KR100503567B1 - Human protooncogene hlc-2 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 HLC-2 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.

Accordingly, the inventors of the present invention have approached the developmental mechanism of lung cancer at the level of the primary cancer gene, and found that the expression of the primary cancer gene named human lung cancer gene 2 (HLC-2) 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 Lung Cancer-related Gene 2 (HLC-2, hereinafter referred to as “HLC-2 Protocogene”), which is the protocogene of the present invention, is described by SEQ ID NO: 1 . It has a full sequence of 596 bp in length.

In the nucleotide sequence of SEQ ID NO: 1 , the open reading frame corresponding to nucleotide numbers 116 to 349 sites (347-349: 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 77 amino acids (hereinafter referred to as "HLC-2 proteomics").

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 DNA sequences encoding protein translation products identical to SEQ ID NO: 2 , having those having at least 80%, preferably at least 90%, most preferably at least 95% homology with SEQ ID NO: 1 it means.

The protein expressed from the oncogene HLC-2 of the present invention is composed of 77 amino acids, has a sequence such as SEQ ID NO: 2, and is about 9 kDa in size.

However, even in the amino acid sequence of the proteins, 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. Thus, the present invention also encompasses polypeptides and fragments thereof having substantially the same amino acid sequence as the carcinogenic proteins, wherein substantially identical polypeptides comprise at least 80%, preferably at least 90% and most preferably at least 95% sequences Means those with homology.

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 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. 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 / HLC-2 / pCEV-LAC was deposited on December 5, 2002 to the Korea Collection for Type Cultures (KCTC) as Accession No. KCTC 10393BP.

In the production of the vector, expression control sequences such as promoters, terminators, self-replicating sequences, secretion signals, and the like 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 lung tissues in Northern blot analysis, whereas overexpression is observed in lung cancer tissues, metastatic lung cancer tissues and lung cancer cell lines. Is judged. In addition, since expression is increased in metastatic lung cancer tissue, cancer metastasis that induces cancer metastasis is considered to be a related gene. In addition to epithelial tissues such as lung cancer, the primary cancer gene of the present invention is 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 part of the primary cancer gene as a probe to hybridize with the nucleic acid isolated from the body fluids of the subject and then detect the subject 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 probes with radioisotopes or enzymes can easily confirm the presence of genes. 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 the protein binding site of the mRNAs. 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 present invention also encompasses a method of treating or preventing cancer by administering to the patient a therapeutically effective amount of the antisense gene of the present invention within the scope thereof, and at the same time, also used for the treatment of cancer metastasis.

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 ODNs may be mixed with poly-L-lysine derivatives by electrostatic attraction according to the methods of JS Kim et al., J. Controlled Release 53: 175-182, 1998, and the mixture may be Intravenously

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. The antibody of the present invention is a monoclonal antibody or polyclonal according to a conventional method known in the art, using a protein having an amino acid sequence of SEQ ID NO: 2 expressed from the protogene HLC-2 of the present invention or a fragment thereof. It can be prepared as a raw antibody, using these antibodies enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), sandwich assay, polyacrylamide gel 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 Western blot or immunoblot on the subject.

In addition, cancer cell lines capable of continuously proliferating can be established by using the primary cancer gene of the present invention, and these cell lines are formed by, for example, tumor tissues formed in nude mice using fibroblasts transduced with the primary cancer gene. It can be prepared from. These 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 Tumor Cell Culture and Isolation of Total RNA

(Step 1) Tumor Cell Culture

For differential development of mRNA, normal lung tissue and lung cancer patients previously untreated with chemotherapy and radiation were taken at the time of operation. A549 (American Type Culture Collection; ATCC Number CCL-185) was used as a differential lung cancer cell line.

The cells obtained from the tissues obtained above and from the A549 lung cancer cell line were obtained from Weimouth's MB 752 containing 2 mM glutamine, 100 IU / ml penicillin, 100 μg / ml streptomycin and 10% fetal bovine serum (Gibco, USA). Proliferation in / 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 lung tissue, primary lung cancer tissue, metastasized lung cancer tissue and A549 cells obtained in step 1 using the 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 an nucleotide sequence set forth in 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.

Subsequently, 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) described as SEQ ID NO: 4 in H-APs 1 to 32 PCR was performed using the H-AP32 primer having the base 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 224 base pair (bp) sized band, L224 cDNA (325-548 bp of SEQ ID NO: 1 ) was cut out from the dried gel. After eluting L244 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. Reamplified.

Example 3 Cloning

The reamplified L224 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 reamplified L244 PCR product obtained in Example 2, 1 μl of the pGEM-T EASY vector (50 ng), 1 μl of T4 DNA ligase 10 × buffer and T4 DNA ligase (3 weiss units / μl; T4 DNA ligase , 1 μl of Promega) was added to a 0.5 ml test tube, and distilled water was added to make 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-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. 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, the colonies of which insertion was confirmed, that is, transformed Escherichia coli JM109 / L224, were selected and 10 ml of territorial broth (900 ml of TDW, 12 g of bacto-tryptone, and 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) to separate the L224 plasmid DNA from E. coli transformed according to the manufacturer's instructions.

A part of the separated plasmid DNA was treated with Eco RI enzyme, followed by electrophoresis on a 2% gel to confirm that the L224 subsequence was inserted into the plasmid.

Example 5 DNA Sequence Analysis

After amplifying the L224 PCR product obtained in Example 2 according to a conventional method, the cloned and re-amplified L224 PCR fragments were 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 nucleotide sequence of the DNA corresponds to nucleotide numbers 325 to 548 of SEQ ID NO: 1 , referred to herein as "L224".

The 224 bp cDNA fragment obtained above, ie L224, using 5 'random primers H-AP2 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 was confirmed to be different in normal lung tissue, left lung cancer tissue, metastatic lung cancer tissue metastasized from left to right, and A549 lung cancer cells. As can be seen in Figure 1 , 224 bp cDNA fragment L224 was expressed in lung cancer, metastatic lung cancer tissue and A549 lung cancer cells, but not in normal lung tissue.

Example 6 Total cDNA Sequence Analysis of HLC-2 Protogenes

^The bacteriophage λgt11 human lung fetal fibroblast cDNA library (Miki, T. et al. , Gene 83: 137-146, 1989) was screened using ³² P-labeled L224 as a probe. From the human lung fetal fibroblast cDNA library, a total HLC-2 cDNA clone with 596 bp inserted into the pCEV-LAC vector was obtained.

HLC-2 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 HLC-2 gene was linked by T4 DNA ligation to prepare HLC-2 plasmid DNA, and the linked clones were transformed into E. coli DH5α.

The obtained DH5α / HLC-2 / pCEV-LAC, according to the provisions of the Budapest Treaty on the International Approval of Microbial Deposits in Patent Procedures, was dated 5 December 2002 by the Korea Biotechnology Research Institute Gene Bank (Korea Collection for Type Cultures). KCTC) as Deposit No. KCTC 10393BP. The entire sequence of HLC-2 consisting of 596 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 116-349, predicted to encode a protein consisting of 77 amino acids of SEQ ID NO: 2 do.

Example 7 Northern Blot Analysis of the HLC-2 Gene in Various Cells

As in Example 1, normal lung tissue, left lung cancer tissue, metastatic lung cancer tissue metastasized from left lung to right lung, A549, NCI-H2009 (American Type Culture Collection; ATCC Number CRL-5911), NCI-H441 (American Type Total RNA was extracted from culture collection; ATCC Number HTB-174) lung cancer cell lines.

To determine the expression level of the HLC-2 gene, 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). The blots were hybridized with ³² P-labeled random primed L224 partial 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 HLC-2 primary cancer genes in normal lung tissue, lung cancer tissue, lung cancer metastatic tissue and lung cancer cell lines (A549, NCI-H2009, and NCI-H441). As can be seen in Figure 2a , gene expression was significantly increased in lung cancer tissues, lung cancer metastatic tissues and lung cancer cell lines A549, NCI-H2009, NCI-H441 lung cancer cell lines, but the expression level is very low in normal lung tissue or Not observed. In FIG. 3 , normal fever is normal lung tissue, cancer fever is lung cancer tissue, metastasis fever is lung cancer metastatic tissue and A549 fever, NCI-H2009 fever and NCI-H441 fever are lung cancer cell lines. Respectively. 3B shows the presence of mRNA by hybridizing the same sample with β-actin probe.

Figure 3a shows the expression of HLC-2 prototypic genes 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 , HLC-2 mRNA (about 0.6 kb) is weakly expressed only in muscle and heart tissues.

Figure 4a shows the results of Northern blot analysis confirming the expression of HLC-2 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 , HLC-2 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.

<Example 8> Determination of the amount of protein expressed after transfection of E. coli HLC-2 gene

The entire HLC-2 primary oncogene of SEQ ID NO: 1 was inserted into the Bam HI / Sal I restriction enzyme site in the multiple cloning site of the pET-32b (+) vector (Novagen, Germany), and the resulting pET-32b (+) / HLC-2 vector was transfected into E. coli BL21 (ATCC 47092), respectively. Expression proteins Trx.Tag, His.Tag, and S.Tag are inserted at the front of the multiple cloning site 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).

FIG. 5 shows the results of SDS-PAGE expression of the protein of E. coli BL21 strain transfected with pET-32b (+) / HLC-2 vector. A fusion protein band of about 30 kDa was clearly observed after IPTG induction. This 30 kDa fusion protein contains about 21 kDa Trx, Tag, His, Tag, and S. Tag proteins and about 9 kDa HLC-2 protein inserted into the pET-32b (+) / HLC-2 vector. have.

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) confirming the expression of L224 DNA fragment in normal lung tissue, left lung cancer tissue, metastasized lung cancer tissue from left to right and A549 lung cancer cell line. ,

Figure 2a is a northern blot confirming the expression of HLC-2 primary cancer gene of the present invention in normal lung tissue, left lung cancer tissue, metastatic lung cancer tissue metastasized from left lung to right lung, A549, NCI-H2009, NCI-H441 lung cancer cell lines Shows the results of the analysis,

FIG. 2B shows the results obtained by hybridizing the same samples as those of FIG. 2A with β-actin,

Figure 3a shows the Northern blot analysis results confirming the expression of the HLC-2 primary cancer gene of the present invention in normal human 12-row multiple tissues,

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

Figure 4a shows the Northern blot analysis results confirming the expression of the HLC-2 primary oncogene of the present invention in human cancer cell lines,

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

Figure 5 is the result of electrophoresis of sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) the size of the protein expressed before and after IPTG induction after transduction of the HLC-2 primary cancer gene of the present invention .

<110> KIM, Jin Woo <120> HUMAN PROTOONCOGENE HLC-2 AND PROTEIN ENCODED THEREIN <130> FPD / 200212-0008 <160> 4 <170> KopatentIn 1.71 <210> 1 <211> 596 <212> DNA <213> Human Lung Cancer-related Gene 2 (HLC-2) <400> 1 gaaaccgcgc agaggaggag gagaaagctg accgcttagg cccgggtagt ggtcgtcgtg 60 gttttccttg tagttcgtgg tctgagacca ggcctcaagt ggaaacggcg tcaccatgat 120 cgcacggcgg aacccagaac ccttacggtt tctgccggat gaggcccgga gcctgccccc 180 gcccaagctg accgacccgc ggctcctcta catcggcttc ttgggctact gctccggcct 240 gattgataac gtgatccggc ggaggccgat cgcgacggct ggtttgcatc gccagcttct 300 atatattacg gccttttttt tttgctggat attatcttgt aaaacgtgaa gactacctgt 360 atgctgtgag ggaccgtgaa atgtttggat atatgaaatt acatccagag gattttcctg 420 aagaagataa gaaaacatat ggtgaaattt ttgaaaaatt ccatccaata cgttgaagtc 480 ttcaaaatgc ttgctccagt ttcactgata cctgctgttt ctgaatttga tggaacatgt 540 ttcttatgac agttgaagct tatgctaatc tgtatgttga caccttgtaa ttaaaa 596 <210> 2 <211> 77 <212> PRT <213> HLC-2 protein <400> 2 Met Ile Ala Arg Arg Asn Pro Glu Pro Leu Arg Phe Leu Pro Asp Glu 1 5 10 15 Ala Arg Ser Leu Pro Pro Pro Lys Leu Thr Asp Pro Arg Leu Leu Tyr 20 25 30 Ile Gly Phe Leu Gly Tyr Cys Ser Gly Leu Ile Asp Asn Val Ile Arg 35 40 45 Arg Arg Pro Ile Ala Thr Ala Gly Leu His Arg Gln Leu Leu Tyr Ile 50 55 60 Thr Ala Phe Phe Phe Cys Trp Ile Leu Ser Cys Lys Thr 65 70 75 <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-AP32 <400> 4 aagcttcctg caa 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, A human primary oncogene comprising the nucleotide sequence corresponding to base numbers 83 to 442 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 / HLC-2 / pCEV-LAC (Accession No .: KCTC 10393BP). A method for preparing a protein having an amino acid sequence of SEQ ID NO: 2 using the microorganism of claim 6. Cancer diagnostic kit comprising the protein of claim 1. A cancer diagnostic kit comprising the original oncogene of any one of claims 2 to 4. delete delete