KR20060090781A - Human protooncogene and protein encoded by same, and expression vector containing same - Google Patents

Abstract

The present invention relates to a novel primary cancer gene and a protein encoded thereby, the primary cancer gene of the present invention is a novel gene for the diagnosis of cancer, including leukemia, uterine cancer, lymphoma, colon cancer, lung cancer, skin cancer, etc. And the like can be effectively used.

Description

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

Figure 1a shows the results of differential development reverse transcription polymerase chain reaction (DDRT-PCR) confirmed the expression of L699 in normal lung tissue, left lung cancer tissue, metastasized lung cancer tissue transitioned from left to right and A549 lung cancer cells; FIG. 1B shows the results of differentially developed reverse transcriptase polymerase chain reaction (DDRT-PCR) confirming the expression of CA325 DNA fragment in normal cervical tissue, cervical tumor tissue, metastatic lymph node tumor tissue and CUMC-6 cancer cells; FIG. 1c shows the results of differentially developed reverse transcriptase polymerase chain reaction (DDRT-PCR) confirming the expression of CA273 DNA fragment in normal cervical tissue, cervical tumor tissue, metastatic lymph node tumor tissue and CUMC-6 cancer cells; FIG. 1D Figure 1e shows the results of the differential development reverse transcriptase polymerase chain reaction (DDRT-PCR) confirming the expression of L667 in normal lung tissue, left lung cancer tissue, metastatic lung cancer tissue transitioned from left to right and A549 lung cancer cells; Differential development reverse transcriptase polymerase chain reaction (DDRT-) confirming the expression of L668 in normal lung tissue, left lung cancer tissue, metastasized lung cancer tissue from left to right, and A549 lung cancer cells PCR results; Fig. 1F shows differentially developed reverse transcriptase polymerase chain reaction (DDRT-) confirming the expression of L211 in normal lung tissue, left lung cancer tissue, metastasized lung cancer tissue from left to right, and A549 lung cancer cells. Showing the results of PCR); Figure 1g shows the results of differential development reverse transcriptase polymerase chain reaction (DDRT-PCR) confirmed the expression of L722 in normal lung tissue, left lung cancer tissue, metastasized lung cancer tissue transitioned from left to right and A549 lung cancer cells; Figure 1h shows the results of differential development reverse transcription polymerase chain reaction (DDRT-PCR) confirmed the expression of L752 in normal lung tissue, left lung cancer tissue, metastasized lung cancer tissue metastasized from left to right and A549 lung cancer cells; FIG. 1i shows the results of differentially developed reverse transcriptase polymerase chain reaction (DDRT-PCR) confirming the expression of L1003 in normal lung tissue, left lung cancer tissue, metastatic lung cancer tissue transitioned from left to right, and A549 lung cancer cells; Figure 1j shows the results of differential reverse transcription polymerase chain reaction (DDRT-PCR) confirming the expression of HP90-8115 DNA fragments in normal cervical tissue, cervical tumor tissue, metastatic lymph node tumor tissue and CUMC-6 cancer cells. .

FIG. 2A shows the results of Northern blot analysis confirming the expression of PIG5 primary cancer genes in normal lung tissue, left lung cancer tissue, metastatic lung cancer tissue metastasized from left lung to right lung, A549, NCI-H2009, and NCI-H441 lung cancer cell lines. FIG. 2B shows the results obtained by hybridizing the same samples as β-actin with FIG. 2A; FIG. 3A shows the PIG6 proto-oncogene of the invention in normal cervical tissue, cervical cancer tissue, cervical metastatic lymph node tissue and cervical cancer cell lines. 3 shows a result obtained by hybridizing the same samples as in FIG. 3A with β-actin, and FIG. 4A shows normal cervical tissue, cervical cancer tissue, and cervical metastatic lymph node tissue. And Northern blot analysis results confirming the expression of the PIG7 primary cancer gene of the present invention in cervical cancer cell lines, Figure 4b Results obtained by hybridizing the same samples as 4a with β-actin, Figure 5A shows normal lung tissue, left lung cancer tissue, metastatic lung cancer tissue metastasized from left lung to right lung, A549, NCI-H2009, NCI-H441 lung cancer cell line Shows a result of Northern blot analysis confirming the expression of the PIG11 primary cancer gene; Figure 5B shows the result obtained by hybridizing the same samples as β-actin in Figure 5A; FIG. 6A shows the results of Northern blot analysis confirming the expression of PIG16 primary cancer genes in normal lung tissue, left lung cancer tissue, metastatic lung cancer tissue metastasized from left lung to right lung, and A549, NCI-H2009, and NCI-H441 lung cancer cell lines. FIG. 6B shows the results obtained by hybridizing the same samples as in FIG. 6A with β-actin; FIG. 7A shows the results of Northern blot analysis confirming the expression of PIG17 primary cancer gene in normal lung tissue, left lung cancer tissue, metastatic lung cancer tissue metastasized from left lung to right lung, A549, NCI-H2009, NCI-H441 lung cancer cell lines. FIG. 7B shows the results obtained by hybridizing the same samples as in FIG. 7A with β-actin; FIG. 8A shows the results of Northern blot analysis confirming the expression of PIG19 primary cancer genes in normal lung tissue, left lung cancer tissue, metastatic lung cancer tissue metastasized from left lung to right lung, A549, NCI-H2009, and NCI-H441 lung cancer cell lines. FIG. 8B shows the results obtained by hybridizing the same samples as in FIG. 8A with β-actin; Figure 9A shows the results of Northern blot analysis confirming the expression of PIG20 primary cancer genes in normal lung tissue, left lung cancer tissue, metastatic lung cancer tissue metastasized from left lung to right lung, A549, NCI-H2009, NCI-H441 lung cancer cell lines FIG. 9B shows the results obtained by hybridizing the same samples as β-actin with FIG. 9A;

10A shows a result of Northern blot analysis confirming the expression of PIG21 primary cancer gene in normal lung tissue, left lung cancer tissue, metastatic lung cancer tissue metastasized from left lung to right lung, A549, NCI-H2009, and NCI-H441 lung cancer cell lines. FIG. 10B shows the results obtained by hybridizing the same samples as in FIG. 10A with β-actin; 11 is a promyelocytic 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 results confirming the expression of HCCRBP2 primary cancer gene in lung cancer cell line A549 and melanoma skin cancer cell line G361, Figure 12a shows normal cervical tissue, cervical cancer tissue, cervical metastatic lymph node tissue and cervical cancer cell line These results show the results of Northern blot analysis confirming the expression of the TRG2 primary cancer gene of the present invention, Figure 12b shows the results obtained by hybridizing the same samples as β-actin Figure 12a.

FIG. 13A shows the results of Northern blot analysis confirming the expression of PIG5 primary cancer gene in normal human 12-row multiple tissues; FIG. 13B shows the result obtained by hybridizing the same sample as that of FIG. 13A with β-actin; FIG.

Figure 14a shows the results of Northern blot analysis confirming the expression of the PIG6 primary cancer gene of the present invention in normal human 12-row multiple tissues, Figure 14b shows the results obtained by hybridizing the same sample as β-actin 14a ,

FIG. 15a shows the results of Northern blot analysis confirming the expression of the PIG7 primary cancer gene of the present invention in normal human 12-row multiple tissues, and FIG. 15b shows the result obtained by hybridizing the same sample as in FIG. 15a with β-actin. ,

Figure 16A shows the results of Northern blot analysis confirming the expression of the PIG11 primary cancer gene in normal human 12-row multiple tissues; Figure 16B shows the results obtained by hybridizing the same sample as in Figure 16A with β-actin;

FIG. 17A shows the results of Northern blot analysis confirming the expression of PIG16 primary cancer gene in normal human 12-row multiple tissues; FIG. 17B shows the result obtained by hybridizing the same sample as that of FIG. 17A with β-actin; FIG.

FIG. 18A shows the results of Northern blot analysis confirming the expression of PIG17 primary cancer gene in normal human 12-row multiple tissues; FIG. 18B shows the result obtained by hybridizing the same sample as FIG. 18A with β-actin; FIG.

FIG. 19A shows the results of Northern blot analysis confirming the expression of PIG19 primary cancer gene in normal human 12-row multiple tissues; FIG. 19B shows the result obtained by hybridizing the same sample as that of FIG. 19A with β-actin;

FIG. 20A shows the results of Northern blot analysis confirming the expression of PIG20 prostate cancer gene in normal human 12-row multiple tissues; FIG. 20B shows the result obtained by hybridizing the same sample as in FIG. 20A with β-actin; FIG.

FIG. 21A shows the results of Northern blot analysis confirming the expression of PIG21 prostate cancer gene in normal human 12-row multiple tissues; FIG. 21B shows the result obtained by hybridizing the same sample as FIG. 21A with β-actin; FIG.

22 is a Northern blot analysis confirming the expression of HCCRBP2 primary cancer genes in normal human 12-row multiple tissues, ie brain, heart, skeletal muscle, colon, thymus, spleen, kidney, liver, small intestine, placenta, lung and peripheral blood leukocytes. The results,

Figure 23a shows the results of the Northern blot analysis confirming the expression of the TRG2 primary cancer gene of the present invention in normal human 12-row multiple tissues, Figure 23b shows the results obtained by hybridizing the same sample as β-actin in Figure 23a .

FIG. 24A shows the results of Northern blot analysis confirming the expression of PIG5 primary cancer gene in human cancer cell lines; FIG. 24B shows the result obtained by hybridizing the same sample as in FIG. 24A with β-actin; FIG.

FIG. 25a shows the results of Northern blot analysis confirming the expression of the PIG6 primary oncogene of the present invention in human cancer cell lines. FIG. 25b shows the result obtained by hybridizing the same sample as in FIG. 25a with β-actin. FIG.

FIG. 26a shows a result of Northern blot analysis confirming the expression of the PIG7 primary cancer gene of the present invention in human cancer cell lines, and FIG. 26b shows the result obtained by hybridizing the same sample as in FIG. 26a with β-actin. FIG.

FIG. 27A shows the results of Northern blot analysis confirming the expression of the PIG11 primary cancer gene in human cancer cell lines; FIG. 27B shows the result obtained by hybridizing the same sample as that of FIG. 27A with β-actin; FIG.

FIG. 28A shows the results of Northern blot analysis confirming the expression of PIG16 primary cancer gene in human cancer cell lines; FIG. 28B shows the result obtained by hybridizing the same sample as that of FIG. 28A with β-actin;

FIG. 29A shows the results of Northern blot analysis confirming the expression of PIG17 primary cancer gene in human cancer cell lines; FIG. 29B shows the result obtained by hybridizing the same sample as FIG. 29A with β-actin; FIG.

FIG. 30A shows the results of Northern blot analysis confirming the expression of PIG19 primary cancer gene in human cancer cell lines; FIG. 30B shows the result obtained by hybridizing the same sample as in FIG. 30A with β-actin; FIG.

FIG. 31A shows the results of Northern blot analysis confirming the expression of PIG20 primary cancer gene in human cancer cell lines; FIG. 31B shows the result obtained by hybridizing the same sample as FIG. 31A with β-actin; FIG.

FIG. 32A shows the results of Northern blot analysis confirming the expression of PIG21 primary cancer gene in human cancer cell lines; FIG. 32B shows the result obtained by hybridizing the same sample as FIG. 32A with β-actin; FIG.

33 shows the results of Northern blot analysis confirming the expression of the HCCRBP2 primary cancer gene in uterine cancer tissues, the bottom of Figure 33 shows the results obtained by hybridizing the same samples as β-actin top of Figure 33,

34 shows the results of Northern blot analysis confirming the expression of HCCRBP2 primary cancer gene in colorectal cancer tissues, the bottom of FIG. 34 shows the results obtained by hybridizing the same samples as β-actin in the top of FIG.

35 shows the results of Northern blot analysis confirming the expression of the HCCRBP2 primary cancer gene in leukemia tissues, the bottom of FIG. 35 shows the results obtained by hybridizing the same samples as β-actin in the top of FIG.

Figure 36a shows the results of Northern blot analysis confirming the expression of the TRG2 primary oncogene of the present invention in human cancer cell lines, Figure 36b shows the result obtained by hybridizing the same sample as in Figure 36a with β-actin,

Figure 37a shows the size of the protein expressed before and after the induction of L-Arabinose after transduction of the PIG5 primary cancer gene of the present invention in sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) Results of electrophoresis; FIG. 37B shows the size of the protein expressed before and after L-Arabinose induction after transduction of the PIG6 pro-oncogene of the present invention into E. coli. Electrophoresis on SDS-PAGE); Figure 37c shows the size of the protein expressed before and after the induction of L-Arabinose after transduction of the E. coli PIG7 gene of the present invention in sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) Results of electrophoresis; FIG. 37D shows the size of the protein expressed before and after the induction of L-Arabinose after transduction of the PIG11 pro-oncogene of the present invention into E. coli. Electrophoresis on SDS-PAGE; FIG. 37E shows the size of protein expressed in sodium dodecyl sulfate- before and after L-Arabinose induction after transduction of the PIG16 pro-oncogene of the present invention into E. coli. Results of electrophoresis on polyacrylamide gel (SDS-PAGE); FIG. 37F shows the transduction of PIG17 proteomic gene of the present invention into Escherichia coli before induction of L-Arabinose. And the result of electrophoresis of the size of the protein to be expressed on sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE); FIG. 37 g shows the transfection of E. coli with the PIG19 proto-oncogene of the present invention. -Arabinose) electrophoresis of the size of the protein expressed before and after induction on sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE); FIG. 37H shows that the PIG20 proto-oncogene of the present invention was transduced into E. coli. The size of the protein expressed before and after L-Arabinose induction was electrophoresed on sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE); FIG. 37I shows the PIG21 proto-oncogene of the present invention. The size of the protein expressed before and after induction of L-Arabinose after transduction in L-Arabinose was determined in sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE). Electrophoresis results; Figure 37j shows the result of electrophoresis of the protein expressed before and after IPTG induction after transduction of HCCRBP2 prostate gene into Escherichia coli; Figure 37K shows the transduction of E. coli TRG2 prototypic gene of the present invention. The size of the protein expressed before and after L-Arabinose induction is the result of electrophoresis on sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE).

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 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 many biological phenomena.

For example, the mRNA differential display method proposed by Liang and Pardee (see supra), currently regulates transcriptional control related to genes and apoptosis related to tumor suppressor genes or cell cycles. It is effectively used to search for transcriptional regulatory genes, etc., and is also used in various ways to investigate the interrelationship of 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 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); and 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 gene plays an important role in the etiology of many cancers, and it is required to identify it.

Accordingly, the present inventors have found that the mechanism of development of lung cancer and cervical cancer at the level of the primary cancer gene revealed that the expression of a human proliferative gene named primary cancer gene is specifically increased only in cancer cells. The primary cancer genes can be effectively used for diagnosis of various cancers such as leukemia, uterine cancer, lymphoma, colon cancer, lung cancer and skin cancer.

Accordingly, it is 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 genes or fragments thereof and the microorganisms transformed thereby.

It is still another object of the present invention to provide proteins and fragments thereof encoded by the primary cancer genes.

Still another object of the present invention is to provide a kit for diagnosing cancer, including the primary cancer genes or fragments thereof.

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

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

According to this another object, the present invention provides a protein having a amino acid sequence of SEQ ID NO: 2 or a fragment thereof.

The present invention also provides a primary cancer gene or fragment thereof having the nucleotide sequence of SEQ ID NO: 5.

The present invention provides a protein or fragment thereof having the amino acid sequence of SEQ ID NO: 6.

The present invention also provides a primary cancer gene or fragment thereof having the nucleotide sequence of SEQ ID NO: 9.

The present invention provides a protein or fragment thereof having the amino acid sequence of SEQ ID NO: 10.

The present invention also provides a primary cancer gene or fragment thereof having the nucleotide sequence of SEQ ID NO: 13.

The present invention provides a protein having a amino acid sequence of SEQ ID NO: 14 or a fragment thereof.

The present invention also provides a primary cancer gene or fragment thereof having the nucleotide sequence of SEQ ID NO: 17.

The present invention provides a protein or fragment thereof having the amino acid sequence of SEQ ID NO: 18.

The present invention also provides a primary cancer gene having a nucleotide sequence of SEQ ID NO: 21 or a fragment thereof.

The present invention provides a protein having a amino acid sequence of SEQ ID NO: 22 or a fragment thereof.

The present invention also provides a primary cancer gene or fragment thereof having the nucleotide sequence of SEQ ID NO: 25.

The present invention provides a protein having a amino acid sequence of SEQ ID NO: 26 or a fragment thereof.

The present invention also provides a primary cancer gene or fragment thereof having the nucleotide sequence of SEQ ID NO: 29.

The present invention provides a protein or fragment thereof having the amino acid sequence of SEQ ID 30.

The present invention also provides a primary cancer gene or fragment thereof having the nucleotide sequence of SEQ ID NO: 33.

The present invention provides a protein or fragment thereof having the amino acid sequence of SEQ ID 34.

In accordance with the above object, the present invention provides a primary cancer gene or fragment thereof having the nucleotide sequence of SEQ ID NO: 37.

The present invention provides a protein or fragment thereof having the amino acid sequence of SEQ ID 38.

The present invention also provides a primary cancer gene or fragment thereof having the nucleotide sequence of SEQ ID NO: 39.

The present invention provides a protein or fragment thereof having the amino acid sequence of SEQ ID 40.

In accordance with another object, the present invention provides a cancer diagnostic kit comprising the original cancer gene or a fragment thereof.

According to another object, the present invention provides a cancer diagnostic kit comprising the primary cancer protein or fragment thereof.

Hereinafter, the present invention will be described in detail.

One. PIG  5

Human proliferation-inducing gene 5 (PIG5, hereinafter referred to as “PIG5 original oncogene”), the protocogene of the present invention, is 1009 bp in length as shown in SEQ ID NO: 1. Has the entire sequence

In the nucleotide sequence of SEQ ID NO: 1, the open reading frame corresponding to the nucleotide numbers 9 to 746 site (744-746: end codon) is the entire protein coding region and the amino acid sequence derived therefrom is SEQ ID NO: It is shown in Figure 2 and consists of 245 amino acids (hereinafter referred to as "PIG5 protein").

The base sequence of SEQ ID NO: 1 was registered in the NIH GenBank database of the National Institutes of Health as registration number AY236486 (published: December 31, 2004). Homo sapiens cDNA FLJ12453 fis, clone NT2RM1000430, moderately similar to Homo sapiens erythroblast macrophage protein EMP mRNA gene and BC006470 were identified to be similar to some gene sequences. Homo sapiens cDNA FLJ12453 gene is known in gene sequence but has not yet been identified.

This study led to the discovery of the PIG5 primary oncogene, which is highly expressed in many human tumors, including lung cancer, while in very low levels in normal tissues.

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-mentioned primary cancer gene of SEQ ID NO: 1 and the nucleotide sequence substantially identical to the primary 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 PIG5 of the present invention is composed of 245 amino acids, has a sequence such as SEQ ID NO: 2, and is about 27 kDa in size.

However, even in the amino acid sequence of the proteins, substitution, addition or deletion of amino acids may be made within a range that does not affect the function of the protein, and only a part of the protein may be used depending on the purpose. Such modified amino acid sequences are also within the scope of the present invention. Thus, the present invention also encompasses polypeptides and fragments thereof having substantially the same amino acid sequence as the carcinogenic proteins, wherein substantially identical polypeptides comprise at least 80%, preferably at least 90% and most preferably at least 95% sequences Means those with homology.

2. PIG  6

Human proliferation-inducing gene 6 (PIG6, hereafter referred to as “PIG6 proto-gene”), the protocogene of the present invention, is the entire 2,964 bp length set forth in SEQ ID NO: 5. Has a nucleotide sequence

In the nucleotide sequence of SEQ ID NO: 5, the open reading frame corresponding to the nucleotide number 293 to 2302 site (2300-2302: end codon) is the entire protein coding region and the amino acid sequence derived therefrom is SEQ ID NO: It is shown at 6 and consists of 669 amino acids (hereinafter referred to as "PIG6 protein").

The nucleotide sequence of SEQ ID NO: 5 was registered in the NIH GenBank database of the National Institutes of Health as registration number AY236487 (published: December 31, 2004). Homo sapiens HLC-6 mRNA gene, NM_003971 Homo sapiens sperm associated antigen 9 (SPAG9), and transcript variant gene were found to be similar. The HLC-6 gene has only been identified on the chromosome and has not yet been identified. The SPAG9 gene is overexpressed in testicular haploid germ and reported to be related to infertility (Shankar, S., et. al . , Biochem . Biophys . Res . Comm ., 243 , 561-565 (1998); Yasuoka, H., et al ., J. Immunol ., 171 , 6883-6890 (2003)). However, this study has identified PIG6 primary oncogenes, which are highly expressed in human tumors, including uterine cancer, and very low in normal tissues.

 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 nucleotide sequence as the above oncogene of SEQ ID NO: 5. Substantially identical polynucleotides refer to those DNAs encoding the same protein translation product as SEQ ID NO: 6, having at least 80%, preferably at least 90%, most preferably at least 95% homology with SEQ ID NO: 5 do.

The protein expressed from the proto-oncogene of the present invention consists of 669 amino acids and has an amino acid sequence set forth in SEQ ID NO: 6 and is about 72 kDa in size. However, even in the amino acid sequence of the protein, substitution, addition or deletion of amino acids may be made within a range that does not affect the function of the protein, and only a part of the protein may be used depending on the purpose. Such modified amino acid sequences are also within the scope of the present invention. Accordingly, the present invention also encompasses polypeptides and fragments thereof having substantially the same amino acid sequence as the carcinogenic protein, wherein substantially identical polypeptides are at least 80%, preferably at least 90% and most preferably at least 95% sequences It means things with same sex.

3. PIG  7

Human proliferation-inducing gene 7, PIG7, hereinafter referred to as “PIG7 prooncogene,” the protocogene of the present invention is the entire 4,301 bp length set forth in SEQ ID NO: 9. Has a nucleotide sequence

In the nucleotide sequence of SEQ ID NO: 9, the open reading frame corresponding to the nucleotide numbers 3536 to 3792 sites (3790-3792: end codon) is the entire protein coding region and the amino acid sequence derived therefrom is SEQ ID NO: It is shown at 10 and consists of 78 amino acids (hereinafter referred to as "PIG7 protein").

The base sequence of SEQ ID NO: 9 was registered in the NIH GenBank database of the National Institutes of Health as No. AY236488 (published: December 31, 2004). Homo sapiens chromosome 18, clone RP11-54G14 gene and gene sequence were confirmed to be similar. The clone RP11-54G14 gene has only been identified on the chromosome and its function has not been identified. However, this study identified PIG7 primary oncogenes, which are highly expressed in various human tumors, including uterine cancer, and very low in various normal tissues.

 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 nucleotide sequence as the above oncogene of SEQ ID NO: 9. Substantially identical polynucleotides refer to those DNAs encoding the same protein translation product as SEQ ID NO: 10 and having at least 80%, preferably at least 90%, most preferably at least 95% homology with SEQ ID NO: 9 do.

The protein expressed from the proto-oncogene of the present invention consists of 78 amino acids, has an amino acid sequence set forth in SEQ ID NO: 10, and is about 9 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 comprise at least 80%, preferably at least 90% and most preferably at least 95% sequences Means those with homology.

4. PIG  11

Human proliferation-inducing gene 11 (PIG11, hereinafter referred to as “PIG11 original oncogene”), which is a protocogene of the present invention, has a total length of 1038 bp as shown in SEQ ID NO: 13. Has a nucleotide sequence

In the nucleotide sequence of SEQ ID NO: 13, an open reading frame corresponding to nucleotide numbers 50 to 931 sites (929-931: end codon) is the entire protein coding region, and the amino acid sequence derived therefrom is SEQ ID NO: It is shown at 14 and consists of 293 amino acids (hereinafter referred to as “PIG11 protein”).

The nucleotide sequence of SEQ ID NO: 13 was registered in the NIH GenBank database of the National Institutes of Health as registration number AY258284 (published: December 31, 2004). As a result of sequencing, BC014991 was registered in this database. Homo sapiens N-methylpurine-DNA glycosylase gene sequence was confirmed to match. The N-methylpurine-DNA glycosylase gene is known as an enzyme that exhibits resistance from DNA damaging agents (O 'Connor, TR and Laval, J., Biochem. Biophys . Res . Comm ., 176 , 1170- 1177 (1991); Wyatt, MD, et al ., Bioessays , 21 , 668-676 (1999)). However, contrary to the previously reported functions, this study uncovered the PIG11 proto-oncogene, which is highly expressed in various human tumors, including lung cancer, and very low in various normal tissues.

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 having the same base sequence as those of the primary cancer gene of SEQ ID NO: 13 and the primary cancer gene of SEQ ID NO: 13. 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 PIG11 of the present invention is composed of 293 amino acids, has a sequence such as SEQ ID NO: 14, and is about 32 kDa in size.

However, even in the amino acid sequence of the proteins, substitution, addition or deletion of amino acids may be made within a range that does not affect the function of the protein, and only a part of the protein may be used depending on the purpose. Such modified amino acid sequences are also within the scope of the present invention. Thus, the present invention also encompasses polypeptides and fragments thereof having substantially the same amino acid sequence as the carcinogenic proteins, wherein substantially identical polypeptides comprise at least 80%, preferably at least 90% and most preferably at least 95% sequences Means those with homology.

5. PIG  16

Human proliferation-inducing gene 16 (PIG16, hereinafter referred to as “PIG16 proto-oncogene”), which is a protocogene of the present invention, is 1682 bp in length as shown by SEQ ID NO: 17. Has the entire sequence

In the nucleotide sequence of SEQ ID NO: 17, the open reading frame corresponding to nucleotide numbers 696 to 1577 sites (1575-1577: end codon) is the entire protein coding region and the amino acid sequence derived therefrom is SEQ ID NO: It is shown at 18 and consists of 293 amino acids (hereinafter referred to as “PIG16 protein”).

The base sequence of SEQ ID NO: 17 is registered in the NIH GenBank database of the National Institutes of Health as registration number AY305873 (published: December 31, 2004). As a result of sequencing analysis, BC014991 is registered in this database.

Homo sapiens N-methylpurine-DNA glycosylase gene was found to be similar to some gene sequences. The N-methylpurine-DNA glycosylase gene has been reported to have a function of repairing damaged DNA (Chakravarti, D., et al., J. Biol . Chem ., 266 , 15710-15715 (1991); O'Brien, PJ and Ellenberger, T., Biochemistry , 42 , 12418-12429 (2003)). However, contrary to the previously reported functions, this study uncovered the PIG16 primary oncogene, which is highly expressed in various human tumors including lung cancer and very low in various normal tissues.

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. Various modifications may be made in the coding region within the coding region, and various modifications or modifications may be made within the range not affecting the expression of the gene even in parts 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 having the same base sequence as the above cancer gene of SEQ ID NO: 17 and the primary cancer gene of SEQ ID NO: 17. 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 PIG16 of the present invention is composed of 293 amino acids, has a sequence such as SEQ ID NO: 18, and is about 32 kDa in size.

However, even in the amino acid sequence of the proteins, substitution, addition or deletion of amino acids may be made within a range that does not affect the function of the protein, and only a part of the protein may be used depending on the purpose. Such modified amino acid sequences are also within the scope of the present invention. Thus, the present invention also encompasses polypeptides and fragments thereof having substantially the same amino acid sequence as the carcinogenic proteins, wherein substantially identical polypeptides comprise at least 80%, preferably at least 90% and most preferably at least 95% sequences Means those with homology.

6. PIG  17

Human proliferation-inducing gene 17 (PIG17, hereinafter referred to as “PIG17 original oncogene”), a protocogene of the present invention, is 626 bp in length as shown in SEQ ID NO: 21. Has the entire sequence

In the nucleotide sequence of SEQ ID NO: 21, an open reading frame corresponding to nucleotide numbers 59 to 610 sites (608-610: end codon) is the entire protein coding region, and the amino acid sequence derived therefrom is SEQ ID NO: It is shown at 22 and consists of 183 amino acids (hereinafter referred to as “PIG17 protein”).

The base sequence of SEQ ID NO: 21 is registered in the NIH GenBank database of the National Institutes of Health as No. AY336092 (published: December 31, 2004). As a result of sequencing analysis, the sequence of NM_016454 is registered in this database. Homo sapiens hypothetical protein LOC51234 (LOC51234) gene, CRCR446 No. Pongo pygmaeus mRNA; The gene sequence of cDNA DKFZp468K0411 (from clone DKFZp468K0411) and Homo sapiens HSPC184 mRNA gene of AF151018 was confirmed to be identical. The HSPC184 gene has been shown to exist in CD34 + hematopoietic stem / progenitor cells and its function has not been reported yet (Zhang, QH., Et . al ., Genome Res., 10 , 1546-1560 (2000)). This study led to the discovery of the PIG17 proto-oncogene, which is highly expressed in many human tumors, including lung cancer, but very low in several normal tissues.

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. Various modifications may be made in the coding region within the coding region, and various modifications or modifications may be made within the range not affecting the expression of the gene even in parts 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 said gene having the same base sequence as said primary cancer gene of SEQ ID NO: 21 and the primary cancer gene of SEQ ID NO: 21. 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 PIG17 of the present invention is composed of 183 amino acids, has a sequence such as SEQ ID NO: 22, and is about 20 kDa in size.

However, even in the amino acid sequence of the proteins, substitution, addition or deletion of amino acids may be made within a range that does not affect the function of the protein, and only a part of the protein may be used depending on the purpose. Such modified amino acid sequences are also within the scope of the present invention. Thus, the present invention also encompasses polypeptides and fragments thereof having substantially the same amino acid sequence as the carcinogenic proteins, wherein substantially identical polypeptides comprise at least 80%, preferably at least 90% and most preferably at least 95% sequences Means those with homology.

7. PIG  19

Human proliferation-inducing gene 19 (PIG19, hereinafter referred to as “PIG19 original oncogene”), which is the protocogene of the present invention, is 1031 bp in length as shown by SEQ ID NO: 25. Has the entire sequence

In the nucleotide sequence of SEQ ID NO: 25, the open reading frame corresponding to nucleotide number 32 to 1030 site (1028-1030: end codon) is the entire protein coding region and the amino acid sequence derived therefrom is SEQ ID NO: It is shown in 26 and consists of 332 amino acids (hereinafter referred to as “PIG19 protein”).

The base sequence of SEQ ID NO: 25 was registered in the NIH GenBank database of the National Institutes of Health as Registration No. AY423727 (published: December 31, 2004). Homo sapiens lactate dehydrogenase A (LDHA) gene and gene sequence were confirmed to match. The LDHA gene converts lactate to pyruvate during aerobic glycolysis and is reported to be highly expressed in the mammary gland during pregnancy and lactation (Tsujibo, H., et. al ., Eur . J. Biochem ., 147 , 9-15 (1985); Li, X., et al ., Biochem. Biophys . Res . Commu . 320 , 625-634 (2004). However, contrary to the previously reported functions, this study uncovered the PIG19 proto-oncogene, which is highly expressed in various human tumors including lung cancer and very low in various normal tissues.

However, due to the degeneracy of the codons or in consideration of the preferred codons in the organism to express the primary cancer gene, the primary cancer gene of the present invention is within a range that does not change the amino acid sequence of the carcinogenic protein expressed from the coding region. Various modifications may be made to the coding region, and various modifications or modifications may be made within a range that does not affect the expression of the gene in parts 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 having the same base sequence as those of the primary cancer gene of SEQ ID NO: 25 and the primary cancer gene of SEQ ID NO: 25. 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 PIG19 of the present invention is composed of 332 amino acids, has a sequence such as SEQ ID NO: 26, and is about 37 kDa in size.

However, even in the amino acid sequence of the proteins, substitution, addition or deletion of amino acids may be made within a range that does not affect the function of the protein, and only a part of the protein may be used depending on the purpose. Such modified amino acid sequences are also within the scope of the present invention. Thus, the present invention also encompasses polypeptides and fragments thereof having substantially the same amino acid sequence as the carcinogenic proteins, wherein substantially identical polypeptides comprise at least 80%, preferably at least 90% and most preferably at least 95% sequences Means those with homology.

8. PIG  20

Human proliferation-inducing gene 20 (PIG20, hereinafter referred to as “PIG20 progeny gene”), the protocogene of the present invention, is 526 bp in length as shown in SEQ ID NO: 29. Has the full sequence

In the nucleotide sequence of SEQ ID NO: 29, the open reading frame corresponding to the nucleotide number 1 to 498 sites (496-498: end codon) is the entire protein coding region and the amino acid sequence derived therefrom is SEQ ID NO: It is shown at 30 and consists of 165 amino acids (hereinafter referred to as “PIG20 protein”).

The base sequence of SEQ ID No. 29 was registered as registered number AY423728 in the NIH GenBank database of the National Institutes of Health (published: December 31, 2004). Homo sapiens reticulocalbin 1, EF-hand calcium binding domain (RCN1) gene and gene sequence were confirmed to be identical. The reticulocalbin 1 gene has been reported as a calcium-binding protein present in the endoplasmic reticulum (Ozawa, M. and Muramatsu, TJ Biol. Chem., 268, 699-705 (1993); Ozawa, MJ Biochem (Tokyo), 117, 1113-1119 (1995)). However, contrary to the previously reported functions, this study uncovered the PIG20 proto-oncogene, which is highly expressed in various human tumors, including lung cancer, and very low in various normal tissues.

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 having the same base sequence as those of the primary cancer gene of SEQ ID NO: 29 and the primary cancer gene of SEQ ID NO: 29. 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 PIG20 of the present invention is composed of 165 amino acids, has a sequence such as SEQ ID NO: 30, and is about 19 kDa in size.

However, even in the amino acid sequence of the proteins, substitution, addition or deletion of amino acids may be made within a range that does not affect the function of the protein, and only a part of the protein may be used depending on the purpose. Such modified amino acid sequences are also within the scope of the present invention. Thus, the present invention also encompasses polypeptides and fragments thereof having substantially the same amino acid sequence as the carcinogenic proteins, wherein substantially identical polypeptides comprise at least 80%, preferably at least 90% and most preferably at least 95% sequences Means those with homology.

9. PIG  21

Human proliferation-inducing gene 21 (PIG21, hereinafter referred to as “PIG21 original oncogene”), which is a protocogene of the present invention, has a length of 965 bp as shown in SEQ ID NO: 33. Has the entire sequence

In the nucleotide sequence of SEQ ID NO: 33, the open reading frame corresponding to the nucleotide numbers 146 to 961 site (959-961: end codon) is the entire protein coding region and the amino acid sequence derived therefrom is SEQ ID NO: It is shown at 34 and consists of 271 amino acids (hereinafter referred to as “PIG21 protein”).

The base sequence of SEQ ID NO: 33 was registered as Registration Number AY336089 in the NIH GenBank Database of the National Institutes of Health (published: December 31, 2004). full-length cDNA clone CS0DA008YE03 of Neuroblastoma of Homo sapiens (human) gene, CR616147 protein, beta polypeptide 2-like 1, and mRNA (cDNA clone IMAGE: 4705256) genes were found to be similar in some gene sequences. The function of these genes has not been reported yet, but this study led to the discovery of the PIG21 proto-oncogene, which is highly expressed in many human tumors, including lung cancer, and very low in several normal tissues.

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 having the same base sequence as those of the primary cancer gene of SEQ ID NO: 33 and the primary cancer gene of SEQ ID NO: 33. 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 PIG21 of the present invention is composed of 271 amino acids, has a sequence such as SEQ ID NO: 34, and is about 30 kDa in size.

However, even in the amino acid sequence of the proteins, substitution, addition or deletion of amino acids may be made within a range that does not affect the function of the protein, and only a part of the protein may be used depending on the purpose. Such modified amino acid sequences are also within the scope of the present invention. Thus, the present invention also encompasses polypeptides and fragments thereof having substantially the same amino acid sequence as the carcinogenic proteins, wherein substantially identical polypeptides comprise at least 80%, preferably at least 90% and most preferably at least 95% sequences Means those with homology.

10. HCCR BP2

The novel primary cancer gene (protooncogene) of the present invention is named HCCRBP2 Human Cervical Cancer 1 protooncogene, HCCR-1, as described in Korean Patent Application No. 2000-16757, filed by Applicant, having a full base sequence of 626 bp as shown in SEQ ID NO: 37 "HCCR-1 primary cancer gene").

The base sequence of SEQ ID No. 37 is registered as Registration No. AY323819 in the NIH GenBank database of the National Institutes of Health (published: December 31, 2004). Homo sapiens tumor protein D52-like 2 (TPD52L2), transcript variant 5 gene and gene BC006804 Homo sapiens tumor protein D52-like 2, transcript variant 5, mRNA (cDNA clone MGC: 5064 IMAGE: 3446037) gene and gene sequence This similarity was confirmed. However, this study identified HCCRBP2 proto-oncogenes, which are highly expressed in various human tumors, including uterine cancer, and very low in various normal tissues.

     In the nucleotide sequence of SEQ ID NO: 37, the open reading frame corresponding to the nucleotide numbers 9 to 356 site is the entire protein coding region, and the amino acid sequence derived therefrom is shown in SEQ ID NO: 38 and consists of 115 amino acids. (Hereinafter referred to as "HCCRBP2 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 and fragments of said genes having base sequences substantially the same as the original cancer gene of SEQ ID NO: 37 above. Substantially identical polynucleotides refer to 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 consists of 115 amino acids, has a sequence such as SEQ ID NO: 38, 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 "HCCRBP2 (HCCR-binding protein 2)" 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.

11. TRG  2

Human transformation-related gene 2 (hereinafter referred to as TRG2), which is the protocogene of the present invention, has an entire sequence of 2,302 bp in length as set forth in SEQ ID NO: 39.

In the nucleotide sequence of SEQ ID NO: 39, the open reading frame corresponding to nucleotide numbers 747 to 2066 (2064-2066: end codon) is the entire protein coding region, and the amino acid sequence derived therefrom is SEQ ID NO: It is shown at 40 and consists of 439 amino acids (hereinafter referred to as the "TRG2 protein").

The base sequence of SEQ ID NO: 39 was registered as Registration No. AY170823 in the NIH GenBank Database of the National Institutes of Health (published: December 30, 2004). Homo sapiens cDNA: FLJ22058 fis, clone HEP10089, highly similar to HUMRANBP2 Ran-binding protein 2 gene, L41840 Homo sapiens nucleoporin (NUP358) gene gene and D42063 human mRNA for RanBP2 (Ran-binding protein 2 gene) ) Similarity between the gene and the gene sequence was confirmed. Ran-binding protein 2 (RanBP2) genes are known as the cytoplasmically exposed nucleoporin, Nup358 gene with peptide repeats, Ran-GTP binding sites, zinc fingers, a cyclophilin A homologous domain, and leucine-rich regions ((Wu, J. , et al ., J. Biol . Chem ., 270 , 14209-13213 (1995); Yokoyama, N., et al ., Nature , 376 , 184-188). However, this study identified TRG2 proto-oncogenes, which are highly expressed in various human tumors, including uterine cancer, and very low in various normal tissues.

 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 nucleotide sequence as the above oncogene of SEQ ID NO: 39. Substantially identical polynucleotides refer to DNAs encoding protein translation products identical to SEQ ID NO: 40, having those having at least 80%, preferably at least 90%, most preferably at least 95% homology with SEQ ID NO: 39 do.

The protein expressed from the primary oncogene of the present invention consists of 439 amino acids and has an amino acid sequence set forth in SEQ ID NO: 40 and is about 48 kDa in size. However, even in the amino acid sequence of the protein, substitution, addition or deletion of amino acids may be made within a range that does not affect the function of the protein, and only a part of the protein may be used depending on the purpose. Such modified amino acid sequences are also within the scope of the present invention. Accordingly, the present invention also encompasses polypeptides and fragments thereof having substantially the same amino acid sequence as the carcinogenic protein, wherein substantially identical polypeptides are at least 80%, preferably at least 90% and most preferably at least 95% sequences It means things with same sex.

The primary 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. 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.

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 depending on the type of host cell to produce the primary cancer genes or proteins.

Genes of the present invention are rarely identified in normal lung tissues by Northern blot analysis, whereas overexpression of lung cancer tissues, metastatic lung cancer tissues and lung cancer cell lines is confirmed. Is judged. In addition, the increased expression in metastatic lung cancer tissues has been found to be related to cancer metastasis causing cancer metastasis. In addition to epithelial tissues such as lung cancer, the primary cancer genes of the present invention are highly expressed in many other cancer tumors such as leukemia, uterine cancer, lymphoma, colorectal cancer, lung cancer and skin cancer. Therefore, the primary cancer genes of the present invention are considered to be common carcinogens for the development of various cancers, and can be effectively used for diagnosis of various cancers, production of transformed animals, and the like.

The method for diagnosing cancer using the primary cancer genes is, for example, by using all or a part of the primary cancer genes as probes to hybridize with nucleic acids isolated from the body fluids of the subject and then detecting them by various methods known in the art. Determining whether the subject has the primary cancer genes of the present invention. Labeling the probes with radioisotopes or enzymes can easily confirm the presence of genes. Accordingly, the present invention also provides a kit for diagnosing cancer comprising all or part of the primary cancer genes.

The transgenic animal can be produced by introducing the primary cancer genes 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 prior to 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.

Proteins derived from the primary cancer genes 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 expressed from the primary cancer genes of the present invention or fragments thereof. Subjects by subjects such as enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), sandwich assay, western blot on polyacryl gel or immunoblot. Cancer can be diagnosed by checking whether the protein is expressed in a bodily fluid sample.

In addition, cancer cell lines capable of continuously proliferating can be established by using the primary cancer genes of the present invention, and these cell lines are formed by, for example, tumor tissues formed in nude mice using fibroblasts transduced with the primary cancer genes. It can be prepared from. These cancer cell lines can be usefully used for the search for anticancer agents.

Hereinafter, the present invention will be described in detail by way of examples, but the present invention is not limited thereto.

Example  1: Tumor Cell Culture and Total RNA Separation of

1-1: PIG  5, PIG  11, PIG  16, PIG  17, PIG  19, PIG  20, PIG  21

(Step 1) Tumor Cell Culture

For differential development of mRNA, normal lung tissue and lung cancer patients who had not previously been treated with chemotherapy and radiation were taken primary lung cancer tissue and cancer tissue metastasized to the right lung during surgery. In the differential development, A549 (American Type Culture Collection; ATCC Number CCL-185) was used as a human lung cancer cell line.

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 used cells that exhibited 95% or more viability when stained with trypan blue dye as cells during exponential growth (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.).

1-2: PIG  6, PIG  7, TRG  2

(Step 1) culturing tumor cells

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

Cells obtained from the tissues obtained above and from the CUMC-6 cell line were obtained from Waymouth's MB containing 2 mM glutamine, 100 IU / ml penicillin, 100 μg / ml streptomycin and 10% fetal bovine serum (Gibco, USA). Proliferation in 752/1 culture (Gibco). The cultured cells used in the experiments were cells of exponential proliferation which showed viability of at least 95% by trypan blue staining (Freshney, "Culture of Animal Cells: A Manual of Basic Technique" 2nd). Ed., AR Liss, New York, 1987).

(Step 2) RNA isolation and differential development of mRNA

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

1-3: yeast teeth hybrid  By analytical method HCCR BP2 Cloning

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). 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. Strains selected therefrom were cultured in SD / -uracil / glucose media, and then the HCCR-1 gene was transferred to the pLexA vector. Vectors prepared by inserting the Bam HI and Sal I sites were transformed into these yeast strains as baits. In order to confirm that the HCCR-1 gene cloned into the pLexA vector is 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 assay (Breeden, L. & Nasmyth, K., Cold) to confirm the binding of bait and library Spring Harbor Symposium Quant . Bio. 50: 643-650, 1985). 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 transformant, transformed into E. coli DH5α, and the DNA was extracted to treat Hind III to obtain a clone having a desired size of about 0.6 kb.

Example 2: Differential expansion RT-PCR (differential display reverse transcription (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.

2-1: PIG  5

First, H-T11G (5'-AAGCTTTTTTTTTTTG-3 ') fixed primer (RNAimage) having an nucleotide sequence set forth in SEQ ID NO: 3 as an oligo-dT primer immobilized on 0.2 μg of total RNA obtained in step 1 of Example 1 kit, Genhunter, Cor., MA, USA) was used to reverse transcription.

Subsequently, in the presence of 0.5 mM [α-35S] dATP (1200 Ci / mmole), the same fixed primers and random 5 ′ 11 primers (RNAimage primer sets 1-5, H-AP9 primers among H-APs 1 to 40 (sequences) No. 4) PCR was performed using (5'-AAGCTTCATTCCG-3 ') The conditions of PCR reaction were 40 seconds at 95 ° C., 2 minutes at 40 ° C., 40 seconds at 72 ° C., and 40 times. The reaction was further reacted at 72 ° C. for 5 minutes for 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 differently expressed degrees.

A 192 base pair (bp) sized band, L699 cDNA (SEQ ID NO: 778-969 bp) was cut out from the dried gel. After eluting the L699 cDNA by heating for 15 minutes, PCR was carried out under the same primers and the same conditions as above except that [α-35S] labeled dATP (1200 Ci / mmole) and 20 μM dNTP were not used. I was.

2-2: PIG  6

First, an H-T11A fixed primer (5'-AAGCTTTTTTTTTTTTTA-3 ', RNAimage kit having a nucleotide sequence set forth in SEQ ID NO: 7 as an oligo-dT primer immobilized on 0.2 μg of total RNA obtained in step 1 of Example 1) , Genhunter, Cor., MA, USA) was used to perform reverse transcription.

Subsequently, in the presence of 0.5 mM [α-35S] dATP (1200 Ci / mmole), the same fixed primers and random 5 ′ 11 primers (RNAimage primer sets 1-5) described as SEQ ID NO: 8 in H-APs 1-40 PCR was performed using H-AP32 primer (5'-AAGCTTCCTGCAA-3 ') having a nucleotide sequence. The PCR reaction conditions were 40 seconds at 95 ° C., 40 minutes at 40 ° C. for 2 minutes, and 40 seconds at 72 ° C. for 5 minutes at 72 ° C. for final extension.

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

A 392 base pair (bp) sized band, CA325 cDNA (SEQ ID NO: 2485-2876 bp) was cut out from the dried gel. After eluting CA325 cDNA by heating for 15 minutes, PCR was performed under the same primers and the same conditions as above except that [α-35S] labeled dATP (1200 Ci / mmole) and 20 μM dNTP were not used. Amplified.

2-3: PIG  7

First, an H-T11A fixed primer (5'-AAGCTTTTTTTTTTTATA-3 ', RNAimage kit having a nucleotide sequence of SEQ ID NO: 11 as an oligo-dT primer immobilized on 0.2 μg of total RNA obtained in step 1 of Example 1) , Genhunter, Cor., MA, USA) was used to perform reverse transcription.

Then, in the presence of 0.5 mM [α-35S] dATP (1200 Ci / mmole), the same fixed primers and random 5 ′ 11 primers (RNAimage primer sets 1-5) described as SEQ ID NO: 12 in H-APs 1-40 PCR was performed using H-AP27 primer (5'-AAGCTTCTGCTGG-3 ') having a nucleotide sequence. The PCR reaction conditions were 40 seconds at 95 ° C., 40 minutes at 40 ° C. for 2 minutes, and 40 seconds at 72 ° C. for 5 minutes at 72 ° C. for final extension.

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

A 368 base pair (bp) sized band, CA273 cDNA (3762-4129 bp of SEQ ID NO: 9) was cut out from the dried gel. After elution of CA273 cDNA by heating for 15 minutes, PCR was performed under the same primers and the same conditions as above except that [α-35S] labeled dATP (1200 Ci / mmole) and 20 μM dNTP were not used. Amplified.

2-4: PIG  11

First, oligo-dT primers immobilized on 0.2 μg of total RNA obtained in step 1 of Example 1 having the nucleotide sequence set forth in SEQ ID NO: 15

 Reverse transcription was performed using H-T11G (5′-AAGCTTTTTTTTTTTA-3 ′) fixed primers (RNAimage kit, Genhunter, Cor., MA, USA).

Then, in the presence of 0.5 mM [α-35S] dATP (1200 Ci / mmole), the same fixed primers and random 5 ′ 11 primers (RNAimage primer sets 1-5, H-AP11 primers among H-APs 1 to 40 (sequences) PCR was performed using No. 16) (5'-AAGCTTCGGGTAA-3 ') The conditions of the PCR reaction were 40 seconds at 95 ° C., 2 minutes at 40 ° C., 40 seconds at 72 ° C., and 40 times. The reaction was further reacted at 72 ° C. for 5 minutes for 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 differently expressed degrees.

A 203 base pair (bp) sized band, L667 cDNA (SEQ ID NOs: 796-998 bp) was cut out from the dried gel. After heating for 15 minutes to elute the L667 cDNA, PCR was carried out under the same primers and the same conditions as above except that [α-35S] labeled dATP (1200 Ci / mmole) and 20 μM dNTP were not used. I was.

2-5: PIG  16

First, oligo-dT primers immobilized on 0.2 μg of total RNA obtained in step 1 of Example 1 having the nucleotide sequence set forth in SEQ ID NO: 19.

 Reverse transcription was performed using H-T11C (5′-AAGCTTTTTTTTTTTC-3 ′) fixed primers (RNAimage kit, Genhunter, Cor., MA, USA).

Subsequently, in the presence of 0.5 mM [α-35S] dATP (1200 Ci / mmole), the same fixed primers and random 5 ′ 11 primers (RNAimage primer sets 1-5, H-AP16 primers (SEQ ID NOs: 1-40) PCR was performed using No. 20) (5'-AAGCTTTAGAGCG-3 ') PCR reaction conditions were 40 seconds at 95 ° C., 2 minutes at 40 ° C. and 40 seconds at 72 ° C. for 40 times. The reaction was further reacted at 72 ° C. for 5 minutes for 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 differently expressed degrees.

A 322 base pair (bp) sized band, L668 cDNA (1277-1598 bp of SEQ ID NO: 17) was cut out from the dried gel. After heating for 15 minutes to elute the L668 cDNA, PCR was carried out under the same primers and the same conditions as above, except that [α-35S] labeled dATP (1200 Ci / mmole) and 20 μM dNTP were not used. I was.

2-6: PIG  17

First, oligo-dT primers immobilized on 0.2 µg of total RNA obtained in step 1 of Example 1 having the nucleotide sequence set forth in SEQ ID NO: 23

 Reverse transcription was performed using H-T11C (5′-AAGCTTTTTTTTTTTC-3 ′) fixed primers (RNAimage kit, Genhunter, Cor., MA, USA).

Subsequently, in the presence of 0.5 mM [α-35S] dATP (1200 Ci / mmole), the same fixed primers and random 5 ′ 11 primers (RNAimage primer sets 1-5, H-AP18 primers among H-APs 1 to 40 (sequences) No. 24) PCR was performed using (5'-AAGCTTAGAGGCA-3 ') The conditions of the PCR reaction were 40 seconds at 95 ° C., 2 minutes at 40 ° C., 40 seconds at 72 ° C., and 40 times. The reaction was further reacted at 72 ° C. for 5 minutes for 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 differently expressed degrees.

A 211 base pair (bp) sized band, L211 cDNA (389-599 bp of SEQ ID NO: 21) was cut out from the dried gel. After heating for 15 minutes to elute L211 cDNA, PCR was performed under the same primers and the same conditions as above except that [α-35S] -labeled dATP (1200 Ci / mmole) and 20 μM dNTP were not used. I was.

2-7: PIG  19

First, oligo-dT primers immobilized on 0.2 μg of total RNA obtained in step 1 of Example 1 having the nucleotide sequence set forth in SEQ ID NO: 27

 Reverse transcription was performed using H-T11G (5′-AAGCTTTTTTTTTTTG-3 ′) fixed primers (RNAimage kit, Genhunter, Cor., MA, USA).

Subsequently, in the presence of 0.5 mM [α-35S] dATP (1200 Ci / mmole), the same fixed primers and random 5 ′ 11 primers (RNAimage primer sets 1-5, H-AP16 primers (SEQ ID NOs: 1-40) No. 28) PCR was performed using (5'-AAGCTTTAGAGCG-3 ') The conditions of the PCR reaction were 40 seconds at 95 ° C., 2 minutes at 40 ° C. and 40 seconds at 72 ° C. for 40 times. The reaction was further reacted at 72 ° C. for 5 minutes for 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 differently expressed degrees.

A 233 base pair (bp) sized band, L722 cDNA (SEQ ID NO: 777-1009 bp) was cut out from the dried gel. After heating for 15 minutes to elute L722 cDNA, PCR was performed under the same primers and the same conditions as above, except that [α-35S] labeled dATP (1200 Ci / mmole) and 20 μM dNTP were not used. I was.

2-8: PIG  20

First, oligo-dT primers immobilized on 0.2 μg of total RNA obtained in step 1 of Example 1 having the nucleotide sequence set forth in SEQ ID NO: 31.

 Reverse transcription was performed using H-T11G (5′-AAGCTTTTTTTTTTTG-3 ′) fixed primers (RNAimage kit, Genhunter, Cor., MA, USA).

Subsequently, in the presence of 0.5 mM [α-35S] dATP (1200 Ci / mmole), the same fixed primers and random 5 ′ 11 primers (RNAimage primer sets 1-5, H-AP17 primers among H-APs 1 to 40 (sequences) No. 32) PCR was performed using (5'-AAGCTTACCAGGT-3 ') The conditions of the PCR reaction were 40 seconds at 95 ° C., 2 minutes at 40 ° C., 40 seconds at 72 ° C., and 40 times. The reaction was further reacted at 72 ° C. for 5 minutes for 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 differently expressed degrees.

A 211 base pair (bp) sized band, L752 cDNA (304-514 bp of SEQ ID NO: 29) was cut out from the dried gel. After heating for 15 minutes to elute the L752 cDNA, PCR was carried out under the same primers and the same conditions as above except that [α-35S] labeled dATP (1200 Ci / mmole) and 20 μM dNTP were not used. I was.

2-9: PIG  21

First, oligo-dT primers immobilized on 0.2 μg of total RNA obtained in step 1 of Example 1 having the nucleotide sequence set forth in SEQ ID NO: 35.

 Reverse transcription was performed using H-T11C (5′-AAGCTTTTTTTTTTTC-3 ′) fixed primers (RNAimage kit, Genhunter, Cor., MA, USA).

Then, in the presence of 0.5 mM [α-35S] dATP (1200 Ci / mmole), the same fixed primers and random 5 ′ 11 primers (RNAimage primer sets 1-5, H-AP15 primer (SEQ ID NO: 1-40) No. 36) PCR was performed using (5'-AAGCTTACGCAAC-3 ') The PCR reaction conditions were 40 seconds at 95 ° C., 2 minutes at 40 ° C. and 40 seconds at 72 ° C. for 40 seconds. The reaction was further reacted at 72 ° C. for 5 minutes for 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 differently expressed degrees.

A 272 base pair (bp) sized band, L1003 cDNA (665-936 bp of SEQ ID NO: 33) was cut out from the dried gel. After eluting the L1003 cDNA by heating for 15 minutes, PCR was performed under the same primers and the same conditions as above except that [α-35S] labeled dATP (1200 Ci / mmole) and 20 μM dNTP were not used. I was.

2-10: TRG  2

First, H-T11A fixed primer (5'-AAGCTTTTTTTTTTTATA-3 ', RNAimage kit having a nucleotide sequence set forth in SEQ ID NO: 41 as an oligo-dT primer immobilized on 0.2 μg of total RNA obtained in step 1 of Example 1) , Genhunter, Cor., MA, USA).

Then, in the presence of 0.5 mM [α-35S] dATP (1200 Ci / mmole), the same fixed primers and random 5 ′ 11 primers (RNAimage primer sets 1-5) described as SEQ ID NO: 42 in H-AP 1-40 PCR was performed using H-AP32 primer (5'-AAGCTTCCTGCAA-3 ') having a nucleotide sequence. The PCR reaction conditions were 40 seconds at 95 ° C., 40 minutes at 40 ° C. for 2 minutes, and 40 seconds at 72 ° C. for 5 minutes at 72 ° C. for final extension.

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

A 373 base pair (bp) sized band, HP90-811 cDNA (1797-2169 bp of SEQ ID NO: 39) was cut out from the dried gel. After eluting the HP90-811 cDNA by heating for 15 minutes, PCR was performed under the same primers and under the same conditions except that [α-35S] labeled dATP (1200 Ci / mmole) and 20 μM dNTP were not used. By reamplification.

Example 3: Cloning

PIG 5, PIG 6, PIG7 , PIG 11, PIG 16, PIG 17, PIG 19, PIG 20, PIG 21, TRG 2

Reamplified L699 PCR product obtained above; CA325 PCR product; CA273 PCR product; L667 PCR product; L668 PCR product; L211 PCR product; L722 PCR product; L752 PCR product; L1003 PCR product; Alternatively, the HP90-811 PCR product was inserted into the pGEM-T Easy (EASY) vector using the TA cloning system (Promega, USA) according to the manufacturer's method.

(Step 1) Linking reaction

Reamplified L699 PCR product obtained in Example 2; CA325 PCR product; CA273 PCR product; L667 PCR product; L668 PCR product; L211 PCR product; L722 PCR product; L752 PCR product; 2 μl of L1003 PCR product or HP90-811 PCR product, 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, Promega 1 μl was placed in a 0.5 ml test tube, and distilled water was added so that the final volume was 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) to obtain an optical density of about 0.3-0.6 at 600 nm. 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. Colonies of which insertion was identified, ie, transformed E. coli JM109 / L699, to prepare plasmids; JM109 / CA325; JM109 / CA273; JM109 / L667

JM109 / L668; JM109 / L211; JM109 / L699; JM109 / L752; JM109 / L1003; Or JM109 / HP90-811 to select 10 ml of terrific broth (900 ml of TDW, 12 g of bacto-tryptone, 24 g of bacto-yeast extract, 4 ml of glycerol, 0.17 M KH2PO4, 0.72 N K2HPO4 100 in ml).

Example 4: Isolation of Recombinant Plasmid DNA

PIG 5, PIG 6, PIG7 , PIG 11, PIG 16, PIG 17, PIG 19, PIG 20, PIG 21, TRG 2

L699 plasmid DNA from E. coli transformed according to the manufacturer's instructions using the Wizard Plus Minipreps DNA Purificatin Kit, Promega, USA; CA325 plasmid DNA; CA273 plasmid DNA; L667 plasmid DNA; L668 plasmid DNA; L211 plasmid DNA; L722 plasmid DNA; L752 plasmid DNA; L1003 plasmid DNA or HP90-8115 plasmid DNA was isolated.

A portion of the separated plasmid DNA was treated with ECo RI enzyme, followed by electrophoresis on 2% gel to L699; CA325; CA273; L667; L668; L211; L722; L752; L1003; Or it was confirmed that the HP90-8115 subsequence was inserted into the plasmid.

Example 5: DNA sequencing

5-1: PIG  5

After the L699 PCR product obtained in Example 2 was PCR according to a conventional method, the cloned and re-amplified L699 PCR fragment was subjected to a Sequencena version 2.0 DNA sequencing kit (United States Biochemical, Cleveland, OH, USA) was used for sequencing according to dideoxy chain termination.

The base sequence of the DNA corresponds to nucleotide numbers 778 to 969 of SEQ ID NO: 1, referred to herein as "L699".

The 192 bp cDNA fragment obtained above, i.e., L699, using 5 'random primers H-AP9 and 3' H-T11G fixed primers, was differentially developed by reverse transcriptase polymerase chain reaction (DDRT-PCR) and confirmed by electrophoresis. . As shown in FIG. 1A, gene expression by DD was found 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 1a, 699 bp cDNA fragment L699 was expressed in lung cancer, metastatic lung cancer tissue and A549 lung cancer cells, but not in normal lung tissue.

5-2: PIG  6

The CA325 PCR product obtained in Example 2 was amplified according to a conventional method, and then cloned and re-amplified CA325 PCR fragments were prepared using the 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 2485 to 2876 of SEQ ID NO: 5, and is referred to as "CA325" in the present invention.

The 392 bp cDNA fragment obtained above, ie CA325, using 5 'random primer H-AP32 and 3' H-T11A fixed primer, was differentially developed by reverse transcription polymerase chain reaction (DDRT-PCR) and confirmed by electrophoresis. .

As shown in FIG. 1B, gene expression by DD in normal cervical tissue, metastatic lymph node tissue and CUMC-6 cells was found to be different. As can be seen in Figure 1b, the 392 bp cDNA fragment CA325 was expressed in cervical cancer, metastatic lymph node tissue and CUMC-6 cancer cells, but the expression was very weak in normal tissues.

5-3: PIG  7

After amplifying the CA273 PCR product obtained in Example 2 according to a conventional method, the cloned and re-amplified CA273 PCR fragment was prepared using a sequencer 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 3762 to 4129 of SEQ ID NO: 9, and is referred to as "CA273" in the present invention.

The 368 bp cDNA fragment obtained above, ie CA273, using 5 'random primers H-AP27 and 3' H-T11A fixed primers was subjected to differential reverse reverse transcription polymerase chain reaction (DDRT-PCR) and confirmed by electrophoresis. .

As shown in FIG. 1C, gene expression by DD in normal cervical tissue, metastatic lymph node tissue and CUMC-6 cells was found to be different. As can be seen in Figure 1c, the 368 bp cDNA fragment CA273 was expressed in cervical cancer, metastatic lymph node tissue and CUMC-6 cancer cells, but the expression was very weak in normal tissues.

5-4: PIG  11

After the L667 PCR product obtained in Example 2 was PCR according to a conventional method, the cloned and re-amplified L667 PCR fragment was subjected to a Sequencena version 2.0 DNA sequencing kit (United States Biochemical, Cleveland, OH, USA) was used for sequencing according to dideoxy chain termination.

The base sequence of the DNA corresponds to nucleotide numbers 796 to 998 of SEQ ID NO: 13, referred to herein as "L667".

The 203 bp cDNA fragment obtained above, ie, L667, using 5 'random primers H-AP11 and 3' H-T11A immobilized primers, was subjected to differential reverse transcription polymerase chain reaction (DDRT-PCR) and confirmed by electrophoresis. . As shown in FIG. 1D, gene expression by DD was found 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 FIG. 1D, L667, a 203 bp cDNA fragment, was expressed in lung cancer, metastatic lung cancer tissue and A549 lung cancer cells, but not in normal lung tissue.

5-5: PIG  16

After the L668 PCR product obtained in Example 2 was PCR according to a conventional method, the cloned and re-amplified L668 PCR fragment was subjected to a Sequenase version 2.0 DNA Sequencing kit (United States Biochemical, Cleveland, OH, USA) was used for sequencing according to dideoxy chain termination.

The base sequence of the DNA corresponds to nucleotide numbers 1277 to 1598 of SEQ ID NO: 17, referred to herein as "L668".

The 322 bp cDNA fragment obtained above, L668, using 5 'random primers H-AP16 and 3' H-T11C immobilized primers, was subjected to differential reverse reverse transcriptase polymerase chain reaction (DDRT-PCR) and confirmed by electrophoresis. . As shown in FIG. 1E, gene expression by DD was found 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 1e, 322 bp cDNA fragment L668 was expressed in lung cancer, metastatic lung cancer tissue and A549 lung cancer cells, but not in normal lung tissue.

5-6: PIG  17

After PCR of the L211 PCR product obtained in Example 2 according to a conventional method, the cloned and re-amplified L211 PCR fragments were subjected to a Sequenase version 2.0 DNA Sequencing kit (United States Biochemical, Cleveland, OH, USA) was used for sequencing according to dideoxy chain termination.

The base sequence of the DNA corresponds to nucleotide numbers 389 to 599 of SEQ ID NO: 21, referred to herein as "L211".

The 211 bp cDNA fragment obtained above, L211, using 5 'random primers H-AP18 and 3' H-T11C fixed primers, was subjected to differential reverse transcription polymerase chain reaction (DDRT-PCR) and confirmed by electrophoresis. . As shown in FIG. 1F, gene expression by DD was found 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 1f, 211 bp cDNA fragment L211 was expressed in lung cancer, metastatic lung cancer tissue and A549 lung cancer cells, but not in normal lung tissue.

5-7: PIG  19

After PCR of the L722 PCR product obtained in Example 2 according to a conventional method, the cloned and re-amplified L722 PCR fragments were subjected to a Sequenase version 2.0 DNA Sequencing kit (United States Biochemical, Cleveland). , OH, USA) was used to sequence analysis according to dideoxy chain termination (dideoxy chain termination).

The base sequence of the DNA corresponds to nucleotide numbers 777 to 1009 of SEQ ID NO: 25, referred to herein as "L722".

The 233 bp cDNA fragment obtained above, i.e., L722, using 5 'random primers H-AP16 and 3' H-T11G fixed primers was subjected to differential reverse reverse transcription polymerase chain reaction (DDRT-PCR) and confirmed by electrophoresis. . As shown in FIG. 1G, gene expression by DD was found 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 1g, 233 bp cDNA fragment L722 was expressed in lung cancer, metastatic lung cancer tissue and A549 lung cancer cells, but not in normal lung tissue.

5-8: PIG  20

After PCR of the L752 PCR product obtained in Example 2 according to a conventional method, the cloned and re-amplified L752 PCR fragments were subjected to a Sequenase version 2.0 DNA Sequencing kit (United States Biochemical, Cleveland, OH, USA) was used for sequencing according to dideoxy chain termination.

The base sequence of the DNA corresponds to nucleotide numbers 304 to 514 of SEQ ID NO: 29, referred to herein as "L752".

The 211 bp cDNA fragment obtained above, i.e., L752, using 5 'random primers H-AP17 and 3' H-T11G immobilized primers, was subjected to differential reverse reverse transcription polymerase chain reaction (DDRT-PCR) and confirmed by electrophoresis. . As shown in FIG. 1H, gene expression by DD was found 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 FIG. 1H, the 211 bp cDNA fragment L752 was expressed in lung cancer, metastatic lung cancer tissue and A549 lung cancer cells, but very weakly expressed in normal lung tissue.

5-9: PIG  21

After PCR of the L1003 PCR product obtained in Example 2 according to a conventional method, the cloned and re-amplified L1003 PCR fragments were subjected to a Sequenase version 2.0 DNA Sequencing kit (United States Biochemical, Cleveland, OH, USA) was used for sequencing according to dideoxy chain termination.

The base sequence of the DNA corresponds to nucleotide numbers 665 to 936 of SEQ ID NO: 33, referred to herein as "L1003".

The 272 bp cDNA fragment obtained above, i.e., L1003, using 5 'random primers H-AP15 and 3' H-T11C fixed primers was subjected to differential reverse transcription polymerase chain reaction (DDRT-PCR) and confirmed by electrophoresis. . As shown in FIG. 1I, gene expression by DD was found 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 1i, L2723, a 272 bp cDNA fragment, was expressed in lung cancer, metastatic lung cancer tissue and A549 lung cancer cells, but very weakly expressed in normal lung tissue.

5-10: TRG  2

The HP90-811 PCR product obtained in Example 2 was amplified according to a conventional method, and then cloned and re-amplified HP90-811 PCR fragments were prepared using a sequencer DNA 2.0 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 1797 to 2169 of SEQ ID NO: 39, which is referred to as "HP90-811" in the present invention.

The 373 bp cDNA fragment obtained above, HP90-811, using 5 'random primers H-AP32 and 3' H-T11A fixed primers was subjected to differential reverse transcription polymerase chain reaction (DDRT-PCR) and subjected to electrophoresis. Confirmed.

As shown in FIG. 1J, gene expression by DD in normal cervical tissue, metastatic lymph node tissue and CUMC-6 cells was found to be different. As can be seen in Figure 1j, HP-90-811, a 373 bp cDNA fragment, was expressed in cervical cancer, metastatic lymph node tissue and CUMC-6 cancer cells, but very weak in normal tissue.

Example 6: Whole cDNA sequence analysis of the primary oncogene

6-1: PIG  5

Bacteriophage λgt11 human lung fetal fibroblast cDNA library using 32P-labeled L699 as a probe (Miki, T. et. al . , Gene , 83 : 137-146 (1989)) were screened for full-length genes with L699 cDNA sequences. Two types of full-length genes were obtained from the human lung fetal fibroblast cDNA library, and one obtained a full PIG5 cDNA clone with 1009 bp inserted into the pCEV-LAC vector. GeneBank) (published December 31, 2004).

PIG5 clones inserted into the λpCEV vectors from phage were isolated in the form of ampicillin resistant pCEV-LAC phagemid vectors by Not I cleavage (see above).

The pCEV-LAC vector containing the PIG5 gene was linked with T4 DNA ligase to prepare PIG5 plasmid DNA, and E. coli DH5α was transformed with the linked clone.

The entire sequence of PIG5 consisting of 1009 bp is shown in SEQ ID NO: 1.

In the nucleotide sequence of SEQ ID NO: 1, the full open reading frame of the proto-oncogene of the present invention corresponds to nucleotides 9 to 746, predicted to encode a protein consisting of the 245 amino acids of SEQ ID NO: 2. do.

6-2: PIG  6

Bacteriophage λgt11 human lung fetal fibroblast cDNA library using 32P-labeled CA325 as a probe (Miki, T. et. al . , Gene 83: 137-146, 1989). From the human lung fetal fibroblast cDNA library, a full PIG6 cDNA clone with 2964 bp inserted into the pCEV-LAC vector was obtained and registered with GeneBank of the United States on February 13, 2003 under the accession number AY236487. Scheduled date: December 31, 2004).

PIG6 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 PIG6 gene was linked with T4 DNA ligase to prepare PIG6 plasmid DNA and transformed into E. coli DH5α with the linked clone.

The entire nucleotide sequence of PIG6 consisting of 22964 bp is shown in SEQ ID NO: 5.

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 293 to 2302, predicted to encode a protein consisting of 669 amino acids of SEQ ID NO: 6 do.

6-3: PIG  7

Bacteriophage λgt11 human lung fetal fibroblast cDNA library using 32P-labeled CA273 as a probe (Miki, T. et. al . , Gene 83: 137-146, 1989). From the human lung fetal fibroblast cDNA library, a full PIG7 cDNA clone with 4301 bp inserted into the pCEV-LAC vector was obtained and registered with GeneBank of the United States on February 13, 2003 under the accession No. AY236488. Scheduled date: December 31, 2004).

PIG7 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 PIG7 gene was linked with T4 DNA ligase to prepare PIG7 plasmid DNA, and transformed into E. coli DH5α with the linked clone.

The entire nucleotide sequence of PIG7 consisting of 4301 bp is shown in SEQ ID NO: 9.

In the nucleotide sequence of SEQ ID NO: 9, the entire open reading frame of the proto-oncogene of the present invention corresponds to nucleotides 3556-3792, predicted to encode a protein consisting of 78 amino acids of SEQ ID NO: 10. do.

6-4: PIG  11

Bacteriophage lambdagt11 human lung fetal fibroblast cDNA library using 32P-labeled L667 as a probe (Miki, T. et. al . , Gene , 83 : 137-146 (1989)) were screened for full-length genes with L667 cDNA sequences. Two full-length genes were obtained from the human lung fetal fibroblast cDNA library, and one obtained a full PIG11 cDNA clone into which 1038 bp was inserted into the pCEV-LAC vector, which was obtained from US Genbank (AY258284) on February 24, 2003. GeneBank) (published December 31, 2004).

The PIG11 clone inserted into the λpCEV vector from phage was isolated in the form of an ampicillin resistant pCEV-LAC phagemid vector by Not I cleavage (see above).

The pCEV-LAC vector containing the PIG11 gene was linked with T4 DNA ligase to prepare PIG11 plasmid DNA, and E. coli DH5α was transformed with the linked clone.

The entire sequence of PIG11 consisting of 1038 bp is shown in SEQ ID NO: 13.

In the nucleotide sequence of SEQ ID NO: 13, the full open reading frame of the proto-oncogene of the present invention corresponds to nucleotides 50 to 931, predicted to encode a protein consisting of 293 amino acids of SEQ ID NO: 14. do.

6-5: PIG  16

Bacteriophage λgt11 human lung fetal fibroblast cDNA library using 32P-labeled L668 as a probe (Miki, T. et. al . , Gene , 83 : 137-146 (1989)) were screened for full-length genes with L668 cDNA sequences. Two kinds of full-length genes were obtained from the human lung fetal fibroblast cDNA library, and one obtained a full PIG16 cDNA clone with 1682 bp inserted into the pCEV-LAC vector, which was obtained by US Genebank (AY305873) on May 24, 2003. GeneBank) (published December 31, 2004).

PIG16 clones inserted into λpCEV vectors from phage were isolated in the form of ampicillin resistant pCEV-LAC phagemid vectors by Not I cleavage (see above).

The pCEV-LAC vector containing the PIG16 gene was linked with T4 DNA ligase to prepare PIG16 plasmid DNA, and the linked clones were transformed into E. coli DH5α.

The total sequence of PIG16 consisting of 1682 bp is shown in SEQ ID NO: 17.

In the nucleotide sequence of SEQ ID NO: 17, the full open reading frame of the proto-oncogene of the present invention corresponds to nucleotides 696-1577, predicted to encode a protein consisting of 293 amino acids of SEQ ID NO: 18. do.

6-6: PIG  17

Bacteriophage λgt11 human lung fetal fibroblast cDNA library using 32P-labeled L211 as a probe (Miki, T. et. al . , Gene , 83 : 137-146 (1989)), and obtained full-length genes with L211 cDNA sequence. Two kinds of full-length genes were obtained from the human lung fetal fibroblast cDNA library, and one obtained a full PIG17 cDNA clone with 626 bp inserted into the pCEV-LAC vector, which was obtained by the US Genebank (AY336092) on July 4, 2003. GeneBank) (published December 31, 2004).

PIG5 clones inserted into the λpCEV vectors from phage were isolated in the form of ampicillin resistant pCEV-LAC phagemid vectors by Not I cleavage (see above).

The pCEV-LAC vector containing the PIG17 gene was linked with T4 DNA ligase to prepare PIG17 plasmid DNA and transformed with E. coli DH5α with the linked clone.

The complete sequence of PIG17, consisting of 626 bp, is shown in SEQ ID NO: 21.

In the nucleotide sequence of SEQ ID NO: 21, the entire open reading frame of the proto-oncogene of the present invention corresponds to nucleotides 59 to 610, which encodes a protein consisting of 183 amino acids of SEQ ID NO: 22. It is predicted.

6-7: PIG  19

Bacteriophage λgt11 human lung fetal fibroblast cDNA library using 32P-labeled L722 as a probe (Miki, T. et. al . , Gene , 83 : 137-146 (1989)), and obtained full-length genes with L722 cDNA sequence. Two kinds of full-length genes were obtained from the human lung fetal fibroblast cDNA library, and one obtained a full PIG19 cDNA clone into which 1031 bp was inserted in the pCEV-LAC vector, which was obtained by the US Genebank (AY423727) on September 26, 2003. GeneBank) (published December 31, 2004).

The PIG19 clone inserted into the λpCEV vector from phage was isolated in the form of an ampicillin resistant pCEV-LAC phagemid vector by Not I cleavage (see above).

The pCEV-LAC vector containing the PIG19 gene was linked with T4 DNA ligation to prepare PIG19 plasmid DNA, and the linked clones were transformed into E. coli DH5α.

The complete sequence of PIG19, consisting of 1031 bp, is shown in SEQ ID NO: 25.

In the nucleotide sequence of SEQ ID NO: 25, the full open reading frame of the proto-oncogene of the present invention corresponds to nucleotides 32 to 1030, predicted to encode a protein consisting of 332 amino acids of SEQ ID NO: 26. do.

6-8: PIG  20

Bacteriophage λgt11 human lung fetal fibroblast cDNA library using 32P-labeled L752 as a probe (Miki, T. et. al . , Gene , 83 : 137-146 (1989)) were screened for full-length genes with L752 cDNA sequences. Two kinds of full-length genes were obtained from the human lung fetal fibroblast cDNA library, and one obtained a full PIG20 cDNA clone with 526 bp inserted into the pCEV-LAC vector, which was obtained from US Genebank (AY423728) on September 27, 2003. GeneBank) (published December 31, 2004).

PIG21 clones inserted into the λpCEV vectors from phage were isolated in the form of ampicillin resistant pCEV-LAC phagemid vectors by Not I cleavage (see above).

The pCEV-LAC vector containing the PIG20 gene was linked with T4 DNA ligase to prepare PIG20 plasmid DNA, and the linked clones were transformed into E. coli DH5α.

The entire sequence of PIG20, consisting of 526 bp, is shown in SEQ ID NO: 29.

In the nucleotide sequence of SEQ ID NO: 29, the entire open reading frame of the far-oncogene of the present invention corresponds to nucleotides 1 to 498, and predicted to encode a protein consisting of 165 amino acids of SEQ ID NO: 30. do.

6-9: PIG  21

Bacteriophage lambdagt11 human lung fetal fibroblast cDNA library using 32P-labeled L1003 as a probe (Miki, T. et. al . , Gene , 83 : 137-146 (1989)) screened for full-length genes with L1003 cDNA sequences. Two kinds of full-length genes were obtained from the human lung fetal fibroblast cDNA library, and one obtained a full PIG21 cDNA clone in which 965 bp was inserted into the pCEV-LAC vector. GeneBank) (published December 31, 2004).

PIG21 clones inserted into the λpCEV vectors from phage were isolated in the form of ampicillin resistant pCEV-LAC phagemid vectors by Not I cleavage (see above).

The pCEV-LAC vector containing the PIG21 gene was linked with T4 DNA ligase to prepare PIG21 plasmid DNA and transformed with E. coli DH5α with the linked clone.

The total sequence of PIG21 consisting of 965 bp is shown in SEQ ID NO: 33.

In the nucleotide sequence of SEQ ID NO: 33, the entire open reading frame of the proto-oncogene of the present invention corresponds to nucleotides 146-961, predicted to encode a protein consisting of 271 amino acids of SEQ ID NO: 34. do.

6-10: HCCR BP  2

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 626 bp shown in SEQ ID NO: 37 was identified as "HCCRBP2", and the entire HCCRBP2 cDNA clone into which this gene was inserted was assigned to GenBank on June 14, 2003 under the registration number AY323819. Registered.

In the nucleotide sequence of SEQ ID NO: 37, the open transcription frame of the primary cancer gene HCCRBP2 of the present invention corresponds to nucleotides 9 to 356, which encodes a protein consisting of 115 amino acids of SEQ ID NO: 38. It is predicted.

The HCCRBP2 gene was linked to the cloning site of the pGEM-T-easy vector (Promega) by T4 DNA ligase to prepare HCCRBP2 expressing plasmid DNA, and transformed into E. coli DH5α (Stratagene) with the prepared plasmid.

6-11: TRG  2

Bacteriophage λgt11 human lung fetal fibroblast cDNA library using 32P-labeled HP90-811 as a probe (Miki, T. et. al . , Gene 83: 137-146, 1989). From the human lung fetal fibroblast cDNA library, a full TRG2 cDNA clone with 2302 bp inserted into the pCEV-LAC vector was obtained and registered with GeneBank of the United States as Gen. No. AY170823 dated 30 October 2002. Scheduled date: December 31, 2004).

TRG2 clone inserted into λpCEV vector from phage was isolated in the form of ampicillin resistant pCEV-LAC phagemid vector by Not I cleavage (Miki, T. et. al . , Gene 83: 137-146, 1989).

The pCEV-LAC vector containing the TRG2 gene was linked with T4 DNA ligase to prepare TRG2 plasmid DNA and transformed into E. coli DH5α with the linked clone.

The entire nucleotide sequence of TRG2 consisting of 2302 bp is shown in SEQ ID NO: 39.

In the nucleotide sequence of SEQ ID NO: 39, the full open reading frame of the proto-oncogene of the present invention corresponds to nucleotides 747 to 2066, predicted to encode a protein consisting of 439 amino acids of SEQ ID NO: 40. do.

Example 7 Northern Blot Analysis of Genes in Various Cells

7-1: PIG  5, PIG  11, PIG  16, PIG  17, PIG  19, PIG  20, PIG  21

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

PIG5; PIG 11; PIG 16; PIG 17; PIG 19; PIG 20; Alternatively, to determine the degree of PIG 21 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). 32P-labeled random primed L699 prepared using the Rediprime II random prime labeling system (Amersham, UK); L667; L668; L211; L722; L752; Or blot hybridized with L1003 partial cDNA probe. 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 PIG5 primary cancer genes in normal lung tissue, lung cancer tissue, lung cancer metastases and lung cancer cell lines (A549, NCI-H2009, and NCI-H441). As can be seen in Figure 2A, significant gene expression was observed in lung cancer tissues, lung cancer metastatic tissues and lung cancer cell lines A549, NCI-H2009, NCI-H441 lung cancer cell lines, but the expression level is very low or observed in normal lung tissue It wasn't. In FIG. 2, normal fever is normal lung tissue, cancer fever is lung cancer tissue, metastasis fever is lung cancer metastatic tissue and A549 fever, NCI-H2009 fever and NCI-H441 fever are lung cancer cell lines. Respectively. 2B shows the presence of mRNA by hybridizing the same sample with β-actin probe.

FIG. 13A shows Northern PIG5 primary cancer gene expression 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 results of the blot analysis are shown. Figure 13B confirms the presence of mRNA by hybridizing the same sample with β-actin probe. As can be seen in Figure 13A, PIG5 mRNA (about 3.0 kb) is weakly expressed only in muscle and heart tissues and very weak or no expression in other normal tissues.

24A shows human cancer cell lines, HL-60, HeLa, K-562, MOLT-4, Raji. SW480, A549 and G361 (Clontech) shows the results of Northern blot analysis confirming the expression of PIG5 primary cancer gene. 24B shows the presence of mRNA by hybridizing the same sample with β-actin probe. As can be seen in Figure 24A, PIG5 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 lymphoma cell line High levels in Raji, colon cancer cell line SW480, lung cancer cell line A549 and skin cancer cell line G361.

5A shows the results of Northern blot analysis confirming the expression of PIG11 primary cancer genes in normal lung tissue, lung cancer tissue, lung cancer metastasis tissue and lung cancer cell lines (A549, NCI-H2009, and NCI-H441). As can be seen in FIG. 5A, significant gene expression was observed in lung cancer tissues, lung cancer metastases, and lung cancer cell lines A549, NCI-H2009, and NCI-H441 lung cancer cell lines, but the expression level was very low or observed in normal lung tissue. It wasn't. In FIG. 5, normal fever is normal lung tissue, cancer fever is lung cancer tissue, metastasis fever is lung cancer metastatic tissue and A549 fever, NCI-H2009 fever and NCI-H441 fever are lung cancer cell lines. Respectively. Figure 5B shows the presence of mRNA by hybridizing the same sample with β-actin probe.

FIG. 16A shows northern expression of PIG11 primary cancer genes in normal human 12-row multiple tissue (Clontech), brain, heart, rhabdomyo, colon, thymus, spleen, kidney, liver, small intestine, placenta, lung and peripheral blood leukocyte tissue The results of the blot analysis are shown. Figure 16B shows the presence of mRNA by hybridizing the same sample with β-actin probe. As can be seen in Figure 16A, PIG11 mRNA (about 1.3 kb) is weakly expressed only in muscle and heart tissues and very weak or no expression in other normal tissues.

27A shows human cancer cell lines, HL-60, HeLa, K-562, MOLT-4, Raji. SW480, A549 and G361 (Clontech) shows the results of Northern blot analysis confirming the expression of PIG11 primary cancer gene. Figure 27B shows the presence of mRNA by hybridizing the same sample with a β-actin probe. As can be seen in Figure 27A, PIG11 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 lymphoma cell line High levels in Raji, colon cancer cell line SW480, lung cancer cell line A549 and skin cancer cell line G361.

Figure 6A shows the results of Northern blot analysis confirming the expression of PIG16 primary cancer genes in normal lung tissue, lung cancer tissue, lung cancer metastasis tissue and lung cancer cell lines (A549, NCI-H2009, and NCI-H441). As can be seen in Figure 6A, significant gene expression was observed in lung cancer tissues, lung cancer metastatic tissues and lung cancer cell lines A549, NCI-H2009, NCI-H441 lung cancer cell lines, but the expression level is very low or observed in normal lung tissue It wasn't. In FIG. 6, normal fever is normal lung tissue, cancer fever is lung cancer tissue, metastasis fever is lung cancer metastatic tissue and A549 fever, NCI-H2009 fever and NCI-H441 fever are lung cancer cell lines. Respectively. Figure 6B confirms the presence of mRNA by hybridizing the same sample with β-actin probe.

FIG. 17A shows Northern PIG16 primary cancer gene expression 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 results of the blot analysis are shown. 17B shows the presence of mRNA by hybridizing the same sample with β-actin probe. As can be seen in Figure 17A, PIG16 mRNA (about 1.3 kb) is weakly expressed only in muscle and heart tissues and very weak or no expression in other normal tissues.

28A shows human cancer cell lines, HL-60, HeLa, K-562, MOLT-4, Raji. SW480, A549 and G361 (Clontech) shows the results of Northern blot analysis confirming the expression of PIG16 primary cancer gene. Figure 28B shows the presence of mRNA by hybridizing the same sample with β-actin probe. As can be seen in Figure 28A, PIG16 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 lymphoma cell line High levels in Raji, colon cancer cell line SW480, lung cancer cell line A549 and skin cancer cell line G361.

Figure 7A shows the results of Northern blot analysis confirming the expression of PIG17 primary cancer genes in normal lung tissue, lung cancer tissue, lung cancer metastasis tissue and lung cancer cell lines (A549, NCI-H2009, and NCI-H441). As can be seen in FIG. 7A, significant gene expression was observed in lung cancer tissues, lung cancer metastases, and lung cancer cell lines A549, NCI-H2009, and NCI-H441 lung cancer cell lines, but the expression level was very low or observed in normal lung tissue. It wasn't. In FIG. 7, normal fever is normal lung tissue, cancer fever is lung cancer tissue, metastasis fever is lung cancer metastatic tissue, and A549 fever, NCI-H2009 fever and NCI-H441 fever are lung cancer cell lines. Respectively. 7B confirms the presence of mRNA by hybridizing the same sample with β-actin probe.

FIG. 18A shows northern expression of PIG17 primary cancer genes in normal human 12-row multiple tissue (Clontech), brain, heart, rhabdomyo, colon, thymus, spleen, kidney, liver, small intestine, placenta, lung and peripheral blood leukocyte tissue. The results of the blot analysis are shown. Figure 18B shows the presence of mRNA by hybridizing the same sample with β-actin probe. As can be seen in Figure 18A, PIG17 mRNA (about 1.3 kb) is weakly expressed only in muscle and heart tissues and very weak or no expression in other normal tissues.

29A shows human cancer cell lines, HL-60, HeLa, K-562, MOLT-4, Raji. SW480, A549 and G361 (Clontech) shows the results of Northern blot analysis confirming the expression of PIG17 primary cancer gene. Figure 29B shows the presence of mRNA by hybridizing the same sample with β-actin probe. As can be seen in Figure 29A, PIG17 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 lymphoma cell line High levels in Raji, colon cancer cell line SW480, lung cancer cell line A549 and skin cancer cell line G361.

8A shows a result of Northern blot analysis confirming the expression of PIG19 primary cancer gene in normal lung tissue, lung cancer tissue, lung cancer metastasis tissue and lung cancer cell lines (A549, NCI-H2009, and NCI-H441). As can be seen in FIG. 8A, significant gene expression was observed in lung cancer tissues, lung cancer metastases, and lung cancer cell lines A549, NCI-H2009, and NCI-H441 lung cancer cell lines, but expression levels were very low or observed in normal lung tissue. It wasn't. In FIG. 8, normal rows represent normal lung tissues, cancer rows represent lung cancer tissues, metastasis rows represent lung cancer metastatic tissues, and columns A549, NCI-H2009, and NCI-H441 are lung cancer cell lines. Respectively. 8B confirms the presence of mRNA by hybridizing the same sample with β-actin probe.

FIG. 19A shows Northern expression of PIG19 primary cancer genes in normal human 12-row multiple tissue (Clontech), brain, heart, rhabdomyo, colon, thymus, spleen, kidney, liver, small intestine, placenta, lung and peripheral blood leukocyte tissue. The results of the blot analysis are shown. 19B confirms the presence of mRNA by hybridizing the same sample with β-actin probe. As can be seen in Figure 19A, PIG19 mRNA (about 1.5 kb) is weakly expressed only in muscle and heart tissues and very weak or no expression in other normal tissues.

30A shows human cancer cell lines, HL-60, HeLa, K-562, MOLT-4, Raji. SW480, A549 and G361 (Clontech) shows the results of Northern blot analysis confirming the expression of PIG19 primary cancer gene. Figure 30B shows the presence of mRNA by hybridizing the same sample with β-actin probe. As can be seen in Figure 30A, PIG19 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 lymphoma cell line High levels in Raji, colon cancer cell line SW480, lung cancer cell line A549 and skin cancer cell line G361.

Figure 9A shows the results of Northern blot analysis confirming the expression of PIG20 primary cancer genes in normal lung tissue, lung cancer tissue, lung cancer metastatic tissue and lung cancer cell lines (A549, NCI-H2009, and NCI-H441). As can be seen in Figure 9A, significant gene expression was observed in lung cancer tissues, lung cancer metastatic tissues and lung cancer cell lines A549, NCI-H2009, NCI-H441 lung cancer cell lines, but the expression level was very low in normal lung tissue. In FIG. 9, normal fever is normal lung tissue, cancer fever is lung cancer tissue, metastasis fever is lung cancer metastatic tissue and A549 fever, NCI-H2009 fever and NCI-H441 fever are lung cancer cell lines. Respectively. 9B shows the presence of mRNA by hybridizing the same sample with β-actin probe.

FIG. 20A shows Northern expression of PIG20 primary cancer genes in normal human 12-row multiple tissue (Clontech), brain, heart, rhabdomyo, colon, thymus, spleen, kidney, liver, small intestine, placenta, lung and peripheral blood leukocyte tissue. The results of the blot analysis are shown. Figure 20B shows the presence of mRNA by hybridizing the same sample with β-actin probe. As can be seen in FIG. 20A, PIG20 mRNA (about 2.5 kb) is weakly expressed only in muscle, heart, and placental tissues and very weak or absent in other normal tissues.

31A shows human cancer cell lines, HL-60, HeLa, K-562, MOLT-4, Raji. SW480, A549 and G361 (Clontech) shows the results of Northern blot analysis confirming the expression of PIG20 primary cancer gene. Figure 31B shows the presence of mRNA by hybridizing the same sample with β-actin probe. As can be seen in Figure 31A, PIG20 was expressed at high levels in HeLa uterine cancer cell line, colon cancer cell line SW480, lung cancer cell line A549 and skin cancer cell line G361, but is a promyeloid leukemia cell line HL-60, chronic myeloid leukemia cell line K-562, lymphocyte leukemia cell line, MOLT-4, and Burkitt's lymphoma cell line, Raji, had very low or no expression.

Figure 10A shows the results of Northern blot analysis confirming the expression of PIG21 primary cancer genes in normal lung tissue, lung cancer tissue, lung cancer metastasis tissue and lung cancer cell lines (A549, NCI-H2009, and NCI-H441). As can be seen in Figure 10A, significant gene expression was observed in lung cancer tissues, lung cancer metastatic tissues and lung cancer cell lines A549, NCI-H2009, NCI-H441 lung cancer cell lines, but the expression level was very low in normal lung tissue. In FIG. 10, normal rows represent normal lung tissue, cancer rows represent lung cancer tissues, metastasis rows represent lung cancer metastatic tissues, and columns A549, NCI-H2009, and NCI-H441 are lung cancer cell lines. Respectively. 10B shows the presence of mRNA by hybridizing the same sample with β-actin probe.

FIG. 21A shows Northern PIG21 primary cancer gene expression 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 results of the blot analysis are shown. Figure 21B shows the presence of mRNA by hybridizing the same sample with β-actin probe. As can be seen in FIG. 21A, PIG21 mRNA (approximately 1.3 kb) is weakly expressed only in muscle and heart tissues and very weak or absent in other normal tissues.

32A shows human cancer cell lines, HL-60, HeLa, K-562, MOLT-4, Raji. SW480, A549 and G361 (Clontech) shows the results of Northern blot analysis confirming the expression of PIG21 primary cancer gene. Figure 32B shows the presence of mRNA by hybridizing the same sample with β-actin probe. As can be seen in Figure 31A, PIG21 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 lymphoma cell line High levels in Raji, colon cancer cell line SW480, lung cancer cell line A549 and skin cancer cell line G361.

7-2: PIG  6, PIG  7, TRG  2

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

PIG6; To determine the extent of PIG 7 or TRG 2 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 32P-labeled random primed PIG 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 3a shows the results of Northern blot analysis confirming the expression of the PIG6 primary cancer gene in normal cervical tissue, cervical cancer tissue, cervical cancer metastatic lymph node tissue and cervical cancer cell lines (CaSki and CUMC-6). As can be seen in Figure 3a, PIG6 proto-oncogenes have increased gene expression in cervical cancer tissues and cervical cancer cell lines, CaSki and CUMC-6 cell lines, ie overexpressing a dominant PIG6 mRNA transcript of about 4.4 kb. In particular, cervical cancer metastasis lymph node tissue showed the highest expression level and normal tissue expression level was very low. In FIG. 3, normal rows represent normal cervical tissue, cancer rows represent cervical cancer tissues, metastasis rows represent cervical cancer metastatic lymph node tissues, and CaSki rows and CUMC-6 rows, respectively. Uterine cancer cell line. Figure 3b hybridizes the same sample with β-actin probe to confirm the presence of mRNA.

FIG. 14A shows Northern expression of PIG6 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 results of the blot analysis are shown. 14B hybridizes the same sample with β-actin probe to confirm the presence of mRNA. As can be seen in FIG. 14A, PIG6 mRNA ((dominant PIG6 mRNA transcript of about 4.4 kb and PIG6 mRNA transcript of about 8 kb)) is normal brain, heart, rhabdomyo, colon, thymus, spleen, It is weakly expressed in kidney, liver, small intestine, placenta, lung and peripheral blood leukocyte tissues.

Figure 25a shows the results of Northern blot analysis confirming the expression of PIG6 primary oncogenes in human cancer cell lines, HL-60, HeLa, K-562, MOLT-4, Raji, SW480, A549 and G361 (Clontech). 25B shows the presence of mRNA by hybridizing the same sample with β-actin probe. As can be seen in FIG. 25A, PIG6 mRNA ((dominant PIG6 mRNA transcript of about 4.4 kb and PIG6 mRNA transcript of about 8 kb) is a promyeloid leukemia cell line HL-60, HeLa cervical cancer cell line, Very elevated levels were expressed in chronic myeloid leukemia cell line K-562, lymphocyte leukemia cell line MOLT-4, Burkitt's lymphoma cell line Raji, and colorectal cancer cell line SW480.

Figure 4a shows the results of Northern blot analysis confirming the expression of PIG7 primary cancer genes in normal cervical tissue, cervical cancer tissue, cervical cancer metastatic lymph node tissue and cervical cancer cell lines (CaSki and CUMC-6). As can be seen in Figure 4a, PIG7 primary cancer gene gene expression was increased in the cervical cancer tissue and cervical cancer cell lines CaSki and CUMC-6 cell line, that is, over 7.5 kb dominant PIG7 mRNA transcript overexpressed In particular, cervical cancer metastasis lymph node tissue showed the highest expression level and normal tissue expression level was very low. In FIG. 4, normal columns represent normal cervical tissue, cancer rows represent cervical cancer tissues, metastasis rows represent cervical cancer metastatic lymph node tissues, and CaSki rows and CUMC-6 rows, respectively. Uterine cancer cell line. 4B shows the presence of mRNA by hybridizing the same sample with β-actin probe.

FIG. 15A shows Northern expression of PIG7 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 tissue The results of the blot analysis are shown. 15B shows the presence of mRNA by hybridizing the same sample with β-actin probe. As can be seen in FIG. 15A, PIG7 mRNA (dominant PIG7 mRNA transcript of about 7.5 kb) is normal brain, heart, rhabdomyo, colon, thymus, spleen, kidney, liver, small intestine, placenta, lung and peripheral It is weakly expressed in blood leukocyte tissues.

Figure 26a shows the results of Northern blot analysis confirming the expression of PIG7 primary oncogenes in human cancer cell lines, HL-60, HeLa, K-562, MOLT-4, Raji, SW480, A549 and G361 (Clontech). Figure 26b shows the presence of mRNA by hybridizing the same sample with β-actin probe. As can be seen in FIG. 26A, PIG7 mRNA (dominant PIG7 mRNA transcript of about 7.5 kb) is a promyeloid leukemia cell line HL-60, HeLa uterine cancer cell line, chronic myeloid leukemia cell line K-562, lymphocytic leukemia The cell lines MOLT-4, Burkitt's lymphoma cell line Raji, colorectal cancer cell line SW480, lung cancer cell line A549, and skin cancer cell line G361 were also expressed at significantly increased levels.

Figure 12a shows the results of Northern blot analysis confirming the expression of TRG2 primary cancer genes in normal cervical tissue, cervical cancer tissue, cervical cancer metastatic lymph node tissue and cervical cancer cell lines (CaSki and CUMC-6). As can be seen in Figure 12a, the TRG2 proto-oncogenes have increased gene expression in cervical cancer tissues and cervical cancer cell lines CaSki and CUMC-6 cell lines, ie overexpressing a dominant TRG2 mRNA transcript of about 10 kb. In FIG. 12, normal columns represent normal cervical tissue, cancer columns represent cervical cancer tissues, metastasis columns represent cervical cancer metastatic lymph node tissues, and CaSki columns and CUMC-6 columns. Each represents a uterine cancer cell line. 2B shows the presence of mRNA by hybridizing the same sample with β-actin probe.

23A shows Northern TRG2 proto-oncogene expression 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 results of the blot analysis are shown. FIG. 23B hybridizes the same sample with β-actin probe to confirm the presence of mRNA. As can be seen in Figure 23a, TRG2 mRNA (dominant TRG2 mRNA transcript of about 10 kb) is expressed in normal muscle tissue but normal brain, heart, colon, thymus, spleen, kidney, liver, small intestine , Very weakly expressed in placenta, lung and peripheral blood leukocyte tissues.

Figure 36a shows the results of Northern blot analysis confirming the expression of TRG2 primary oncogenes in human cancer cell lines, HL-60, HeLa, K-562, MOLT-4, Raji, SW480, A549 and G361 (Clontech). 36B shows the presence of mRNA by hybridizing the same sample with β-actin probe. As can be seen in FIG. 36A, TRG2 mRNA (dominant TRG2 mRNA transcript of about 10 kb) was expressed in HeLa uterine cancer cell line, chronic myeloid leukemia cell line K-562, Burkitt lymphoma cell line Raji, colon cancer cell line. It was expressed at increased levels in SW480, lung cancer cell line A549 and skin cancer cell line G361.

7-3: HCCR BP  2

To determine the degree of HCCRBP2 gene expression in normal tissues and cancer cells, commercially available normal human 12-row multiple tissues (Clontech), which extracted normal RNA from 12 organs and blotted onto the nylon membrane (FIG. 22). RNA was extracted from eight types of cancer cells and commercialized human cancer cell 8-column multiple tissues (Clontech Co., Ltd.) (FIG. 11), which were blotted onto the membrane, were compared, and the expression differences were compared. Both membranes hybridized blots with 32P-labeled random primed HCCRBP2 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 22 Expression of HCCRBP2 primary cancer gene in normal human 12-row multiple tissues (Clontech), ie, brain, heart, skeletal muscle, colon, thymus, spleen, kidney, liver, small intestine, placenta, lung and peripheral blood leukocyte tissues Northern blot analysis results confirmed whether the results are shown. Figure 22 confirms the presence of mRNA by hybridizing the same sample with β-actin probe. As can be seen in FIG. 22, the dir 2.4 kb transcript, HCCRBP2 mRNA transcript, was weak or unidentified in several normal tissues.

FIG. 11 shows Northern blot analysis of human cancer cell lines (Clontech Co., Ltd.), ie, HL-60, HeLa, K-562, MOLT-4, Raji, SW480, A549, and G361 cell lines. It is shown. As can be seen in Figure 11, the 2.4 kb transcript of HCCRBP2 is HL-60, a promyelocytic leukemia cell line, HeLa cells, a uterine cancer cell line, K-562, a chronic myeloid leukemia cell line, MOLT-4, a lymphocyte leukemia cell line High levels were expressed in the Rakit, Burkitt lymphoma cell line, SW480, the colorectal cancer cell line, A549, the lung cancer cell line, and the G361 cell line, the skin cancer.

Uterine, colorectal and leukocyte tissues of healthy normal people, and uterine cancers from uterine cancer, colorectal and leukemia patients, using the RNeasy Total RNA Kit (Qiagen) to confirm the expression of HCCRBP2 gene in uterine cancer, colorectal cancer and leukemia, Total RNA was extracted from colorectal cancer and leukemia 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). The blot was hybridized with a 32P-labeled random primed HCCRBP2 whole cDNA probe 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 33a shows the results of Northern blot analysis confirming the expression of HCCRBP2 primary cancer genes in normal cervical tissue, cervical cancer tissue, cervical cancer metastatic lymph node tissue and cervical cancer cell lines (CaSki and CUMC-6). As can be seen in Figure 33a, the HCCRBP2 proto-oncogene has increased gene expression in cervical cancer tissues and cervical cancer cell lines CaSki and CUMC-6 cell lines, that is, overexpression of the dominant HCCRBP2 mRNA transcript of about 2.4 kb. In particular, cervical cancer metastasis lymph node tissue showed the highest expression level and normal tissue expression level was very low. In FIG. 33, normal rows represent normal cervical tissue, cancer rows represent cervical cancer tissues, metastasis rows represent cervical cancer metastatic lymph node tissues, and CaSki rows and CUMC-6 rows, respectively. Uterine cancer cell line. 33B hybridizes the same sample with β-actin probe to confirm the presence of mRNA.

Figure 34a shows the results of Northern blot analysis confirming the expression of HCCRBP2 primary cancer gene in normal colon tissue and colorectal cancer tissues. 34B shows the presence of mRNA by hybridizing the same sample with β-actin probe. As can be seen in FIG. 34A, significant gene expression of 2.4 kb HCCRBP2 mRNA transcript was observed in colon cancer tissues, but was low in normal colon tissues. 34A and 34B, normal fever (N) represents normal colon tissue and dark fever (C) shows colon cancer tissue, respectively.

Figure 35a shows the results of Northern blot analysis confirming the expression of HCCRBP2 primary cancer gene in normal leukocyte tissue and leukemia tissues. 35B shows the presence of mRNA by hybridizing the same sample with β-actin probe. As can be seen in Figure 35a, remarkable gene expression of 2.4 kb HCCRBP2 mRNA transcript was observed in leukemia tissues, but the expression level was low in normal leukocyte tissues. 35A and 35B, normal fever (N) represents normal leukocyte tissue and dark fever (C) represents leukemia tissue, respectively.

Example 8: Determination of the protein expressed after transfection of E. coli

8-1: PIG 5, PIG 6, PIG 7, PIG 11, PIG 16, PIG 17, PIG 19, PIG 20, PIG 21, TRG 2

The PIG5 oncogene of SEQ ID NO: 1; The PIG6 far-oncogene of SEQ ID NO: 5; the PIG7 far-oncogene of SEQ ID NO: 9; PIG 11 of SEQ ID NO: 13; PIG 16 of SEQ ID NO: 17; PIG 17 of SEQ ID NO: 21; PIG 19 of SEQ ID NO: 25; PIG 20 of SEQ ID NO: 29; PIG 21 of SEQ ID NO: 33; Or insert the TRG 2 primary oncogene of SEQ ID NO: 39 into the multi-cloning site of the pBAD / thio-Topo vector (Invitrogen, USA), and then generate the resulting pBAD / thio-Topo / PIG vector to E. coli Top10 (Invitrogen, USA) Was transfected. 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. To this was added 0.5 mM L-Arabinose (L-Arabinose, Sigma) to induce protein production.

E. coli cells in the culture medium before and after L arabinose induction were subjected to ultrasonic grinding and electrophoresed on 12% sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE).

37A shows the results of SDS-PAGE expression of the protein of the E. coli Top10 strain transfected with the pBAD / thio-Topo / PIG5 vector. A fusion protein band of about 42 kDa was clearly observed after the induction of El arabinose. This 42 kDa fusion protein contains about 15 kDa HT-thioredoxin protein and about 27 kDa PIG5 protein inserted into pBAD / thio-Topo / PIG5 vector.

37B shows the expression profile of the E. coli Top10 strain transfected with the pBAD / thio-Topo / PIG6 vector by SDS-PAGE. A fusion protein band of about 87 kDa was clearly observed after El arabinose induction. This 87 kDa fusion protein contains about 15 kDa HT-thioredoxin protein and about 72 kDa PIG6 protein inserted into pBAD / thio-Topo / PIG6 vector.

37C shows the expression profile of the E. coli Top10 strain transfected with the pBAD / thio-Topo / PIG7 vector by SDS-PAGE. A fusion protein band of about 24 kDa was clearly observed after El arabinose induction. This 24 kDa fusion protein contains about 15 kDa HT-thioredoxin protein and about 9 kDa PIG7 protein inserted into pBAD / thio-Topo / PIG7 vector.

37D shows the protein expression pattern of the E. coli Top10 strain transfected with the pBAD / thio-Topo / PIG11 vector by SDS-PAGE. A fusion protein band of about 47 kDa was clearly observed after induction of El arabinose. This 47 kDa fusion protein contains about 15 kDa HT-thioredoxin protein and about 32 kDa PIG11 protein inserted into pBAD / thio-Topo / PIG11 vector.

37E shows SDS-PAGE expression of the protein of the E. coli Top10 strain transfected with the pBAD / thio-Topo / PIG16 vector. As a result, the fusion protein band of about 47 kDa was clearly observed after induction of El arabinose. This 47 kDa fusion protein contains about 15 kDa HT-thioredoxin protein and about 32 kDa PIG16 protein inserted into pBAD / thio-Topo / PIG16 vector.

37F shows the expression pattern of the E. coli Top10 strain transfected with the pBAD / thio-Topo / PIG17 vector by SDS-PAGE. A fusion protein band of about 35 kDa was clearly observed after El arabinose induction. This 35 kDa fusion protein contains about 15 kDa HT-thioredoxin protein and about 20 kDa PIG17 protein inserted into the pBAD / thio-Topo / PIG17 vector.

Figure 37g shows the results of SDS-PAGE expression of the protein of E. coli Top10 strain transfected with pBAD / thio-Topo / PIG19 vector, the fusion protein band of about 52 kDa was clearly observed after El arabinose induction. This 52 kDa fusion protein contains about 15 kDa HT-thioredoxin protein and about 37 kDa PIG19 protein inserted into the pBAD / thio-Topo / PIG19 vector.

FIG. 37h shows the expression profile of the protein of E. coli Top10 strain transfected with the pBAD / thio-Topo / PIG20 vector by SDS-PAGE. A fusion protein band of about 34 kDa was clearly observed after El arabinose induction. This 34 kDa fusion protein contains about 15 kDa HT-thioredoxin protein and about 19 kDa PIG20 protein inserted into pBAD / thio-Topo / PIG20 vector.

37i shows the expression pattern of the E. coli Top10 strain transfected with the pBAD / thio-Topo / PIG21 vector by SDS-PAGE. A fusion protein band of about 45 kDa was clearly observed after El arabinose induction. This 45 kDa fusion protein contains about 15 kDa HT-thioredoxin protein and about 30 kDa PIG21 protein inserted into pBAD / thio-Topo / PIG21 vector.

37K shows the results of SDS-PAGE expression of the protein of the E. coli Top10 strain transfected with the pBAD / thio-Topo / TRG2 vector. As a result, the fusion protein band of about 63 kDa was clearly observed after El arabinose induction. This 63 kDa fusion protein contains about 15 kDa HT-thioredoxin protein and about 48 kDa TRG2 protein inserted into pBAD / thio-Topo / TRG2 vector.

8-2: HCCR BP  2

PGEX 4T-3 vector (Amersham) in which the entire coding region of the HCCRBP2 primary cancer gene corresponding to nucleotides 9 to 356 of SEQ ID NO: 37 and predicted to encode amino acid numbers 1 to 115 of SEQ ID NO: 38 was fused by inserting a multiple cloning site within the BamH I and N ot I restriction site of Pharmacia Biotech Co.) and then producing a pGEX 4T-3 / HCCRBP2 vector, it was transfected them into 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 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. 37j shows the protein expression profile of the E. coli BL21 strain transfected with the pGEX 4T-3 / HCCRBP2 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.

As a novel gene, the primary cancer gene of the present invention can be effectively used for diagnosis of cancer including leukemia, uterine cancer, lymphoma, colorectal cancer, lung cancer, skin cancer, and the production of transgenic animals.

<110> KIM, HYUN KEE <120> HUMAN PROTOONCOGENE AND PROTEIN ENCODED BY SAME AND EXPRESSION          VECTOR CONTAINING SAME <160> 42 <170> KopatentIn 1.71 <210> 1 <211> 1009 <212> DNA <213> Homo sapiens <400> 1 gcttcaagat ggcggtgcag gagtcggcgg ctcagttgtc catgaccctg aaggtccagg 60 agtacccgac cctcaaggtg ccctacgaga cgctgaacaa atgctttcgc gccgctcaga 120 agaacattga ccgggagacc agccacgtca ccatggtggt ggccgagctg gagaagacgt 180 tgagcggctg ccccgccgtg gactccgtgg tcagcctgct ggacggcgtg gtggagaagc 240 tcagcgtcct caagaggaag gcggtggaat ccatccaggc cgaggacgag agcgccaagc 300 tgtgcaagcg ccggatcgag cacctcaaag agcatagcag cgaccagccc gcggcggcca 360 gcgtgtggaa gaggaagcgc atggatcgca tgatggtgga gcacctgctg cgttgcggct 420 actacaacac ggctgtcaag ctggcgcgcc agagcggcat cgagacatgc aagaaagcac 480 ttcagccaag cagaagggag ccagctggac gaggtgcgcc aggccatggg catgctggcc 540 ttcccgcccg acacgcacat ctccccgtac aaggaccttc tggaccctgc acggtggcgg 600 atgctgatcc agcagttccg gtacgacaac taccgactac accagctggg aaacaattct 660 gtgttcaccc tcaccctgca ggccggcctc tcagccatca agacaccaca gtgctacaag 720 gaggacggca gctccaagag ccctgactgc cctgtgtgca gccgctccct gaacaagctg 780 gcgcagcccc tgctcatggc ccactgtgcc aactcccgcc tggtctgcaa gatttctggc 840 gacgtgatga acgagaacaa tccgcccatg atgctgccca acggctacgt ctacggctac 900 aattctctgc tttctatccg tcaagatgat aaagtcgtgt gcccgagaac caaagaagtc 960 ttccacttct cacaagccga gaaggtgtac atcatgtagg ccccacgtc 1009 <210> 2 <211> 245 <212> PRT <213> Homo sapiens <400> 2 Met Ala Val Gln Glu Ser Ala Ala Gln Leu Ser Met Thr Leu Lys Val   1 5 10 15 Gln Glu Tyr Pro Thr Leu Lys Val Pro Tyr Glu Thr Leu Asn Lys Cys              20 25 30 Phe Arg Ala Ala Gln Lys Asn Ile Asp Arg Glu Thr Ser His Val Thr          35 40 45 Met Val Val Ala Glu Leu Glu Lys Thr Leu Ser Gly Cys Pro Ala Val      50 55 60 Asp Ser Val Val Ser Leu Leu Asp Gly Val Val Glu Lys Leu Ser Val  65 70 75 80 Leu Lys Arg Lys Ala Val Glu Ser Ile Gln Ala Glu Asp Glu Ser Ala                  85 90 95 Lys Leu Cys Lys Arg Arg Ile Glu His Leu Lys Glu His Ser Ser Asp             100 105 110 Gln Pro Ala Ala Ala Ser Val Trp Lys Arg Lys Arg Met Asp Arg Met         115 120 125 Met Val Glu His Leu Leu Arg Cys Gly Tyr Tyr Asn Thr Ala Val Lys     130 135 140 Leu Ala Arg Gln Ser Gly Ile Glu Thr Cys Lys Lys Ala Leu Gln Pro 145 150 155 160 Ser Arg Arg Glu Pro Ala Gly Arg Gly Ala Pro Gly His Gly His Ala                 165 170 175 Gly Leu Pro Ala Arg His Ala His Leu Pro Val Gln Gly Pro Ser Gly             180 185 190 Pro Cys Thr Val Ala Asp Ala Asp Pro Ala Val Pro Val Arg Gln Leu         195 200 205 Pro Thr Thr Pro Ala Gly Lys Gln Phe Cys Val His Pro His Pro Ala     210 215 220 Gly Arg Pro Leu Ser His Gln Asp Thr Thr Val Leu Gln Gly Gly Arg 225 230 235 240 Gln Leu Gln Glu Pro                 245 <210> 3 <211> 16 <212> DNA <213> Artificial Sequence <220> <223> H-T11G primer <400> 3 aagctttttt tttttg 16 <210> 4 <211> 13 <212> DNA <213> Artificial Sequence <220> <223> H-AP9 primer <400> 4 aagcttcatt ccg 13 <210> 5 <211> 2964 <212> DNA <213> Homo sapiens <400> 5 gaagcctgaa ccacctgtta atctgaagta caatgcaccc acgtctcatg ttactccgtc 60 cgtcaagaaa agaagcagca ccttatctca gctccctggg gataagtcca aagcctttga 120 tttccttagt gaagaaactg aagctagttt agcctcacgc agagaacaaa agagagagca 180 gtatcgtcag gtaaaagcac atgttcagaa ggaagacggt agagtgcagg cttttggctg 240 gagtctgcct cagaagtaca aacaggtaac caatggtcaa ggtgaaaata agatgaaaaa 300 tttacctgtg cctgtctatc tcagacctct ggatgaaaaa gatacatcaa tgaagctgtg 360 gtgtgctgtt ggagtcaatt tatctggtgg gaagaccaga gatggtggtt ctgttgttgg 420 agcaagtgta ttttacaagg atgttgctgg tttggataca gaaggcagta aacagcgaag 480 tgcctctcag agtagtttag ataagttaga tcaggaactt aaggaacagc agaaggagtt 540 aaaaaatcaa gaagaattat ccagtctagt ttggatctgt accagcactc attcggctac 600 aaaagttctt attattgatg ctgttcaacc tggcaacatc ctagacagtt tcactgtttg 660 caactctcat gttctgtgca ttgcaagtgt gccaggtgca cgagaaacag actaccctgc 720 aggagaagat ctttcagaat ctggtcaggt agacaaagca tctttatgtg gaagtatgac 780 aagcaacagc tcagcagaga cagacagcct gttaggaggc atcacagtgg ttggttgttc 840 tgcagaaggt gtgacgggag ctgccacttc ccctagtaca aatggtgctt ctccagtgat 900 ggataaacca ccagaaatgg aagcagaaaa tagtgaggtt gatgaaaatg ttccaacagc 960 agaagaagca actgaagcta cagaagggaa tgcggggtca gctgaagaca cagtggacat 1020 ctcccaaact ggcgtctaca cagagcatgt ctttacagat cctttgggag ttcagatccc 1080 agaagacctc tccccagtgt atcagtcgag caatgactca gatgcatata aagatcaaat 1140 atcagtactg ccaaatgaac aagacttggt gagagaagaa gcccagaaaa tgagtagtct 1200 tttaccaact atgtggcttg gagctcaaaa tggctgtttg tatgtccatt catctgtagc 1260 ccagtggagg aaatgtctcc attccattaa acttaaagat tcgattctca gtattgtaca 1320 cgtgaaggga atcgtgttag tagccctggc tgacggcacc cttgcaatct ttcacagagg 1380 agtggatggg cagtgggatt tgtcaaacta tcacctctta gaccttggac ggcctcatca 1440 ttccatccgt tgcatgactg tggtacatga caaagtctgg tgtggctata ggaacaaaat 1500 ctatgtggtg cagccaaagg ccatgaaaat agagaaatct tttgatgcac atcccaggaa 1560 ggagagccaa gtgcgacagc ttgcgtgggt gggggatggc gtgtgggtct ccattcgctt 1620 ggattctacg ctccgtctct atcatgcaca cacttatcaa catctacagg atgtggacat 1680 tgagccttat gtaagcaaaa tgttaggtac tggaaaactg ggcttctctt ttgtgagaat 1740 tacagctctt atggtgtctt gtaatcgttt gtgggtgggg acaggaaatg gtgtcattat 1800 ctccatccca ttgacagaaa ccgtaatcct ccaccaggga cgtttactgg ggctgagggc 1860 aaataaaacc tcaggtgtac caggaaatcg tcctggaagt gtaatccgtg tatatggtga 1920 tgaaaacagt gataaagtga ctccagggac atttataccc tattgttcaa tggcacatgc 1980 acagctttgc ttccatgggc accgggatgc tgtgaaattc tttgtggcag tcccaggtca 2040 agtcatcagc ccacaaagta gcagtagtgg cacggatctg acgggtgaca aagcagggcc 2100 atctgcacag gagcctggta gtcagacgcc cttgaagtct atgcttgtca tcagtggagg 2160 agagggctac atcgacttcc gaatgggtga tgaaggtgga gaatcagaac ttcttggaga 2220 ggatcttcca cttgaacctt ctgtcaccaa agcagaaagg agtcacttga tagtgtggca 2280 agtgatgtat ggcaatgagt gagcccatgg gaaacaggtg gagatgggga agccgtctct 2340 tctgcatggt ttattttccc tctatccttt tatttaatgc tcttttgtga gataagtttc 2400 accacataat gtgtgagcat tttttcctgt taactttata ttacaaaatc cgttctacca 2460 taacaataca gaggaactag ctgtgttact gcaccagtgt tataggtaac ttcagtatat 2520 tatgaacaaa tcaaagaatg tttacttcct gcaaactggt gaattataga aagcaatcca 2580 gatgtggttt actctgccac agtctaatgt cattcacttc atttgatggg gtcacttgtt 2640 agctgtcact aataatggaa ttaatgggaa acacttgata aatgaaactg taccgttaaa 2700 tacaatagct gagttttccc cagtgtattg taaaattgac acacactaat acaagatgga 2760 ttatcaggat gtatctaaat gtcccgagag ggttaaaagc taactgtaaa ttactttaac 2820 tttcactttc aaatctgaca aatcttgttt atatggtata taaaactttg ttttcatcag 2880 atttgggggg gttttatatt aaagaaatta accctaaaaa aaaaaaaaaa aaaaaaaaaa 2940 aaaaaaaaaa aaaaaaaaaa aaaa 2964 <210> 6 <211> 669 <212> PRT <213> Homo sapiens <400> 6 Met Lys Asn Leu Pro Val Pro Val Tyr Leu Arg Pro Leu Asp Glu Lys   1 5 10 15 Asp Thr Ser Met Lys Leu Trp Cys Ala Val Gly Val Asn Leu Ser Gly              20 25 30 Gly Lys Thr Arg Asp Gly Gly Ser Val Val Gly Ala Ser Val Phe Tyr          35 40 45 Lys Asp Val Ala Gly Leu Asp Thr Glu Gly Ser Lys Gln Arg Ser Ala      50 55 60 Ser Gln Ser Ser Leu Asp Lys Leu Asp Gln Glu Leu Lys Glu Gln Gln  65 70 75 80 Lys Glu Leu Lys Asn Gln Glu Glu Leu Ser Ser Leu Val Trp Ile Cys                  85 90 95 Thr Ser Thr His Ser Ala Thr Lys Val Leu Ile Ile Asp Ala Val Gln             100 105 110 Pro Gly Asn Ile Leu Asp Ser Phe Thr Val Cys Asn Ser His Val Leu         115 120 125 Cys Ile Ala Ser Val Pro Gly Ala Arg Glu Thr Asp Tyr Pro Ala Gly     130 135 140 Glu Asp Leu Ser Glu Ser Gly Gln Val Asp Lys Ala Ser Leu Cys Gly 145 150 155 160 Ser Met Thr Ser Asn Ser Ser Ala Glu Thr Asp Ser Leu Leu Gly Gly                 165 170 175 Ile Thr Val Val Gly Cys Ser Ala Glu Gly Val Thr Gly Ala Ala Thr             180 185 190 Ser Pro Ser Thr Asn Gly Ala Ser Pro Val Met Asp Lys Pro Pro Glu         195 200 205 Met Glu Ala Glu Asn Ser Glu Val Asp Glu Asn Val Pro Thr Ala Glu     210 215 220 Glu Ala Thr Glu Ala Thr Glu Gly Asn Ala Gly Ser Ala Glu Asp Thr 225 230 235 240 Val Asp Ile Ser Gln Thr Gly Val Tyr Thr Glu His Val Phe Thr Asp                 245 250 255 Pro Leu Gly Val Gln Ile Pro Glu Asp Leu Ser Pro Val Tyr Gln Ser             260 265 270 Ser Asn Asp Ser Asp Ala Tyr Lys Asp Gln Ile Ser Val Leu Pro Asn         275 280 285 Glu Gln Asp Leu Val Arg Glu Glu Ala Gln Lys Met Ser Ser Leu Leu     290 295 300 Pro Thr Met Trp Leu Gly Ala Gln Asn Gly Cys Leu Tyr Val His Ser 305 310 315 320 Ser Val Ala Gln Trp Arg Lys Cys Leu His Ser Ile Lys Leu Lys Asp                 325 330 335 Ser Ile Leu Ser Ile Val His Val Lys Gly Ile Val Leu Val Ala Leu             340 345 350 Ala Asp Gly Thr Leu Ala Ile Phe His Arg Gly Val Asp Gly Gln Trp         355 360 365 Asp Leu Ser Asn Tyr His Leu Leu Asp Leu Gly Arg Pro His His Ser     370 375 380 Ile Arg Cys Met Thr Val Val His Asp Lys Val Trp Cys Gly Tyr Arg 385 390 395 400 Asn Lys Ile Tyr Val Val Gln Pro Lys Ala Met Lys Ile Glu Lys Ser                 405 410 415 Phe Asp Ala His Pro Arg Lys Glu Ser Gln Val Arg Gln Leu Ala Trp             420 425 430 Val Gly Asp Gly Val Trp Val Ser Ile Arg Leu Asp Ser Thr Leu Arg         435 440 445 Leu Tyr His Ala His Thr Tyr Gln His Leu Gln Asp Val Asp Ile Glu     450 455 460 Pro Tyr Val Ser Lys Met Leu Gly Thr Gly Lys Leu Gly Phe Ser Phe 465 470 475 480 Val Arg Ile Thr Ala Leu Met Val Ser Cys Asn Arg Leu Trp Val Gly                 485 490 495 Thr Gly Asn Gly Val Ile Ile Ser Ile Pro Leu Thr Glu Thr Val Ile             500 505 510 Leu His Gln Gly Arg Leu Leu Gly Leu Arg Ala Asn Lys Thr Ser Gly         515 520 525 Val Pro Gly Asn Arg Pro Gly Ser Val Ile Arg Val Tyr Gly Asp Glu     530 535 540 Asn Ser Asp Lys Val Thr Pro Gly Thr Phe Ile Pro Tyr Cys Ser Met 545 550 555 560 Ala His Ala Gln Leu Cys Phe His Gly His Arg Asp Ala Val Lys Phe                 565 570 575 Phe Val Ala Val Pro Gly Gln Val Ile Ser Pro Gln Ser Ser Ser Ser             580 585 590 Gly Thr Asp Leu Thr Gly Asp Lys Ala Gly Pro Ser Ala Gln Glu Pro         595 600 605 Gly Ser Gln Thr Pro Leu Lys Ser Met Leu Val Ile Ser Gly Gly Glu     610 615 620 Gly Tyr Ile Asp Phe Arg Met Gly Asp Glu Gly Gly Glu Ser Glu Leu 625 630 635 640 Leu Gly Glu Asp Leu Pro Leu Glu Pro Ser Val Thr Lys Ala Glu Arg                 645 650 655 Ser His Leu Ile Val Trp Gln Val Met Tyr Gly Asn Glu             660 665 <210> 7 <211> 16 <212> DNA <213> Artificial Sequence <220> <223> H-T11A primer <400> 7 aagctttttt ttttta 16 <210> 8 <211> 13 <212> DNA <213> Artificial Sequence <220> <223> H-AP32 primer <400> 8 aagcttcctg caa 13 <210> 9 <211> 4301 <212> DNA <213> Homo sapiens <400> 9 cggcggaatt gtcgacggcc attaccaatc gcgaaaccaa ttataacact agggaaaaat 60 ttgtagcgga tggcagtgtt gaaggcaaat gtaaacataa gggtaatggt ctgtcatggc 120 ttttagaaaa agatgataga gttcatcatt attttgcctt catctttgtt aaggacagaa 180 aattccctga caggtgggca agtatcaggt tacctatttt ttattccttt ggtacaaaag 240 ggttgaacgt caggctaaaa aagcagccat gcatttatta ttaagcattt tctaccgaca 300 aggcactgtg ctaggtactg taatcctacc ataagtaggt aggtatttct tccactgtaa 360 atcatagggg tttgctgttt tatgtgagtt agcctcttcc ccttgtctga gcattcctca 420 ggggaggtca cctgtgaggt tcccagaact gtagtttttt ttaccagggt gttgtatttg 480 gagggggagg aggactcggc tcaaaagagc tagctggctc tccagtgttc agaggtgagt 540 ccacgatact cttaccacaa tttggaagtt tgtgaatctt tttaaagaac taatcaatct 600 ctaatagcat tgaggttgta cctacatatt aagttgaatg gactgttcta tttaaaaaat 660 aaacaactag acaattaact agtttattaa cctatcacaa ttgaattttt ttttaatttt 720 cagtcttaac acatttttta aaatgtatta aagtaataca ttgtagtagt aggattatat 780 actccttggc tgagaattcc aagtactgtg gttctactgt ttagtggaaa actctggaag 840 ttaaaatata gaatatgaga ggaggctttt ttataatggg catcattgtg tggaaaatga 900 cccatgtgaa tacaaatatt tcctagttca gagattttgg ttatatctgg tgcttggatc 960 aagtttaaaa atggaaggtg agattttgca tgagcctatt aaaaagcata gtaataaatg 1020 caaggccagc tggtggaaaa gtgaggcaga atggagcttg tttataggtt ttctgataac 1080 aattataaaa aatgtgcttt atagattaag atttattgaa gtataaatat gtagtaatga 1140 tataatgtat tttaagttat acaagaaaat gtagggactt ttgtttgggt ctttttctct 1200 ttgtggctga ggggaaacaa gtcagtgtcc aataaagctg taaactcctc tgctctaaga 1260 taaatgatgt gatttattta tttataactg gcttctttcc aagtaggttt tcaggtggca 1320 tatttggaag acggctggaa tgacagaatt cttgtatcag agtaggtaag aagggagcaa 1380 cctctcaatg gctattatgt catgcttttt aagatcgtat gcggttccta tatacaagga 1440 agcttccctg tggtagattt gtcataaatg ccaaagatat ttggtaatgt gagtgtagaa 1500 aaagtagtat gagggttggc aaatactgtt tttgtcttgg cagctctaat atctgcattg 1560 ttcagaaagg attctgaggc taaggcaaag ctctgtggga aaaagggact ggaccaaaaa 1620 aaactggatg gtggcacaca agaagaggaa atgatgagat gtgtactttc tatctctggt 1680 taggcttagt ccccactaga caaattgatt ttaaattcca tccacacatt cattacccac 1740 acagataatc atagaaattt gggggagtgc ctagcttctg atgaagtggt gatatggcag 1800 ttgccaagca gtggcatcgc cagagtatct gtttggttag caaatgagca gtcattttag 1860 gtcatgcaga ttgctgatat ctgcccagta gccactgagc atttgctggt tttttcttct 1920 ggctttcttg gaggttaagc tctctgtagt catacccagt tggtacttga tctttagcaa 1980 tatgtctcat attcatgtaa attgaaggga gggttacatg tactgaaata atctgcatgc 2040 taggcattgg cttagacacc gtacctatct cacttagatt tgtggactag gaaagcaaga 2100 ttcagagatc atgtgacttg catgtggcct agagataaaa ttcaaatctg gttctgtaga 2160 ctccagggac atattcacca tgccatgggt ggtggctatt aaaccttgat aaatttgtgt 2220 ttatggttaa caaatgtgaa agctattaaa cattgctggt ttgaattttt tacagtgcag 2280 aaatgtaaaa tgaaaaagga tatttccttt cacagtgtta ccgagaagtc atgataattt 2340 cgtttgttct tccagattta ggcatatact tatttaatca ataatgtgtt aacagctgac 2400 acctgtggtt gctgtgacag gcactatttg aagtgcttta tcatggatta actcttaatc 2460 ctcagctacc gtatacagta ggacataacc ccatttcaca tgcactacac tgagacttgc 2520 ctcctctccc cccacattga agatgttctt ttttcataac tatatactat tccattgcat 2580 gaatattctg taatttattt aatcccctat ggattgataa ttaggttcat tatagataga 2640 agtgtaatta acattcctgt acatgtattt tgctacttgt gtgggtattt ctgtaggatg 2700 aataactaga aatttattgg atcaggtttc acatttgcag ttttgaaaac tactaccaaa 2760 aagatttcac caatttacaa ctccatcatt agtaagaatg cctgtttgcc tatagtctgc 2820 caaccctgaa tccttaaaaa tttttgccaa tctggtaggc aaaatttctt tcttttcttt 2880 gaatattaat gaggaggaac atcttttcat gtttcttggc catttgcatt tcctattatg 2940 aattgctttt gcccattttc ctttttttaa ttatgaaagt ctaatgacta ccttctcatt 3000 gtataaaaaa cacagttctt tgaatagaga gacccttttc tccaatgcta ccaatcacat 3060 tccacttacc acagtttaac atacatcctc tagtcacctt tccgtacgaa tatacataca 3120 cataaaaaca ctttttacat aaataggatc tcatattctg tagcttttta aaattttggt 3180 ctcaaaaaaa gataacaggt ctttaaattt ctttaatggt tgaatatgat taaatactat 3240 gaaaatgcca ttatttattc ccttaatttt tttcctctcg ctattacatt gccaaagtaa 3300 acatcctatt cagatgtctt tgtgcatgtg tgtgaatatt tctttagtct ggagtccagt 3360 aaggtggatt tttggatcaa agggtttgtt ctctgtccac cttcagtctt cccaaaggcc 3420 ttcataactg tattttcacc aagtgtatgg agaatgttca tttccccata taaccatacc 3480 tacacttgat agtttttatc tgttgggcga aaaagaacct tttcttattt tgcatttccc 3540 tgattataaa aaaaaatggt gagattgggg ttattttcat gtttattggc catttatagt 3600 ttactgtgga ttgtttgtat cccttacctg ctttctattg ggttatgtgt ggatatattg 3660 tttttatttg ttcagcatct ccttccccat cttctggtaa cacaaccttt atttatttgt 3720 ggggaaccta ttccctgtgg cttaggtgag catgtgacca ggcctggcct cctgagtccc 3780 acagcttcct agccacagtg ataaaagaat gggtatataa cttaagccag gctaaggaaa 3840 gcccttaaca gaacttctgc tggaactact ggaaagaagg ctttatggag atcccaggaa 3900 ccaaggacca tgtaagcctg aatttgtgcc atgtggagag agtctgtctg aggagaaact 3960 cggatgctag cagaaatgga aagagaacta agttctgatg tcatttttct ggaggcccta 4020 gatccagctg tgcctaaagc ctgccctacc tccggacttt aaagttttgt gagccaataa 4080 agtccctttc ttgtttaaga taattgaatt gagtttctgt tctgattaat ataggttatt 4140 tgtattttct tattgatttg tagaaaacct ttgtaatttt aaattctaga ctttatgcac 4200 tatataagtt aataaaatta gcatggcctt ccatgaaaaa aaaaaaaaaa aaaaaaaaaa 4260 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa a 4301 <210> 10 <211> 78 <212> PRT <213> Homo sapiens <400> 10 Met Val Arg Leu Gly Leu Phe Ser Cys Leu Leu Ala Ile Tyr Ser Leu   1 5 10 15 Leu Trp Ile Val Cys Ile Pro Tyr Leu Leu Ser Ile Gly Leu Cys Val              20 25 30 Asp Ile Leu Phe Leu Phe Val Gln His Leu Leu Pro His Leu Leu Val          35 40 45 Thr Gln Pro Leu Phe Ile Cys Gly Glu Pro Ile Pro Cys Gly Leu Gly      50 55 60 Glu His Val Thr Arg Pro Gly Leu Leu Ser Pro Thr Ala Ser  65 70 75 <210> 11 <211> 16 <212> DNA <213> Artificial Sequence <220> <223> H-T11A primer <400> 11 aagctttttt ttttta 16 <210> 12 <211> 13 <212> DNA <213> Artificial Sequence <220> <223> H-AP27 primer <400> 12 aagcttctgc tgg 13 <210> 13 <211> 1038 <212> DNA <213> Homo sapiens <400> 13 gctccgcgcc ggggtccgag tcccacgaag ccccggcccg agccgccgga tgcccgcgcg 60 cagcggggcc cagttttgcc gacggatggg gcaaaagaag cagcgaccag ctagagcagg 120 gcagccacac agctcgtccg acgcagccca ggcacctgca gagcagccac acagctcgtc 180 cgatgcagcc caggcacctt gccccaggga gcgctgcttg ggaccgccca ccactccggg 240 cccataccgc agcatctatt tctcaagccc aaagggccac cttacccgac tggggttgga 300 gttcttcgac cagccggcag tccccctggc ccgggcattt ctgggacagg tcctagtccg 360 gcgacttcct aatggcacag aactccgagg ccgcatcgtg gagaccgagg catacctggg 420 gccagaggat gaagccgccc actcaagggg tggccggcag accccccgca accgaggcat 480 gttcatgaag ccggggaccc tgtacgtgta catcatttac ggcatgtact tctgcatgaa 540 catctccagc cagggggacg gggcttgcgt cttgctgcga gcactggagc ccctggaagg 600 tctggagacc atgcgtcagc ttcgcagcac cctccggaaa ggcaccgcca gccgtgtcct 660 caaggaccgc gagctctgca gtggcccctc caagctgtgc caggccctgg ccatcaacaa 720 gagctttgac cagagggacc tggcacagga tgaagctgta tggctggagc gtggtcccct 780 ggagcccagt gagccggctg tagtggcagc agcccgggtg ggcgtcggcc atgcagggga 840 gtgggcccgg aaacccctcc gcttctatgt ccggggcagc ccctgggtca gtgtggtcga 900 cagagtggct gagcaggaca cacaggcctg agcaaagggc ctgcccagac aagatttttt 960 aattgtttaa aaaccgaata aatgttttat ttctagaaaa aaaaaaaaaa aaaaaaaaaa 1020 aaaaaaaaac aaaaaaaa 1038 <210> 14 <211> 293 <212> PRT <213> Homo sapiens <400> 14 Met Pro Ala Arg Ser Gly Ala Gln Phe Cys Arg Arg Met Gly Gln Lys   1 5 10 15 Lys Gln Arg Pro Ala Arg Ala Gly Gln Pro His Ser Ser Ser Asp Ala              20 25 30 Ala Gln Ala Pro Ala Glu Gln Pro His Ser Ser Ser Asp Ala Ala Gln          35 40 45 Ala Pro Cys Pro Arg Glu Arg Cys Leu Gly Pro Pro Thr Thr Pro Gly      50 55 60 Pro Tyr Arg Ser Ile Tyr Phe Ser Ser Pro Lys Gly His Leu Thr Arg  65 70 75 80 Leu Gly Leu Glu Phe Phe Asp Gln Pro Ala Val Pro Leu Ala Arg Ala                  85 90 95 Phe Leu Gly Gln Val Leu Val Arg Arg Leu Pro Asn Gly Thr Glu Leu             100 105 110 Arg Gly Arg Ile Val Glu Thr Glu Ala Tyr Leu Gly Pro Glu Asp Glu         115 120 125 Ala Ala His Ser Arg Gly Gly Arg Gln Thr Pro Arg Asn Arg Gly Met     130 135 140 Phe Met Lys Pro Gly Thr Leu Tyr Val Tyr Ile Ile Tyr Gly Met Tyr 145 150 155 160 Phe Cys Met Asn Ile Ser Ser Gln Gly Asp Gly Ala Cys Val Leu Leu                 165 170 175 Arg Ala Leu Glu Pro Leu Glu Gly Leu Glu Thr Met Arg Gln Leu Arg             180 185 190 Ser Thr Leu Arg Lys Gly Thr Ala Ser Arg Val Leu Lys Asp Arg Glu         195 200 205 Leu Cys Ser Gly Pro Ser Lys Leu Cys Gln Ala Leu Ala Ile Asn Lys     210 215 220 Ser Phe Asp Gln Arg Asp Leu Ala Gln Asp Glu Ala Val Trp Leu Glu 225 230 235 240 Arg Gly Pro Leu Glu Pro Ser Glu Pro Ala Val Val Ala Ala Ala Arg                 245 250 255 Val Gly Val Gly His Ala Gly Glu Trp Ala Arg Lys Pro Leu Arg Phe             260 265 270 Tyr Val Arg Gly Ser Pro Trp Val Ser Val Val Asp Arg Val Ala Glu         275 280 285 Gln Asp Thr Gln Ala     290 <210> 15 <211> 16 <212> DNA <213> Artificial Sequence <220> <223> H-T11G primer <400> 15 aagctttttt ttttta 16 <210> 16 <211> 13 <212> DNA <213> Artificial Sequence <220> <223> H-AP11 primer <400> 16 aagcttcggg taa 13 <210> 17 <211> 1682 <212> DNA <213> Homo sapiens <400> 17 caagtatcta ttgcttagca tatgtaaagt tgtagtctat atttatgggg ccattgctta 60 aagattataa attatgtaaa tacattaata aattctaagt ttcatttgac attccattga 120 atctcgcacc cagtcttgcg tatgcctgcc cagttttcag cctcttaacg ggagactcaa 180 gcacattggt attgtataaa ggtatagagc acttagctta caatctttaa aggtttctct 240 gccttccctt ctacccaccc gcctcccacc agatcccatc tggaaatcat aataaagaca 300 tatgccactt tgacaaacct gactagtcct tactagcctg agggtaaaag attaagctcc 360 aacctcaagt catttacctg gtcttggtaa taagtttctt ttagcttgta cagcatcctc 420 agaccaactg aggagctttc cttgttaaca atttagctta tctttctgtt tcctttattt 480 ttcccctgcc tctgttagtg gttaacactc ttttccctca gggagcctaa tgaggttttt 540 aatatcatct aaaaataaag cattgaagtg aagttggtga aaaaaaaaaa aaaaaaaaaa 600 aaaaaaaaaa aaaaaaaagg ccgcctcggc cgtcggcggc tgctgggctc cgcgccgggg 660 tccgagtccc acgaagcccc ggcccgagcc gccggatgcc cgcgcgcagc ggggcccagt 720 tttgccgacg gatggggcaa aagaagcagc gaccagctag agcagggcag ccacacagct 780 cgtccgacgc agcccaggca cctgcagagc agccacacag ctcgtccgat gcagcccagg 840 caccttgccc cagggagcgc tgcttgggac cgcccaccac tccgggccca taccgcagca 900 tctatttctc aagcccaaag ggccacctta cccgactggg gttggagttc ttcgaccagc 960 cggcagtccc cctggcccgg gcatttctgg gacaggtcct agtccggcga cttcctaatg 1020 gcacagaact ccgaggccgc atcgtggaga ccgaggcata cctggggcca gaggatgaag 1080 ccgcccactc aaggggtggc cggcagaccc cccgcaaccg aggcatgttc atgaagccgg 1140 ggaccctgta cgtgtacatc atttacggca tgtacttctg catgaacatc tccagccagg 1200 gggacggggc ttgcgtcttg ctgcgagcac tggagcccct ggaaggtctg gagaccatgc 1260 gtcagcttcg cagcaccctc cggaaaggca ccgccagccg tgtcctcaag gaccgcgagc 1320 tctgcagtgg cccctccaag ctgtgccagg ccctggccat caacaagagc tttgaccaga 1380 gggacctggc acaggatgaa gctgtatggc tggagcgtgg tcccctggag cccagtgagc 1440 cggctgtagt ggcagcagcc cgggtgggcg tcggccatgc aggggagtgg gcccggaaac 1500 ccctccgctt ctatgtccgg ggcagcccct gggtcagtgt ggtcgacaga gtggctgagc 1560 aggacacaca ggcctgagca aagggcctgc ccagacaaga ttttttaatt gtttaaaaac 1620 cgaataaatg ttttatttct agaaaaaaaa aaaaaaaaaa aaaaaacaaa aaaaaaaaaa 1680 aa 1682 <210> 18 <211> 293 <212> PRT <213> Homo sapiens <400> 18 Met Pro Ala Arg Ser Gly Ala Gln Phe Cys Arg Arg Met Gly Gln Lys   1 5 10 15 Lys Gln Arg Pro Ala Arg Ala Gly Gln Pro His Ser Ser Ser Asp Ala              20 25 30 Ala Gln Ala Pro Ala Glu Gln Pro His Ser Ser Ser Asp Ala Ala Gln          35 40 45 Ala Pro Cys Pro Arg Glu Arg Cys Leu Gly Pro Pro Thr Thr Pro Gly      50 55 60 Pro Tyr Arg Ser Ile Tyr Phe Ser Ser Pro Lys Gly His Leu Thr Arg  65 70 75 80 Leu Gly Leu Glu Phe Phe Asp Gln Pro Ala Val Pro Leu Ala Arg Ala                  85 90 95 Phe Leu Gly Gln Val Leu Val Arg Arg Leu Pro Asn Gly Thr Glu Leu             100 105 110 Arg Gly Arg Ile Val Glu Thr Glu Ala Tyr Leu Gly Pro Glu Asp Glu         115 120 125 Ala Ala His Ser Arg Gly Gly Arg Gln Thr Pro Arg Asn Arg Gly Met     130 135 140 Phe Met Lys Pro Gly Thr Leu Tyr Val Tyr Ile Ile Tyr Gly Met Tyr 145 150 155 160 Phe Cys Met Asn Ile Ser Ser Gln Gly Asp Gly Ala Cys Val Leu Leu                 165 170 175 Arg Ala Leu Glu Pro Leu Glu Gly Leu Glu Thr Met Arg Gln Leu Arg             180 185 190 Ser Thr Leu Arg Lys Gly Thr Ala Ser Arg Val Leu Lys Asp Arg Glu         195 200 205 Leu Cys Ser Gly Pro Ser Lys Leu Cys Gln Ala Leu Ala Ile Asn Lys     210 215 220 Ser Phe Asp Gln Arg Asp Leu Ala Gln Asp Glu Ala Val Trp Leu Glu 225 230 235 240 Arg Gly Pro Leu Glu Pro Ser Glu Pro Ala Val Val Ala Ala Ala Arg                 245 250 255 Val Gly Val Gly His Ala Gly Glu Trp Ala Arg Lys Pro Leu Arg Phe             260 265 270 Tyr Val Arg Gly Ser Pro Trp Val Ser Val Val Asp Arg Val Ala Glu         275 280 285 Gln Asp Thr Gln Ala     290 <210> 19 <211> 16 <212> DNA <213> Artificial Sequence <220> <223> H-T11C primer <400> 19 aagctttttt tttttc 16 <210> 20 <211> 13 <212> DNA <213> Artificial Sequence <220> <223> H-AP16 primer <400> 20 aagctttaga gcg 13 <210> 21 <211> 626 <212> DNA <213> Homo sapiens <400> 21 tgttgtggag tacgctttgg actgagaagc atcgaggcta taggacgcag ctgttgccat 60 gacggcccag gggggcctgg tggctaaccg aggccggcgc ttcaagtggg ccattgagct 120 aagcgggcct ggaggaggca gcaggggtcg aagtgaccgg ggcagtggcc agggagactc 180 gctctaccca gtcggtcact tggacaagca agtgcctgat accagcgtgc aagagacaga 240 ccggatcctg gtggagaagc gctgctggga catcgccttg ggtcccctca aacagattcc 300 catgaatctc ttcatcatgt acatggcagg caatactatc tccatcttcc ctactatgat 360 ggtgtgtatg atggcctggc gacccattca ggcacttatg gccatttcag ccactttcaa 420 gatgttagaa agttcaagcc agaggtttct tcagggtttg gtctatctca ttgggaacct 480 gatgggtttg gcattggctg tttacaagtg ccagtccatg ggactgttac ctacacatgc 540 atcggattgg ttagccttca ttgagccccc tgagagaatg gagttcagtg gtggaggact 600 gcttttgtga acatgagaaa gcagcg 626 <210> 22 <211> 183 <212> PRT <213> Homo sapiens <400> 22 Met Thr Ala Gln Gly Gly Leu Val Ala Asn Arg Gly Arg Arg Phe Lys   1 5 10 15 Trp Ala Ile Glu Leu Ser Gly Pro Gly Gly Gly Ser Arg Gly Arg Ser              20 25 30 Asp Arg Gly Ser Gly Gln Gly Asp Ser Leu Tyr Pro Val Gly His Leu          35 40 45 Asp Lys Gln Val Pro Asp Thr Ser Val Gln Glu Thr Asp Arg Ile Leu      50 55 60 Val Glu Lys Arg Cys Trp Asp Ile Ala Leu Gly Pro Leu Lys Gln Ile  65 70 75 80 Pro Met Asn Leu Phe Ile Met Tyr Met Ala Gly Asn Thr Ile Ser Ile                  85 90 95 Phe Pro Thr Met Met Val Cys Met Met Ala Trp Arg Pro Ile Gln Ala             100 105 110 Leu Met Ala Ile Ser Ala Thr Phe Lys Met Leu Glu Ser Ser Ser Gln         115 120 125 Arg Phe Leu Gln Gly Leu Val Tyr Leu Ile Gly Asn Leu Met Gly Leu     130 135 140 Ala Leu Ala Val Tyr Lys Cys Gln Ser Met Gly Leu Leu Pro Thr His 145 150 155 160 Ala Ser Asp Trp Leu Ala Phe Ile Glu Pro Pro Glu Arg Met Glu Phe                 165 170 175 Ser Gly Gly Gly Leu Leu Leu             180 <210> 23 <211> 16 <212> DNA <213> Artificial Sequence <220> <223> H-T11C primer <400> 23 aagctttttt tttttc 16 <210> 24 <211> 13 <212> DNA <213> Artificial Sequence <220> <223> H-AP18 primer <400> 24 aagcttagag gca 13 <210> 25 <211> 1031 <212> DNA <213> Homo sapiens <400> 25 attcccgatt ccttttggtt ccaagtccaa tatggcaact ctaaaggatc agctgattta 60 taatcttcta aaggaagaac agacccccca gaataagatt acagttgttg gggttggtgc 120 tgttggcatg gcctgtgcca tcagtatctt aatgaaggac ttggcagatg aacttgctct 180 tgttgatgtc atcgaagaca aattgaaggg agagatgatg gatctccaac atggcagcct 240 tttccttaga acaccaaaga ttgtctctgg caaagactat aatgtaactg caaactccaa 300 gctggtcatt atcacggctg gggcacgtca gcaagaggga gaaagccgtc ttaatttggt 360 ccagcgtaac gtgaacatat ttaaattcat cattcctaat gttgtaaaat acagcccgaa 420 ctgcaagttg cttattgttt caaatccagt ggatatcttg acctacgtgg cttggaagat 480 aagtggtttt cccaaaaacc gtgttattgg aagtggttgc aatctggatt cagcccgatt 540 ccgttacctg atgggggaaa ggctgggagt tcacccatta agctgtcatg ggtgggtcct 600 tggggaacat ggagattcca gtgtgcctgt atggagtgga atgaatgttg ctggtgtctc 660 tctgaagact ctgcacccag atttagggac tgataaagat aaggaacagt ggaaagaggt 720 tcacaagcag gtggttgaga gtgcttatga ggtgatcaaa ctcaaaggct acacatcctg 780 ggctattgga ctctctgtag cagatttggc agagagtata atgaagaatc ttaggcgggt 840 gcacccagtt tccaccatga ttaagggtct ttacggaata aaggatgatg tcttccttag 900 tgttccttgc attttgggac agaatggaat ctcagacctt gtgaaggtga ctctgacttc 960 tgaggaagag gcccgtttga agaagagtgc agatacactt tgggggatcc aaaaggagct 1020 gcaattttaa a 1031 <210> 26 <211> 332 <212> PRT <213> Homo sapiens <400> 26 Met Ala Thr Leu Lys Asp Gln Leu Ile Tyr Asn Leu Leu Lys Glu Glu   1 5 10 15 Gln Thr Pro Gln Asn Lys Ile Thr Val Val Gly Val Gly Ala Val Gly              20 25 30 Met Ala Cys Ala Ile Ser Ile Leu Met Lys Asp Leu Ala Asp Glu Leu          35 40 45 Ala Leu Val Asp Val Ile Glu Asp Lys Leu Lys Gly Glu Met Met Asp      50 55 60 Leu Gln His Gly Ser Leu Phe Leu Arg Thr Pro Lys Ile Val Ser Gly  65 70 75 80 Lys Asp Tyr Asn Val Thr Ala Asn Ser Lys Leu Val Ile Ile Thr Ala                  85 90 95 Gly Ala Arg Gln Gln Glu Gly Glu Ser Arg Leu Asn Leu Val Gln Arg             100 105 110 Asn Val Asn Ile Phe Lys Phe Ile Ile Pro Asn Val Val Lys Tyr Ser         115 120 125 Pro Asn Cys Lys Leu Leu Ile Val Ser Asn Pro Val Asp Ile Leu Thr     130 135 140 Tyr Val Ala Trp Lys Ile Ser Gly Phe Pro Lys Asn Arg Val Ile Gly 145 150 155 160 Ser Gly Cys Asn Leu Asp Ser Ala Arg Phe Arg Tyr Leu Met Gly Glu                 165 170 175 Arg Leu Gly Val His Pro Leu Ser Cys His Gly Trp Val Leu Gly Glu             180 185 190 His Gly Asp Ser Ser Val Pro Val Trp Ser Gly Met Asn Val Ala Gly         195 200 205 Val Ser Leu Lys Thr Leu His Pro Asp Leu Gly Thr Asp Lys Asp Lys     210 215 220 Glu Gln Trp Lys Glu Val His Lys Gln Val Val Glu Ser Ala Tyr Glu 225 230 235 240 Val Ile Lys Leu Lys Gly Tyr Thr Ser Trp Ala Ile Gly Leu Ser Val                 245 250 255 Ala Asp Leu Ala Glu Ser Ile Met Lys Asn Leu Arg Arg Val His Pro             260 265 270 Val Ser Thr Met Ile Lys Gly Leu Tyr Gly Ile Lys Asp Asp Val Phe         275 280 285 Leu Ser Val Pro Cys Ile Leu Gly Gln Asn Gly Ile Ser Asp Leu Val     290 295 300 Lys Val Thr Leu Thr Ser Glu Glu Glu Ala Arg Leu Lys Lys Ser Ala 305 310 315 320 Asp Thr Leu Trp Gly Ile Gln Lys Glu Leu Gln Phe                 325 330 <210> 27 <211> 16 <212> DNA <213> Artificial Sequence <220> <223> H-T11G primer <400> 27 aagctttttt tttttg 16 <210> 28 <211> 13 <212> DNA <213> Artificial Sequence <220> <223> H-AP16 primer <400> 28 aagctttaga gcg 13 <210> 29 <211> 526 <212> DNA <213> Homo sapiens <400> 29 atgctgccac gtgatgagag aagattcaaa gctgcagacc tcaatggtga cctgacagct 60 actcgggagg agttcactgc ctttctgcat cctgaagagt ttgaacatat gaaggaaatt 120 gtggttttgg aaaccctgga ggacatcgac aagaacgggg atgggttcgt ggatcaggat 180 gagtatattg cggatatgtt ttcccatgag gagaatggcc ctgagccaga ctgggtttta 240 tcagaacggg agcagtttaa cgaattccgg gatctgaaca aggacgggaa gttagacaaa 300 gatgagattc gccactggat cctccctcaa gattatgatc acgcacaggc tgaggccagg 360 catctggtat atgaatcaga caaaaacaag gatgagaagc taactaaaga ggaaatattg 420 gagaactgga acatgtttgt tggaagccaa gctaccaatt acggggaaga tctcacaaaa 480 aatcatgatg agctttgata gacactcacc agaatatggc agactg 526 <210> 30 <211> 165 <212> PRT <213> Homo sapiens <400> 30 Met Leu Pro Arg Asp Glu Arg Arg Phe Lys Ala Ala Asp Leu Asn Gly   1 5 10 15 Asp Leu Thr Ala Thr Arg Glu Glu Phe Thr Ala Phe Leu His Pro Glu              20 25 30 Glu Phe Glu His Met Lys Glu Ile Val Val Leu Glu Thr Leu Glu Asp          35 40 45 Ile Asp Lys Asn Gly Asp Gly Phe Val Asp Gln Asp Glu Tyr Ile Ala      50 55 60 Asp Met Phe Ser His Glu Glu Asn Gly Pro Glu Pro Asp Trp Val Leu  65 70 75 80 Ser Glu Arg Glu Gln Phe Asn Glu Phe Arg Asp Leu Asn Lys Asp Gly                  85 90 95 Lys Leu Asp Lys Asp Glu Ile Arg His Trp Ile Leu Pro Gln Asp Tyr             100 105 110 Asp His Ala Gln Ala Glu Ala Arg His Leu Val Tyr Glu Ser Asp Lys         115 120 125 Asn Lys Asp Glu Lys Leu Thr Lys Glu Glu Ile Leu Glu Asn Trp Asn     130 135 140 Met Phe Val Gly Ser Gln Ala Thr Asn Tyr Gly Glu Asp Leu Thr Lys 145 150 155 160 Asn His Asp Glu Leu                 165 <210> 31 <211> 16 <212> DNA <213> Artificial Sequence <220> <223> H-T11G primer <400> 31 aagctttttt tttttg 16 <210> 32 <211> 13 <212> DNA <213> Artificial Sequence <220> <223> H-AP17 primer <400> 32 aagcttacca ggt 13 <210> 33 <211> 965 <212> DNA <213> Homo sapiens <400> 33 gccatgactg agcagatgac ccttcgtggc accctcaagg gccacaacgg ctgggtaacc 60 cagatcgcta ctaccccgca gttcccggac atgatcctct ccgcctctcg tgataagacc 120 atcatcatgt ggaaactgac cagggatgag accaactatg gaattccaca gcgtgctctg 180 cggggtcact cccactttgt tagtgatgtg gttatctcct cagatggcca gtttgccctc 240 tcaggctcct gggatggaac cctgcgcctc tgggatctca caacgcaagg gcaccaccac 300 gaggcgattt gtgggccata ccaaggatgt gctgagtgtg gccttctcct ctgacaaccg 360 gcagattgtc tctggatctc gagataaaac catcaagcta tggaataccc tgggtgtgtg 420 caaatacact gtccaggatg agagccactc agagtgggtg tcttgtgtcc gcttctcgcc 480 caacagcagc aaccctatca tcgtctcctg tggctgggac aagctggtca aggtatggaa 540 cctggctaac tgcaagctga agaccaacca cattggccac acaggctatc tgaacacggt 600 gactgtctct ccagatggat ccctctgtgc ttctggaggc aaggatggcc aggccatgtt 660 atgggatctc aacgaaggca aacaccttta cacgctagat ggtggggaca tcatcaacgc 720 cctgtgcttc agccctaacc gctactggct gtgtgctgcc acaggcccca gcatcaagat 780 ctgggattta gagggaaaga tcattgtaga tgaactgaag caagaagtta tcagtaccag 840 cagcaaggca gaaccacccc agtgcacctc cctggcctgg tctgctgatg gccagactct 900 gtttgctggc tacacggaca acctggtgcg agtgtggcag gtgaccattg gcacacgcta 960 gaagt 965 <210> 34 <211> 271 <212> PRT <213> Homo sapiens <400> 34 Met Arg Pro Thr Met Glu Phe His Ser Val Leu Cys Gly Val Thr Pro   1 5 10 15 Thr Leu Leu Val Met Trp Leu Ser Pro Gln Met Ala Ser Leu Pro Ser              20 25 30 Gln Ala Pro Gly Met Glu Pro Cys Ala Ser Gly Ile Ser Gln Arg Lys          35 40 45 Gly Thr Thr Thr Arg Arg Phe Val Gly His Thr Lys Asp Val Leu Ser      50 55 60 Val Ala Phe Ser Ser Asp Asn Arg Gln Ile Val Ser Gly Ser Arg Asp  65 70 75 80 Lys Thr Ile Lys Leu Trp Asn Thr Leu Gly Val Cys Lys Tyr Thr Val                  85 90 95 Gln Asp Glu Ser His Ser Glu Trp Val Ser Cys Val Arg Phe Ser Pro             100 105 110 Asn Ser Ser Asn Pro Ile Ile Val Ser Cys Gly Trp Asp Lys Leu Val         115 120 125 Lys Val Trp Asn Leu Ala Asn Cys Lys Leu Lys Thr Asn His Ile Gly     130 135 140 His Thr Gly Tyr Leu Asn Thr Val Thr Val Ser Pro Asp Gly Ser Leu 145 150 155 160 Cys Ala Ser Gly Gly Lys Asp Gly Gln Ala Met Leu Trp Asp Leu Asn                 165 170 175 Glu Gly Lys His Leu Tyr Thr Leu Asp Gly Gly Asp Ile Ile Asn Ala             180 185 190 Leu Cys Phe Ser Pro Asn Arg Tyr Trp Leu Cys Ala Ala Thr Gly Pro         195 200 205 Ser Ile Lys Ile Trp Asp Leu Glu Gly Lys Ile Ile Val Asp Glu Leu     210 215 220 Lys Gln Glu Val Ile Ser Thr Ser Ser Lys Ala Glu Pro Pro Gln Cys 225 230 235 240 Thr Ser Leu Ala Trp Ser Ala Asp Gly Gln Thr Leu Phe Ala Gly Tyr                 245 250 255 Thr Asp Asn Leu Val Arg Val Trp Gln Val Thr Ile Gly Thr Arg             260 265 270 <210> 35 <211> 16 <212> DNA <213> Artificial Sequence <220> <223> H-T11C primer <400> 35 aagctttttt tttttc 16 <210> 36 <211> 13 <212> DNA <213> Artificial Sequence <220> <223> H-AP15 primer <400> 36 aagcttacgc aac 13 <210> 37 <211> 626 <212> DNA <213> Homo sapiens <400> 37 gcccgaacat ggactccgcc ggccaagata tcaacctgaa ttctcctaac aaaggtctgc 60 tgcctgactc catgacggat gttcctgtcg acacaggtgt ggctgcccgg actcctgctg 120 ttgagggtct gacagaggct gaggaggagg agctcagggc tgagcttacc aaggtggaag 180 aggaaattgt cactctgcgc caggtcctgg cagccaagga gaggcactgt ggagagctca 240 agaggaggct gggcctctcc accctggggg agctaaaaca gaacctgtcc aggagctggc 300 atgacgtgca ggtctctagc gcctatgtga aaacttctga gaaacttgga gagtgaatga 360 gaaagtgacc cagtcagacc tctacaagaa gactcaggag actctttcac aggcaggaca 420 gaagacttca gctgccctgt ccacagtggg ctctgccatc agcaggaagc ttggagacat 480 gaggaactct gcgaccttca agtcgtttga ggaccgagtt gggaccataa agtctaaggt 540 tgtgggtgac agagggaacg gcagtgacaa cctcccttcc tcagcgggga gtggtgacaa 600 gcccctgtcg gatcccgcac ctttct 626 <210> 38 <211> 115 <212> PRT <213> Homo sapiens <400> 38 Met Asp Ser Ala Gly Gln Asp Ile Asn Leu Asn Ser Pro Asn Lys Gly   1 5 10 15 Leu Leu Pro Asp Ser Met Thr Asp Val Pro Val Asp Thr Gly Val Ala              20 25 30 Ala Arg Thr Pro Ala Val Glu Gly Leu Thr Glu Ala Glu Glu Glu Glu          35 40 45 Leu Arg Ala Glu Leu Thr Lys Val Glu Glu Glu Ile Val Thr Leu Arg      50 55 60 Gln Val Leu Ala Ala Lys Glu Arg His Cys Gly Glu Leu Lys Arg Arg  65 70 75 80 Leu Gly Leu Ser Thr Leu Gly Glu Leu Lys Gln Asn Leu Ser Arg Ser                  85 90 95 Trp His Asp Val Gln Val Ser Ser Ala Tyr Val Lys Thr Ser Glu Lys             100 105 110 Leu Gly Glu         115 <210> 39 <211> 2302 <212> DNA <213> Homo sapiens <400> 39 tttttttttt tttttttttt tttttttttt ttttttttaa agttcagctt tttattgaac 60 atgttataaa agaggtttag tcaaaaagac caaagcccat gtcatcatca gactcctcgg 120 attcttcttt ctttgcttcc actttcttct cctcagctgg agcagcagca gtggaggggg 180 caggacctcc tgctggtgca gcaccagctg ctggagcagg tccaccggcc cctacattgc 240 agatgaggct cccaatgttg acgttggcca gggcctttgc aaacaagcca ggccaaaaag 300 gctcaacatt tacaccggct gctttaatga gggcattgat cttatcctcc gtgactgtca 360 cctcatcgtc gtgcagaatg agggccgagt agatgcaggc gagctcggag acagaggcca 420 tggcgcgggc gagtgtaggg ctggcgctgc cggacgcggt gctagtcgcc ggatgaagtg 480 agggcctcac cccaacgcag ccttagcttc ctcggaagga ccgagcacct tgggaaaaga 540 aaccaacagt tgaagagaag gcaaaagcag atacgttaaa acttccacct acattttttt 600 gtggagtctg tagtgatact gatgaagaca atggaaatgg ggaagacttt caatcagagc 660 ttcaaaaagt tcaggaagct caaaaatctc agacagaaga aataactagc acaactgaca 720 gtgtatatac aggtgggact gaagtgatgg taccttcttt ctgtaaatct gaagaacctg 780 attctattac caaatccgtt agttcaccat ctgtttcctc tgaaactatg gacaaacctg 840 tagatttgtc aactagaaag gaaattgata cagattctac aagccaaggg gaaagcaaga 900 tagtttcatt tggatttgga agtagcacag ggctctcatt tgcagacttg gcttccagta 960 attctggaga ttttgctttt ggttctaaag ataaaaattt ccaatgggca aatactggag 1020 cagctgtgtt tggaacacag tcagtcggaa cccagtcagc cggtaaagtt ggtgaagatg 1080 aagatggtag tgatgaagaa gtagttcata atgaagatat ccattttgaa ccaatagtgt 1140 cactaccaga ggtagaagta aaatctggag aagaagatga agaaattttg tttaaagaga 1200 gagccaaact ttatagatgg gatcgggatg tcagtcagtg gaaggagcgc ggtgttggag 1260 atataaagat tctttggcat acaatgaaga attattaccg gatcctaatg agaagagacc 1320 aggtttttaa agtgtgtgca aaccacgtta ttactaaaac aatggaatta aagcccttaa 1380 atgtttcaaa taatgcttta gtttggactg cctcagatta tgctgatgga gaagcaaaag 1440 tagaacagct tgcagtgaga tttaaaacta aagaagtagc tgattgtttc aagaaaacat 1500 ttgaagaatg tcagcagaat ttaatgaaac tccagaaagg acatgtatca ctggcagcag 1560 aattatcaaa ggagaccaat cctgtggtgt tttttgatgt ttgtgcggac ggtgaacctc 1620 tagggcggat aactatggaa ttattttcaa acattgttcc tcggactgct gagaacttca 1680 gagcactatg cactggagag aaaggctttg gtttcaagaa ttccattttt cacagagtaa 1740 ttccagattt tgtttgccaa ggaggagata tcaccaaaca tgatggaaca ggcggacagt 1800 ccatttatgg agacaaattt gaagatgaaa attttgatgt gaaacatact ggtcctggtt 1860 tactatccat ggccaatcaa ggccagaata ccaataattc tcaatttgtt ataacactga 1920 agaaagcaga acatttggac tttaagcatg tagtatttgg gtttgttaag gatggcatgg 1980 atactgtgaa aaagattgaa tcatttggtt ctcccaaagg gtctgtttgt cgaagaataa 2040 ctatcacaga atgtggacag atataaaatc attgttgttc atagaaaatt tcatctgtat 2100 aagcagttgg attgaagctt agctattaca atttgatagt tatgttcagc ttttgaaaat 2160 ggacgtttcc gatttacaaa tgtaaaattg cagcttatag ctgttgtcac tttttaatgt 2220 gttataattg accttgcatg gtgtgaaata aaagtttaaa cactggtgta aaaaaaaaaa 2280 aaaaaaaaaa aaaaaaaaaa aa 2302 <210> 40 <211> 439 <212> PRT <213> Homo sapiens <400> 40 Met Val Pro Ser Phe Cys Lys Ser Glu Glu Pro Asp Ser Ile Thr Lys   1 5 10 15 Ser Val Ser Ser Pro Ser Val Ser Ser Glu Thr Met Asp Lys Pro Val              20 25 30 Asp Leu Ser Thr Arg Lys Glu Ile Asp Thr Asp Ser Thr Ser Gln Gly          35 40 45 Glu Ser Lys Ile Val Ser Phe Gly Phe Gly Ser Ser Thr Gly Leu Ser      50 55 60 Phe Ala Asp Leu Ala Ser Ser Asn Ser Gly Asp Phe Ala Phe Gly Ser  65 70 75 80 Lys Asp Lys Asn Phe Gln Trp Ala Asn Thr Gly Ala Ala Val Phe Gly                  85 90 95 Thr Gln Ser Val Gly Thr Gln Ser Ala Gly Lys Val Gly Glu Asp Glu             100 105 110 Asp Gly Ser Asp Glu Glu Val Val His Asn Glu Asp Ile His Phe Glu         115 120 125 Pro Ile Val Ser Leu Pro Glu Val Glu Val Lys Ser Gly Glu Glu Asp     130 135 140 Glu Glu Ile Leu Phe Lys Glu Arg Ala Lys Leu Tyr Arg Trp Asp Arg 145 150 155 160 Asp Val Ser Gln Trp Lys Glu Arg Gly Val Gly Asp Ile Lys Ile Leu                 165 170 175 Trp His Thr Met Lys Asn Tyr Tyr Arg Ile Leu Met Arg Arg Asp Gln             180 185 190 Val Phe Lys Val Cys Ala Asn His Val Ile Thr Lys Thr Met Glu Leu         195 200 205 Lys Pro Leu Asn Val Ser Asn Asn Ala Leu Val Trp Thr Ala Ser Asp     210 215 220 Tyr Ala Asp Gly Glu Ala Lys Val Glu Gln Leu Ala Val Arg Phe Lys 225 230 235 240 Thr Lys Glu Val Ala Asp Cys Phe Lys Lys Thr Phe Glu Glu Cys Gln                 245 250 255 Gln Asn Leu Met Lys Leu Gln Lys Gly His Val Ser Leu Ala Ala Glu             260 265 270 Leu Ser Lys Glu Thr Asn Pro Val Val Phe Phe Asp Val Cys Ala Asp         275 280 285 Gly Glu Pro Leu Gly Arg Ile Thr Met Glu Leu Phe Ser Asn Ile Val     290 295 300 Pro Arg Thr Ala Glu Asn Phe Arg Ala Leu Cys Thr Gly Glu Lys Gly 305 310 315 320 Phe Gly Phe Lys Asn Ser Ile Phe His Arg Val Ile Pro Asp Phe Val                 325 330 335 Cys Gln Gly Gly Asp Ile Thr Lys His Asp Gly Thr Gly Gly Gln Ser             340 345 350 Ile Tyr Gly Asp Lys Phe Glu Asp Glu Asn Phe Asp Val Lys His Thr         355 360 365 Gly Pro Gly Leu Leu Ser Met Ala Asn Gln Gly Gln Asn Thr Asn Asn     370 375 380 Ser Gln Phe Val Ile Thr Leu Lys Lys Ala Glu His Leu Asp Phe Lys 385 390 395 400 His Val Val Phe Gly Phe Val Lys Asp Gly Met Asp Thr Val Lys Lys                 405 410 415 Ile Glu Ser Phe Gly Ser Pro Lys Gly Ser Val Cys Arg Arg Ile Thr             420 425 430 Ile Thr Glu Cys Gly Gln Ile         435 <210> 41 <211> 16 <212> DNA <213> Artificial Sequence <220> <223> H-T11A primer <400> 41 aagctttttt ttttta 16 <210> 42 <211> 13 <212> DNA <213> Artificial Sequence <220> <223> H-AP32 primer <400> 42 aagcttcctg caa 13

Claims (6)

Human prooncoprotein having the amino acid sequence of SEQ ID 38. A human primary cancer gene comprising the nucleotide sequence corresponding to base numbers 9 to 356 of SEQ ID NO: 37 encoding the primary cancer protein of claim 1. The human primary cancer gene according to claim 2, wherein the gene has a nucleotide sequence of SEQ ID NO: 37. A vector comprising the proto-oncogene of any one of claims 2 to 3. Claim 1 cancer and cancer metastasis diagnostic kit comprising the primary cancer protein. A kit for diagnosing cancer and cancer metastasis comprising the proto-oncogene of any one of claims 2 to 3.

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