinese Academy of Medical Sciences 1989, 11 (5 ): 331 -337), using primer 1 and primer 2 to obtain a DNA fragment containing an EcoRI recognition site. , Kozak sequence, α-factor leader peptide, EGF and 8 amino acid arms consisting of glycine and serine. Using the plasmid _P UC-E4orf4 as a template, the DNA fragment amplified with primer 3 and primer 4 contained 8 amino acid arms consisting of glycine and serine, E4orf4 and E coRI recognition sites. Further, using two DNA fragments as a template, the first annealing is performed to make the five amino acid bases of the two amino acid arms complementary, and then the primers 1 and the primers 4 are used for amplification, thereby obtaining the EcoRI recognition sites at both ends and containing the complete Α-factor leader peptide-EGF-E4orf4 gene fragment. The gene fragment was digested with EcoRI and ligated into the pUC18 plasmid vector, and the resulting recombinant clone was pUC-EGF-E4orf4, as shown in FIG. The sequencing results confirmed that it was completely consistent with the design. Example 2: Enzyme digestion identification of recombinant plasmid pUC-EGF-E4orf4

Identification method: 2μ _δ recombinant plasmid pUC-EGF-E4orf4 was added to EcoRI enzyme for digestion for 2h at 37 °C, 1.0% agarose gel electrophoresis, ethidium bromide staining showed pUC18 vector fragment and encoding α-factor leader peptide-EGF_E4orf4 total 261 Approximately 800 bp DNA cloned fragments of amino acids, 2 terminators, Kozak sequences and EcoRI sites.

The restriction enzyme digestion of the plasmid is shown in Fig. 2. In the figure: Lane 1: λϋΝΑ/ΗΐηάΠΙ, the fragments are: 23130 bp, 9414 bp, 6557 bp, 4361 bp, 2322 bp, 2021 bp, 564 bp. Lane 2: Recombinant plasmid pUC-EGF-E4orf4. Lane 3: pUC-EGF-E4orf4/EcoRI 2.69 kb (vector), 800 bp (insert). Lane 4: DNA Marker DL2000, the fragments from large to small are: 2000 bp, 1000 bp, 750 bp, 500 bp, 250 bp, 100 bp. Example 3: Site-directed mutagenesis of recombinant plasmid pUC-EGF-E4orf4

 Since the construction of multi-copy expression vectors requires the use of the same tail enzymes Bglll and BamHI for the tandem junction of expression units, the exogenous gene fragments cannot contain the tangent points of Bglll and BamHI. The E4orf4 gene fragment contains a Bglll recognition site, which is removed by site-directed mutagenesis, but does not alter the amino acid sequence.

 The site-directed mutagenesis was performed using Stratagene's kit (QuickCliangeTM Site-Directed Mutagenesis Kit). The strategy is shown in Figure 3. The primer sequences for site-directed mutagenesis are as follows:

Primer 1 :

 5,-CGG AGA CGC AGA TCG GTT TGT CAC GCC CGC-3'

5,-GCG GGC GTG ACA AAC CGA TCT GCG TCT CCG-3' The plasmid pUC-EGF-E4orf4 obtained in Example 1 was used as a template, and after PCR amplification of site-directed mutagenesis, Escherichia coli DH5a was transformed, and the transformant was subjected to DNA sequence. Analysis confirmed that the 228th base of the E4orf4 gene changed from T to G. The pUC-EGF-E4orf4 plasmid was digested with EcoRI to obtain a gene fragment encoding the a-factor leader peptide-EGF_E4orf4, and ligated with the EcoRI-linearized yeast expression vector pA0815 (purchased from Iiivitrogen) to obtain the recombinant plasmid pAO-EGF. -E4orf4 (Figure 5). Example 4: Restriction enzyme digestion of recombinant plasmid pAO-EGF-E4orf4

The pA0815 vector AOX1 promoter was the EcoRI cloning site at the 944 bp downstream. After the recombinant plasmid pAO-EGF-E4orf4 was digested with EcoRI, an insert of about 800 bp was observed. The recombinant plasmid pAO-EGF-E4orf4 was screened by restriction enzyme digestion to identify the forward clone. There is a Pstl tangent point at the 24th base of the a-factor leader peptide, and a Pstl cut at the 6858th base of the pA0815 vector. Point, recombinant plasmid pAO-EGF-E4orf4 After digestion with Pstl, two fragments of 1.8 kb and 6.7 kb were obtained by forward cloning, and two fragments of 2.6 kb and 5.9 kb were obtained by reverse cloning.

 The electrophoresis results of the restriction enzyme digestion of the plasmid are shown in Fig. 6. In the figure: Lane 1: DNA molecular weight marker DL2000, the fragments are from 2000 bp to 100 bp, 750 bp, 500 bp, 250 bp, and 100 bp. Lane 2: pAO-EGF-E4orf4/EcoRI, 7.7 kb (vector), 800 bp (insert). Lane 3: pAO-EGF-E4orf4/PstI, 6.7 kb, 1.8 kb. Lane 4: pAO-EGF-E4orf4. Lane 5: The DNA/HindllL fragments were: 23130 bp, 9414 bp, 6557 bp, 4361 bp, 2322 bp, 202 lbp, 564 bp in descending order. Example 5: Construction of a three-copy expression vector pAO-3EGF-E4orf4

 The pAO-EGF-E4orf4 obtained in Example 3 was excised with Bglll and BamHI to contain the AOX1 promoter, the α-factor leader peptide-EGF-E4orf4 coding sequence and the AOX1 terminator fragment, so that Bglll and Bglll were used in vitro. BamHI was digested. At this time, only the dimer of Bglll and BamHI was formed in the reaction system, that is, the fragments of Bglll and BamHI at both ends of the fragment could not be cleaved again. The reaction was ligated into the BamHI site of alkaline phosphatase-treated pAO-EGF-E4orf4, and transformed into DH5a receptor bacteria. Three-copy EGF-E4orf4 expression unit containing the same direction was obtained after recombinant DNA digestion and identification. Recombinant pAO-3EGF-E4orf4. The plasmid construction map is shown in Figure 7. The plasmid restriction map is shown in Figure 8.

After pG0815 digestion with Bglll and BamHI, three fragments of 4028 bp, 2405 bp and 1279 bp were generated. The pAO-EGF-E4orf4 was digested with Bglll and BamHI to produce three fragments of 4028 bp, 2405 bp and 2079 bp. The last fragment was inserted into the 1279 fragment due to the 800 bp fragment. After digestion with Bglll and BamHI, pAO-3EGF-E4orf4 will produce triploids from 6237 bp, 4028 bp, 2405 bp in large to small, and the 2237 bp fragment in 6237 bp. In Figure 8: Lane 1: DNA/Hindlll, the fragments from big to small are: 23130 bp, 9414 bp, 6557 bp. 4361 bp, 2322 bp, 202 lbp, 564 bp. Lane 2: pA0815/BglII+BamHI, the fragments from large to small are: 4028 bp, 2405 bp, 1279 bp. Lane 3: pAO-EGF-E4orf4/ Bglll+BamHI, the fragments from large to small are: 4028 bp, 2405 bp, 2079 bp. Lane 4: pAO-3EGF-E4orf4/ Bglll+BamHI, the fragments from large to small are: 6237 bp, 4028 bp, 2405 bp. Example 6: Preparation, transformation and screening of host bacteria

 A methylotrophic yeast (i.e., Pichiapastoris, Pichia pastoris) was used as an expression host. Methylotrophic yeast has many advantages: (1) Contains the unique AOX1 (alcohol oxidase) promoter, which can be tightly regulated by methanol. (2) There is a complete fermentation method, which can be continuously cultured at a high density, and the protein expression yield is high. (3) It is highly viable, easy to handle, and can be grown in inexpensive non-selective media. To this end, the multicopy EGF-E4orf4 expression vector pAO-3EGF-E4orf4 obtained in Example 5, which was obtained by the present invention, was transformed, screened and expressed as follows.

 The materials used in Examples 6 to 9 are:

 10X YNB: 134 g of yeast base containing ammonium sulfate but no amino acid was dissolved in 1000 ml of water, sterile filtered and used.

 500X biotin (0.02% biotin): 20mg VitH is dissolved in 100ml water, filtered after sterile filtration and stored at 4°C.

 10X D (20% glucose): 200 g of glucose is dissolved in 1000 ml of water, sterile filtered, and stored at 4 ° C until use.

 10X M (5% methanol): 5ml of methanol dissolved in 95ml of water, sterile filtered, stored at 4 ° C for use.

10X GY (10% glycerol): 100ml glycerol is dissolved in 900ml water, autoclaved, and stored at room temperature for use. 1M phosphate buffer, pH 6.0: mixed 132ml 1M K ₂ HP04, 868ml 1M

 BMG and BMMY: lOOmM phosphate buffer pH 6.0, 1.34% YNB, 1% glycerol or 0.5% methanol, 0.00004% biotin, BMMY also contains 1% yeast extract, 2% peptone.

 YPD medium: 1% yeast extract, 2% peptone, 2% glucose.

 MD and MM: 1.34% YNB, 0.00004% biotin, 2% glucose or 0.5% methanol.

 The preparation and linearization of the expression vector 3EGF-E4orf4:

Preparation of medium amount of plasmid DNA: 50 ml of LB medium containing 100 μ _§ ampicillin/ml was inoculated with the strain containing the expression vector, and cultured at 37 ° C overnight, and the cells were collected by centrifugation, and then the plasmid purification reagent of QIAGEN was used. The plasmid was extracted and purified, and 10 g of pAO-3EGF-E4orf4 was completely digested with Bglll, extracted with phenol-chloroform-isoamyl alcohol, ethanol precipitated, and lyophilized for use. Example 7: pAO-3EGF-E4orf4 transformed host strain

Pichia pastoris GS115 (his4-) was purchased from Invitrogen, inoculated on YPD plates, and cultured at 30 ° C for 2 days to isolate monoclonals. The monoclonal was inoculated in 5 ml of YPD liquid medium, and cultured at 30 ° C until the OD was about 1.3-1.5. The cells were collected by centrifugation, rinsed with sterile cooling water, centrifuged, and suspended in 1 ml of ice-cold 1 M sorbitol solution. 80 μΐ of the host strain was mixed with 5 μl of 10 μ _§ linearized pAO-3EGF-E4orf4 prepared in Example 6 in an electric punching cup, and precooled at 0 ° C for 5 minutes. The Bio-Rad Gene Punch (Gene-phiserll) program was set up to generate 7500 V/cm, 10 ms electrical pulses to complete the transformation. Immediately add 1 ml of ice-cold 1 M sorbitol in an electric punching cup, take a 200 μl-packed MD culture plate, and incubate at 30 ° C until the recombinant appeared. Example 8: Screening of Mut ⁺ and Mut ^s transformants

The methylotrophic yeast contains two AOX genes, AOX1 and AOX2, respectively. The expression vector is linearized with a different enzyme (e.g., BglII, Notl or Sail, Stul, etc.) and integrated into the position of the AOX1 or HIS4 gene of the yeast genome. When the yeast AOX1 gene is replaced and lost, a Mut ^s phenotype (methanol utilization slow) is generated, which will initiate the synthesis of AOX using the weak AOX2 gene and grow slowly in methanol-containing medium. The wild-type yeast grows normally in methanol-containing medium and is called the Mut+ phenotype.

Prepare MD-Sepharose plate and MM-Sepharose plate. In the case of using GS115- Albumin His^uf) and GS 115-Gal (His+Mut ⁺ ) as controls, the transformants obtained in Example 7 were sequentially symmetrically inoculated from MM plate to MD plate with a sterile toothpick. The growth of the transformants was observed by culturing on both plates at 30 °C. The Mut ^s phenotype grew slowly in methanol-containing medium and the Mut+ phenotype grew normally in methanol-containing medium, see Figure 9. The transformant of His ⁺ Mut ^{+ was} picked as a genetic engineering strain, named GS115-3EGF-E4orf4, and the expression of EGF-E4orf4 was observed. Example 9: Expression of EGF-E4orf4

The genetically engineered strain GS115-3EGF-E4orf4 obtained in Example 8 was cultured in a shake flask. Inoculate the cells in a single or 80 °C culture in 25nil BMG medium, culture at 30 °C for 18-24 hours, and OD _{6 (K)} two 10~12. Collect the cells by centrifugation and transfer to 250ml BMMY medium. The culture was cultured at 30 ° C, and methanol was added at a concentration of 1% every 24 hours to induce expression. The optimal induction time was determined to be 96 to 120 h. The culture supernatant was collected by centrifugation, and the EGF-E4orf4 fusion protein was detected. Example 10: Speculation of recombinant EGF-E4orf4 fusion protein

The recombinant EGF-E4orf4 fusion protein obtained in the above Example 9 was analyzed by SDS-PAGE protein electrophoresis. 15% separation using the Bio-Rad protein electrophoresis unit Gum, collected culture supernatant, added protein electrophoresis sample buffer, denatured at 100 ° C for 5 min, applied to electrophoresis, 100 V constant pressure, electrophoresis to bromophenol blue gel, stop electrophoresis, stain with silver nitrate.

 The results are shown in Fig. 10. According to the molecular weight standard of the protein, the molecular weight of the EGF-E4orf4 fusion protein is about 20 kD, which is in accordance with the molecular weight of the EGF-E4orf4 fusion protein of amino acid composition. In Figure 10: Lane 1 : GS115-3 EGF-E4orf4 expression supernatant, the arrow shows the EGF-E4orf4 fusion protein band. Lane 2: Protein low molecular weight standards, the fragments from large to small are: 43kD, 29kD, 18.4kD, 14.3kD, 6.2kD.

 Western Blot analysis was also performed. The recombinant EGF-E4orf4 fusion protein was first separated by SDS-PAGE and electrotransferred to a nitrocellulose membrane. First, rabbit anti-human EGF polyclonal antibody (purchased from Santa Cruz) or rabbit anti-E4orf4 protein antiserum was added. The inventors prepared according to the conventional method) were incubated with a nitrocellulose membrane at room temperature for 1 hour, and then washed with HRP-labeled goat anti-rabbit IgG antibody at room temperature for 1 hour, rinsed and detected by ECL chemiluminescence.

 The results are shown in Figure 11. It can be seen that the EGF-E4orf4 fusion protein is positive for human EGF antibody. In Figure 11: Lane 1 : Protein low molecular weight standards, the fragments from large to small are: 43kD, 29kD, 18.4kD, 14.3kD, 6.2kD. Lane 2-3: GS115-3 EGF-E4orf4 expression supernatant, primary antibody is rabbit anti-human EGF polyclonal antibody. Lane 4: GS115_3EGF_E4orf4 expression supernatant, the primary antibody is rabbit anti-E4orf4 antiserum, and the arrow shows the EGF-E4orf4 fusion protein band. Example 11. Isolation and purification of recombinant EGF-E4orf4 fusion protein

The yeast containing the recombinant EGF-E4orf4 fusion protein obtained in Example 9 was expressed and purified by cation exchange chromatography using a FPLC system of Pharmacia. Column: XK26, chromatography medium: SP Sepharose Fast Flow (strong cation), column volume 50 ml. The yeast expression supernatant was diluted with 6-7 volumes of buffer A (50 mM NaAC/HAC, pH 4.0) and filtered through a 0.45 μηι microporous membrane; using 5 cylinders After accumulating the buffer A, the medium was loaded, the flow rate was 5 ml/min, and after the sample was washed to the baseline with buffer A, the flow rate was adjusted to 3 ml/min, and buffer B with different salt concentrations (bl: 0.5 M). NaCL, 50 mM NaAC/HAC, pH 4.0; B2: 1.0 M NaCL, 50 mM NaAC/HAC, pH 4.0; B3: 1.5 M NaCL, 50 mM NaAC/HAC, pH 4.0) Stepwise elution, collection of eluted peaks, Identification by SDS-PAGE protein electrophoresis. Most of the hybrid proteins were eluted with 0.5 M and 1.0 M NaCl, 50 mM acetate buffer, pH 4.0, followed by 1.5 M NaCl, 50 mM acetate buffer, pH 4.0, which gave better purification. effect. The eluate containing the fusion protein identified by electrophoresis was collected, desalted by dialysis, concentrated by PEG-10000, sterilized by filtration through a 0.22 μm filter, stored at 20 ° C, and quantified by protein. See Figure 12.

 In Figure 12: Lane 1 : Fusion protein EGF-E4orf4 yeast expression supernatant. Lane 2: Protein low molecular weight standards, the fragments from large to small are: 43kD, 29kD, 18.4kD, 14.3kD. Lane 3: Eluate collected by cation exchange chromatography, indicated by the arrow as recombinant EGF-E4orf4 fusion protein. Example 12. Determination of the effect of recombinant EGF-E4orf4 fusion protein on cell viability by MTT colorimetric assay Mitochondrial dehydrogenase of live cells converts dye MTT (dimethylthiazole blue diphenyltetrazolium bromide) to insoluble The color of the purple substance (formazan) particles, which is expressed by the organic solvent DMSO (indicated by the OD value), reflects the metabolic level of living cells. Dead cells have no such enzyme activity. Therefore, within a certain number of cells, the amount of MTT crystal formation is proportional to the number of viable cells. In this case, the experiment was used to test the effect of the fusion protein on cell viability.

Specifically, the monolayer cultured cells were digested with 0.25% trypsin, and boiled into a single cell suspension with a medium containing 10% fetal bovine serum, and seeded in a 96-well culture plate at 5000 cells per well at a volume of 200 per well. μ 1, 37 ° C, 5% C0 ₂ culture, 20-24 hours to be fine After the cells were completely adherent, the supernatant was discarded and replaced with medium containing 5% fetal bovine serum at 200 每, 37 ° C, 5% C0 ₂ per well. The fusion protein EGF-E4orf4 obtained in Example 11 was serially diluted with PBS from the concentration of lng-15 g/ml, and the EGF standard was used as a control, and diluted according to the concentration of the fusion protein EGF-E4orf4, etc., per well. Add the diluted continuous gradient fusion protein EGF-E4orf4 solution and the equivalent molecular weight EGF solution, set 3 parallel wells for each concentration, repeat 2 plates, incubate with cells, 37 °C, 5% C0 ₂ culture 2 - 3 days. Add MTT solution to each well

(5mg/ml) 20 μ ΐ, 37 ° C, 5% C0 ₂ continue to incubate for 4-5 hours, carefully aspirate the culture supernatant in the well, add 100 I DMSO per well, shake for 10 minutes to fully dissolve the crystals . The light absorption value of each well at a wavelength of 570 nm was measured on an enzyme-linked immunosorbent detector. The OD value is averaged and input into the SPSS analysis software. The result is represented by an automatically generated graph, and the abscissa indicates the concentration of the stimulant, which is the final concentration of the fusion protein E4orf4.

(ng/ml), the ordinate is the OD value.

As can be seen in Figure 13, BT325 cells (human brain glioma cells, constructed by Beijing Tiantan Hospital) were co-cultured with the fusion protein EGF-E4orf4 for 44 hours, from 2.5 g/ml to 12.5 μ _§ /ηι1 at higher concentrations. The cell viability continued to decrease. When the fusion protein reached 12.5μ _§ /ιιι1, the highest concentration of the fusion protein used in this experiment, the cell viability was minimized, and the OD _57Q was 0.183, which was far lower than the negative control group without _sputum . Cell viability, its OD ₅₇ . The activity was 0.276, which was completely different from the positive control cells in which EGF was added. At this time, the viability of the cells in the EGF group at the corresponding molecular concentration was maintained at a high level, and the two vitality curves were separated. This result demonstrates that the fusion protein EGF-E4orf4 can enter BT325 cells under the guidance of EGF, and when its concentration reaches a high level, it can produce significant cytotoxic effects on BT325 cells.

As can be seen in Figure 14, MDA-MB-231 cells (human breast cancer cells, ATCC Number: HTB-26) were co-cultured with the fusion protein EGF-E4orf4 for 68 hours, and the cell viability was minimized from the concentration of 500 ng/ml. Below the unstimulated control cell group, at subsequent irritant concentrations (2.5 g/ml-15 g/ml), cell viability is always The control cell group was lower or closer to the unstimulated control group, and the cell viability of the EGF control group was always higher than that of the unstimulated group, indicating that the fusion protein EGF-E4orf4 can inhibit the activity of MDA-MB-231 cells. Example 13. Flow cytometry for detection of apoptosis

During the process of apoptosis, due to the concentration of cytoplasm and chromatin, the nucleus cleaves to produce apoptotic bodies, which leads to changes in the light scattering properties of the cells. Compared to living cells or necrotic cells, apoptotic cells have a special light scattering type - low forward scatter and high side scatter. Flow cytometry is capable of measuring changes in cell light scattering and is therefore widely used for the observation, detection and analysis of apoptosis. According to the results of the MTT colorimetric experiment, the concentration of the fusion protein which is capable of substantially saturating the cell surface is used as the end point, and the concentration of the fusion protein which is capable of substantially saturating the cell surface is the starting point, in this range. Set a series of continuous fusion protein concentrations (10μ ₈ /3 Χ 10 ⁵ cells - 25μ _§ /3 Χ 10 ⁵ cells), co-culture with MDA-MB-231 cells, L15 medium containing 5% FBS, culture 72 Cellular changes were measured by flow cytometry after an hour. The results are shown in Figures 15-19. It can be seen that the fusion protein EGF-E4orf4 is capable of apoptotic MDA-MB-231 cells. Within a certain concentration range, the degree of apoptosis is positively correlated with the concentration of the fusion protein. As the fusion protein is from scratch, apoptosis is also absent; the fusion protein rises from 10μ _§ to 25μ cells from a small amount of apoptosis to obvious apoptosis of the cells until the cells enter the late stage of apoptosis. It can be concluded that the fusion protein EGF-E4orf4 can induce apoptosis in MDA-MB-231 cells, and MDA-MB-231 cells are relatively sensitive to the cytotoxicity of the fusion protein. Apoptosis is evident.

As shown in Fig. 15, MDA-MB-231 cells were cultured for 72 hours, and 92.1% of the cells grew well. As shown in Figure 16: MDA-MB-231 cells were co-cultured with the fusion protein EGF-E4orf4 (10 g/3x10 ⁵ cells), and a small number of cells had apoptosis, accounting for 15.4%.

As shown in Figure 17, MDA-MB-231 cells were co-cultured with the fusion protein (15 g/3x10 ⁵ cells), and 28.2% of the cells were detached from the cell cycle, and the cells developed obvious apoptosis. Moreover, the characteristics of early cell apoptosis can be seen from the figure, that is, a slope generated by apoptotic cells or a sub-peak before the Go / G! peak. This is because in the early and middle stages of apoptosis, nuclear DNA is broken and is degraded into DNA fragments of integer multiples of nucleosomes. The arrow shows a subdiploid peak.

18: MDA- MB- 231 cells and the fusion protein (20μ β _/ 3 Χ 10 ⁵ cells were co-cultured :), showing a 19.1% of apoptosis.

As shown in Figure 19: When the concentration of the fusion protein reached 25 μ _§ /3 Χ 10 ⁵ cells, 27.0% of MDA-MB-231 cells were apoptotic. At this time, apoptosis has entered an advanced stage, and nuclear DNA is degraded into nucleosome-sized DNA fragments, resulting in a peak at a very low caloric absorption value, as indicated by the arrow.

Claims

Rights request  1. A recombinant human epidermal growth factor (EGF)-adenovirus E4orf4 fusion protein, which is human epidermal growth factor, a linker, and an adenovirus E4orf4 protein from the N-terminus to the C-terminus.  2. The fusion protein according to claim 1, wherein the fusion protein has an amino acid sequence as shown in SEQ ID NO: 2, which is composed of a 51 amino acid residue human epidermal growth factor, a 11 amino acid residue linker, and Adenovirus A5 consists of the E4orf4 protein.  3. A fusion gene having a nucleotide sequence encoding the fusion protein of claim 1 or 2.  4. The fusion gene of claim 3 having the sequence of SEQ ID NO: 1.  5. An expression vector comprising the fusion gene of claim 3 or 4 containing at least one copy.  6. The expression vector according to claim 5 which is the plasmid pAO-EGF-E4orf4 shown in Fig. 5.  The expression vector according to claim 5, which is the plasmid pAO-3EGF-E4orf4 shown in Fig. 7.  8. A host cell comprising the fusion gene of claim 3 or 4 or the expression vector of any of claims 5-7.  9. The host cell of claim 8 which is a methylotrophic yeast cell.  10. The host cell according to claim 9, which is Pichia pastoris GS115-3 EGF-E4orf4, and the accession number is CGMCC 0771.  A method of producing the fusion protein of claim 1 or 2, which comprises culturing the host cell of any one of claims 8 to 10 under conditions suitable for expression, and isolating and purifying the expressed fusion protein. 12. A pharmaceutical composition comprising the fusion protein of claim 1 or 2 and a pharmaceutically acceptable carrier. 13. Use of the fusion protein of claim 1 or 2 in the manufacture of a medicament for the treatment of a malignant tumor.  14. The use according to claim 13, wherein said malignant tumor is a tumor in which the p53 gene is absent.