KR100873382B1 - Human protooncogene, protein encoded by same, expression vector containing same, and cell transformed by said vector - Google Patents

Abstract

The present invention relates to a novel primary cancer gene and a protein encoded by the same, more specifically, a gene having a nucleotide sequence represented by SEQ ID NO: 1 or a fragment thereof and a protein having an amino acid sequence represented by SEQ ID NO: 2 or a fragment thereof It is about. In addition, the primary cancer gene of the present invention is for the diagnosis and prevention of various cancers, including liver cancer, leukemia, uterine cancer, malignant lymphoma, colorectal cancer, lung cancer, skin cancer, antisense or RNA interference (RNA interference, RNAi) gene therapy, and transformation It can be effectively used for the production of transgenic animals.

Primary cancer gene, primary cancer protein, transformation, antisense, RNA interference

Description

HUMAN PROTOONCOGENE, PROTEIN ENCODED BY SAME, EXPRESSION VECTOR CONTAINING SAME, AND CELL TRANSFORMED BY SAID VECTOR}

Figure 1 shows the results of the differential development reverse transcription polymerase chain reaction (DDRT-PCR) confirming the expression of HP73 in normal liver tissue, liver cancer tissue, and HepG2 liver cancer cells,

Figure 2 A shows the Northern blot analysis results confirming the expression of GIG47 primary cancer genes in human normal liver, liver cancer and liver cancer cell lines,

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

Figure 3a shows the results of Northern blot analysis confirming the expression of GIG47 primary cancer gene in normal human 12-row multiple tissues,

3 b shows the result obtained by hybridizing the same sample as a in FIG. 3 with β-actin,

Figure 4 a shows the Northern blot analysis results confirming the expression of GIG47 primary cancer gene in human cancer cell lines,

4b shows the result obtained by hybridizing the same sample as a of FIG. 4 with β-actin,

FIG. 5 shows the results of electrophoresis of sodium dodecyl sulphate (SDS) -polyacrylamide gel (PAGE) on the size of proteins expressed before and after L-arabinose induction after transduction of E. coli GIG47 gene will be.

Higher animals, including humans, have about 30,000 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 (programmed cell death). [Liang, P. and A. ratio. AB Pardee, Science, 257 : 967-971, 1992]. Pathological phenomena, such as the development of tumors, are triggered during gene mutation and eventually lead to variations in gene expression. Thus, comparison of gene expression between different cells is a fundamental and effective approach to understanding many biological phenomena.

For example, the mRNA Differential Display (DD) method proposed by Liang and Paddy in the paper mentioned above is currently used to search for tumor suppressor genes, genes involved in the cell division cycle, and transcription control genes related to apoptosis. It is effectively used, and it is also used in various ways to identify the interrelationship between various genes occurring in one cell.

Taken together, the results of several studies on tumor development suggest that several genetic variants, such as loss of chromosomal heterozygosity, activation of primary cancer genes, and inactivation of p53 genes and other tumor suppressor genes, can be identified. It has been reported that tumors develop by accumulating in tissues [Bishop Jay. Bishop, JM, Cell, 64 : 235-248, 1991; Hunter, T., Cell, 64 : 249-270, 1991], reported that 10-30% of cancers are caused by the activation of the primary cancer genes by amplification of the primary cancer genes.

In particular, the issue of the activation of the primary cancer gene plays an important role in the etiology of many cancers, it is required to reveal this.

Accordingly, the present inventors have completed the present invention by confirming that the oncogenic genes of human cancer proliferation-inducing genes 47 and GIG47 are specifically expressed only in cancer cells. The GIG 47 primary cancer gene can be effectively used for the diagnosis, prevention and treatment of various cancers such as liver cancer, leukemia, uterine cancer, malignant lymphoma, colorectal cancer, lung cancer and skin cancer.

It is an object of the present invention to provide novel primary cancer genes and fragments thereof.

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

It is another object of the present invention to provide a recombinant vector comprising the primary cancer gene or fragment thereof and a cell transformed thereby to provide a technique for mass production of the gene or fragment thereof and the protein or fragment thereof.

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

Still another object of the present invention is to provide a kit for diagnosing cancer, which comprises an antibody against the protein or fragment thereof.

It is another object of the present invention to provide a gene to be used for gene therapy, such as antisense polynucleotides and interfering RNA (RNAi) molecules, which prevent or treat cancer by inhibiting or inhibiting the expression of the protein encoded in the primary cancer gene. .

Still another object of the present invention is to provide a gene therapy agent using the antisense polynucleotide and RNAi molecule.

In order to achieve the above object, the present invention provides a nucleotide sequence encoding a primary cancer protein having an amino acid sequence represented by SEQ ID NO: 2, preferably a primary cancer gene having a nucleotide sequence of SEQ ID NO: 1.

According to another object of the present invention, the present invention provides a recombinant vector comprising the primary cancer gene or a fragment thereof and a host cell transformed thereby.

The present invention also provides a primary cancer protein or fragment thereof having the amino acid sequence represented by SEQ ID NO: 2.

According to another object, the present invention provides a kit for diagnosing cancer, including the primary cancer gene or a fragment thereof, for examining the presence of the primary cancer gene or a fragment thereof in a sample.

The present invention provides a kit for diagnosing cancer, comprising a polyclonal or monoclonal antibody having specificity for the primary cancer protein or fragment thereof, and a diagnostic kit for examining the presence of the primary cancer protein or fragment thereof present in a sample. to provide.

The present invention also has an nucleotide sequence complementary to all or part of the mRNA sequence transcribed from the primary cancer gene, the antisense polynucleotide or mRNA of the primary cancer gene that binds to the mRNA to inhibit the expression of the protein from the primary cancer gene Provided are double stranded interfering RNA (RNAi) molecules with homologous sequences.

The present invention provides a pharmaceutical composition for the genetic treatment and prevention of cancer comprising the antisense polynucleotide and RNAi molecule as an active ingredient.

Hereinafter, the present invention will be described in detail.

Protocogene according to the present invention (protooncogene) is a human cell proliferation induction gene 47 (hereinafter abbreviated as "GIG47" primary cancer gene), a gene encoding a protein having an amino acid sequence represented by SEQ ID NO: 2, preferably The primary cancer gene and fragments thereof may have a total length of 517 to 703 bp, and are preferably genes having a 703 bp long nucleotide sequence represented by SEQ ID NO: 1.

Specifically, the open reading frame corresponding to the 17th to 535th nucleotide site (533-535: end codon) of the entire nucleotide sequence of SEQ ID NO: 1 is a protein coding region, and the amino acid sequence encoded therefrom is It is the amino acid sequence shown in SEQ ID NO: 2, and consists of a total of 172 amino acids (hereinafter abbreviated as "GIG47 primary cancer protein").

The primary cancer gene of the present invention does not change the amino acid sequence of the carcinogenic protein to be expressed in consideration of the wobbling code according to the degeneracy of the codon and the preferred codon in the organism to express the primary cancer gene. A variety of nucleotide sequences may be varied in a range that does not range, and various modifications or modifications may be made in a range that does not affect the expression of genes in portions other than the primary cancer protein coding region, and such modified sequences may also be used in the original cancer of the present invention. Included in the range of genes.

Therefore, the primary cancer gene of the present invention may be a polynucleotide having a nucleotide sequence substantially the same as the nucleotide sequence represented by SEQ ID NO: 1 and fragments thereof. Substantially identical polynucleotides mean having at least 80%, preferably at least 90% and most preferably at least 95% sequence homology with respect to the nucleotide sequence represented by SEQ ID NO: 1.

The protein expressed from the primary cancer gene of the present invention consists of 172 amino acids, has an amino acid sequence represented by SEQ ID NO: 2, and is about 19 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 the substantially identical polypeptide is at least 80%, preferably 90%, relative to the amino acid sequence represented by SEQ ID NO: 2 above As mentioned above, most preferably, it has 95% or more of sequence homology.

The primary cancer genes and proteins of the present invention may be isolated from human cancer tissue or synthesized according to known DNA or peptide synthesis methods. In addition, the thus prepared gene 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, yeast, animal cells and plant cells. Using the transformed host cell as described above, the primary cancer gene DNA of the present invention can be replicated in large quantities, or the primary cancer protein or fragment thereof can be produced in large quantities.

Expression vectors of the present invention can be prepared by combining expression control sequences, such as promoters, terminators, self-replicating sequences, secretion signals selected according to the type of host cell to produce the primary cancer gene or protein.

As confirmed by Northern blot and the like, the expression of the gene of the present invention is hardly confirmed in normal liver tissue, while in the liver cancer tissue and liver cancer cell lines overexpression of the gene was confirmed. That is, the primary cancer gene of the present invention is determined to be a potent oncogenic gene that induces liver cancer. Furthermore, in addition to the liver cancer tissue, the primary cancer gene of the present invention is a variety of leukemia, uterine cancer, malignant lymphoma, colorectal cancer, lung cancer, skin cancer, etc. It is expressed at high levels in cancer tumors. Therefore, the primary cancer gene of the present invention is considered to be a carcinogen common to the occurrence of various cancers, and thus can be effectively used for diagnosis, prevention and treatment of various various cancers. In addition, the primary cancer gene of the present invention can be effectively used for the production of transformed animals and antisense gene therapy or RNA interference gene therapy for the diagnosis, prevention and treatment of the cancer described above.

It is possible to diagnose cancer using the primary cancer gene. For example, all or part of the primary cancer gene may be used as a probe to hybridize with nucleic acid isolated from the body fluid of the subject, and then the probe may be detected by various methods known in the art. And determining whether the subject has the primary cancer gene of the present invention by detecting whether a complex is formed. The probe may be labeled with a radioisotope, an enzyme, or the like to easily confirm complex formation. The present invention also provides a kit for diagnosing cancer comprising all or part of the original cancer gene.

Transgenic animals may be prepared by introducing the primary cancer genes of the present invention into mammals, for example, rodents such as rats, and the transduction of the genes may be performed at the fertilized egg stage before at least 8 cell stages. It is preferable to make. 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 antisense polynucleotides that are effective for gene therapy. The antisense polynucleotide is a single-stranded polynucleotide having a sequence having complementarity with some or all of the mRNA transcribed from the primary cancer gene sequence represented by SEQ ID NO: 1. It is a sequence capable of binding to the protein binding site of the mRNA to destroy the open reading frame (ORF) of the primary cancer gene, and by introducing these antisense polynucleotides into the body of the patient to prevent or treat cancer caused by the expression of the primary cancer gene Can be. The antisense polynucleotides can be produced by any method known in the art.

Antisense gene therapy of the present invention is to administer the antisense polynucleotide to the patient in a conventional manner to prevent the expression of the primary cancer gene. For example, the antisense gene therapy method is described in J.S. JS Kim, et al. , J. Controlled Release , 53 : 175-182, 1998 ”, wherein antisense ODN is mixed with a poly-L-lysine derivative by electrostatic attraction and the mixture Intravenously to a patient.

The present invention also provides an anticancer composition comprising the antisense polynucleotide of the present invention as an active ingredient and a pharmaceutically acceptable carrier, excipient or other additives as necessary. The anticancer composition of the present invention is preferably formulated as an injection. The amount of antisense polynucleotide administered can be determined by considering various related factors such as the type of symptom to be treated, the route of administration, the age and weight of the patient, and the severity of the symptom.

The present invention also provides interfering RNA (RNAi) effective for gene therapy. RNA interference refers to a phenomenon in which when double-stranded RNA (dsRNA) enters a cell, only mRNA having a homologous nucleotide sequence thereof is selectively degraded in the post-transcriptional step. RNA interference has been reported in several animals, such as fruit flies, mice, tripanosoma, and planaria, since reports of this phenomenon have been confirmed to occur in insects, and have been reported to be involved in gene silencing in plants [Sharp, Blood. . a. Etc. (Sharp, PA et.al.), Genes Dev., 13, 139-41, 1999]. Recently, in mammals, if the dsRNA is processed to 21-22 small sizes, it is possible to avoid degradation of the dsRNA due to protein kinase activation by interferon in animal cells, thus successfully inducing RNA interference. It has been confirmed that it is possible, and there is an active research to silencing many genes related to the development of the disease [Elbash, S. M. Elbashir, SM et. Al., Nature , 411; 494-8, 2001; Elbashi, S. M. Et al . , Genes Dev ., 15 ; 188-200, 2001; Rossell, K. Rosel, KF and George, S., Nucleic acids Res ., 31 ; 4417-4424, 2003; Judy, L. Et al ., Judy, L., et al ., Trends Mol Med ., 9 ; 397-403, 2003; Michael, bee. M., et al ., Michael, BM, et al ., Methods , 30 ; 287-8, 2003]. However, these RNA interference phenomena are known to cause severe cytotoxic reactions in mammalian cells, and these side effects are avoided when using short (19-22 nucleotides long) coherent RNA (siRNA). It is known that the gene expression is very large and specific. M., et al ., Nature , 411 ; 494-8, 2001; Elbashi, S. M., et al., Genes Dev ., 15 ; 188-200, 2001].

Thus, the interfering RNA according to the present invention can be prepared from a double-stranded RNA molecule having a nucleotide sequence having complementarity with some or all of the mRNA transcribed from the primary cancer gene sequence represented by SEQ ID NO: 1. Specific manufacturing methods are in accordance with methods known in the art.

The present invention also provides an anticancer composition comprising the interfering RNA of the present invention as an active ingredient and a pharmaceutically acceptable carrier, excipient or other additives as necessary. The anticancer composition of the present invention is preferably formulated as an injection. The amount of interfering RNA administered can be determined by considering various related factors such as the type of symptom to be treated, the route of administration, the age and weight of the patient, and the severity of the symptom.

Proteins derived from the primary cancer genes of the present invention can be usefully used for producing antibodies as diagnostic tools. Monoclonal antibodies or polyclonal antibodies can be prepared according to conventional methods known in the art using a protein having an amino acid sequence represented by SEQ ID NO: 2 expressed from a primary cancer gene of the present invention or a fragment thereof. A body fluid sample of a subject using the antibodies by methods known in the art, such as enzyme immunoassay (ELISA), radioimmunoassay (RIA), sandwich assay (Sandwich Assay), Western blot or immunoblot on polyacryl gel. Cancer can be diagnosed by examining whether the protein is expressed.

In addition, cancer cell lines capable of continuously proliferating can be established by using the primary cancer gene of the present invention. Such cell lines can be formed by, for example, tumor tissues formed in nude mice using fibroblasts transduced with the primary cancer gene. 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, but the present invention is not to be construed as being limited thereto.

Example  1: Tumor Cell Culture and Isolation of Total RNA

(Step 1) Tumor Cell Culture

For differential development of mRNA, primary liver tissue was taken at the time of tissue biopsy from normal liver tissue from patients undergoing liver tissue biopsy, and from liver cancer patients previously untreated with chemotherapy and radiation. HepG2 (American Type Culture Collection) liver cancer cell line was used for the differential development. The cultured cells used in the experiments were exponentially proliferating cells that survived 95% or more when stained with trypan blue dye (Freshney, “Culture of Animal Cells”, A Manual). of Basic Technique , 2nd edition , AR Liss, New York, 1987].

(Step 2) RNA isolation and differential development of mRNA

Total RNA was extracted from normal liver tissue, primary liver cancer tissue, and HepG2 cells obtained in step 1 using the commercially available system RNeasy Total RNA Kit (Qiagen, Germany). DNA contaminants were removed from RNA using a Message clean kit (GenHunter, USA).

Example  2: differential development Reverse transcription  Polymerase chain reaction DDRT - 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-T11C fixed primer (RNAimage kit, Genhunter, USA) of SEQ ID NO: 3 as an oligo-dT primer immobilized on 0.2 μg of total RNA obtained in step 1 of Example 1.

Then, in the presence of 0.5 mM [α-35S] dATP (1200 Ci / mmole), the above-described fixed primer and random 5-terminal 13 primer (RNAimage primer set 1-5, H-AP23 primer in H-AP 1-40) ( PCR was performed using SEQ ID NO: 4). 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 the final extension.

After PCR-amplified fragments were dissolved in a 6% polyacrylamide sequencing gel, radiographs were used to identify the positions of the bands showing different degrees of differential expression.

A 282 base pair (bp) size band HP73 cDNA (346-627 site of SEQ ID NO: 1) was cut out of the dried gel. HP73 cDNA was eluted by heating for 15 minutes and then reamplified by PCR under the same primers and the same conditions except that [α-35S] labeled dATP (1200 Ci / mmole) and 20 μM dNTP were not used. I was.

Example  3: Cloning

The reamplified HP73 PCR product obtained above was inserted into pGEM-T easy (EASY) vector using the TA cloning system (Promega, USA) according to the manufacturer's method.

(Step 1) Linking reaction

2 μl of re-amplified HP73 PCR product obtained in Example 2, 1 μl of pGEM-T easy vector (50 ng), 1 μl of T4 DNA ligase 10X buffer and 3 weiss units / ul (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

E. coli JM109 (Promega, WI, USA) was prepared in 10 ml of LB broth (10 g of bacto-tryptone, 5 g of bacto-yeast extract, 5 g of NaCl) at an optical density of about 0.3-0.6. 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. 800 μl of 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 Mg2 +, 1 ml of 2 M glucose) The mixture was added and incubated for 45 minutes in a rotary shaking incubator at 37 ° C. at 220 rpm.

25 μl of X-gal (stored in 40 mg / ml dimethylformamide) was diffused into a glass rod in an ampicillin-added LB plate, previously placed in a 37 ° C. incubator, and 25 μl of the 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, i.e., transformed E. coli JM109 / HP73, was selected and 10 ml of territorial broth (900 ml of TDW, 12 g of bacto-tryptone, 12 g of bacto-). 24 g of yeast extract, 4 ml of glycerol, 0.17 M KH 2 PO 4, 0.72 N K 2 HPO 4 100 ml).

Example  4: Isolation of Recombinant Plasmid DNA

HP73 plasmid DNA was isolated from transformed E. coli using the Wizard Plus Minipreps DNA Purificatin Kit (Promega, USA) 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 HP73 subsequence was inserted into the plasmid.

Example  5: DNA sequencing

The HP73 PCR product obtained in Example 2 was PCR according to a conventional method, and then cloned and re-amplified HP73 PCR fragments were sequenced into a Sequenase version 2.0 DNA Sequencing kit (United States Biochemical, USA). ) Was subjected to sequencing according to dideoxy chain termination.

The nucleotide sequence of the DNA corresponds to nucleotide numbers 346 to 627 of the nucleotide sequence represented by SEQ ID NO: 1, and was named "HP73" herein.

The 282 bp cDNA fragment, ie, HP73, obtained above using 5-terminal random primer H-AP23 and 3-terminal H-T11C fixed primer, was identified by differential development reverse transcriptase polymerase chain reaction (DDRT-PCR) and confirmed by electrophoresis. As shown in FIG. 1, gene expression by DD in normal liver tissue, liver cancer tissue and HepG2 cells was found to be different. As can be seen in Figure 1, 282 bp cDNA fragment HP73 was expressed in liver cancer and HepG2 cancer cells, but not or weak in normal tissues.

Example  6: GIG47 Wonam Gene  Full cDNA Sequence Analysis

Bacteriophage λgt11 human lung fetal fibroblast cDNA library using ³² P-labeled HP73 as a probe [Mickey T. et al. (Miki, T. et al. , Gene, 83 : 137-146, 1989). From the human lung fetal fibroblast cDNA library, a total GIG47 cDNA clone with 703 bp inserted into the pCEV-LAC vector was obtained and registered with GeneBank, USA, on September 24, 2004 as AY762102 (published date: 2006 December 22). The AY762102 gene is Homo sapiens neuron derived neurotrophic factor (NENF), other genes registered with Gene Bank, Homo sapiens neuron derived neurotrophic factor (ACENCESS NM_013349). Homo sapiens mRNA for SCIRP10-related protein variant (clone: KAT11637) genes, sapiens secreted protein of unknown function (SPUF), mRNA, compte cds gene, ACCESSION AK223135 And sequence similarity. However, the function of these genes or their association with diseases such as cancer has not been revealed or reported. Accordingly, the present inventors have confirmed that the primary cancer gene, ie, the AY762102 gene, according to the present invention is deeply involved in the development of various cancers of humans including liver cancer.

The present inventors confirmed that the expression of the GIG47 primary cancer gene was greatly increased in various human tissues including liver cancer, while the expression was weak or absent in various human normal tissues.

The entire sequence of GIG47, consisting of 703 bp, is as shown in SEQ ID NO: 1. In the nucleotide sequence represented by SEQ ID NO: 1, the entire open reading frame of the primary oncogene of the present invention corresponds to nucleotides 17 to 535, which encodes a protein consisting of 172 amino acids represented by SEQ ID NO: 2.

Example  7: in various cells GIG47  Northern of genes Blot  analysis

As in Example 1, total RNA was extracted from normal liver tissue, liver cancer tissue, and liver cancer cell line HepG2.

To determine the degree of GIG47 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). The blots were hybridized with ³² P-labeled random primed HP73 cDNA probes prepared using the Ready Prime 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.

2A shows a result of Northern blot analysis confirming the expression of GIG47 primary cancer gene in normal liver tissue, liver cancer tissue and liver cancer cell lines (HepG2). As can be seen in A of FIG. 2, significant gene expression was observed in hepatocellular tissues and HepG2 cell lines, which are liver cancer cell lines, but was not or weak in normal tissues. 2B shows the presence of mRNA by hybridizing the same sample with β-actin probe.

Figure 3a shows the expression of the GIG47 primary cancer gene in normal human 12-row multiple tissue (Clontech), brain, heart, rhabdomyo, colon, thymus, spleen, kidney, liver, small intestine, placenta, lung and peripheral blood leukocyte tissue. 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, GIG47 mRNA (about 1.2 kb) was not expressed or very weak in many other normal tissues as in normal liver tissue and only weakly expressed in normal heart.

4 a shows human cancer cell lines, HL-60, HeLa, K-562, MOLT-4, Raji. SW480, A549 and G361 (Clontech) shows the Northern blot analysis results confirming the expression of the GIG47 primary cancer gene. 4b shows the presence of mRNA by hybridizing the same sample with β-actin probe. As can be seen in Figure 4a, GIG47 is a promyeloid leukemia cell line HL-60, HeLa cervical cancer cell line, chronic myeloid leukemia cell line K-562, lymphocyte leukemia cell line MOLT-4, Burkitt The expression was strong in the lymphoma cell line Raji, colon cancer cell line SW480, lung cancer cell line A549, and skin cancer cell line G361. At the same time, about 5.0 kb of GIG47 mRNA is also very weakly expressed.

Example 8: GIG47 Sizing of Proteins Expressed After Transfection of E. Coli by Proto-Genes

The entire GIG47 primary cancer gene having the nucleotide sequence represented by SEQ ID NO: 1 was inserted into the multi-cloning site of the pBAD / thio-Topo vector (Invitrogen, USA), and the resulting pBAD / thio-Topo / GIG47 vector was transferred to E. coli Top10. (Invitrogen, USA). In front of the multi-cloning site of the pBAD / thio-Topo vector, expression proteins HT-Thioredoxin are inserted. The transfected E. coli was shaken in LB broth, then diluted to 1/100 and incubated for another 3 hours. 0.5 mM L-arabinose (Sigma) was added thereto to induce protein production. E. coli cells in culture medium before and after L-arabinose induction were subjected to ultrasonic grinding and electrophoresed on 12% sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE). 5 shows the results of SDS-PAGE expression of the protein of E. coli Top10 strain transfected with pBAD / thio-Topo / GIG47 vector. A fusion protein band of about 34 kDa was clearly observed after L-arabinose induction. This 34 kDa fusion protein contains about 15 kDa HT-thioredoxine protein and about 19 kDa GIG47 protein inserted into pBAD / thio-Topo / GIG47 vector.

The primary cancer gene of the present invention can be effectively used for the diagnosis of various cancers including liver cancer, leukemia, uterine cancer, malignant lymphoma, colorectal cancer, lung cancer, skin cancer, etc., the production of transgenic animals, antisense gene therapy, RNA interference gene therapy and the like. .

<110> KIM, hyun kee <120> HUMAN PROTOONCOGENE, PROTEIN ENCODED BY SAME, EXPRESSION VECTOR          CONTAINING SAME, AND CELL TRANSFORMED BY SAID VECTOR <160> 4 <170> KopatentIn 1.71 <210> 1 <211> 703 <212> DNA <213> Homo sapiens <220> <221> CDS (222) (17) .. (532) <220> <221> terminator <222> (533) .. (535) <400> 1 gcgctgcgcg ctcacc atg gtg ggc ccc gcg ccg cgg cgg cgg ctg cgg 49                       Met Val Gly Pro Ala Pro Arg Arg Arg Leu Arg                         1 5 10 ccg ctg gca gcg ctg gcc ctg gtc ctg gcg ctg gcc ccg ggg ctg ccc 97 Pro Leu Ala Ala Leu Ala Leu Val Leu Ala Leu Ala Pro Gly Leu Pro              15 20 25 aca gcc cgg gcc ggg cag aca ccg cgc cct gcc gag cgg ggg ccc cca 145 Thr Ala Arg Ala Gly Gln Thr Pro Arg Pro Ala Glu Arg Gly Pro Pro          30 35 40 gtg cgg ctt ttc acc gag gag gag ctg gcc cgc tat ggc ggg gag gag 193 Val Arg Leu Phe Thr Glu Glu Glu Leu Ala Arg Tyr Gly Gly Glu Glu      45 50 55 gaa gat cag ccc atc tac ttg gca gtg aag gga gtg gtg ttt gat gtc 241 Glu Asp Gln Pro Ile Tyr Leu Ala Val Lys Gly Val Val Phe Asp Val  60 65 70 75 acc tcc gga aag gag ttt tat gga cga gga gcc ccc tac aat gcc ttg 289 Thr Ser Gly Lys Glu Phe Tyr Gly Arg Gly Ala Pro Tyr Asn Ala Leu                  80 85 90 acg ggg aag gac tcc act aga ggg gta gcc aag atg tcc ttg gat cct 337 Thr Gly Lys Asp Ser Thr Arg Gly Val Ala Lys Met Ser Leu Asp Pro              95 100 105 gca gac ctc acc cat gac act acg ggt ctc acg gcc aag gaa ctg gag 385 Ala Asp Leu Thr His Asp Thr Thr Gly Leu Thr Ala Lys Glu Leu Glu         110 115 120 gcc ctg gat gag gtc ttc acc aaa gtg tac aaa gcc aaa tac ccc atc 433 Ala Leu Asp Glu Val Phe Thr Lys Val Tyr Lys Ala Lys Tyr Pro Ile     125 130 135 gtc ggc tac act gcc cgg aga att ctc aat gag gat ggc agc cct aac 481 Val Gly Tyr Thr Ala Arg Arg Ile Leu Asn Glu Asp Gly Ser Pro Asn 140 145 150 155 ctg gac ttc aag cct gaa gac cag ccc cat ttt gac atc aag gat gag 529 Leu Asp Phe Lys Pro Glu Asp Gln Pro His Phe Asp Ile Lys Asp Glu                 160 165 170 ttc tgatgttc cccctgcagg agcaggttct tgggagcgtg aggcaggaag 580 Phe acactaggtg ctgaatctcc tgcaaaactg gctgcctgga ggccctgagc cacccagatc 640 tgaataaaac agatgcttac cctggaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 700 aaa 703 <210> 2 <211> 172 <212> PRT <213> Homo sapiens <400> 2 Met Val Gly Pro Ala Pro Arg Arg Arg Leu Arg Pro Leu Ala Ala Leu   1 5 10 15 Ala Leu Val Leu Ala Leu Ala Pro Gly Leu Pro Thr Ala Arg Ala Gly              20 25 30 Gln Thr Pro Arg Pro Ala Glu Arg Gly Pro Pro Val Arg Leu Phe Thr          35 40 45 Glu Glu Glu Leu Ala Arg Tyr Gly Gly Glu Glu Glu Asp Gln Pro Ile      50 55 60 Tyr Leu Ala Val Lys Gly Val Val Phe Asp Val Thr Ser Gly Lys Glu  65 70 75 80 Phe Tyr Gly Arg Gly Ala Pro Tyr Asn Ala Leu Thr Gly Lys Asp Ser                  85 90 95 Thr Arg Gly Val Ala Lys Met Ser Leu Asp Pro Ala Asp Leu Thr His             100 105 110 Asp Thr Thr Gly Leu Thr Ala Lys Glu Leu Glu Ala Leu Asp Glu Val         115 120 125 Phe Thr Lys Val Tyr Lys Ala Lys Tyr Pro Ile Val Gly Tyr Thr Ala     130 135 140 Arg Arg Ile Leu Asn Glu Asp Gly Ser Pro Asn Leu Asp Phe Lys Pro 145 150 155 160 Glu Asp Gln Pro His Phe Asp Ile Lys Asp Glu Phe                 165 170 <210> 3 <211> 16 <212> DNA <213> Artificial Sequence <220> <223> H-TIIC anchored oligo-dT primer <400> 3 aagctttttt tttttc 16 <210> 4 <211> 13 <212> DNA <213> Artificial Sequence <220> <223> H-AP23, 5 'random primer <400> 4 aagcttggct atg 13  

Claims (18)

delete delete delete delete delete delete delete delete delete delete Cancer diagnostic kit for testing for the presence of the gene in a sample, including all or part of a primary cancer gene having a nucleotide sequence encoding a protein having an amino acid sequence represented by SEQ ID NO: 2 or a nucleotide sequence represented by SEQ ID NO: 1 . The diagnostic kit of claim 11, wherein the cancer diagnostic kit further comprises a detection label. A cancer diagnostic kit for examining the presence of a primary cancer protein represented by SEQ ID NO: 2, comprising an antibody specific for a primary cancer protein having an amino acid sequence represented by SEQ ID NO: 2. The cancer diagnostic kit of claim 13, wherein the antibody is in a form associated with a detection label. An antisense polynucleotide that inhibits the expression of a GIG 47 primary cancer protein encoded by a nucleotide sequence encoding a protein having an amino acid sequence represented by SEQ ID NO: 2 or a primary cancer gene having a nucleotide sequence represented by SEQ ID NO: 1. An interference RNA (RNAi) molecule that inhibits the expression of a GIG 47 primary cancer protein encoded by a nucleotide sequence encoding a protein having an amino acid sequence represented by SEQ ID NO: 2 or a primary cancer gene having a nucleotide sequence represented by SEQ ID NO: 1. A cancer suppression composition for inhibiting the expression of a primary cancer gene having a nucleotide sequence encoding a protein having an amino acid sequence represented by SEQ ID NO: 2 comprising an antisense polynucleotide according to claim 15.  A cancer suppression composition for inhibiting the expression of a primary cancer gene having a nucleotide sequence encoding a protein having an amino acid sequence represented by SEQ ID NO: 2 comprising an interference RNA molecule according to claim 16.

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