KR100503565B1 - Human protooncogene hlc-3 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 diagnosis of cancer including uterine cancer, lymphoma, colorectal cancer, lung cancer, skin cancer, etc., production of transgenic animals, and anti-cancer gene therapy and anti-cancer metastasis therapy, including anti-sense gene therapy.

Description

HUMAN PROTOONCOGENE HLC-3 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 3 (HLC-3) 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.

Still another object of the present invention is to provide a double-stranded RNA having the characteristics of a short fragment having a nucleotide sequence equivalent to mRNA derived from the primary oncogene or a fragment thereof.

Another object of the present invention to provide an anti-cancer and anti-transition composition for RNAi gene therapy comprising the double-stranded RNA.

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 anti-cancer and anti-transition composition comprising the anti-sense gene as an active ingredient.

In accordance with another object, the present invention provides a double-stranded RNA having the characteristics of a short fragment having a nucleotide sequence equivalent to mRNA derived from the primary oncogene or fragment thereof.

According to another object, the present invention provides an anti-cancer and anti-metastatic composition for RNAi gene therapy comprising the double-stranded RNA.

Hereinafter, the present invention will be described in detail.

Human Lung Cancer Oncogene 3 (HLC-3, hereinafter referred to as “HLC-3 Protocogene”), which is the protocogene of the present invention, is 2,390 bp described in SEQ ID NO: 1 Has the entire nucleotide sequence of length

In the nucleotide sequence of SEQ ID NO: 1 , the open reading frame corresponding to the nucleotide numbers 590 to 2,062 site (2,060-2,062: 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 490 amino acids (hereinafter referred to as "HLC-3 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 DNA sequences encoding protein translation products identical to SEQ ID NO: 2 , having those having sequence homology of at least 80%, preferably at least 90%, most preferably at least 95% with SEQ ID NO: 1 it means.

The protein expressed from the proto-oncogene of the present invention consists of 490 amino acids, has an amino acid sequence set forth in SEQ ID NO: 2 , and is about 56 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% 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 / HLC-3 / pCEV-LAC was deposited on May 30, 2003 to the Korea Collection for Type Cultures (KCTC) as Accession No. KCTC 10479BP.

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 lung tissues in Northern blot analysis, whereas overexpression is observed in lung cancer tissues, metastatic lung cancer tissues and lung cancer cell lines, thus a potent oncogenic gene that induces lung cancer. Judging by In addition, increased expression in metastatic lung cancer tissues has been shown to be a cancer metastasis-related gene that causes cancer metastasis. 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 oncogenes of the present invention are considered to be common carcinogens for the development of various cancers, diagnosis of various cancers, preparation of transformed animals and anti-sense gene therapy, RNAi (RNA interference) genes. It can be effectively used in various gene therapy therapies including treatment.

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 provides double stranded RNA useful for RNAi (RNA interference) gene therapy.

RNAi is introduced into the cell and double-stranded RNA (dsRNA) is introduced into the cell is converted into short RNA of 21 to 23 bp, the homologous mRNA is degraded by the intracellular enzyme complex that recognizes the gene of the gene It is a phenomenon that expression is suppressed, and is a general mechanism for regulating genes in vivo, and it is used for developmental processes, virus resistance, and tissue-specific gene targeting in vivo (Fire, AS, et al., Nature 391: 806). -811, 1998; Montgomery and Fire, Trends Genetics 14: 255-258, 1998).

The double stranded RNA of the present invention has a nucleotide sequence homologous to the mRNA sequence transcribed from the primary oncogene of the present invention, and may be a fragment thereof having the same length as the mRNA sequence or having a length of 21 to 23 bp.

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 or double stranded RNA of the invention within the scope.

In the 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 can then be administered intravenously to the patient.

In the case of double stranded RNA, for example, 50 ug of double stranded RNA was mixed in 1 ml of phosphate buffer according to the method of E. Song et al., Nature Medicine 9: 347-351, 2003. The mixture can then be administered to the vein three times at 8 hour intervals by hydrodynamic transfection (G. Zhang et al., Hum. Gene Ther. 10: 1735-1737, 1999).

The scope of the present invention also includes anticancer and anti-metastatic compositions comprising as an active ingredient the antisense gene or double stranded RNA of the present invention together with a pharmaceutically acceptable carrier, excipient or optionally other additives. The pharmaceutical composition of the present invention is preferably formulated as an injection.

The amount of antisense gene or double stranded RNA that is 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 confirming whether the protein is expressed in a 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, 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, reverse transcription was performed using H-T11G fixed primers (RNAimage kit, Genhunter, Cor., MA, USA) as oligo-dT primers immobilized on 0.2 μg of total RNA obtained in step 1 of Example 1.

Subsequently, in the presence of 0.5 mM [α- ³⁵ S] dATP (1200 Ci / mmole), using the same fixed primer and random 5 ′ 11 primer (RNAimage primer sets 1-4) H-AP7 primer in H-AP 1 to 32 PCR was performed. 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 162 base pair (bp) sized band, L72 cDNA (1,842-2,003 bp of SEQ ID NO: 1 ) was cut out from the dried gel. After eluting the L72 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 L72 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 L72 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, the colonies of which insertion was confirmed, i.e., transformed E. coli JM109 / L72, were selected to make 10 ml of territorial broth (900 ml of TDW, 12 g of bacto-tryptone, 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) to separate the L72 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 L72 subsequence was inserted into the plasmid.

Example 5 DNA Sequence Analysis

After amplifying the L72 PCR product obtained in Example 2 according to a conventional method, the cloned and re-amplified L72 PCR fragment was prepared using 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 1,842 to 2,003 of SEQ ID NO: 1 , and is referred to as "L72" in the present invention.

The 162 bp cDNA fragment obtained above, i.e., L72, using 5 'random primers H-AP7 and 3' H-T11G immobilized primers, was subjected to differential development 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 , 162 bp cDNA fragment L72 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-3 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 L72 as a probe. From the human lung fetal fibroblast cDNA library, a full HLC-3 cDNA clone with 2,390 bp inserted into the pCEV-LAC vector was obtained and registered with GeneBank of the United States as Gen. No. AY117688 dated 3 June 2002. (Expected date of release: July 1, 2003).

HLC-3 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-3 gene was linked with T4 DNA ligase to prepare HLC-3 plasmid DNA, and transformed into E. coli DH5α with the linked clone.

The obtained DH5α / HLC-3 / pCEV-LAC, according to the provisions of the Budapest Treaty on the International Approval of Microbial Deposits in Patent Procedures, was dated May 30, 2003. KCTC) as Deposit No. KCTC 10479BP. The entire sequence of HLC-3 consisting of 2,390 bp is shown in SEQ ID NO: 1 .

In the nucleotide sequence of SEQ ID NO: 1 , the entire open reading frame of the proto-oncogene of the present invention corresponds to nucleotides 590 to 2,062, predicted to encode a protein consisting of 490 amino acids of SEQ ID NO: 2 do.

Example 7 Northern Blot Analysis of the HLC-3 Gene in Various Cells

As in Example 1, total RNA from normal lung tissue, left lung cancer tissue, metastatic lung cancer tissue metastasized from left lung to right lung, A549 and NCI-H358 (American Type Culture Collection; ATCC Number CRL-5807) lung cancer cell lines Extracted.

To determine the degree of HLC-3 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 HLC-3 whole 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-3 primary oncogenes in normal lung tissue, lung cancer tissue, lung cancer metastases and lung cancer cell lines (A549 and NCI-H358). As can be seen in Figure 2a , HLC-3 primary cancer gene gene expression was significantly increased in lung cancer tissue, lung cancer metastasis tissue and A549 and NCI-H358 lung cancer cell lines, but expression level is very low or observed in normal lung tissue It wasn't. In FIG. 2 , normal rows represent normal lung tissues, cancer rows represent lung cancer tissues, metastasis rows represent lung cancer metastatic tissues, and A549 and NCI-H358 columns represent lung cancer cell lines, respectively. 2B shows the presence of mRNA by hybridizing the same sample with β-actin probe.

Figure 3a shows the expression of the HLC-3 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 , HLC-3 mRNA (approximately 2.4 kb) is expressed only in muscle and heart tissues and weakly expressed in other brain and placental tissues.

Figure 4a shows the results of Northern blot analysis confirming the expression of HLC-3 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-3 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 mRNAs of about 1.4 kb and about 0.8 kb, which are smaller than this, were also expressed.

<Example 8> Determination of the size of the protein expressed after transfection of E. coli HLC-3 gene

The entire HLC-3 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-3 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). FIG. 5 shows the results of SDS-PAGE expression of the protein of E. coli BL21 strain transfected with pET-32b (+) / HLC-3 vector. A fusion protein band of about 77 kDa was clearly observed after IPTG induction. This 77 kDa fusion protein contains about 21 kDa Trx, Tag, His, Tag, and S. Tag proteins and about 56 kDa HLC-3 protein inserted into the pET-32b (+) / HLC-3 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 production of animals and anti-cancer gene therapy, including anti-sense gene therapy, and anti-cancer metastasis therapy.

Figure 1 shows the results of differential reverse transcription polymerase chain reaction (DDRT-PCR) confirmed the expression of L72 DNA fragment in normal lung tissue, left lung cancer tissue, metastasized lung cancer tissue from left to right and A549 lung cancer cells ,

Figure 2a shows the results of Northern blot analysis confirming the expression of HLC-3 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 and NCI-H358 lung cancer cell lines Will,

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

Figure 3a shows the Northern blot analysis results confirming the expression of the HLC-3 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 HLC-3 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 proteins expressed before and after IPTG induction after transduction of E. coli HLC-3 gene of the present invention .

<110> KIM, Jin-Woo <120> HUMAN PROTOONCOGENE HLC-3 AND PROTEIN ENCODED THEREIN <130> FPD / 200306-0001 <160> 2 <170> KopatentIn 1.71 <210> 1 <211> 2390 <212> DNA <213> Homo sapiens <220> <221> CDS (222) (590) .. (2059) <400> 1 accaatcgcg aaaccgcccg acatccctcc tcctcagccg gactgcggtg attttaggag 60 tctacaggag gagcagtcgc gccccacgac agcgggttct tcccctggcg ggccagcccg 120 ggctcccccc taccaagagc ctccatgggg tggccctgcc acagccccct acagcttaga 180 gaccctgaag ggcgggacta tccttggcac ccgtagcttg aaagggacga gttactgcct 240 tttcgggagg ctgtctggct gcgacgtgtg cctggagcac ccttcggtgt ctcggtacca 300 cgcagtgctg cagcacaggg cgtccggccc tgacggagaa tgcgacagca acgggccggg 360 cttctacctc tacgatctgg gaagcaccca tggcactttt ctcaacaaaa ctcgcatccc 420 acctcgcacc tactgtcgag tccacgttgg gcatgttggt cgctttggag gcagcacccg 480 gctctttatc ctgcagggac cagaggaaga ccgagaggca gaatccgagt taacagtaac 540 acagttgaag gaattgcgca agcagcagca aatattgttg gagaagaag atg cta gga 598 Met leu gly One gaa gac tca gat gaa gaa gag gaa atg gat acc tct gaa agg aag ata 646 Glu Asp Ser Asp Glu Glu Glu Glu Met Asp Thr Ser Glu Arg Lys Ile 5 10 15 aat gct ggt agc caa gat gat gag atg ggt tgc acc tgg gga atg gga 694 Asn Ala Gly Ser Gln Asp Asp Glu Met Gly Cys Thr Trp Gly Met Gly 20 25 30 35 gaa gat gca gta gag gat gat gct gaa gag aac cct att gtc tta gag 742 Glu Asp Ala Val Glu Asp Asp Ala Glu Glu Asn Pro Ile Val Leu Glu 40 45 50 ttt cag cag gaa agg gag gcc ttt tat ata aag gat ccc aaa aag gct 790 Phe Gln Gln Glu Arg Glu Ala Phe Tyr Ile Lys Asp Pro Lys Lys Ala 55 60 65 ctc caa ggc ttt ttt gac cga gaa gga gaa gaa tta gaa tat gaa ttt 838 Leu Gln Gly Phe Phe Asp Arg Glu Gly Glu Glu Leu Glu Tyr Glu Phe 70 75 80 gat gaa cag gga cat agc act tgg ctc tgc agg gtg aga tta cct gtg 886 Asp Glu Gln Gly His Ser Thr Trp Leu Cys Arg Val Arg Leu Pro Val 85 90 95 gac gat tca act gga aaa caa ctg gtg gct gag gcc att cac tca gga 934 Asp Asp Ser Thr Gly Lys Gln Leu Val Ala Glu Ala Ile His Ser Gly 100 105 110 115 aag aaa aaa gaa gca atg atc cag tgc tca ttg gaa gct tgt cgg att 982 Lys Lys Lys Glu Ala Met Ile Gln Cys Ser Leu Glu Ala Cys Arg Ile 120 125 130 ctt gat act ttg gga ttg att cgg cag gaa gca gta tct cgg aaa agg 1030 Leu Asp Thr Leu Gly Leu Ile Arg Gln Glu Ala Val Ser Arg Lys Arg 135 140 145 aaa gcc aag aac tgg gaa gat gaa gac ttt tat gat agt gat gat gac 1078 Lys Ala Lys Asn Trp Glu Asp Glu Asp Phe Tyr Asp Ser Asp Asp Asp 150 155 160 aca ttt ctt gat agg act ggc ctg att gag aag aag cgt ctg aac aga 1126 Thr Phe Leu Asp Arg Thr Gly Leu Ile Glu Lys Lys Arg Leu Asn Arg 165 170 175 atg aag aag gct ggc aag att gat gag aag cca gag acc ttt gaa tca 1174 Met Lys Lys Ala Gly Lys Ile Asp Glu Lys Pro Glu Thr Phe Glu Ser 180 185 190 195 ttg gtt gca aaa tta aat gat gct gaa agg gaa ctt tct gaa att tct 1222 Leu Val Ala Lys Leu Asn Asp Ala Glu Arg Glu Leu Ser Glu Ile Ser 200 205 210 gag aga ttg aaa gcc tca agc caa gtt cta tca gag tct cca tct cag 1270 Glu Arg Leu Lys Ala Ser Ser Gln Val Leu Ser Glu Ser Pro Ser Gln 215 220 225 gat tct tta gat gcg ttc atg tca gaa atg aaa tca ggc agt aca tta 1318 Asp Ser Leu Asp Ala Phe Met Ser Glu Met Lys Ser Gly Ser Thr Leu 230 235 240 gat ggt gtg tcc cgg aag aaa ctt cac ctg aga act ttt gaa ctg agg 1366 Asp Gly Val Ser Arg Lys Lys Leu His Leu Arg Thr Phe Glu Leu Arg 245 250 255 aaa gaa caa cag aga ctt aaa ggg tta ata aaa att gta aag cca gca 1414 Lys Glu Gln Gln Arg Leu Lys Gly Leu Ile Lys Ile Val Lys Pro Ala 260 265 270 275 gag att cca gaa cta aaa aag act gaa act cag act aca ggt gca gaa 1462 Glu Ile Pro Glu Leu Lys Lys Thr Glu Thr Gln Thr Thr Gly Ala Glu 280 285 290 aac aaa gct aaa aag ctt aca ttg cct cta ttt ggt gcc atg aaa gga 1510 Asn Lys Ala Lys Lys Leu Thr Leu Pro Leu Phe Gly Ala Met Lys Gly 295 300 305 gga agc aaa ttc aaa tta aaa act gga aca gta ggg aag tta ccc ccc 1558 Gly Ser Lys Phe Lys Leu Lys Thr Gly Thr Val Gly Lys Leu Pro Pro 310 315 320 aag cgt cca gaa ctc cct cca act cta atg aga atg aaa gat gag cct 1606 Lys Arg Pro Glu Leu Pro Pro Thr Leu Met Arg Met Lys Asp Glu Pro 325 330 335 gaa gta gaa gag gag gag gaa gag gaa gag gaa gaa gag aaa gaa aag 1654 Glu Val Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Lys Glu Lys 340 345 350 355 gag gag cat gaa aag aaa aaa ctg gag gat gga agc ctc agt agg cca 1702 Glu Glu His Glu Lys Lys Lys Leu Glu Asp Gly Ser Leu Ser Arg Pro 360 365 370 cag cca gag ata gag cca gaa gca gca gtg cag gaa atg agg cct ccc 1750 Gln Pro Glu Ile Glu Pro Glu Ala Ala Val Gln Glu Met Arg Pro Pro 375 380 385 aca gat ctc aca cat ttt aaa gaa acc caa acc cat gaa aac atg tct 1798 Thr Asp Leu Thr His Phe Lys Glu Thr Gln Thr His Glu Asn Met Ser 390 395 400 caa ctt agc gag gaa gaa cag aat aaa gat tat caa gac tgt agc aaa 1846 Gln Leu Ser Glu Glu Glu Gln Asn Lys Asp Tyr Gln Asp Cys Ser Lys 405 410 415 acc act tca ttg tgc gca gga ccc tca gca tca aag aat gaa tat gag 1894 Thr Thr Ser Leu Cys Ala Gly Pro Ser Ala Ser Lys Asn Glu Tyr Glu 420 425 430 435 aaa agc aga ggt gaa ttg aag aaa aag aaa aca cct ggt cca ggc aaa 1942 Lys Ser Arg Gly Glu Leu Lys Lys Lys Lys Thr Pro Gly Pro Gly Lys 440 445 450 ctt cca cca aca ctt tct tcc aaa tat cct gaa gat gac cca gac tac 1990 Leu Pro Pro Thr Leu Ser Ser Lys Tyr Pro Glu Asp Asp Pro Asp Tyr 455 460 465 tgt gtg tgg gtc cca cct gaa ggt caa agt gga gat ggc aga acc cat 2038 Cys Val Trp Val Pro Pro Glu Gly Gln Ser Gly Asp Gly Arg Thr His 470 475 480 ctt aat gac aag tat ggc tat t gattgcttca gaatcccaaa agaaaacctt 2090 Leu Asn Asp Lys Tyr Gly Tyr 485 490 gtggaccatg tgacatggaa tatttgggat aatgtatcaa attgaatggc cagagaagtt 2150 tagatgatta tttgtaagat ctggtgactg gcttttcgtt ctgtgttctt ggcttcctaa 2210 atttatctgc ccatatgatt ctcatgcatt tgatatttat gtttaaaagt gtttatatat 2270 gtatgtaaaa agggaaccat atgttttgag aatttgtaaa gtgagagaca tgatcctatt 2330 aaaataagaa ggcaaaaatg taaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 2390 2390 <210> 2 <211> 490 <212> PRT <213> Homo sapiens <400> 2 Met Leu Gly Glu Asp Ser Asp Glu Glu Glu Glu Met Asp Thr Ser Glu 1 5 10 15 Arg Lys Ile Asn Ala Gly Ser Gln Asp Asp Glu Met Gly Cys Thr Trp 20 25 30 Gly Met Gly Glu Asp Ala Val Glu Asp Asp Ala Glu Glu Asn Pro Ile 35 40 45 Val Leu Glu Phe Gln Gln Glu Arg Glu Ala Phe Tyr Ile Lys Asp Pro 50 55 60 Lys Lys Ala Leu Gln Gly Phe Phe Asp Arg Glu Gly Glu Glu Leu Glu 65 70 75 80 Tyr Glu Phe Asp Glu Gln Gly His Ser Thr Trp Leu Cys Arg Val Arg 85 90 95 Leu Pro Val Asp Asp Ser Thr Gly Lys Gln Leu Val Ala Glu Ala Ile 100 105 110 His Ser Gly Lys Lys Lys Glu Ala Met Ile Gln Cys Ser Leu Glu Ala 115 120 125 Cys Arg Ile Leu Asp Thr Leu Gly Leu Ile Arg Gln Glu Ala Val Ser 130 135 140 Arg Lys Arg Lys Ala Lys Asn Trp Glu Asp Glu Asp Phe Tyr Asp Ser 145 150 155 160 Asp Asp Asp Thr Phe Leu Asp Arg Thr Gly Leu Ile Glu Lys Lys Arg 165 170 175 Leu Asn Arg Met Lys Lys Ala Gly Lys Ile Asp Glu Lys Pro Glu Thr 180 185 190 Phe Glu Ser Leu Val Ala Lys Leu Asn Asp Ala Glu Arg Glu Leu Ser 195 200 205 Glu Ile Ser Glu Arg Leu Lys Ala Ser Ser Gln Val Leu Ser Glu Ser 210 215 220 Pro Ser Gln Asp Ser Leu Asp Ala Phe Met Ser Glu Met Lys Ser Gly 225 230 235 240 Ser Thr Leu Asp Gly Val Ser Arg Lys Lys Leu His Leu Arg Thr Phe 245 250 255 Glu Leu Arg Lys Glu Gln Gln Arg Leu Lys Gly Leu Ile Lys Ile Val 260 265 270 Lys Pro Ala Glu Ile Pro Glu Leu Lys Lys Thr Glu Thr Gln Thr Thr 275 280 285 Gly Ala Glu Asn Lys Ala Lys Lys Leu Thr Leu Pro Leu Phe Gly Ala 290 295 300 Met Lys Gly Gly Ser Lys Phe Lys Leu Lys Thr Gly Thr Val Gly Lys 305 310 315 320 Leu Pro Pro Lys Arg Pro Glu Leu Pro Pro Thr Leu Met Arg Met Lys 325 330 335 Asp Glu Pro Glu Val Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu 340 345 350 Lys Glu Lys Glu Glu His Glu Lys Lys Lys Leu Glu Asp Gly Ser Leu 355 360 365 Ser Arg Pro Gln Pro Glu Ile Glu Pro Glu Ala Ala Val Gln Glu Met 370 375 380 Arg Pro Pro Thr Asp Leu Thr His Phe Lys Glu Thr Gln Thr His Glu 385 390 395 400 Asn Met Ser Gln Leu Ser Glu Glu Glu Gln Asn Lys Asp Tyr Gln Asp 405 410 415 Cys Ser Lys Thr Thr Ser Leu Cys Ala Gly Pro Ser Ala Ser Lys Asn 420 425 430 Glu Tyr Glu Lys Ser Arg Gly Glu Leu Lys Lys Lys Lys Thr Pro Gly 435 440 445 Pro Gly Lys Leu Pro Pro Thr Leu Ser Ser Lys Tyr Pro Glu Asp Asp 450 455 460 Pro Asp Tyr Cys Val Trp Val Pro Pro Glu Gly Gln Ser Gly Asp Gly 465 470 475 480 Arg Thr His Leu Asn Asp Lys Tyr Gly Tyr 485 490

Claims (13)

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 590 to 2062 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-3 / pCEV-LAC (Accession No .: KCTC 10479BP). 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 delete