KR20020083776A - Human protooncogene and protein encoded therein - Google Patents

Abstract

PURPOSE: Provided are a human protooncogene which can be useful for diagnosis of various cancers and anti-sense genetic therapeutics and a protein encoded thereby. CONSTITUTION: The human protooncogene HCC-6 derived from human cervical cancer has the nucleotide sequence of SEQ ID NO: 1. Alternatively, the human protooncogene HCC-6 may have the nucleotide sequence of from base 174 to base 317 of SEQ ID NO: 1. A protein encoded by the human protooncogene HCC-6 of SEQ ID NO: 1 has the amino acid sequence of SEQ ID NO: 2. A vector pCEV-LAC contains the human protooncogene HCC-6 of SEQ ID NO: 1. A transformant E. coli DH5α/HCC-6(KCTC 0989BP) is produced by transforming with the vector pCEV-LAC. A kit for diagnosis of cancer containing the human protooncogene HCC-6 of SEQ ID NO: 1. A kit for diagnosis of cancer contains the protein of SEQ ID NO: 2. An anti-sense gene contains the nucleotide sequence complementary to the total or partial mRNA sequence transcribed from the human protooncogene HCC-6 of SEQ ID NO: 1 and binds with the mRNA to inhibit expression of the human protooncogene HCC-6.

Description

HUMAN PROTOONCOGENE AND PROTEIN ENCODED THEREIN}

The present invention relates to a novel proto-oncogene and a protein encoded by it, and more particularly, to a novel primary cancer derived from human cervical cancer, which shows no homology with existing reported primary cancer genes. Gene HCC-6 and the protein encoded thereby.

Higher animals, including humans, have about 100,000 genes, but only about 15% of them are expressed in each individual. Thus, which gene is selected and expressed determines all phenomena of life: development, differentiation, homeostasis, response to stimuli, cell division cycle regulation, aging and apoptosis (program cell death). (Liang, P. and AB Pardee, Science, 257: 967-971, 1992).

Pathological phenomena, such as tumorigenesis, are caused by genetic mutations that eventually lead to changes in gene expression. Thus, comparison of gene expressions between different cells is a fundamental and effective approach to understanding various biological phenomena.

For example, the mRNA differential display method proposed by Liang, P. and AB Pardee, Science, 257: 967-971, 1992 currently uses tumor suppressor genes or cell division cycles. It is effectively used to search for genes related to the cycle and transcriptional regulatory genes related to apoptosis, and is also used for various correlations of various genes occurring in one cell.

Taken together, the results of several studies on tumor development suggest that various genetic changes, such as loss of chromosomal heterozygosity, activation of primary cancer genes, and inactivation of other tumor suppressor genes, including the p53 gene, may be associated with tumor tissue. Has been reported to cause tumors in humans (Bishop, JM, Cell, 64: 235-248, 1991; Hunter, T., Cell, 64: 249-270, 1991). In addition, 10 to 30% of cancers are reported to be caused by activation of the primary cancer gene by amplification of the primary cancer gene.

The activation of primary cancer genes plays an important role in the etiology of many cancers, and it is very urgent to identify them for the prevention and treatment of cancers.

Accordingly, the present inventors have approached the developmental mechanism of cervical cancer at the level of primary cancer, and revealed that the primary cancer gene named human cervical cancer gene 6 (HCC-6) is specifically increased only in cancer cells. The present invention has been completed by suggesting that it can be effectively used for the diagnosis, prevention and treatment of various cancers, such as lymphoma, colorectal cancer, and skin cancer.

It is an object of the present invention to provide novel primary cancer genes and proteins encoded thereby that can be effectively used for the diagnosis, prevention and treatment of various cancers.

Figure 1 shows the results of differential development reverse transcription polymerase chain reaction (DDRT-PCR) confirmed the expression of CA245 in cervical normal tissue, cervical tumor tissue, metastatic lymph node tumor tissue and CUMC-6 cancer cells,

Figure 2a shows the Northern blot analysis results confirming the expression of the HCC-6 primary cancer gene of the present invention in uterine cancer tissues,

Figure 2b shows the results obtained by hybridizing the same samples as in Figure 2a with β-actin,

Figure 3a shows the Northern blot analysis results confirming the expression of the HCC-6 primary cancer gene of the present invention in normal human 12-row multiple tissues,

Figure 3b shows the results obtained by hybridizing the same samples as in Figure 3a with β-actin,

Figure 4a shows the results of Northern blot analysis confirming the expression of the HCC-6 primary cancer gene of the present invention in human cancer cell lines,

Figure 4b shows the result obtained by hybridizing the same samples as in Figure 4a with β-actin,

FIG. 5 shows the results of electrophoresis of SDS-PAGE (sodium dodecylsulfate acrylamide gel electrophorasis) gels with the size of proteins expressed before and after IPTG induction after HCC-6 primary cancer gene of the present invention.

In order to achieve the above object, the present invention provides a novel primary cancer gene derived from human cervical cancer and fragments thereof.

In addition, the present invention provides an expression vector comprising the primary cancer gene or fragment thereof and a microorganism transformed with the expression vector.

In addition, the present invention provides a protein encoded by the primary cancer gene and fragments thereof.

The present invention also provides a kit for diagnosing cancer comprising the original cancer gene or fragment thereof or comprising the protein or fragment thereof.

Finally, the present invention provides an antisense gene having a base sequence complementary to mRNA derived from the primary cancer gene or fragment thereof and a method for treating or preventing cancer using the same.

Hereinafter, the present invention will be described in detail.

The present invention provides novel primary cancer genes derived from human cervical cancer and fragments thereof.

The novel primary cancer gene (protooncogene) was named as human cervical cancer gene 6 (Human Cervical Cancer proto-oncogene 5, HCC-6, hereinafter abbreviated as 'HCC-6 primary cancer gene') and is described by SEQ ID NO: 1. Has a total sequence of 913 bp in length.

In the nucleotide sequence of SEQ ID NO: 1, the open reading frame corresponding to nucleotide numbers 174 to 317 (315-317: end codon) is the entire protein coding region, and the amino acid sequence derived therefrom is SEQ ID NO: 2 It consists of 47 amino acids described (hereinafter abbreviated as 'HCC-6 protein').

However, due to the degeneracy of the codons or in view of the preferred codons in the organism to express the primary cancer gene, the HCC-6 primary cancer gene of the present invention does not change the amino acid sequence of the carcinogenic protein expressed from the coding region. Various modifications may be made to the coding region within a range that does not occur, and various modifications or modifications may be made within the range that does not affect the expression of the gene even in portions other than the coding region, and such modified genes are also included in the scope of the present invention. . Accordingly, the present invention encompasses polynucleotides having fragment sequences that are substantially identical to those of the HCC-6 primary cancer gene of SEQ ID NO: 1. By substantially identical polynucleotides are meant those having sequence homology of at least 80%, preferably at least 90% and most preferably at least 95%.

The HCC-6 protein expressed from the HCC-6 primary cancer gene of the present invention is composed of 47 amino acids, has the same sequence as SEQ ID NO: 2, and is about 5 kDa in size. However, even in the amino acid sequence of the HCC-6 protein, substitution, addition or deletion of amino acids may be made within a range that does not affect the function of the protein, and only a part of the protein may be used according to the purpose, and thus the modified amino acid Sequences are also within the scope of the present invention. Accordingly, the present invention encompasses polypeptides and fragments thereof having substantially the same amino acid sequence as the HCC-6 carcinogenic protein, wherein substantially identical polypeptides are at least 80%, preferably at least 90%, most preferably at least 95% By those having sequence homology.

The HCC-6 primary cancer gene and protein of the present invention may be isolated from human cancer tissue or synthesized according to known DNA or peptide synthesis methods. In addition, the gene thus prepared may be inserted into a microorganism expression vector known in the art to prepare an expression vector, and then introduced into a suitable host cell, for example, E. coli or yeast cell. Using the transformed host as described above, the DNA of the gene of the present invention can be replicated in large quantities or a large amount of protein can be produced.

In the present invention, as a preferred embodiment, the vector containing the HCC-6 primary cancer gene is transfected into E. coli DH5α, and the E. coli DH5α / HCC-6 / pCEV-LAC obtained in this way is dated April 10, 2001. It was deposited in the Korea Institute for Engineering Culture (KCTC) (Accession Number: KCTC 0989BP).

When constructing a vector, appropriate expression control sequences such as promoters, terminators, self-replicating sequences, and secretion signals may be appropriately selected depending on the type of host cell intended to produce the HCC-6 primary cancer gene or protein. You can choose and combine.

The HCC-6 primary cancer gene of the present invention is hardly expressed in normal cervical tissues by Northern blot analysis, whereas the overexpression of cervical cancer tissues and cervical cancer cell lines is confirmed, thus it is a potent inducing uterine cancer. It is considered to be a carcinogenic gene. In addition to epithelial tissue such as cervical cancer, the HCC-6 primary cancer gene of the present invention is highly expressed in many other cancer tumors such as leukemia, uterine cancer, lymphoma, colon cancer, and skin cancer (see FIGS. 2-4). Therefore, the HCC-6 primary cancer gene of the present invention is considered to be a carcinogenic gene common to the development of various cancers, and can be effectively used for diagnosis of various cancers, production of transformed animals, and anti-sense gene therapy. Can be.

The method for diagnosing cancer using the HCC-6 primary cancer gene, for example, using all or a portion of the primary cancer gene as a probe (hybridization) after hybridization with the nucleic acid separated from the body fluids of the subject in the art Detecting them by a variety of known methods to determine whether the subject has the HCC-6 primary cancer gene of the present invention. At this time, if the probe is labeled with radioisotopes or enzymes, the presence of genes can be easily confirmed. Accordingly, the present invention also provides a kit for diagnosing cancer comprising all or part of the HCC-6 primary cancer gene.

The transgenic animal can be produced by introducing the HCC-6 primary cancer gene of the present invention into a mammal, for example, a rodent animal such as a rat, and the gene is preferably introduced at the fertilized egg stage before at least 8 cell stages. . The transgenic animals thus prepared may be usefully used for the search for anticancer substances such as carcinogenic substances or antioxidants.

The present invention also provides anti-sense genes that are effective in gene therapy and have base sequences complementary to mRNA derived from the HCC-6 primary cancer gene or fragment thereof.

In the present invention, the "anti-sense gene" is a polynucleotide having a sequence complementary to some or all of the mRNA transcribed from the primary cancer gene of SEQ ID NO: 1 or a part thereof, and binds to the protein binding site of the mRNA Thus, by introducing a DNA sequence having a sequence capable of destroying an open reading frame (ORF) of a primary cancer gene into a patient's body, cancer caused by the expression of the primary cancer gene can be prevented or treated.

The present invention also encompasses methods within the scope of treating or preventing cancer by administering to a patient a therapeutically effective amount of the HCC-6 antisense gene of the present invention.

In the HCC-6 antisense gene therapy of the present invention, the antisense gene of the present invention is administered to a patient in a conventional manner to prevent the expression of the primary cancer gene. For example, antisense HCC-6 is mixed with a poly-L-lysine derivative by electrostatic attraction according to the method of JS Kim et al., J. Controlled Release , 53: 175-182, 1998, The mixture may be administered intravenously to the patient.

The scope of the present invention also includes an anticancer pharmaceutical composition comprising the HCC-6 antisense gene of the present invention as an active ingredient together with a pharmaceutically acceptable carrier, excipient or other additives as the case may be. The anticancer pharmaceutical composition of the present invention is preferably formulated as an injection.

The amount of HCC-6 antisense gene that is actually administered is determined by considering various related factors such as the condition being treated, the route of administration, the age and weight of the patient, and the severity of the condition.

Proteins derived from the HCC-6 primary cancer gene of the present invention can be usefully used for producing antibodies as a diagnostic tool. The antibody of the present invention is a monoclonal antibody (monoclonal anticody) according to a conventional method known in the art using a protein having a amino acid sequence of SEQ ID NO: 2 expressed from the HCC-6 primary cancer gene of the present invention or a fragment thereof Or as a polyclonal antibody, using these antibodies known enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), sandwich assay (sandwich) known in the art. Cancer can be diagnosed by confirming whether the protein is expressed in a bodily fluid sample of a subject by a method such as assay), Western blot or immunoblot analysis on a polyacrylamide gel.

In addition, cancer cell lines capable of continuously proliferating can be established using the HCC-6 primary cancer gene of the present invention, and these cell lines are formed in nude mice or the like, for example, using fibroblasts transduced with the primary cancer gene. It can be prepared from tumor tissue. Such cancer cell lines can be usefully used for the search for anticancer agents.

Hereinafter, the present invention will be described in detail by way of examples.

The following examples are merely illustrative of the present invention, but the content of the present invention is not limited by the examples.

Example 1 Tumor Cell Culture and Isolation of Total RNA

<1-1> Tumor Cell Culture

For differential development of mRNA, normal cervical tissue from hysterectomy patients and primary cervical and metastatic lymph node tumors during surgery from uterine cancer patients who have not previously received chemotherapy and radiation therapy It was. The human cervical cancer cell line used for differentiation was CUMC-6 (Kim, JW et al., Gynecol. Oncol. , 62: 230-240, 1996).

Cells obtained from the tissues described above and from the CUMC-6 cell line were obtained from Waymouth's MB containing 2 mM glutamine, 100 IU / ml penicillin, 100 μg / ml streptomycin and 10% fetal bovine serum (Gibco, USA). Proliferation in 752/1 culture (Gibco). The cultured cells used in the experiments were cells during exponential growth using cells that showed 95% or more viability when stained with trypan blue dye (Freshney, "Culture of Animal Cells: A Manual of Basic Technique"). 2nd Ed., AR Liss, New York, 1987).

<1-2> Isolation of RNA and Differentiation of mRNA

Normal cervical tissue, primary cervical tumor tissue, metastatic lymph node tumor tissue and CUMC-6 cells obtained in Example <1-1> using RNeasy Total RNA Kit (Qiagen Inc., Germany), a commercially available system Total RNA was extracted from. DNA contaminants were removed from RNA using a Message clean kit (GenHunter Corp., Brookline, Mass.).

Example 2 Differential Display Reverse Transcription Polymerase Chain Reaction (differential display reverse transcription, DDRT-PCR)

Differentiation reverse transcription was performed with a slight modification of the reverse transcription-polymerase chain reaction (RT-PCR) described by Liang and Paddy (Liang, P. and AB Pardee, Science, 257: 967-971, 1992).

First, 0.2 μg of total RNA obtained in Example <1-1> was used as a template, and three primers of H-T11G, H-T11C, or H-T11A (RNAimage kit, Genhunter, Cor. , MA, USA), reverse transcription was performed using the H-T11A primer set forth in SEQ ID NO: 3.

Then, the same fixed primer of SEQ ID NO: 3 and random 5 'primer of SEQ ID NO: 4 (RNAimage primer sets 1-4, H-AP 1-32s) in the presence of 0.5 mM [α- ³⁵ S] dATP (1200 Ci / mmole) PCR was performed using H-AP24 primer). The PCR reaction conditions were 40 seconds at 95 ° C., 40 minutes at 40 ° C. for 2 minutes, and 40 seconds at 72 ° C. for 5 minutes at 72 ° C. for final extension.

The PCR fragments amplified therefrom were subjected to electrophoresis on 6% polyacrylamide sequencing gel, and radiographs were used to identify the positions of bands showing different degrees of expression.

From the dried sequencing gel, 147 bp band, CA245 cDNA (SEQ ID NO: 705-851 bp) fragments were cut out and heated for 15 minutes to elute CA245 cDNA, followed by [α- ³⁵ S] dATP (1200 Ci / mmole) and 20 μM. Amplification was performed under the same primers and PCR conditions as above except that dNTP was not used.

Example 3 Cloning

The reamplified CA245 PCR product obtained above was inserted into the pGEM-T Easy vector using the TA cloning system (Promega, USA) according to the manufacturer's instructions.

<3-1> Connection reaction

2 μl of the re-amplified CA245 PCR product obtained in Example 2, 1 μl of pGEM-T Easy vector (50 ng), 1 μl of T4 DNA ligase 10 × buffer and T4 DNA ligase (3 weiss units / μl; T4 1 μl of DNA ligase, Promega) was added to a 0.5 ml test tube, and distilled water was added to obtain a final volume of 10 μl. The ligation reaction mixture was incubated overnight at 14 ° C.

<3-2> TA cloning transformation

TA cloning transformation was performed according to the following steps.

First, E. coli JM109 (Promega, WI, USA) was subjected to absorbance at 600 nm in 10 ml of LB medium (10 g of bacto-tryptone, 5 g of bacto-yeast extract, 5 g of NaCl) to about 0.3-0.6. Incubate until. The culture mixture was left on ice for about 10 minutes and then centrifuged at 4,000 rpm for 10 minutes at 4 ° C to discard the supernatant and recover only the cells. The recovered cell pellets were exposed to 10 ml of ice-cold 0.1 M CaCl ₂ for about 30 minutes to 1 hour to prepare competent cells. The resultant was centrifuged again at 4,000 rpm for 10 minutes to discard the supernatant, and the cells were collected and suspended in 2 ml of ice-cold 0.1 M CaCl ₂ .

200 μl of competent cell suspension was transferred to a new microfuge tube, and 2 μl of the ligation reaction product prepared in <3-1> was added thereto. The mixture was incubated in a water bath at 42 ° C. for 90 seconds and then quenched at 0 ° C. Here, SOC medium (2.0 g of bacto-tryptone, 0.5 g of bacto-yeast extract, 1 ml of 1 M NaCl, 0.25 ml of 1 M KCl, 97 ml of TDW, 1 ml of 2M Mg ^2+, 1 ml of 2 M glucose) 800 Μl was added and the mixture was incubated for 45 minutes in a rotary shake incubator at 37 ° C. at 220 rpm.

25 μl of X-gal (stored in 40 mg / ml dimethylformamide) was spread on a glass rod in an ampicillin-added LB plate, previously placed in a 37 ° C. incubator, and then 25 μl of transformed cells were added thereto. After adding the plate again with a glass rod and incubated overnight at 37 ℃. Three to four white colonies formed after the incubation were selected and inoculated on the LB plates to which ampicillin was added to incubate. To prepare the plasmid, a colony of which insertion was confirmed, that is, transformed Escherichia coli JM109 / CA245, was selected and 10 ml of territorial broth (900 ml of TDW, 12 g of bacto-tryptone, 12 g of bacto-yeast extract 24). g, glycerol 4 ml, 0.17 M KH ₂ PO ₄ , 0.72 NK ₂ HPO ₄ 100 ml).

Example 4 Isolation of Recombinant Plasmid DNA

Wizard Plus Mini repseu program by using the DNA purification kit (Wizard ^TM Plus Minipreps DNA Purificatin Kit, Promega, USA) were isolated CA245 plasmid DNA from E. coli transformed according to the manufacturer's instructions.

Part of the isolated plasmid DNA was treated with ECoRI restriction enzyme, followed by electrophoresis on 2% gel to confirm that the CA245 subsequence was inserted into the plasmid.

Example 5 DNA Sequence Analysis

After amplifying the CA245 PCR product obtained in Example 2 according to a conventional method, the cloned and re-amplified CA245 PCR fragment was subjected to a Sequenase version 2.0 DNA Sequencing kit (United States Biochemical, Cleveland). , OH, USA) was used to sequence analysis according to the dideoxy chain termination (dideoxy chain termination).

The base sequence of the DNA corresponds to nucleotide numbers 705 to 851 of SEQ ID NO: 1, and the inventors named the DNA fragment "CA245".

Differential Expansion Reverse Transcription Polymerase Chain Reaction (DDRT-PCR) of the 147 bp cDNA fragment obtained above, ie CA245, using the 3 'H-T11A fixed primer of SEQ ID NO: 3 and the 5' random primer H-AP24 of SEQ ID NO: 4 And confirmed by electrophoresis. As a result, gene expression by developmental differentiation was confirmed in normal cervical tissue, metastatic lymph node tissue, and CUMC-6 cells (FIG. 1). As can be seen in Figure 1, 147 bp cDNA fragment CA245 was expressed in cervical cancer, metastatic lymph node tissue and CUMC-6 cancer cells, but not in normal tissues.

Example 6 Analysis of Total cDNA Sequences of HCC-6 Primary Cancer Genes

^The bacteriophage λgt11 human lung fetal fibroblast cDNA library (Miki, T. et al . , Gene, 83: 137-146, 1989) was screened using ³² P-labeled CA245 as a probe. From the human lung fetal fibroblast cDNA library, a full cDNA clone with 913 bp inserted into the pCEV-LAC vector was obtained and named HCC-6, and was registered in GenBank, USA on April 5, 2001 (GenBank NO). : AF368271; published May 1, 2002).

HCC-6 clones inserted into λpCEV vectors from phage were isolated in the form of ampicillin resistant pCEV-LAC phagemid vectors by Not I cleavage (Miki, T. et al . , Gene, 83: 137-146, 1989).

The pCEV-LAC vector containing the HCC-6 gene was linked with T4 DNA ligase to prepare HCC-6 plasmid DNA, and E. coli DH5α was transformed with the linked clone.

E. coli transformant DH5α / HCC-6 / pCEV-LAC obtained therefrom was deposited in the Korea Collection for Type Cultures (KCTC) on April 10, 2001 (Accession No .: KCTC 0989BP). ).

The entire sequence of HCC-6 consisting of 913 bp is shown in SEQ ID NO: 1.

In the nucleotide sequence of SEQ ID NO: 1, the full transcription reading frame of the HCC-6 primary cancer gene of the present invention corresponds to nucleotides 174 to 317, which encodes a protein consisting of 47 amino acids of SEQ ID NO: 2 It is predicted.

Example 7 Northern Blot Analysis of HCC-6 Gene in Various Cells

As in Example 1 above, total RNA was extracted from normal cervical tissue, cervical cancer tissue and cervical cancer cell lines CaSki (ATCC CRL 1550) and CUMC-6.

To determine the degree of HCC-6 gene expression, 20 μg of denatured total RNA extracted from each tissue and cell line was electrophoresed on a 1% formaldehyde agarose gel and then transferred to a nylon membrane (Boehringer-Mannheim, Germany). Nylon membranes were hybridized using ³² P-labeled random primed HCC-6 whole cDNA prepared as a probe using the Rediprime II random prime labeling system (Amersham, UK). Northern blot analysis was repeated twice and the results were quantified using densitometry and normalized to β-actin.

Figure 2a shows the results of Northern blot analysis confirming the expression of HCC-6 primary cancer genes in normal cervical tissue, cervical cancer tissue and cervical cancer cell lines (CaSki and CUMC-6). As shown in FIG. 2A, significant gene expression was observed in the cervical cancer tissues and the cervical cancer cell lines CaSki and CUMC-6 cell lines, but the expression level was not very low or observed in normal tissues. FIG. 2B hybridizes the same sample with β-actin probe to confirm the presence of mRNA. In FIG. 2, normal lanes represent normal cervical tissue, cancer lanes represent cervical cancer tissues, and CaSki lanes and CUMC-6 lanes represent uterine cancer cell lines, respectively.

Figure 3a shows the expression of HCC-6 primary cancer gene in normal human 12-row multiple tissue (Clontech), brain, heart, rhabdomyo, colon, thymus, spleen, kidney, liver, small intestine, placenta, lung and leukocyte tissues Northern blot analysis results. 3b hybridizes the same sample with β-actin probe to confirm the presence of mRNA. As shown in Figure 3a, HCC-6 mRNA (about 1.0 kb) is not expressed or very weakly expressed in various normal tissues.

Figure 4a shows the results of Northern blot analysis confirmed the expression of HCC-6 primary cancer genes in human cancer cell lines, HL-60, HeLa, K-562, MOLT-4, Raji, SW480, A549 and G361 (Clontech). 4b shows the presence of mRNA by hybridizing the same sample with β-actin probe. As shown in Figure 4a, HCC-6 is a promyeloid leukemia cell line HL-60, HeLa cervical cancer cell line, chronic myeloid leukemia cell line K-562, lymphocyte leukemia cell line MOLT-4, Burkitt lymphoma cell line High levels in Raji, colorectal cancer cell line SW480 and skin cancer cell line G361.

Example 8 Determination of the size of protein expressed after transfection of HCC-6 primary cancer gene into E. coli

The entire HCC-6 primary cancer gene of SEQ ID NO: 1 was inserted into multiple cloning sites of the pGEX-4T-3 vector (Amersham Pharmacia), and the resulting pGEX-4T-3 / HCC-6 vector was transformed into Escherichia coli BL21 (ATCC 47092). Infected. An expression protein, glutathione-S-transferase (GST), is inserted in front of the multiple cloning site of the pGEX-4T-3 vector. The transfected E. coli was shaken in LB medium, then diluted to 1/100 and incubated for another 3 hours. 1 mM of isopropyl β-D-thiogalactopyranose was added thereto to induce protein production.

E. coli cells in culture medium before and after IPTG induction were pulverized ultrasonically, followed by electrophoresis on a 12% SDS-PAGE gel. Figure 5 shows the results of SDS-PAGE expression of the protein expression of E. coli BL21 strains transfected with the pGEX-4T-3 / HCC-6 vector, a fusion protein band of about 31 kDa was clearly observed after IPTG induction. The 31 kDa fusion protein contains about 26 kDa GST protein and about 5 kDa HCC-6 protein inserted into the pGEX-4T-3 vector.

As described above, the HCC-6 primary cancer gene of the present invention is a novel primary cancer gene having no homology with existing reported oncogenic genes and various cancers including leukemia, uterine cancer, lymphoma, colon cancer, lung cancer, skin cancer, and the like. It can be effectively used for the diagnosis, the preparation of transgenic animals and the anti-sense gene therapy.

Claims (11)

A primary cancer gene or fragment thereof having the nucleotide sequence of SEQ ID NO: 1. The cancer gene or fragment thereof according to claim 1, which has a nucleotide sequence corresponding to base numbers 174 to 317 of SEQ ID NO: 1. A protein having a amino acid sequence of SEQ ID NO: 2 or a fragment thereof. A vector comprising the primary cancer gene of claim 1 or a fragment thereof. Microorganism transformed by the vector of claim 4. The microorganism according to claim 5, wherein the microorganism is E. coli DH5α / HCC-6 transformed with the pCEV-LAC vector containing the primary cancer gene of claim 1 (Accession No .: KCTC 0989BP). A method for producing a protein or fragment thereof having an amino acid sequence of SEQ ID NO: 2 using the microorganism of claim 5 or 6. A cancer diagnostic kit comprising the original cancer gene of claim 1 or 2 or a fragment thereof. A diagnostic kit for cancer comprising the protein of claim 3 or a fragment thereof. An anti-sense gene having a nucleotide sequence complementary to all or part of an mRNA sequence transcribed from the primary cancer gene or fragment thereof of claim 1 or 2, and binding to the mRNA to inhibit the expression of the primary cancer gene or fragment thereof. Anti-cancer pharmaceutical composition containing the anti-sense gene of claim 10 as an active ingredient.