KR100472747B1 - Human protooncogene and protein encoded therein - Google Patents

Abstract

The present invention relates to a new primary cancer gene and a protein encoded by the primary cancer gene. The primary cancer gene of the present invention is a novel gene having no homology with existing reported primary cancer genes, such as breast cancer, ovarian cancer, leukemia, lymphoma, uterine cancer, It can be effectively used for diagnosis of cancer including lung cancer, colorectal cancer, skin cancer and the like, preparation of transgenic animals and anti-sense gene therapy.

Description

HUMAN PROTOONCOGENE AND PROTEIN ENCODED THEREIN}

The present invention relates to novel genes and proteins encoded thereby that have no homology with existing reported cancer genes.

Higher animals, including humans, have about 100,000 genes, but only about 15% of them are expressed in each individual. Thus, depending on which gene is selected and expressed, all phenomena of life, including development, differentiation, homeostasis, response to stimuli, cell division cycle control, aging, and apoptosis (programmed cell death), are determined. (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 expression between different cells is a fundamental and effective approach to understanding various biological phenomena.

Taken together, studies on tumor development suggest that several genetic changes, such as loss of chromosomal heterozygosity, activation of primary cancer genes, and inactivation of other tumor suppressor genes, including the p53 gene, may be 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 caused by the activation of the primary cancer gene by amplification of the primary cancer gene. Activation of the primary cancer genes plays an important role in the etiology of many cancers, and it is required to identify them.

Therefore, the present inventors have applied for a patent application by discovering that the primary cancer gene named human cervical cancer 1 (HCCR-1) is specifically expressed only in cancer cells. Patent Application No. 2000-16757, March 31, 2000). Proto-cancerous genes cause changes in the qualitative and quantitative expression of corresponding protein products through structural variations (Weinberg, RA, Cancer Research 49: 3713-3721, 1989). Cancer proteins destroy signal transduction pathways at the cell surface, cytoplasm, or nucleus. In the absence of tumor suppressor gene products, cancer proteins, along with other cancer proteins, promote cell proliferation and promote the cell cycle. It disrupts and also disrupts apoptosis mechanisms (Todd, R., Anticancer Research 19: 4729-4746, 2000).

Yeast two-hybrid methods to characterize these proteins and their interactions have been invented since 1989 (Fields, S. and Song, O., Nature 340: 245-246, 1989). The mechanism of death (Wallach, D. et al., Curr Opin Immunol. 10: 131-136, 1998) and various genome systems have been used to identify life phenomena (Pandey, A. and Mann, M.). , Nature 405: 837-846, 2000). The inventors have identified a new primary cancer gene KG-18 gene using the yeast E-hybrid method and reported it to the US Gene Bank to be notified of the new gene (GenBank Accession No .; AF 441865).

The primary cancer gene can be effectively used for the diagnosis, prevention and treatment of various cancers such as breast cancer, ovarian cancer, leukemia, lymphoma, uterine cancer, lung cancer, colon cancer, skin cancer and the like.

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

Another object of the present invention is to provide a recombinant vector comprising the primary cancer gene or a fragment thereof and a microorganism transformed thereby.

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

Another object of the present invention is to treat or prevent cancer using the antisense gene.

According to one aspect of the present invention, a novel primary cancer gene or fragment thereof having a nucleotide sequence of SEQ ID NO: 1 is provided.

According to another aspect of the present invention, there is provided a recombinant vector comprising the primary cancer gene or a fragment thereof and a microorganism transformed thereby.

According to another aspect of the invention, a protein or fragment thereof having an amino acid sequence of SEQ ID NO: 2 is provided.

The novel protocogene of the present invention (protooncogene), named KG-18, has a full base sequence of 523 bp length as indicated by SEQ ID NO: 1, and is filed by the applicant of Korean Patent Application No. 2000 Human cervical cancer 1 described in -16757 (Human Cervical Cancer 1 protooncogene, HCCR-1, hereinafter referred to as "HCCR-1 primary cancer gene").

In the nucleotide sequence of SEQ ID NO: 1, the open reading frame corresponding to the nucleotide number 4 to 339 site is the entire protein coding region, and the amino acid sequence derived therefrom is shown in SEQ ID NO: 2 and is represented by 111 amino acids. (Hereinafter referred to as "KG-18 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 portions other than the coding region, and such modified genes are also included in the scope of the present invention. Accordingly, the present invention also encompasses polynucleotides having fragments of the above genes and bases having substantially the same base sequence as the original cancer gene of SEQ ID NO: 1. By substantially identical polynucleotides are meant those having sequence homology of at least 80%, preferably at least 90% and most preferably at least 95%.

The protein expressed from the primary cancer gene of the present invention is composed of 111 amino acids, has a sequence such as SEQ ID NO: 2, and is about 12 kd in size. The protein having the property of binding to a protein encoded by the HCCR-1 primary cancer gene is referred to as "HCCR-BP (HCCR-binding protein)" or "KG-18 protein" in the present invention. However, even in the amino acid sequence of the protein, substitution, addition or deletion of amino acids may be made within a range that does not affect the function of the protein, and only a part of the protein may be used depending on the purpose. Such modified amino acid sequences are also within the scope of the present invention. Accordingly, the present invention also encompasses polypeptides and fragments thereof having substantially the same amino acid sequence as the carcinogenic protein, wherein substantially identical polypeptides are at least 80%, preferably at least 90% and most preferably at least 95% sequences It means things with same sex.

The primary 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 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. Cells transformed with the vector containing the KG-18 primary cancer gene or fragment thereof are referred to as "KG-18 cells".

Such a transformed host can be used to replicate the gene DNA of the present invention in large quantities or to mass produce proteins. For example, a pGEM-T vector (Promega) was used to transfect the E. coli DH5α gene into the E. coli DH5α gene. The E. coli DH5α / KG-18 / pGEM-T thus obtained was transfected. As of date, it was deposited with the Korea Collection for Type Cultures (KCTC) as Korea Deposit No. KCTC 10107BP.

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

The gene of the present invention is expressed in normal human tissues, such as Northern blot, such as the brain, heart, skeletal muscle, large intestine, thymus, spleen, kidney, liver, small intestine, placenta, lung and peripheral blood While little is confirmed, overexpression is confirmed in breast cancer, ovarian cancer, leukemia, lymphoma, uterine cancer, lung cancer, colon cancer, and skin cancer cell lines, and thus is considered to be a potent oncogenic gene causing the cancers. Transfection of this gene into normal 293 human embryonic kidney (HEK) shows a cell shape that is completely different in size and shape from that of normal cells. Analysis of the new cell types under an optical microscope reveals the morphological findings of tumor cells. That is, carcinoma was confirmed at the time of hematoxylin-eosin staining.

In addition, when the normal 293 natal kidney cells transduced with the primary cancer gene of the present invention were inoculated into the gastric site of nude mice, the formation of the mass was observed from about 14 days, and 1.5 cm × 1.5 cm after 21 days. Tumors of size form. The formed mass was confirmed carcinoma upon hematoxylin-eosin staining.

Therefore, the primary cancer gene 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 may include, for example, various methods known in the art, for example, after hybridizing with nucleic acids isolated from the body fluids of a subject using all or part of the primary cancer gene as a probe. Including the reverse transcription polymerase chain reaction, Northern blotting (Northern blotting) and the like to detect whether the subject has a 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 primary cancer gene of the present invention into a mammal, for example, a rodent animal such as a rat, and the gene is preferably introduced at the fertilized egg stage before at least 8 cell stage. 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. In the present invention, the "anti-sense gene" is a polynucleotide having a sequence complementary to some or all of the mRNA that is transcribed from the primary cancer gene of SEQ ID NO: 1 or a part thereof, the protein binding site of the mRNA By introducing a DNA sequence having a sequence capable of binding to and destroying an open reading frame (ORF) of the primary cancer gene into the body of the patient, cancer caused by the expression of the primary cancer gene can be prevented or treated.

The present invention also encompasses within its scope therapeutically effective amounts of the antisense genes of the present invention and includes compositions for the treatment or prevention of cancer by administration to a patient.

In antisense gene therapy with the compositions of the invention, a composition comprising an antisense gene of the invention is administered to a patient in a conventional manner to prevent the expression of the primary cancer gene. For example, antisense oligodeoxynucleotides (ODN) are mixed with poly-L-lysine derivatives by electrostatic attraction according to the method of JS Kim et al., J. Controlled Release 53: 175-182, 1998 The mixture is then administered intravenously to the patient.

The anticancer composition of the present invention contains the antisense gene of the present invention as an active ingredient 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 actually administered can be determined by considering 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 cancer genes of the present invention can be usefully used for producing antibodies as diagnostic tools. The antibody of the present invention is used as a monoclonal antibody or a polyclonal antibody 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 primary cancer gene of the present invention or a fragment thereof. These antibodies can be used to prepare enzyme linked immunosorbent assays (ELISAs), radioimmunoassays (RIAs), sandwich assays, western blots on polyacryl gels, or the like. Cancer can be diagnosed by confirming whether the said protein was expressed in the bodily fluid sample of a subject by methods, such as an immune blot.

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 are formed in nude mice. 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 limited thereto.

Example 1 Yeast E-Hybrid Assay

To find a protein that binds to the protein product of the human primary cancer gene HCCR-1 gene (Genebank Accession No .: AF195651), we use the matchmaker LexA Two-Hybrid System (Clontech. Laboratories). Experiments were performed using existing reported methods (Golemis, EA, et al. , Current Protocols in Molecular Biology John Wiley & Sons, Inc. Chapters 20.0 and 20.1, 1996).

The strains and vectors used in the following experiments were those included in the catalog # K1609-1 kit from Clontech.

After transforming the p8op-lacZ vector into EGY48, a yeast strain, strains were selected by growing them on SD / -uracil / glucose plates, which are synthetic dropout medium without uracil. The strains selected therefrom were cultured in SD / -uracil / glucose media, and the vector prepared by inserting the HCCR-1 gene into the Bam HI and Sal I sites of the pLexA vector was bait. Were transformed. In order to confirm that the HCCR-1 gene cloned into the pLexA vector was properly expressed, Western blotting was performed using LexA antibody to obtain a band at a desired size of 64 to 65 kDa. The identified colonies were cultured in SD / -uracil, -histidine / glucose medium and then transformed back into pBD42AD vector, an AD fusion library derived from human fetal brain. Colony lifting assays (Breeden, L. & Nasmyth, K., Cold Spring Harbor Symposium Quant. Bio. 50: 643-650, 1985) were performed to confirm that the bait and the library bind. When the bait and library combine, blue colonies form on the plate containing the X-gal. After culturing yeast, DNA was extracted using glass beads, transformed to E. coli KC8 by electroporation, and plated on M9 minimal medium to select transformants. . The plasmid DNA was extracted from the obtained transformants, transformed into E. coli DH5α, and the DNA was extracted to treat Hind III to obtain a clone having a desired size of about 0.5 kb.

Example 2 Total cDNA Sequence Analysis of the KG-18 Proon Cancer Gene

The clones obtained in Example 1 were sequenced according to dideoxy chain termination using a Sequencena version 2.0 DNA sequencing kit (United States Biochemical) according to a conventional method. Analysis was performed. As a result, the primary cancer gene consisting of 523 bp shown in SEQ ID NO: 1 was identified and referred to as "KG-18", and the entire KG-18 cDNA clone into which this gene was inserted was registered on Nov. 2, 2001, with the registration no. Registered with GenBank.

In the nucleotide sequence of SEQ ID NO: 1, the full transcription reading frame of the primary cancer gene KG-18 of the present invention corresponds to nucleotides 4 to 339, which encodes a protein consisting of 111 amino acids of SEQ ID NO: 2 It is expected to.

The KG-18 gene was linked to a cloning site of the pGEM-T-easy vector (Promega) to T4 DNA ligase to prepare KG-18 expressing plasmid DNA, and the E. coli DH5α (Stratagene) was transfected with the prepared plasmid. Switched.

The obtained Escherichia coli DH5α / KG-18 / pGEM-T was deposited on November 8, 2001, at the Korea Biotechnology Research Institute Gene Bank as Accession No. KCTC 10107BP.

Example 3 Northern Blot Analysis of the KG-18 Gene in Various Cells and Tissues

To determine the degree of KG-18 gene expression in normal tissues and cancer cells, commercially available normal human 12-row multiple tissues (Clontech), each of which extracted normal RNA from 12 organs and blotted onto a nylon membrane (Fig. 1a and 1b) and commercialized human cancer cell 8-column multiple tissues (Clontech Co., Ltd.) (FIGS. 2A and 2B) extracted RNAs from 8 kinds of cancer cells and blotted onto the membranes were compared to compare the expression differences . Both membranes hybridized blots with ³² P-labeled random primed KG-18 full cDNA probes prepared using the Rediprime II random prime labeling system (Amersham). Northern blot analysis was repeated twice, and the results were quantified using densitometry and normalized to β-actin.

FIG. 1A shows KG-18 primary cancer genes in normal human 12-row multiple tissues (Clontech), ie brain, heart, skeletal muscle, large intestine, thymus, spleen, kidney, liver, small intestine, placenta, lung and peripheral blood leukocyte tissue Northern blot analysis results confirm that the expression is shown. 1B shows the presence of mRNA by hybridizing the same sample with β-actin probe. As can be seen in Figure 1a, 0.5 kb transcript, KG-18 mRNA transcript (transcript) was not found in several normal tissues.

Figure 2a is a Northern blot analysis confirming the expression of the KG-18 primary cancer gene in human cancer cell lines (Clontech), that is, HL-60, HeLa, K-562, MOLT-4, Raji, SW480, A549 and G361 cell lines The results are shown. 2B shows the presence of mRNA by hybridizing the same sample with β-actin probe. As can be seen in Figure 2a, 0.5 kb transcript of KG-18 is HL-60, a promyeloid cell leukemia cell line, HeLa cells, uterine cancer cell line, K-562, a chronic myeloid leukemia cell line, MOLT-4, a lymphocyte leukemia cell line , Burkitt lymphoma cell line Raji, colon cancer cell line SW480, lung cancer cell line A549 and skin cancer melanoma cell line G361 cell lines.

In addition, approximately 7.5 kb transcripts, presumed to be transcripts containing the KG-18 primary cancer gene, were also HL-60, a promyelocytic leukemia cell line, HeLa cell, a uterine cancer cell line, K-562, a lymphoid leukemia cell line, and lymphocyte leukemia. High levels were expressed in cell lines MOLT-4, Burkitt's lymphoma cell line Raji, colon cancer cell line SW480, lung cancer cell line A549, and skin cancer melanoma cell line G361 cell lines.

Obtained from healthy normal ovaries, breast and lung tissues, and ovarian, breast and lung cancer patients using the RNeasy Total RNA Kit (Qiagen) to confirm the expression of the KG-18 gene in ovarian, breast and lung cancers. Total RNA was extracted from ovarian cancer, breast cancer and lung cancer tissues. 20 μg of total RNA extracted from each cancer tissue was electrophoresed on a 1% formaldehyde agarose gel and then transferred to a nylon membrane (Boehringer-Mannheim). Blots were hybridized with ³² P-labeled random primed KG-18 full cDNA probes prepared using the Rediprime II random prime labeling system (Amersham). Northern blot analysis was repeated twice, and the results were quantified using densitometry and normalized to β-actin.

Figure 3a shows the results of Northern blot analysis confirming the expression of the KG-18 primary cancer gene in normal ovarian tissue, ovarian cancer tissues. 3b shows the presence of mRNA by hybridizing the same sample with β-actin probe. As can be seen in Figure 3a, significant gene expression of 0.5 kb KG-18 mRNA transcript was observed in ovarian cancer tissues, but low expression in normal ovarian tissues. 3A and 3B, normal fever (N) represents normal ovarian tissue and dark fever (C) represents ovarian cancer tissue, respectively.

Figure 4a shows the northern blot analysis results confirming the expression of the KG-18 primary cancer gene in normal breast tissue and breast cancer tissues. 4b shows the presence of mRNA by hybridizing the same sample with β-actin probe. As can be seen in FIG. 4A, significant gene expression of 0.5 kb KG-18 mRNA transcript was observed in breast cancer tissues, but was low in normal breast tissues. 4A and 4B, normal fever (N) represents normal breast tissue and dark fever (C) represents breast cancer tissue, respectively.

Figure 5a shows the results of Northern blot analysis confirming the expression of the KG-18 primary cancer gene in normal lung tissue and lung cancer tissues. Figure 5b shows the presence of mRNA by hybridizing the same sample with β-actin probe. As can be seen in FIG. 5A, significant gene expression of 0.5 kb KG-18 mRNA transcript was observed in lung cancer tissues, but was low in normal lung tissues. 5A and 5B, normal fever (N) represents normal lung tissue and dark fever (C) represents lung cancer tissue, respectively.

Example 4 Preparation of Expression Vectors and Transformed Cells

(Step 1) Preparation of a Vector Containing KG-18

Expression vectors containing the coding region of KG-18 were prepared as follows.

In case of subsequent western blotting on pcDNA3 (Invitrogen) eukaryotic expression vector, glutathione-S transferase was first treated by Hind III and Kpn I restriction enzymes to confirm gene insertion. GST) gene was introduced. After the fusion vector pcDNA3-GST was treated with restriction enzymes BamH I and Xba I, the KG-18 gene (corresponding to 4 to 339 bases in SEQ ID NO: 1) having the entire coding sequence was linked to the vector. The resulting pcDNA3 / GST / KG-18 expression vector was transduced into 293 human-born renal epithelial cells (ATCC CRL 1573) using lipofectamine (Gibco BRL) and DMEM containing G418 (Gibco BRL) Gibco BRL) was selected in the medium. 293 cells transduced with the KG-18 gene were referred to as "KG-18 cells".

(Step 2) 293 human-born renal epithelial cells transduced with KG-18 primary cancer gene

Normal 293 epithelial cells and 293 cells expressing KG-18 (KG-18 cells) obtained in step 1) were cultured monolayer in DMEM medium containing 10% serum, and then the growth pattern was determined by phase contrast microscopy, Olympus) and the results are shown in Figures 6a and 6b.

As shown in FIG. 6A, normal 293 cells, differentiated epithelial cells, are spindle cells with long, slender nuclei and poor cytoplasm. When KG-18 is expressed in KG-18 cells, the morphology of the cells was changed to a polygonal shape with an ovoid nucleus and a large cytoplasm as shown in FIG. 6B.

Monolayer cultured KG-18 cells with hematoxylin-eosin (H & E) showed staining of the nucleus polymorphism, distinct nucleolus and granular chromatin and tumor giant cells. Atypical mitosis was observed (FIG. 7).

Example 5 Investigation of Tumor Formation Ability of KG-18 Proon Cancer Gene in Animals

Human 293 cells (KG-18 cells) transfected with 5 × 10 ⁶ primary cancer gene KG-18 were examined in 10 nude mice at 5 weeks of age (athymic nu / nu on BALB / c background; Daeduk Chemical Research Institute). Each was injected subcutaneously at the gastric site. Nude mice were sacrificed when subcutaneous tumors reached 1.5-2.5 cm in size.

As shown in FIG. 8, all 10 mice injected with KG-18 cells showed clear tumors after 21 days. Nude mice transplanted with KG-18 cells exhibit carcinoma characteristics. Figure 9 shows the morphological characteristics after staining the mass formed by 293 cells transduced with KG-18 pro-oncogenes in hematoxylin-eosin in nude mice, showing typical carcinoma cells isolated by fibrous interstitial tissue. Indicates.

<Example 6> Determination of the size of protein expressed after transfection of E. coli KG-18 primary cancer gene

PGEX 4T-3 vector in which the entire coding region of the KG-18 primary cancer gene corresponding to nucleotide numbers 4 to 339 of SEQ ID NO: 1 and predicted to encode amino acid numbers 1 to 111 of SEQ ID NO: 2 is fused to the GST gene PGEX 4T-3 / KG-18 vector was prepared by inserting the BamH I and Not I restriction enzyme sites in the multiple cloning site of Amersham Pharmacia Biotech, and transfected with E. coli BL21 (ATCC 47092). The transfected Escherichia coli was shaken for 16 hours at 37 ° C. in LB culture, and then the culture was diluted to 1/100 and incubated for another 3 hours. 1 mM isopropyl β-D-thiogalactopyranose (Isopropyl beta-D-thiogalacto-pyranoside, IPTG, Sigma) was added thereto to induce protein production.

Culture samples were taken before and after IPTG induction and E. coli cells were sonicated and electrophoresed on a 12% SDS-PAGE gel. 10 shows the protein expression pattern of the E. coli BL21 strain transfected with pGEX 4T-3 / KG-18 vector by SDS-PAGE. A protein band of about 38 kDa was clearly observed after IPTG induction. This 38 kDa fusion protein contains a GST protein of about 26 kDa expressed from the gene of the pGEX 4T-3 vector.

The primary cancer gene of the present invention is a novel gene that does not show any homology with existing reported oncogenic genes, and is used for the diagnosis of cancer, transgenic animals, including breast cancer, ovarian cancer, leukemia, lymphoma, uterine cancer, lung cancer, colon cancer, skin cancer, and the like. And anti-sense gene therapy.

Figure 1a shows the expression of the KG-18 primary cancer gene in normal human 12-row multiple tissues, namely brain, heart, skeletal muscle, colon, thymus, spleen, kidney, liver, small intestine, placenta, lung and peripheral blood leukocytes. Shows the results of the blot analysis,

Figure 1b shows the results obtained by hybridizing the same samples as in Figure 1a with β-actin,

Figure 2a is a promyeloid leukemia cell line HL-60, uterine cancer cell line HeLa, chronic myeloid leukemia cell line K-562, lymphocyte leukemia cell line MOLT-4, Burkitt lymphoma cell line Raji, colon cancer cell line SW480 , Northern blot analysis of the lung cancer cell line A549 and melanoma skin cancer cell line G361 to confirm whether the expression of the KG-18 primary cancer gene,

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 KG-18 primary cancer gene in ovarian cancer tissues,

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

Figure 4a shows the Northern blot analysis results confirming the expression of the KG-18 primary cancer gene in breast cancer tissues,

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

Figure 5a shows the Northern blot analysis results confirming the expression of the KG-18 primary cancer gene in lung cancer tissues,

FIG. 5b shows the result obtained by hybridizing the same samples as in FIG. 5a with β-actin,

Figure 6a is a single phase culture of wild-type 293 human-born kidney cells after observation of the growth pattern under a phase contrast microscope,

Figure 6b is a monolayer culture of 293 cells transduced with KG-18 primary cancer gene and observed the growth pattern under a phase contrast microscope,

7 is a monolayer culture of 293 cells transduced with the KG-18 prostate gene and stained with hematoxylin-eosin to observe the morphological characteristics of the cells.

Figure 8 confirms the tumorigenic capacity of 293 cells transduced with KG-18 primary cancer gene in nude mice,

Figure 9 shows the morphological characteristics after staining with hematoxylin-eosin the mass formed by 293 cells transduced with KG-18 prostate gene in nude mice,

Figure 10 is the result of electrophoresis on the SDS-PAGE gel the size of the protein expressed before and after IPTG induction after transduction of the KG-18 primary cancer gene in E. coli.

<110> KIM, Jin Woo <120> HUMAN PROTOONCOGENE AND PROTEIN ENCODED THEREIN <130> FPD / 200202-0009 <160> 2 <170> KopatentIn 1.71 <210> 1 <211> 523 <212> DNA <213> Homo sapiens <220> <221> CDS (222) (4) .. (336) <223> cervical cancer oncogene binding protein <400> 1 gat atg tcc gtg gag aca ctg tgg aaa gtc tgg acc gag ctc ttg 45 Met Ser Val Glu Thr Leu Trp Lys Val Trp Thr Glu Leu Leu 1 5 10 gat gtt ctt gga ctt gac gtc tcc aac ctg tcc cag tat ttc agc cca 93 Asp Val Leu Gly Leu Asp Val Ser Asn Leu Ser Gln Tyr Phe Ser Pro 15 20 25 30 gcc tcg gtg tcc agc agc ccg gcc cgc gcg ctc ctg ctg gtc ggc gtc 141 Ala Ser Val Ser Ser Ser Pro Ala Arg Ala Leu Leu Leu Val Gly Val 35 40 45 gtc ctc ctg gcc tac tgg ttc ttg tcc ctg acc ctg ggc ttc act ttc 189 Val Leu Leu Ala Tyr Trp Phe Leu Ser Leu Thr Leu Gly Phe Thr Phe 50 55 60 agc gtc ctg cac gtg gtg ttc ggc cgc ttc ttc tgg atc gtg cgg tcg 237 Ser Val Leu His Val Val Phe Gly Arg Phe Phe Trp Ile Val Arg Ser 65 70 75 tcc tgt ttt cca tgt cct gcg tgt aca tcc tgc aca agt acg agg gcg 285 Ser Cys Phe Pro Cys Pro Ala Cys Thr Ser Cys Thr Ser Thr Arg Ala 80 85 90 agc cgg aga acg cgg tgc tgc cgc tgt gct tcg tgg tgg ccg tct act 333 Ser Arg Arg Thr Arg Cys Cys Arg Cys Ala Ser Trp Trp Pro Ser Thr 95 100 105 110 tca tgac cgggcccatg ggcttctact ggcgaagcag tcccagcggc cccagcaacc 390 Ser ccagcaaccc cagcgtggag gagaagctgg agcacctgga gaagcaggtc agactgctca 450 acatccgtct caaccgggtg ctcgagagcc tggaccgctc caaggacaag tgaaggtcag 510 ccggccgggc ggg 523 <210> 2 <211> 111 <212> PRT <213> Homo sapiens <400> 2 Met Ser Val Glu Thr Leu Trp Lys Val Trp Thr Glu Leu Leu Asp Val 1 5 10 15 Leu Gly Leu Asp Val Ser Asn Leu Ser Gln Tyr Phe Ser Pro Ala Ser 20 25 30 Val Ser Ser Ser Pro Ala Arg Ala Leu Leu Leu Val Gly Val Val Leu 35 40 45 Leu Ala Tyr Trp Phe Leu Ser Leu Thr Leu Gly Phe Thr Phe Ser Val 50 55 60 Leu His Val Val Phe Gly Arg Phe Phe Trp Ile Val Arg Ser Ser Cys 65 70 75 80 Phe Pro Cys Pro Ala Cys Thr Ser Cys Thr Ser Thr Arg Ala Ser Arg 85 90 95 Arg Thr Arg Cys Cys Arg Cys Ala Ser Trp Trp Pro Ser Thr Ser 100 105 110

Claims (9)

Human KG-18 primary cancer gene having the nucleotide sequence of SEQ ID NO: 1. A protein encoded by the KG-18 primary cancer gene of claim 1 and having an amino acid sequence of SEQ ID NO: 2. A vector comprising the primary cancer gene of claim 1. Microorganisms transformed by the vector of claim 3. The method of claim 4, wherein Microorganism that is Escherichia coli DH5α / KG-18 / pGEM-T (Accession No .: KCTC 10107BP). A method for preparing a protein having an amino acid sequence of SEQ ID NO: 2 by culturing the microorganism of claim 4 or 5. Cancer diagnostic kit comprising the original cancer gene of claim 1. Cancer diagnostic kit comprising the protein of claim 2. delete