KR100503564B1 - Human protooncogene hcc-9 and protein encoded therein - Google Patents

Abstract

The present invention relates to a novel far-oncogene and a protein encoded by the same, wherein the far-oncogene of the present invention has no homology with existing reported far-oncogenes and is a novel gene that exhibits metastasis-inducing ability as leukemia, It can be effectively used for the diagnosis of cancer including uterine cancer, lymphoma, colorectal cancer, lung cancer, skin cancer, etc., production of transgenic animals and anti-sense gene therapy.

Description

HUMAN PROTOONCOGENE HCC-9 AND PROTEIN ENCODED THEREIN}

The present invention relates to novel far-oncogenes and proteins encoded by them, which show no homology with previously reported far-oncogenes and exhibit cancer metastasis.

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, Liang, P. and AB Pardee, Science 257: 967-971, 1992) suggested that the differential display method of mRNA is currently used to search for tumor suppressor genes, cell cycle-related genes, and apoptosis-related transcriptional regulatory genes. It is effectively used, and is also used in a variety of ways to correlate various genes in a single cell.

Taken together, studies on tumor development suggest that several genetic changes, such as loss of chromosomal heterozygosity, activation of proto-oncogenes, and inactivation of other tumor suppressor genes, including the p53 gene, may be present in tumor tissues. It has been reported to accumulate and cause human tumors (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 activated by the amplification of the primary cancer gene, activation of the primary cancer gene plays an important role in the etiology of many cancers, it is required to reveal this.

Therefore, the inventors of the present invention have approached the developmental mechanism of cervical cancer at the level of the primary cancer, and found that the primary cancer gene named human cervical cancer gene 9 (HCC-9) is specifically increased only in cancer cells. The primary cancer gene can be effectively used for the diagnosis, prevention and treatment of various cancers such as leukemia, uterine cancer, lymphoma, colon cancer, lung cancer and skin cancer.

It is an object of the present invention to provide novel far-oncogenes and fragments thereof.

Another object of the present invention is to provide a recombinant vector comprising the above-described oncogene or a fragment thereof and a microorganism transformed thereby.

Still another object of the present invention is to provide a protein and fragment thereof encoded by the primary cancer gene.

Still another object of the present invention is to provide a kit for diagnosing cancer, comprising the original oncogene or a fragment thereof.

Still another object of the present invention is to provide a kit for diagnosing cancer comprising the protein or fragment thereof.

Still another object of the present invention is to provide an antisense gene having a base sequence complementary to mRNA derived from the primary cancer gene or fragment thereof.

Still another object of the present invention is to provide an anticancer and antimetastatic composition comprising the antisense gene.

In accordance with the above object, the present invention provides a far-oncogene or a fragment thereof having a nucleotide sequence of SEQ ID NO: 1 .

According to the above another object, the present invention provides a recombinant vector and the microorganism transformed by the above-described oncogene or a fragment thereof.

In accordance with another object, the present invention provides a protein or fragment thereof having an amino acid sequence of SEQ ID NO: 2 .

In accordance with another object, the present invention provides a kit for diagnosing cancer, including the primary cancer gene or a fragment thereof.

In accordance with another object, the present invention provides a kit for diagnosing cancer, comprising the original cancer protein or fragment thereof.

According to another object of the present invention, the present invention has a base sequence complementary to all or part of the mRNA sequence transcribed from the primary cancer gene or fragment thereof, and binds to the mRNA to inhibit the expression of the primary cancer gene or fragment. Provide sense genes.

In accordance with another object, the present invention provides an anticancer and anti-transition composition comprising the anti-sense gene as an active ingredient.

Hereinafter, the present invention will be described in detail.

Human cervical cancer gene 9 (Human Cervical Cancer proto-oncogene 9, HCC-9, hereinafter referred to as the "HCC-9 primary oncogene") which is the protocogene of the present invention is described by SEQ ID NO: 1 It has a full base sequence of 2,314 bp in length.

In the nucleotide sequence of SEQ ID NO: 1 , the open reading frame corresponding to the nucleotide number 150 to 623 region (621-623: end codon) is the entire protein coding region and the amino acid sequence derived therefrom is SEQ ID NO: It is shown in 2 and consists of 157 amino acids (hereinafter referred to as "HCC-9 protein").

However, due to the degeneracy of the codon or in consideration of the preferred codon in the organism to express the primary cancer gene, the primary cancer gene of the present invention does not change the amino acid sequence of the carcinogenic protein expressed from the coding region. In the coding region, various modifications may be made, and various modifications or modifications may be made within a range that does not affect the expression of genes in parts other than coding regions, and such modified genes are also included in the scope of the present invention. Accordingly, the present invention also encompasses polynucleotides and fragments of said genes having base sequences substantially identical to that of the original oncogene of SEQ ID NO: 1 . Substantially identical polynucleotides are DNAs encoding a protein translation product identical to SEQ ID NO: 2 , meaning those having sequence homology of at least 80%, preferably at least 90%, most preferably at least 95% with SEQ ID NO: 1 do.

The protein expressed from the proto-oncogene of the present invention consists of 157 amino acids and has an amino acid sequence set forth in SEQ ID NO: 2 and is about 17 kDa in size. However, even in the amino acid sequence of the 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 depending on the purpose. Such modified amino acid sequences are also within the scope of the present invention. Accordingly, the present invention also encompasses polypeptides and fragments thereof having substantially the same amino acid sequence as the carcinogenic protein, wherein substantially identical polypeptides are at least 80%, preferably at least 90% and most preferably at least 95% sequences It means things with same sex.

The primary oncogenes and proteins of the present invention may be isolated from human cancer tissues 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 an appropriate 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. For example, in the present invention, the gene of the present invention is inserted into the expression vector pCEV-LAC (Miki, T. et al., Gene 83: 137-146, 1989) and then transfected into E. coli DH5α. Escherichia coli DH5α / HCC-9 / pCEV-LAC was deposited on December 5, 2002 to the Korea Collection for Type Cultures (KCTC) as Accession No. KCTC 10395BP.

In the production of the vector, expression control sequences such as promoters and terminators, self-replicating sequences, secretion signals, etc. may be appropriately selected and combined according to the type of host cell to produce the primary cancer gene or protein.

Genes of the present invention are rarely identified in normal cervical tissues in Northern blot analysis, whereas overexpression of cervical cancer tissues and cervical cancer cell lines is confirmed, and thus, it is determined to be a potent oncogenic gene causing uterine cancer. do. In addition, the expression of cancer metastasis in lymph node tissues is increased, it turns out that the cancer metastasis-related gene causing cancer metastasis. In addition to epithelial tissues such as cervical cancer, the primary cancer genes of the present invention are highly expressed in many other cancer tumors such as leukemia, uterine cancer, lymphoma, colon cancer, lung cancer and skin cancer. Therefore, the primary oncogene of the present invention is considered to be a common carcinogen for the development of various cancers, and can be effectively used for diagnosis of various cancers, production of transformed animals and anti-sense gene therapy.

The method for diagnosing cancer using the primary cancer gene, for example, by using all or a part of the primary cancer gene as a probe, hybridizes with nucleic acid isolated from the body fluid of the subject, and then detects it by various methods known in the art. It includes a process of determining whether or not the primary cancer gene of the present invention. Labeling the probe with a radioisotope or enzyme can easily confirm the presence of the gene. Accordingly, the present invention also provides a kit for diagnosing cancer comprising all or part of the original cancer gene.

The transgenic animal can be produced by introducing the proto-oncogene 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 for gene therapy. As used herein, an "anti-sense gene" is a polynucleotide having a sequence complementary to some or all of the mRNA transcribed from the proto-oncogene or part thereof of SEQ ID NO: 1 , which binds to a protein binding site of the mRNA Thus, by introducing a DNA sequence having a sequence capable of destroying an open reading frame (ORF) of the primary cancer gene into the patient's body, cancer caused by the expression of the primary cancer gene can be prevented or treated.

The invention also encompasses a method for treating or preventing cancer or cancer metastasis by administering to a patient a therapeutically effective amount of an antisense gene of the invention within the scope.

In the 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 proto-oncogene. For example, antisense oligodeoxynucleotides (ODN) are mixed with poly-L-lysine derivatives by electrostatic attraction according to the method of JS Kim et al., J. Controlled Release 53: 175-182, 1998 The mixture is then administered intravenously to the patient.

The scope of the present invention also includes anticancer compositions comprising the antisense gene of the present invention as an active ingredient together with a pharmaceutically acceptable carrier, excipient or optionally other additives. It is preferable to formulate the pharmaceutical composition of the present invention into an injection.

The amount of antisense gene actually administered is determined in consideration of various related factors such as the condition to be treated, the route of administration, the age and weight of the patient and the severity of the condition.

Proteins derived from the primary oncogenes of the present invention can be usefully used for producing antibodies as diagnostic tools. Antibodies of the present invention can be prepared as monoclonal antibodies or polyclonal antibodies according to conventional methods known in the art, using proteins or fragments thereof having the amino acid sequence of SEQ ID NO: 2 expressed from the primary oncogenes of the present invention. Using these antibodies, enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), sandwich assay, western blot on polyacrylamide gels are known in the art. Alternatively, cancer can be diagnosed by checking whether the protein is expressed in the body fluid sample of the subject by an immunoblot or the like.

In addition, cancer cell lines capable of continuously proliferating can be established using the primary cancer gene of the present invention. For example, tumor cells formed in nude mice or the like using fibroblasts transduced with the primary cancer gene can be formed. It can be prepared from. 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.

However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited to the following examples.

Example 1 Culture of Tumor Cells and Isolation of Total RNA

(Step 1) culturing tumor cells

For differential development of mRNA, primary cervical tumor tissues and metastases during surgery from uterine myomas undergoing hysterectomy and from uterine cervical tissue and from uterine cancer patients previously untreated with chemotherapy and radiation Lymph node tumor tissue was taken. For differential development, CUMC -6 (Kim, JW et al. , Gynecol. Oncol. 62: 230-240, 1996) was used as a human cervical cancer cell line.

Cells obtained from the tissues obtained 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 of exponential proliferation which showed viability of at least 95% by trypan blue staining (Freshney, "Culture of Animal Cells: A Manual of Basic Technique" 2nd). Ed., AR Liss, New York, 1987).

(Step 2) RNA isolation and differential development of mRNA

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

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

Differential development reverse transcription was performed with a slight modification of the reverse transcription-polymerase chain reaction (RT-PCR) described by Liang and Paddy.

First, H-T11A fixed primer (RNAimage kit, Genhunter, Cor., MA,) having a nucleotide sequence described as SEQ ID NO: 3 as an oligo-dT primer immobilized on 0.2 μg of total RNA obtained in step 1 of Example 1 USA) was used to perform reverse transcription.

Then, in the presence of 0.5 mM [α- ³⁵ S] dATP (1200 Ci / mmole), the same fixed primers and random 5 ′ 11 primers (RNAimage primer sets 1-4) are described as SEQ ID NO: 4 in H-APs 1 to 32. PCR was performed using H-AP11 primer having a nucleotide sequence. 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 fragments amplified by PCR were dissolved in a 6% polyacrylamide sequencing gel, and radiographs were used to identify the positions of the bands showing different degrees of expression.

A band of at least 250 base pairs (bp) in size, MA11 cDNA (1711-1960 bp of SEQ ID NO: 1 ) was cut out from the dried gel. After eluting MA11 cDNA by heating for 15 minutes, PCR was performed under the same primers and the same conditions as above except that [α- ³⁵ S] labeled dATP (1200 Ci / mmole) and 20 μM dNTP were not used. Amplified.

Example 3 Cloning

The reamplified MA11 PCR product obtained above was inserted into the pGEM-T EASY vector using the TA cloning system (Promega, USA) following the manufacturer's method.

(Step 1) Linking reaction

2 μl of the re-amplified MA11 PCR product obtained in Example 2, 1 μl of the pGEM-T EASY vector (50 ng), 1 μl of T4 DNA ligase 10X buffer and T4 DNA ligase (3 weiss units / μl; T4 DNA ligase, 1 μl of Promega) was placed in a 0.5 ml test tube, and distilled water was added to give a final volume of 10 μl. The ligation reaction mixture was incubated overnight at 14 ° C.

(Step 2) TA Cloning Transformation

Escherichia coli JM109 (Promega, WI, USA) was subjected to an optical density value of about 0.3 to 0.6 at 600 nm in 10 ml of LB broth (10 g of bacto-tryptone, 5 g of bacto-yeast extract, 5 g of NaCl). Incubate until. The culture mixture was left on ice for about 10 minutes and then centrifuged at 4000 rpm for 10 minutes at 4 ° C to discard the supernatant and collect the cells. The collected 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 4000 rpm for 10 minutes at 4 ° C. 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 step 1 was added thereto. The mixture was incubated for 90 seconds in a 42 ° C. water bath and then quenched at 0 ° C. Here, SOC medium (2.0 g of bacto-trypton, 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 2 M 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. Diffused into a glass rod again and incubated overnight at 37 ℃. Three to four white colonies formed after incubation were selected and each selected clones were planted in LB plates to which ampicillin was added. To prepare the plasmid, a colony of which insertion was confirmed, that is, transformed Escherichia coli JM109 / MA11, was selected and 10 ml of territorial broth (900 ml of TDW, 12 g of bacto-tryptone, 12 g of bacto-yeast). 24 g of extract, 4 ml of glycerol, 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 MA11 plasmid DNA from E. coli transformed according to the manufacturer's instructions.

Part of the isolated plasmid DNA was treated with ECo RI enzyme, followed by electrophoresis on a 2% gel to confirm that the MA11 subsequence was inserted into the plasmid.

Example 5 DNA Sequence Analysis

After amplifying the MA11 PCR product obtained in Example 2 according to a conventional method, the cloned and re-amplified MA11 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 dideoxy chain termination (dideoxy chain termination).

The base sequence of the DNA corresponds to nucleotide numbers 1711 to 1960 of SEQ ID NO: 1 , and is referred to as "MA11" in the present invention.

The 250 bp cDNA fragment obtained above, i.e., MA11, using 5 'random primers H-AP11 and 3' H-T11A fixed primers, was differentially developed reverse transcription polymerase chain reaction (DDRT-PCR) and confirmed by electrophoresis. .

As shown in FIG . 1 , gene expression by DD in normal cervical tissue, metastatic lymph node tissue and CUMC-6 cells was found to be different. As can be seen in Figure 1 , 250 bp cDNA fragment MA11 was expressed in cervical cancer, metastatic lymph node tissue and CUMC-6 cancer cells, but not in normal tissues.

Example 6 Total cDNA Sequence Analysis of HCC-9 Proto-Gen Gene

^The bacteriophage λgt11 human lung fetal fibroblast cDNA library (Miki, T. et al. , Gene 83: 137-146, 1989) was screened using ³² P-labeled MA11 as a probe. A total HCC-9 cDNA clone with 2314 bp inserted into the pCEV-LAC vector was obtained from the human lung fetal fibroblast cDNA library, and was registered in GeneBank, USA, on December 26, 2001, with the registration number AY071927. (Expected date of release: January 1, 2003).

HCC-9 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-9 gene was linked with T4 DNA ligase to prepare HCC-9 plasmid DNA, and transformed into E. coli DH5α with the linked clone.

DH5α / HCC-9 / pCEV-LAC obtained above, according to the provisions of the Budapest Treaty on the International Approval of Deposit of Microorganisms in Patent Procedure, was dated Dec. 5, 2002 by the Korea Biotechnology Research Institute Gene Bank (Korea Collection for Type Cultures). , KCTC) was deposited as Accession No. KCTC 10395BP.

The entire sequence of HCC-9 consisting of 2314 bp is shown in SEQ ID NO: 1 .

In the nucleotide sequence of SEQ ID NO: 1 , the full open reading frame of the proto-oncogene of the present invention corresponds to nucleotides 150 to 623 and is predicted to encode a protein consisting of 157 amino acids of SEQ ID NO: 2 do.

Example 7 Northern Blot Analysis of HCC-9 Gene in Various Cells

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

To determine the extent of HCC-9 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). Blots were hybridized with ³² P-labeled random primed HCC-9 full cDNA probes prepared 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-9 primary cancer genes in normal cervical tissue, cervical cancer tissue, cervical cancer metastatic lymph node tissue and cervical cancer cell lines (CaSki and CUMC-6). As can be seen in Figure 2a , the HCC-9 primary cancer gene was significantly increased gene expression in cervical cancer tissues, cervical cancer metastatic lymph node tissue and cervical cancer cell lines CaSki and CUMC-6 cell lines, but the expression level in normal tissues Was very low or not observed. In FIG. 2 , normal rows represent normal cervical tissue, cancer rows represent cervical cancer tissues, metastasis rows represent cervical cancer metastatic lymph node tissues, and CaSki rows and CUMC-6 rows represent uterine cancer cell lines. Represent each. 2B shows the presence of mRNA by hybridizing the same sample with β-actin probe.

Figure 3a shows the expression of HCC-9 primary oncogenes in normal human 12-row multiple tissue (Clontech), brain, heart, rhabdomyo, colon, thymus, spleen, kidney, liver, small intestine, placenta, lung and peripheral blood leukocyte tissues. The confirmed Northern blot analysis is shown. 3B shows the presence of mRNA by hybridizing the same sample with β-actin probe. As can be seen in Figure 3a , HCC-9 mRNA (about 2.3 kb) is weakly expressed in several normal tissues, including muscle, kidney and heart tissues.

Figure 4a shows the Northern blot analysis results confirming the expression of HCC-9 primary oncogenes 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 can be seen in Figure 4a , HCC-9 is a promyeloid leukemia cell line HL-60, HeLa uterine cancer cell line, chronic myeloid leukemia cell line K-562, lymphocyte leukemia cell line MOLT-4, Burkitt It was expressed at high levels in the lymphoma cell lines Raji, colon cancer cell line SW480, lung cancer cell line A549 and skin cancer cell line G361. In addition, it was confirmed that mRNA of about 1.2 kb, which is smaller than this, was expressed in addition to 2.3 kb.

Example 8 Determination of the size of protein expressed after transfection of E. coli HCC-9

The entire HCC-9 primary oncogene of SEQ ID NO: 1 was inserted into the BamH I / Sal I restriction enzyme site in the multiple cloning site of the pET-32b (+) vector (Novagen, Germany) and the resulting pET-32b (+) / HCC-9 vector was transfected into E. coli BL21 (ATCC 47092). Expression proteins Trx.Tag, His.Tag, and S.Tag are inserted in front of the multicloning region of the pET-32b (+) vector. The transfected E. coli was shaken in LB broth, 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 sonicated and electrophoresed on 12% sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE). 5 shows the results of SDS-PAGE expression of the protein of the E. coli BL21 strain transfected with the pET-32b (+) / HCC-9 vector. A fusion protein band of about 38 kDa was clearly observed after IPTG induction. This 38 kDa fusion protein contains about 21 kDa Trx, Tag, His, Tag, and S. Tag proteins and about 17 kDa HCC-9 protein inserted into the pET-32b (+) / HCC-9 vector. It is.

As described above, the primary cancer gene of the present invention is a novel gene that does not show any homology with existing reported carcinogens, and is used for diagnosis and transformation of cancers including leukemia, uterine cancer, lymphoma, colon cancer, lung cancer, skin cancer, and the like. It can be effectively used for the preparation of animals and anti-sense gene therapy.

Figure 1 shows the results of differential reverse transcription polymerase chain reaction (DDRT-PCR) confirmed the expression of MA11 DNA fragments in normal cervical tissue, cervical tumor tissue, metastatic lymph node tumor tissue and CUMC-6 cancer cells,

Figure 2a shows the results of Northern blot analysis confirming the expression of HCC-9 primary oncogenes of the present invention in normal cervix, cervical cancer tissue, cervical metastatic lymph node tissue and cervical cancer cell lines,

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

Figure 3a shows the results of Northern blot analysis confirming the expression of the HCC-9 primary cancer gene of the present invention in normal human 12-row multiple tissues,

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

Figure 4a shows the Northern blot analysis results confirming the expression of the HCC-9 primary oncogene of the present invention in human cancer cell lines,

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

FIG. 5 shows the results of electrophoresis on sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) of the protein expressed before and after IPTG induction after transduction of HCC-9 primary oncogene of the present invention. FIG. .

<110> KIM, Jin Woo <120> HUMAN PROTOONCOGENE HCC-9 AND PROTEIN ENCODED THEREIN <130> FPD / 200212-0007 <160> 4 <170> KopatentIn 1.71 <210> 1 <211> 2314 <212> DNA <213> Human Cervical Cancer proto-oncogene 9 (HCC-9) <400> 1 ggccattacc aatcgcgaaa cctccgggtg tctggaggct gtggccgttt tgttttcttg 60 gctaaaatcg ggggagtgag gcgggccggc gcggcgcgac accgggctcc ggaaccactg 120 cacgacgggg ctggactgac ctgaaaaaaa tgtctggatt tctagagggc ttgagatgct 180 cagaatgcat tgactggggg gaaaagcgca atactattgc ttccattgct gctggtgtac 240 tattttttac aggctggtgg attatcatag atgcagctgt tatttatccc accatgaaag 300 atttcaacca ctcataccat gcctgtggtg ttatagcaac catagccttc ctaatgatta 360 atgcagtatc gaatggacaa gtccgaggtg atagttacag tgaaggttgt ctgggtcaaa 420 caggtgctcg catttggctt ttcgttggtt tcatgttggc ctttggatct ctgattgcat 480 ctatgtggat tctttttgga ggttatgttg ctaaagaaaa agacatagta taccctggaa 540 ttgctgtatt tttccagaat gccttcatct tttttggagg gctggttttt aagtttggcc 600 gcactgaaga cttatggcag tgaacacatc tgatttccca cagcacaaca gccctgcatg 660 ggtttgtttg tttttttact gctcactccc aaccttttgt aatgccattt tctaaactta 720 tttctgagtg tagtctcagc ttaaagttgt gtaatactaa aatcacgaga acacctaaac 780 aacaaccaaa aatctattgt ggtatgcact tgattaactt ataaaatgtt agaggaaact 840 ttcacatgaa taatttttgt caaattttat catggtataa tttgtaaaaa taaaaagaaa 900 ttacaaaaga aattatggat ttgtcaatgt aagtatttgt catatctgag gtccaaaacc 960 acaatgaaag tgctctgaag atttaatgtg tttattcaaa tgtggtctct tctgtgtcaa 1020 atgttaaatg aaatataaac attttttagt ttttaaaata ttccgtggtc aaaattcttc 1080 ctcactataa ttggtattta cttttaccaa aaattctgtg aacatgtaat gtaactggct 1140 tttgagggtc tcccaagggg tgagtggacg tgttggaaga gagaagcacc atggtccagc 1200 caccaggctc cctgtgtccc ttccatggga aggtcttccg ctgtgcctct cattccaagg 1260 gcaggaagat gtgactcagc catgacacgt ggttctggtg ggatgcacag tcactccaca 1320 tccaccactg aaggaaagga aaaaagggca gagacttgac actccagtct tagacagggg 1380 acaatttctt tgtagttgtt ctgataataa actgggttcc atgttgcatt tcatctgtgt 1440 actgaaccac tagcctaata gaccagcacg gtcaactaga atgacaaaca ttagctactg 1500 ggaactcttg ttgtctcttc ctcttcagaa ctcttgtccc cccaggctcc ccacctctgc 1560 aagatgggat acactcttca ggaccacagt ttgaaatgtc tttcataaaa tgtgctacaa 1620 ttagccttcg gaatatttgc tgggtgattt aaatgtgtgg gtctcatttc catgctagcc 1680 atggtcatct gacagtctct acactgtgaa tattgcctgg tgatcaagac tctcctcaaa 1740 gaaatgactt gctgtcatcc cacatgaact cctgatgttt tttctacaaa agtccataaa 1800 atgtgaaaac tggagaagat cttagaggtt gaagcccacc ttctcttttc acataggagg 1860 gaacagacca tggaaattta aacggcttcc tcacggtcac agaactagtt ttttaatcct 1920 caggcagtgc atccccccac cctacaactg agcacaacct ctttccccac agtgcaattc 1980 agaatatgct cagggaatgc cagccacctt gtaaaactgc tgggagaaaa gcatgattcc 2040 cacaaggact aagtatcagt gatttgtaat tttcctgttt tgtattatct gctttgctga 2100 tgtagacaag agttaactga gtagcatgct ttattaagca tgagaaagaa tcttaagaat 2160 tgtcaataaa attaacccaa aactttaata atgtgtctgt aaccaagaaa atattgatag 2220 catcatccta atgaaactaa acatttattt taaacttatt aaattgactc ttaaactaaa 2280 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaa 2314 <210> 2 <211> 157 <212> PRT <213> HCC-9 <400> 2 Met Ser Gly Phe Leu Glu Gly Leu Arg Cys Ser Glu Cys Ile Asp Trp 1 5 10 15 Gly Glu Lys Arg Asn Thr Ile Ala Ser Ile Ala Ala Gly Val Leu Phe 20 25 30 Phe Thr Gly Trp Trp Ile Ile Ile Asp Ala Ala Val Ile Tyr Pro Thr 35 40 45 Met Lys Asp Phe Asn His Ser Tyr His Ala Cys Gly Val Ile Ala Thr 50 55 60 Ile Ala Phe Leu Met Ile Asn Ala Val Ser Asn Gly Gln Val Arg Gly 65 70 75 80 Asp Ser Tyr Ser Glu Gly Cys Leu Gly Gln Thr Gly Ala Arg Ile Trp 85 90 95 Leu Phe Val Gly Phe Met Leu Ala Phe Gly Ser Leu Ile Ala Ser Met 100 105 110 Trp Ile Leu Phe Gly Gly Tyr Val Ala Lys Glu Lys Asp Ile Val Tyr 115 120 125 Pro Gly Ile Ala Val Phe Phe Gln Asn Ala Phe Ile Phe Phe Gly Gly 130 135 140 Leu Val Phe Lys Phe Gly Arg Thr Glu Asp Leu Trp Gln 145 150 155 <210> 3 <211> 16 <212> DNA <213> Artificial Sequence <220> <223> H-T11A <400> 3 aagctttttt ttttta 16 <210> 4 <211> 13 <212> DNA <213> Artificial Sequence <220> <223> H-AP11 <400> 4 aagcttcggg taa 13

Claims (12)

Human prooncoprotein having the amino acid sequence of SEQ ID NO: 2 . A human primary oncogene encoding a protein of SEQ ID NO: 2 . The method of claim 2, Human primary oncogene comprising the nucleotide sequence corresponding to base numbers 195 to 1916 of SEQ ID NO: 1 . The method of claim 2, Human primary oncogene, characterized by having the nucleotide sequence of SEQ ID NO: 1 . A vector comprising the proto-oncogene of any one of claims 2 to 4. A microorganism transformed with the vector of claim 5. The method of claim 6, Microorganism that is Escherichia coli DH5α / HCC-9 / pCEV-LAC (Accession No .: KCTC 10395BP). A method for preparing a protein having an amino acid sequence of SEQ ID NO: 2 using the microorganism of claim 6. A cancer diagnostic kit comprising the original oncogene of any one of claims 2 to 4. Cancer diagnostic kit comprising the protein of claim 1. delete delete