JP2007224009A - Agent for prevention or treatment of cancer - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an agent for the prevention or treatment of colon cancer. <P>SOLUTION: A compound or its salt inhibiting the activity of a protein containing an amino acid sequence identical or essentially identical to the amino acid sequence expressed by sequence number 1, sequence number 3, sequence number 5, sequence number 7, sequence number 9, sequence number 11, sequence number 13, sequence number 15, sequence number 17, sequence number 19, sequence number 21, sequence number 23 or sequence number 25, a compound or its salt inhibiting the expression of the protein gene, an antisense polynucleotide containing a base sequence or a part thereof complementary or essentially complementary to the base sequence of a polynucleotide encoding the protein or its partial peptide, an antibody to the protein, etc., can be used as an agent for the prevention or treatment of colon cancer, etc., an apoptosis promoting agent for colon cancer cell, a growth suppressing agent for colon cancer cell, an agent for suppressing the metastasis and recurrence of colon cancer, etc. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to cancer preventive / therapeutic agents and diagnostic agents, screening for cancer preventive / therapeutic agents, and the like.

  The large intestine consists of the appendix, ascending colon, transverse colon, descending colon, sigmoid colon, and rectum. The part from the appendix to the ascending colon and a part of the transverse colon is called the right large intestine, and the part from the transverse colon to the descending colon, the sigmoid colon, and the rectum is called the left large intestine. There is a difference between the right and left colons. For example, the right large intestine receives blood from the superior mesenteric artery, while the left large intestine receives blood from the inferior mesenteric artery. In addition, it has been reported that the length of the crypt differs between the left and right large intestines (Acta pathologica japonica, 39, 725-730, 1989).

  There are differences in colorectal cancer that develops in the right and left colons. For example, P53 mutation is found in about 50% of the left colorectal cancer, whereas it is about 30% in the right colorectal cancer (International Journal of Cancer, 74, 664-669, 1997). Many things on the left (Cancer Epidemiology, Biomarkers and Prevention, 12, 755-762, 2003) have been reported. P53 mutation and K-ras mutation are considered to be extremely important steps in the development process of colon cancer (adenoma-carcinoma sequence) (Cell, 61, 759-767, 1990). Conversely, the frequency of microsatellite instability is frequently observed in right colon cancer (Cancer Research, 53, 5849-5852, 1993).

  In recent years, cDNA microarrays and DNA chips have been developed, and it has become possible to comprehensively analyze gene expression. In particular, it is an effective analytical technique for analyzing the genetic level of multifactorial diseases such as cancer, and plays an important role in advancing disease research, particularly cancer research, from a clinical viewpoint. From a microarray analysis using tumor tissue and normal tissue of colorectal cancer patients, genes highly expressed in colon cancer have been reported (Cancer Research, 61, 3544-3549, 2001). In addition, genes that can be used as criteria for determining the colorectal cancer Dukes' classification have been reported (Cancer Research, Vol. 62, 4352-4363, 2002). In addition, a report has been made on the high expression of colorectal cancer obtained from microarray analysis, and the relationship with colorectal cancer was examined by experiments using siRNA (Clinical Cancer Research, Vol. 9, pages 931-946). 2003).

  Moreover, it has been reported that the expression pattern of the gene is different between the right colon and the left colon (Non-patent Document 1: Cancer Epidemiology, Biomarkers and Prevention, Vol. 12, 755-762, 2003). Reference is also made to genes with different expression in the left and right colon cancers (Non-Patent Document 2: Gut, 54, 374-384, 2005). However, focusing on the early stage of onset, examples of the search for targets involved in the development of colorectal cancer by conducting gene expression and functional analysis based on the position (left and right) of the tumor large intestine tissue including adenoma and carcinoma No.

LEFTB was cloned as a gene that is expressed only on the left side in a developing mouse embryo, and then a human homolog was cloned (RefSeq Accession No. NM # 020997; Non-Patent Document 3: American Journal of Human Genetics, Vol. 64, 712). -721, 1999).
From experiments using knockout mice, LEFTB is known to play an important role in the determination of left and right (Non-Patent Document 4: Cell, 94, 287-297, 1998).

  FOXA2 is a transcription factor belonging to the forkhead box a superfamily (RefSeq Accession No. NM # 021784; Non-Patent Document 5: Developmental Biology, 250, 1-23, 2002). Increased expression of FOXA2 in liver cancer has been reported (Non-Patent Document 6: Journal of Hepatology, 40, 212-218, 2004). It has also been reported that FOXA2 is expressed in the H358 lung cancer cell line, and growth inhibition and apoptosis are induced (Non-patent Document 7: Cancer Research, 64, 4137-4147, 2004).

  S100P was cloned from the placenta as a gene having homology with S100 (RefSeq Accession No. NM # 005980; Non-Patent Document 8: European Journal of Biochemistry, 207, 541-547, 1992). Increased expression of S100P accompanying malignant transformation of prostate cancer (Non-patent document 9: Cancer Research, 62, 1256-1260, 2002), high expression in pancreatic cancer (Non-patent document 10: American Journal of Pathology, 160, 1239-1249 (2002)). Moreover, it is suggested that the expression of S100P may be related to mammary epithelial cells (Non-patent Document 11: International Journal of Oncology, 16, 231-40, 2000). Furthermore, it has been reported from the site-specific expression analysis of the normal large intestine that the expression of S100P is high in the normal caecum and that the expression is increased in the large intestine (Non-patent Document 12: Gut, 54, 374-384, Year 2005).

  CTSE is an aspartic protease cloned from a cDNA library of gastric cancer (RefSeq Accession No. NM # 001910; Non-Patent Document 13: The Journal of Biological Chemistry, 264, 16748-16753, 1989). In human gastric cancer, it has been reported that cancer malignancy and CTSE expression are correlated (Non-patent Document 14: Human pathology, 27, 184-190, 1996). In addition, there is a report as one of genes whose expression is increased in intraductal papillary mucinous tumor (Non-patent Document 15: American Journal of Pathology, 160, 1745-1754, 2002). Conversely, it has been reported that the expression of CTSE and the progression of bladder cancer are inversely correlated (Non-patent Document 16: Clinical Cancer Research, 11, 4414-4429, 2005).

NOX1 is a NADPH oxidase cloned as a homologous gene to gp91phox, and overexpression of NOX1 in mouse NIH3T3 cells has been reported to enhance cell proliferation (RefSeq Accession No. NM # 007052; Patent Document 17: Nature, 401, 79-82, 1999).
Since NADPH oxidase generates reactive oxygen species, it is considered that NOX1 is involved in cancer, and that NOX1 is necessary for cell canceration by Ras oncogene (Non-patent Document 18: Cancer Reearch, Vol. 64) 3580-3485, 2004), high expression in prostate cancer (Non-Patent Document 19: The Prostate, 62, 200-207, 2005), high expression in colon benign tumors and well-differentiated adenocarcinoma (non- Patent Document 20: Cancer Letters, Vol. 221, pp. 97-104, 2005) has been reported.

  PITX2 was cloned as a causative gene of Rieger syndrome, characterized by facial bone abnormalities and showing autosomal dominant inheritance (RefSeq Accession No. NM # 000325, Nature Genetics, 14, 392-399, 1996). PITX2 has been reported to be involved in organ asymmetry (Nature, 394, 545-551, 1998). Moreover, it is a transcription factor downstream of the Wnt / beta-catenin pathway, and it has been suggested to regulate the expression of c-Myc, cyclin D1, and cyclin D2 (Non-patent Document 21: Proceedings of the National Academy of Sciences of the United States of America, 100, 3245-3250, 2003).

  HOXB13 was cloned in 1996 as a gene belonging to the HOX family, which is a transcription factor that plays a central role in morphogenesis in the developmental differentiation process of multicellular animals (RefSeq Accession No. NM # 006361, Development, Vol. 122, 2475-2484, 1996). In breast cancer, HOXB13 expression has been reported to correlate with poor prognosis (Cancer Cell, 5, 607-616, 2004). On the other hand, HOXB13 has been reported to suppress the androgen signal and suppress the growth of prostate cancer cell lines by androgen (Non-patent Document 22: Cancer Research, 64, 9185-9192, 2004).

  CDH3 was found in the mouse placenta as a novel cadherin molecule that is an adhesion molecule, after which a human gene was identified (RefSeq Accession No. NM # 001793; Journal of Cell Biology, 109, 1787-1794, 1989) . CDH3 is expressed in aberrant crypt foci and is suggested to be involved in the early stage of colorectal cancer development (Non-patent Document 23: Gut, 50, 513-519, 2002). It has also been reported that there is a correlation between CDH3 expression and poor prognosis of breast cancer (Cancer, 86, 1263-1272, 1999).

  KLK10 is a secreted serine protease cloned as a gene whose expression decreases in human mammary epithelial cells upon transformation by irradiation (RefSeq Accession No. NM # 002776; Cancer Research, Vol. 56, 3371-3379). Page, 1996). Indeed, it has been reported that expression has disappeared in the majority of advanced breast cancer patients (Clinical Cancer Research, 7, 3393-3398, 2001). On the other hand, it has been reported that serum KLK10 in ovarian cancer patients is elevated (Clinica Chimica Acta, 306, 111-118, 2001). Furthermore, it has been reported that KLK10 expression is also increased in colorectal cancer and pancreatic cancer (Non-patent Document 24: Anticancer Research, 24, 43-51, 2004).

  DUOX2 was cloned from a human thyroid cell cDNA library as NADPH oxidase (RefSeq Accession No. NM # 014080; The Journal of Biological Chemistry, 275, 23227-23233, 2000). It has been suggested that DUOX2 is involved in the synthesis of thyroid hormone, and patients with hypothyroidism having mutations in DUOX2 have been reported (Non-patent Document 25: New England Journal of Medicine, Vol. 347, pages 95-102, 2002).

  S100A2 was cloned from breast cancer cells as a gene homologous to S100 (RefSeq Accession No. NM # 005978; Proceedings of the National Academy of Sciences of the United States of America, 89, 2504-2508, 1992) ). Increased expression of S100A2 in gastric cancer and ovarian cancer has been reported (Cancer Resarch, 61, 3869-3876, 2001). On the other hand, a decrease in S100A2 expression in prostate cancer has been suggested (Journal of Clinical Oncology, 21, 106-112, 2003). In addition, decreased expression in lung cancer has been reported (Non-patent Document 26: Cancer Science, 96, 844-857, 2005).

  GABRP was identified as a GABA receptor subunit by EST database search (RefSeq Accession No. NM # 001015618; The Journal of Biological Chemistry, 272, 15346-15350, 1997). It has been reported that GABRP is highly expressed in breast tissue and decreased in breast cancer tissue (Non-patent Document 27: International Journal of Cancer, 118, 1453-1459, 2006).

  MSLN was identified as the antigen of antibody Mab K1, which recognizes ovarian cancer and mesothelioma (RefSeq Accession No. NM # 005823; Proceedings of the National Academy of Sciences of the United States of America, 93, 136-140). Page, 1996). MSLN is expressed on the cell surface by a GPI anchor and is suggested to be involved in cell adhesion (The Journal of Biological Chemistry, 279, 9990-9198, 2004). MSLN has been reported to be highly expressed in ovarian cancer, mesothelioma, and pancreatic cancer (Non-patent Document 28: Clinical Cancer Research, 10, 3937-3942, 2004).

  FAIM2 was identified as a molecule that binds to FAS and suppresses FAS-mediated apoptosis signals (RefSeq Accession No. NM # 012306; Proceedings of the National Academy of Sciences of the United States of America, 96, 12667- 12672, 1999). It has been reported that resistance to FASL-induced apoptosis in cerebellar granule neurons is borne by FAIM2 (Non-patent Document 29: The Journal of Neuroscience, 25, 6765-6774, 2005).

  TACSTD2 was cloned as a glycoprotein expressed on the cell surface (RefSeq Accession No. NM # 002353; International of Journal of Cancer, 62, 610-618, 1995). It has been suggested that TACSTD2 is upregulated in the early stages of lung cancer development (Non-patent Document 30: Cancer Science, 96, 668-675, 2005).

CEACAM1 was cloned from a cDNA library of human normal large intestine (RefSeq Accession No. NM # 001712; Proceedings of the National Academy of Sciences of the United States of America, 85, 6959-6963, 1988). CEACAM1 has been reported to decrease in expression in colorectal cancer (Non-patent Document 31: Proceedings of the National Academy of Sciences of the United States of America, 90, 10744-10748, 1993). On the other hand, high expression of CEACAM1 has been reported in non-small cell lung cancer (Clinical Cancer Research, 9, 2260-2266, 2003).
Cancer Epidemiology, Biomarkers and Prevention, 12, 755-762, 2003 Gut, 54, 374-384, 2005 American Journal of Human Genetics, 64, 712-721, 1999 Cell, 94, 287-297, 1998 Developmental Biology, 250, 1-23, 2002 Journal of Hepatology, 40, 212-218, 2004 Cancer Research, 64, 4137-4147, 2004 European Journal of Biochemistry, 207, 541-547, 1992 Cancer Research, 62, 1256-1260, 2002 American Journal of Pathology, 160, 1239-1249, 2002 International Journal of Oncology, 16, 231-40, 2000 Gut, 54, 374-384, 2005 The Journal of Biological Chemistry, 264, 16748-16753, 1989 Human pathology, 27, 184-190, 1996 American Journal of Pathology, 160, 1745-1754, 2002 Clinical Cancer Research, 11, 4414-4429, 2005 Nature, 401, 79-82, 1999 Cancer Reearch, 64, 3580-3485, 2004 The Prostate, 62, 200-207, 2005 Cancer Letters, 221: 97-104, 2005 Proceedings of the National Academy of Sciences of the United States of America, 100, 3245-3250, 2003 Cancer Research, 64, 9185-9192, 2004 Gut, 50, 513-519, 2002 Anticancer Research, 24, 43-51, 2004 New England Journal of Medicine, 347, 95-102, 2002 Cancer Science, 96, 844-857, 2005 International Journal of Cancer, 118, 1453-1459, 2006 Clinical Cancer Research, 10, 3937-3942, 2004 The Journal of Neuroscience, 25, 6765-6774, 2005 Cancer Science, 96, 668-675, 2005 Proceedings of the National Academy of Sciences of the United States of America, 90, 10744-10748, 1993

  There is a strong demand for a preventive / therapeutic agent for colon cancer that is safe and excellent in therapeutic effect, an inhibitor for metastasis / relapse, an apoptosis promoter for colon cancer cells, and a growth inhibitor for colon cancer cells.

  In order to solve the above-mentioned problems, the present inventors analyzed an expressed gene based on information such as the position of a large intestine sample, and performed gene expression analysis of a tumor sample including an adenoma and a carcinoma. Based on the expression level of HOXB13, PITX2, etc., the left and right sides of the large intestine are classified. (3) CDH3, KLK10, DUOX2, S100A2, GABRP, MSLN, FAIM2 and TACSTD2 are up-regulated in right and left tumors, and (4) CEACAM1 is expressed in right and left tumors. The present inventors have found that the expression is reduced and have completed the present invention.

That is, the present invention
[1] SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: : 19, SEQ ID NO: 21, SEQ ID NO: 23 or a compound containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 25 or a partial peptide thereof or a salt thereof Or a preventive / therapeutic agent or a metastasis / recurrence inhibitor of colorectal cancer comprising the salt thereof.
[1a] SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: : 19, SEQ ID NO: 21, SEQ ID NO: 23, or a preventive / therapeutic agent for colorectal cancer comprising a compound or salt thereof that inhibits the activity of the protein consisting of the amino acid sequence represented by SEQ ID NO: 23 or the salt thereof Or a metastasis / recurrence inhibitor.
[2] SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: : 19, SEQ ID NO: 21, SEQ ID NO: 23 or SEQ ID NO: 25 inhibits the expression of a protein comprising the same or substantially the same amino acid sequence as that shown in FIG. A preventive / therapeutic agent or inhibitor of metastasis / recurrence of colorectal cancer, comprising the compound or salt thereof
[2a] SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: : Prevention of colorectal cancer comprising a compound or a salt thereof that inhibits the expression of the protein or its salt gene comprising the amino acid sequence represented by SEQ ID NO: 21, SEQ ID NO: 21, or SEQ ID NO: 25 Therapeutic agent or metastasis / recurrence inhibitor.
[3] SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: : 19, SEQ ID NO: 21, SEQ ID NO: 23, or a nucleotide sequence of a polynucleotide encoding a protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 25 or a partial peptide thereof An antisense polynucleotide containing a complementary or substantially complementary base sequence or a part thereof.
[4] A medicament comprising the antisense polynucleotide according to [3] above.

[5] The medicament described in [4] above, which is a preventive / therapeutic agent for colon cancer or an inhibitor for metastasis / recurrence.
[6] SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: : 19, SEQ ID NO: 21, SEQ ID NO: 23 or siRNA or shRNA against a polynucleotide encoding a protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 25 or a partial peptide thereof A medicament comprising:
[6a] SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: : A pharmaceutical comprising siRNA or shRNA against a polynucleotide encoding a protein consisting of the amino acid sequence represented by SEQ ID NO: 21, SEQ ID NO: 23 or SEQ ID NO: 25.
[7] The medicament of the above-mentioned [6], which is a preventive / therapeutic agent for colon cancer or an inhibitor for metastasis / recurrence.
[8] SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: : 19, SEQ ID NO: 21, SEQ ID NO: 23 or SEQ ID NO: 25 containing a protein containing the same or substantially the same amino acid sequence as shown in FIG. A preventive / therapeutic agent or a metastasis / relapse inhibitor for colon cancer.
[8a] SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: : 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 23 Relapse inhibitor.

[9] SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: : 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, or a protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 27 or a partial peptide thereof or a salt thereof A diagnostic agent for colorectal cancer comprising an antibody.
[9a] SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: : 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25 or SEQ ID NO: 27, a diagnostic agent for colorectal cancer comprising an antibody against the protein consisting of the amino acid sequence represented by SEQ ID NO: 27 or a partial peptide thereof or a salt thereof .
[10] SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: : 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25 or SEQ ID NO: 27 or a partial peptide encoding a protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 27 A diagnostic agent for colorectal cancer comprising a nucleotide.
[11] SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: : 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, or a protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 27 or a partial peptide thereof or a salt thereof A method for diagnosing colorectal cancer, comprising using an antibody, or a polynucleotide encoding the protein or a partial peptide thereof.

[12] SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: : 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, or a protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 27 or a partial peptide thereof or a salt thereof Use as a diagnostic marker for colorectal cancer.
[13] SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: : 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, or a protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 27 or a partial peptide thereof or a salt thereof A screening method for a medicament for the prevention / treatment of colorectal cancer or the suppression of metastasis / recurrence, characterized by being used.
[14] SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: : 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, or a protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 27 or a partial peptide thereof or a salt thereof A screening kit for a drug for prevention / treatment of colorectal cancer or suppression of metastasis / recurrence characterized by containing.
[15] SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: : 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25 or SEQ ID NO: 27 or a partial peptide encoding a protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 27 A method for screening a drug for preventing or treating colorectal cancer or inhibiting metastasis or recurrence, which comprises using a nucleotide.
[16] SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: : 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25 or SEQ ID NO: 27 or a partial peptide encoding a protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 27 A screening kit for a drug for preventing or treating colorectal cancer or suppressing metastasis or recurrence, which comprises a nucleotide.
[17] SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: : 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, or a protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 27 or a partial peptide thereof or a salt thereof A screening method for a drug for preventing or treating colorectal cancer or inhibiting metastasis or recurrence, which comprises measuring activity.
[18] SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: : 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25 or a protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 27 or a partial peptide thereof or a salt thereof A method for screening a drug for preventing or treating colorectal cancer or suppressing metastasis or recurrence, which comprises measuring the amount.

[19] SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: : Inhibits the activity of a protein containing the same or substantially the same amino acid sequence represented by SEQ ID NO: 21, SEQ ID NO: 23 or SEQ ID NO: 25 or a partial peptide thereof or a salt thereof, Alternatively, a method for preventing or treating colorectal cancer, or a method for suppressing metastasis or recurrence, which comprises inhibiting the expression of a gene of the protein or a partial peptide thereof or a salt thereof.
[20] For mammals, (i) SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, a protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23 or SEQ ID NO: 25 or a partial peptide thereof Or a compound or salt thereof that inhibits the activity of the salt, (ii) a compound or salt thereof that inhibits the expression of the gene of the protein or partial peptide or salt thereof, and (iii) the protein or partial peptide or salt thereof. Complementary or substantially complementary to the base sequence of an antibody, or (iv) a polynucleotide encoding the above protein or a partial peptide thereof Complementary base sequence or features that, colorectal cancer prevention and treatment or metastatic or recurrent suppression methods administering an effective amount of an antisense polynucleotide comprising a part thereof.

[21] (i) SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, for producing a preventive / therapeutic agent for colon cancer or a metastasis / recurrence inhibitor SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, or the same or substantially the amino acid sequence represented by SEQ ID NO: 25 A compound or a salt thereof that inhibits the activity of a protein containing the same amino acid sequence or a partial peptide thereof or a salt thereof, (ii) a compound or a salt thereof that inhibits the expression of the gene of the protein or a partial peptide thereof or a salt thereof, (Iii) an antibody against the protein or a partial peptide thereof or a salt thereof; or (iv) a polynucleotide encoding the protein or a partial peptide thereof. Use of an antisense polynucleotide comprising a complementary or substantially complementary base sequence or a portion thereof to the base sequence of Reochido.
[22] SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: : 19, SEQ ID NO: 21, SEQ ID NO: 23 or a compound containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 25 or a partial peptide thereof or a salt thereof Or a colon cancer cell apoptosis promoter or colon cancer cell growth inhibitor comprising a salt thereof.
[23] SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: : 19, SEQ ID NO: 21, SEQ ID NO: 23 or SEQ ID NO: 25 inhibits the expression of a protein comprising the same or substantially the same amino acid sequence as that shown in FIG. A colorectal cancer cell apoptosis promoter or colorectal cancer cell growth inhibitor comprising the compound or a salt thereof.
[24] The medicament of the above-mentioned [4], which is a colon cancer cell apoptosis promoter or a colon cancer cell growth inhibitor.
[25] The medicament of the above-mentioned [6], which is a colon cancer cell apoptosis promoter or a colon cancer cell growth inhibitor.
[26] SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: : 19, SEQ ID NO: 21, SEQ ID NO: 23 or SEQ ID NO: 25 containing a protein containing the same or substantially the same amino acid sequence as shown in FIG. A colon cancer cell apoptosis promoter or colon cancer cell growth inhibitor.

[27] SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: : 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, or a protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 27 or a partial peptide thereof or a salt thereof A screening method for a medicament for promoting apoptosis of colorectal cancer cells or suppressing growth of colorectal cancer cells, which is characterized by using.
[28] SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: : 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, or a protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 27 or a partial peptide thereof or a salt thereof A screening kit for a pharmaceutical for promoting apoptosis of colon cancer cells or suppressing proliferation of colon cancer cells, characterized by comprising.
[29] SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: : 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25 or SEQ ID NO: 27 or a partial peptide encoding a protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 27 A method for screening a drug for promoting apoptosis of colorectal cancer cells or suppressing growth of colorectal cancer cells, comprising using a nucleotide.
[30] SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: : 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25 or SEQ ID NO: 27 or a partial peptide encoding a protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 27 A screening kit for a medicament for promoting apoptosis of colorectal cancer cells or suppressing growth of colorectal cancer cells, comprising a nucleotide.

[31] SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: : Inhibits the activity of a protein containing the same or substantially the same amino acid sequence represented by SEQ ID NO: 21, SEQ ID NO: 23 or SEQ ID NO: 25 or a partial peptide thereof or a salt thereof, Alternatively, a method for promoting apoptosis of colon cancer cells or a method for suppressing proliferation of colon cancer cells, characterized by inhibiting expression of the gene of the protein or a partial peptide thereof or a salt thereof.
[32] For mammals, (i) SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, a protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23 or SEQ ID NO: 25 or a partial peptide thereof Or a compound or salt thereof that inhibits the activity of the salt, (ii) a compound or salt thereof that inhibits the expression of the gene of the protein or partial peptide or salt thereof, and (iii) the protein or partial peptide or salt thereof. Complementary or substantially complementary to the base sequence of an antibody, or (iv) a polynucleotide encoding the above protein or a partial peptide thereof Complementary base sequence or features that, proapoptotic method or antiproliferative methods of colon cancer cells in colon cancer cells by administering an effective amount of an antisense polynucleotide comprising a part thereof.
[33] (i) SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: for producing a colon cancer cell apoptosis promoter or colon cancer cell growth inhibitor 9, the same as the amino acid sequence represented by SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23 or SEQ ID NO: 25 A compound or a salt thereof that inhibits the activity of a protein or a partial peptide thereof or a salt thereof containing substantially the same amino acid sequence; (ii) a compound or a salt thereof that inhibits the expression of the gene of the protein or a partial peptide thereof or a salt thereof. A salt, (iii) an antibody against the protein or a partial peptide thereof or a salt thereof, or (iv) the protein or a partial peptide thereof Provides, such as the use of antisense polynucleotide comprising a complementary or substantially complementary base sequence or a portion thereof to the base sequence of a polynucleotide that over de.

The above-mentioned “medicine for preventing / treating cancer” is a substance having an action for preventing / treating cancer (eg, synthetic compound, peptide, protein, antibody, non-peptidic compound, fermentation product, cell extract, plant extract, Animal tissue extract, plasma, etc.) itself or a medicine containing the substance may be used.
In the present specification, the term “prevention / treatment of colorectal cancer” is used as a term including the meaning of “suppression of malignant (cancerous) colon polyps”.
The above-mentioned “medicine for promoting apoptosis of cancer cells” may be a substance itself having an effect of promoting apoptosis of cancer cells, and the substance (eg, synthetic compound, peptide, protein, antibody, non-peptidic compound, fermentation product) , A cell extract, a plant extract, an animal tissue extract, plasma and the like.
The above-mentioned “medicine for inhibiting the growth of cancer cells” is a substance having an action of inhibiting the growth of cancer cells (eg, synthetic compounds, peptides, proteins, antibodies, non-peptide compounds, fermentation products, cell extracts, plant extracts, Animal tissue extract, plasma, etc.) itself or a medicine containing the substance may be used.

SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, A protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 21, SEQ ID NO: 23 or SEQ ID NO: 25 has, for example, anti-apoptotic activity, cancer cell proliferation promoting activity and the like A compound that inhibits the activity of the protein or a salt thereof, a compound that inhibits the expression of the gene of the protein or a salt thereof, an antibody against the protein, an antisense polynucleotide of a polynucleotide encoding the protein, siRNA, shRNA, etc. For example, preventive / therapeutic agents for colon cancer, colon cancer metastasis / relapse inhibitors, colon cancer cell apoptosis promoter, colon cancer cell It can be used as a safe medicine as a cell growth inhibitor.
A compound or a salt thereof that promotes the activity of a protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 27, a compound that promotes the expression of the gene of the protein, or a salt thereof, etc. Since it can be expected as a safe pharmaceutical as a preventive / therapeutic agent for colorectal cancer, a metastasis / relapse inhibitor of colorectal cancer, an apoptosis promoter of colorectal cancer cells, a growth inhibitor of colorectal cancer cell cells, the protein is It can be used for screening active pharmaceutical ingredients.

In the present specification, SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, A protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23 or SEQ ID NO: 25 is referred to as “protein A used in the present invention” or Abbreviated as “protein A of the present invention”; a protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 27 is referred to as “protein B used in the present invention” or “protein of the present invention” These proteins may be collectively abbreviated as “proteins used in the present invention” or “proteins of the present invention”.
SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, As an amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25 or SEQ ID NO: 27, for example, SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: : 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 23 Or about 50% or more, preferably about 60% or more, more preferably about 70% or more, more preferably about 80% or more, and particularly preferably about 90% or more. Most preferably about 9 Such an amino acid sequence having a% homology thereof.
The homology of the amino acid sequence was determined using the homology calculation algorithm NCBI BLAST (National Center for Biotechnology Information Basic Local Alignment Search Tool) under the following conditions (expected value = 10; allow gap; matrix = BLOSUM62; filtering = OFF) Can be calculated.

SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, Examples of the protein containing the amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25 or SEQ ID NO: 27 include, for example, the above-mentioned SEQ ID NO: 1, sequence SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 21 The amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 23, SEQ ID NO: 25 or SEQ ID NO: 27 is contained, SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7 , SEQ ID NO: 9, SEQ ID NO: 1. It contains the amino acid sequence represented by SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, or SEQ ID NO: 27. A protein having substantially the same quality of activity as a protein is preferred.
In the case of the protein A of the present invention, the substantially homogeneous activity includes, for example, an anti-apoptotic activity and a cancer cell proliferation promoting activity, and in the case of the protein B of the present invention, for example, an apoptotic activity and a cancer cell proliferation inhibiting activity. . Substantially homogeneous indicates that their properties are qualitatively (eg, physiologically or pharmacologically) homogeneous. Therefore, it is preferable that these activities are equivalent (eg, about 0.01 to 100 times, preferably about 0.1 to 10 times, more preferably 0.5 to 2 times). Quantitative factors such as protein molecular weight may be different.
Measurement of anti-apoptotic activity, cancer cell proliferation promoting activity, apoptotic activity, cancer cell growth inhibitory activity, etc. is a method known per se, for example, “Cultured Cell Experimental Method for Molecular Biology Research (Yodosha)” It can be measured according to a method or a method analogous thereto.

Examples of the protein of the present invention include (i) SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, and sequence. SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25 or 1 or 2 or more in the amino acid sequence represented by SEQ ID NO: 27 (for example, 1 to 2) About 100, preferably about 1-30, preferably about 1-10, more preferably several (1-5) amino acids, (ii) SEQ ID NO: 1, SEQ ID NO: : 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 21 : 23, SEQ ID NO: 25 or SEQ ID NO: 1 or 2 (for example, about 1 to 100, preferably about 1 to 30, preferably about 1 to 10, more preferably number (1 to 5)) amino acids in the amino acid sequence represented by 7 (Iii) SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7 or 1 or more in the amino acid sequence represented by SEQ ID NO: 9 (for example, 1 to 100) About, preferably about 1 to 30, preferably about 1 to 10, more preferably several (1 to 5) amino acids, (iv) SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25 or SEQ ID NO : 1 or 2 or more in the amino acid sequence represented by 27 (for example, about 1 to 100, preferably about 1 to 30, preferably about 1 to 10, more preferably number (1 to 5)) Also included are so-called muteins such as proteins containing amino acid sequences in which are replaced with other amino acids, or (v) amino acid sequences combining them.
When the amino acid sequence is inserted, deleted or substituted as described above, the position of the insertion, deletion or substitution is not particularly limited.

The protein in the present specification has an N-terminus (amino terminus) at the left end and a C-terminus (carboxyl terminus) at the right end according to the convention of peptide designation. SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, The protein used in the present invention including the protein containing the amino acid sequence represented by SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, or SEQ ID NO: 27 has a carboxyl group (—COOH) at the C-terminus. , Carboxylate (—COO ⁻ ), amide (—CONH ₂ ) or ester (—COOR).
Here, as R in the ester, for example, a C _1-6 alkyl group such as methyl, ethyl, n-propyl, isopropyl, n-butyl, for example, a C _3-8 cycloalkyl group such as cyclopentyl, cyclohexyl, etc., for example, phenyl C _6-12 aryl groups such as α-naphthyl, C _7- such as phenyl-C _1-2 alkyl groups such as benzyl and phenethyl or α-naphthyl-C _1-2 alkyl groups such as α-naphthylmethyl ₁₄ aralkyl group, pivaloyloxymethyl group is used.
When the protein used in the present invention has a carboxyl group (or carboxylate) other than the C-terminus, a protein in which the carboxyl group is amidated or esterified is also included in the protein used in the present invention. As the ester in this case, for example, the above C-terminal ester or the like is used.
Furthermore, the protein used in the present invention, the amino acid residue (eg, methionine residue) of the N-terminal amino group protecting group (e.g., formyl group, such as C _1-6 alkanoyl such as acetyl group C _{1- 6} protected with an acyl group, N-terminal glutamine residue generated by cleavage in vivo is pyroglutamine oxidized, and a substituent on the side chain of an amino acid in the molecule (eg, —OH, — SH, amino group, imidazole group, indole group, guanidino group, etc.) are protected with an appropriate protecting group (for example, C _1-6 acyl group such as C _1-6 alkanoyl group such as formyl group, acetyl group, etc.) Or a complex protein such as a so-called glycoprotein having a sugar chain bound thereto.
Specific examples of the protein used in the present invention include, for example, a protein containing the amino acid sequence represented by SEQ ID NO: 1, a protein containing the amino acid sequence represented by SEQ ID NO: 3, and a protein represented by SEQ ID NO: 5. A protein containing the amino acid sequence represented by SEQ ID NO: 7, a protein containing the amino acid sequence represented by SEQ ID NO: 9, and the like.

The partial peptide of the protein used in the present invention is a partial peptide of the protein used in the present invention as described above, and preferably any one having the same properties as the protein used in the present invention described above. It may be a thing.
Specifically, for the purpose of preparing the antibody of the present invention described later, for example, at least 20 or more, preferably 50 or more, more preferably 70 or more of the constituent amino acid sequences of the protein used in the present invention, More preferably, a peptide containing 100 or more, most preferably 200 or more amino acid sequences is used.
Moreover, the partial peptide used by this invention is 1 or 2 or more in the amino acid sequence (preferably about 1-20 pieces, More preferably, about 1-10 pieces, More preferably, it is a number (1-5) pieces). Amino acids are deleted, or 1 or 2 (preferably about 1 to 20, more preferably about 1 to 10, more preferably a number (1 to 5)) of amino acids are present in the amino acid sequence. Or 1 or 2 (preferably about 1 to 20, more preferably about 1 to 10, more preferably several (1 to 5)) amino acids are inserted into the amino acid sequence, or In the amino acid sequence, one or more (preferably about 1 to 20, more preferably about 1 to 10, more preferably several (1 to 5)) amino acids are substituted with other amino acids. The Good.

In the partial peptide used in the present invention, the C-terminus may be any of a carboxyl group (—COOH), a carboxylate (—COO ⁻ ), an amide (—CONH ₂ ), or an ester (—COOR).
Furthermore, the partial peptides used in the present invention include those having a carboxyl group (or carboxylate) in addition to the C terminus, as well as the N-terminal amino acid residues (examples) as in the protein used in the present invention described above. , The amino group of the methionine residue) is protected with a protecting group, the N-terminal side is cleaved in vivo, the resulting glutamine residue is pyroglutamine oxidized, the substituent on the side chain of the amino acid in the molecule Also included are those protected with an appropriate protecting group, or complex peptides such as so-called glycopeptides to which sugar chains are bound.
The partial peptide used in the present invention can also be used as an antigen for antibody production.

Examples of salts of proteins or partial peptides used in the present invention include physiologically acceptable metal salts, ammonium salts, salts with organic bases, salts with inorganic acids, salts with organic acids, basic or acidic amino acids, and the like. And the like. Preferable examples of the metal salt include alkali metal salts such as sodium salt and potassium salt; alkaline earth metal salts such as calcium salt, magnesium salt and barium salt; aluminum salt and the like. Preferable examples of the salt with an organic base include, for example, trimethylamine, triethylamine, pyridine, picoline, 2,6-lutidine, ethanolamine, diethanolamine, triethanolamine, cyclohexylamine, dicyclohexylamine, N, N′-dibenzylethylenediamine. And the like. Preferable examples of the salt with inorganic acid include salts with hydrochloric acid, hydrobromic acid, nitric acid, sulfuric acid, phosphoric acid and the like. Preferable examples of salts with organic acids include formic acid, acetic acid, trifluoroacetic acid, propionic acid, phthalic acid, fumaric acid, oxalic acid, tartaric acid, maleic acid, citric acid, succinic acid, malic acid, methanesulfonic acid And salts with benzoic acid, benzenesulfonic acid, p-toluenesulfonic acid and the like. Preferable examples of salts with basic amino acids include salts with arginine, lysine, ornithine and the like, and preferable examples of salts with acidic amino acids include salts with aspartic acid, glutamic acid and the like. Can be mentioned.
The protein used in the present invention or a partial peptide thereof or a salt thereof can be produced from the above-described human or warm-blooded animal cells or tissues by a known protein purification method, and contains a DNA encoding the protein. It can also be produced by culturing a transformant. Moreover, it can also manufacture according to the below-mentioned peptide synthesis method.
When producing from human or mammalian tissue or cells, the human or mammalian tissue or cells are homogenized and then extracted with acid, etc., and the extract is subjected to chromatography such as reverse phase chromatography or ion exchange chromatography. Can be purified and isolated.

For the synthesis of the protein or partial peptide used in the present invention or a salt thereof, or an amide thereof, a commercially available resin for protein synthesis can be used. Examples of such resins include chloromethyl resin, hydroxymethyl resin, benzhydrylamine resin, aminomethyl resin, 4-benzyloxybenzyl alcohol resin, 4-methylbenzhydrylamine resin, PAM resin, 4-hydroxymethylmethyl. Examples include phenylacetamidomethyl resin, polyacrylamide resin, 4- (2 ′, 4′-dimethoxyphenyl-hydroxymethyl) phenoxy resin, 4- (2 ′, 4′-dimethoxyphenyl-Fmocaminoethyl) phenoxy resin, and the like. it can. Using such a resin, an amino acid having an α-amino group and a side chain functional group appropriately protected is condensed on the resin according to various condensation methods known per se according to the sequence of the target protein. At the end of the reaction, the protein or partial peptide is excised from the resin, and at the same time, various protecting groups are removed, and further, intramolecular disulfide bond forming reaction is performed in a highly diluted solution to obtain the target protein or partial peptide or their amides. To do.
For the above-mentioned condensation of protected amino acids, various activating reagents that can be used for protein synthesis can be used, and carbodiimides are particularly preferable. As the carbodiimide, DCC, N, N′-diisopropylcarbodiimide, N-ethyl-N ′-(3-dimethylaminoprolyl) carbodiimide and the like are used. For activation by these, a protected amino acid is added directly to the resin together with a racemization inhibitor (for example, HOBt, HOBt), or the protected amino acid is activated in advance as a symmetric acid anhydride, HOBt ester or HOBt ester. It can later be added to the resin.

  The solvent used for the activation of the protected amino acid and the condensation with the resin can be appropriately selected from solvents known to be usable for protein condensation reactions. For example, acid amides such as N, N-dimethylformamide, N, N-dimethylacetamide, N-methylpyrrolidone, halogenated hydrocarbons such as methylene chloride and chloroform, alcohols such as trifluoroethanol, dimethyl sulfoxide, etc. Examples include sulfoxides, ethers such as pyridine, dioxane, and tetrahydrofuran, nitriles such as acetonitrile and propionitrile, esters such as methyl acetate and ethyl acetate, and appropriate mixtures thereof. The reaction temperature is appropriately selected from a range that is known to be usable for protein bond forming reactions, and is usually selected from the range of about -20 ° C to 50 ° C. The activated amino acid derivative is usually used in an excess of 1.5 to 4 times. As a result of a test using the ninhydrin reaction, if the condensation is insufficient, sufficient condensation can be performed by repeating the condensation reaction without removing the protecting group. When sufficient condensation is not obtained even if the reaction is repeated, acetylation of the unreacted amino acid with acetic anhydride or acetylimidazole can prevent the subsequent reaction from being affected.

Examples of the protective group for the amino group of the raw material include Z, Boc, t-pentyloxycarbonyl, isobornyloxycarbonyl, 4-methoxybenzyloxycarbonyl, Cl-Z, Br-Z, adamantyloxycarbonyl, and trifluoroacetyl. , Phthaloyl, formyl, 2-nitrophenylsulfenyl, diphenylphosphinothioyl, Fmoc and the like.
The carboxyl group is, for example, alkyl esterified (eg, linear, branched or cyclic alkyl ester such as methyl, ethyl, propyl, butyl, t-butyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, 2-adamantyl, etc. ), Aralkyl esterification (eg, benzyl ester, 4-nitrobenzyl ester, 4-methoxybenzyl ester, 4-chlorobenzyl ester, benzhydryl esterification), phenacyl esterification, benzyloxycarbonylhydrazide, t-butoxy It can be protected by carbonyl hydrazation, trityl hydrazation or the like.
The hydroxyl group of serine can be protected, for example, by esterification or etherification. Examples of groups suitable for esterification include groups derived from carbonic acid such as lower (C _1-6 ) alkanoyl groups such as acetyl groups, aroyl groups such as benzoyl groups, benzyloxycarbonyl groups, and ethoxycarbonyl groups. Used. Examples of the group suitable for etherification include a benzyl group, a tetrahydropyranyl group, and a t-butyl group.
Examples of the protecting group for the phenolic hydroxyl group of tyrosine include Bzl, Cl ₂ -Bzl, 2-nitrobenzyl, Br-Z, t-butyl and the like.
Examples of the protecting group for imidazole of histidine include Tos, 4-methoxy-2,3,6-trimethylbenzenesulfonyl, DNP, benzyloxymethyl, Bum, Boc, Trt, Fmoc, and the like.

Examples of the activated carboxyl group of the raw material include a corresponding acid anhydride, azide, active ester [alcohol (eg, pentachlorophenol, 2,4,5-trichlorophenol, 2,4-dinitrophenol, And esters thereof with cyanomethyl alcohol, paranitrophenol, HONB, N-hydroxysuccinimide, N-hydroxyphthalimide, HOBt) and the like. As the activated amino group of the raw material, for example, a corresponding phosphoric acid amide is used.
Examples of the method for removing (eliminating) the protecting group include catalytic reduction in a hydrogen stream in the presence of a catalyst such as Pd-black or Pd-carbon, anhydrous hydrogen fluoride, methanesulfonic acid, trifluoro. Acid treatment with romethanesulfonic acid, trifluoroacetic acid or a mixture thereof, base treatment with diisopropylethylamine, triethylamine, piperidine, piperazine, etc., reduction with sodium in liquid ammonia, and the like are also used. The elimination reaction by the acid treatment is generally performed at a temperature of about −20 ° C. to 40 ° C. In the acid treatment, for example, anisole, phenol, thioanisole, metacresol, paracresol, dimethyl sulfide, 1,4 -Addition of a cation scavenger such as butanedithiol, 1,2-ethanedithiol, etc. is effective. The 2,4-dinitrophenyl group used as an imidazole protecting group of histidine is removed by thiophenol treatment, and the formyl group used as an indole protecting group of tryptophan is the above 1,2-ethanedithiol and 1,4-butane. In addition to deprotection by acid treatment in the presence of dithiol or the like, it can also be removed by alkali treatment with dilute sodium hydroxide solution, dilute ammonia or the like.

The protection of the functional group that should not be involved in the reaction of the raw material, the protection group, the removal of the protective group, the activation of the functional group involved in the reaction, etc. can be appropriately selected from known groups or known means.
As another method for obtaining an amide form of a protein or partial peptide, for example, first, the α-carboxyl group of the carboxy terminal amino acid is amidated and protected, and then the peptide (protein) chain is moved to the desired chain length on the amino group side. After extending, a protein or partial peptide from which only the N-terminal α-amino group protecting group of the peptide chain has been removed and a protein or partial peptide from which only the C-terminal carboxyl group protecting group has been prepared, The protein or peptide is condensed in a mixed solvent as described above. The details of the condensation reaction are the same as described above. After purifying the protected protein or peptide obtained by condensation, all the protecting groups can be removed by the above method to obtain the desired crude protein or peptide. This crude protein or peptide can be purified using various known purification means, and the main fraction can be lyophilized to obtain an amide of the desired protein or peptide.
In order to obtain a protein or peptide ester, for example, the α-carboxyl group of the carboxy terminal amino acid is condensed with a desired alcohol to form an amino acid ester, and then the desired protein or peptide amide is obtained in the same manner as the protein or peptide amide. An ester of a peptide can be obtained.

The partial peptide used in the present invention or a salt thereof can be produced according to a peptide synthesis method known per se, or by cleaving the protein used in the present invention with an appropriate peptidase. As a peptide synthesis method, for example, either a solid phase synthesis method or a liquid phase synthesis method may be used. That is, the partial peptide or amino acid that can constitute the partial peptide used in the present invention is condensed with the remaining portion, and when the product has a protective group, the target peptide can be produced by removing the protective group. it can. Examples of known condensation methods and protecting group elimination include the methods described in the following (i) to (v).
(I) M. Bodanszky and MA Ondetti, Peptide Synthesis, Interscience Publishers, New York (1966)
(Ii) Schroeder and Luebke, The Peptide, Academic Press, New York (1965)
(Iii) Nobuo Izumiya et al., Basics and Experiments of Peptide Synthesis, Maruzen Co., Ltd. (1975)
(Iv) Haruaki Yajima and Shunpei Sugawara, Biochemistry Experiment Course 1, Protein Chemistry IV, 205, (1977)
(V) Supervised by Haruaki Yajima, Development of follow-up drugs, Volume 14, Peptide synthesis, Hirokawa Shoten Also, after the reaction, usual purification methods such as solvent extraction, distillation, column chromatography, liquid chromatography, recrystallization, etc. Can be combined to purify and isolate the partial peptide used in the present invention. When the partial peptide obtained by the above method is a free form, it can be converted to an appropriate salt by a known method or a method equivalent thereto, and conversely, when obtained as a salt, the known method or a method equivalent thereto. It can be converted to the free form or other salts by methods.

The polynucleotide encoding the protein used in the present invention may be any as long as it contains the base sequence encoding the protein used in the present invention described above. Preferably it is DNA. The DNA may be any of genomic DNA, genomic DNA library, cDNA derived from the cells / tissues described above, cDNA library derived from the cells / tissues described above, and synthetic DNA.
The vector used for the library may be any of bacteriophage, plasmid, cosmid, phagemid and the like. Further, it can also be directly amplified by reverse transcription polymerase chain reaction (hereinafter abbreviated as RT-PCR method) using a total RNA or mRNA fraction prepared from the cells / tissues described above.
Examples of the DNA encoding the protein used in the present invention include SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, DNA containing the base sequence represented by SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26 or SEQ ID NO: 28, or SEQ ID NO: 2 SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22 A nucleotide sequence that hybridizes with the nucleotide sequence represented by SEQ ID NO: 24, SEQ ID NO: 26, or SEQ ID NO: 28 under highly stringent conditions, and includes the aforementioned SEQ ID NO: 1, SEQ ID NO: 3, Arrangement SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 23 Any DNA may be used as long as it encodes a protein having substantially the same properties as the protein containing the amino acid sequence represented by SEQ ID NO: 25 or SEQ ID NO: 27.

Examples of DNA that can hybridize with the base sequence represented by SEQ ID NO: 2 under highly stringent conditions include, for example, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, and SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26 or SEQ ID NO: 28 A homology of about 50% or more, preferably about 60% or more, more preferably about 70% or more, more preferably about 80% or more, particularly preferably about 90% or more, most preferably about 95% or more with the base sequence. For example, DNA containing the nucleotide sequence is used.
The homology of the base sequence was determined using the homology calculation algorithm NCBI BLAST (National Center for Biotechnology Information Basic Local Alignment Search Tool) under the following conditions (expected value = 10; allow gap; filtering = ON; match score = 1; It can be calculated by mismatch score = -3).
Hybridization can be performed according to a method known per se or a method analogous thereto, for example, the method described in Molecular Cloning 2nd (J. Sambrook et al., Cold Spring Harbor Lab. Press, 1989). Moreover, when using a commercially available library, it can carry out according to the method as described in an attached instruction manual. More preferably, it can be carried out according to highly stringent conditions.
The highly stringent conditions are, for example, conditions in which the sodium concentration is about 19 to 40 mM, preferably about 19 to 20 mM, and the temperature is about 50 to 70 ° C., preferably about 60 to 65 ° C. In particular, the case where the sodium concentration is about 19 mM and the temperature is about 65 ° C. is most preferable.
More specifically, as a DNA encoding a protein containing the amino acid sequence represented by SEQ ID NO: 1, a DNA containing a base sequence represented by SEQ ID NO: 2 is represented by SEQ ID NO: 3. As a DNA encoding a protein containing the amino acid sequence, a DNA containing the amino acid sequence represented by SEQ ID NO: 5 such as a DNA containing the base sequence represented by SEQ ID NO: 4 As the DNA encoding the protein containing the amino acid sequence represented by SEQ ID NO: 7 such as the DNA containing the base sequence represented by SEQ ID NO: 6, the base represented by SEQ ID NO: 8 The DNA encoding the protein containing the amino acid sequence represented by SEQ ID NO: 9 such as DNA containing the sequence is represented by SEQ ID NO: 10. Examples of the DNA containing the base sequence and the DNA encoding the protein containing the amino acid sequence represented by SEQ ID NO: 11 include the DNA containing the base sequence represented by SEQ ID NO: 12, As the DNA encoding the protein containing the amino acid sequence represented, the DNA containing the base sequence represented by SEQ ID NO: 14 encodes the protein containing the amino acid sequence represented by SEQ ID NO: 15. Examples of the DNA include a DNA containing the base sequence represented by SEQ ID NO: 16 and the like, and a DNA encoding a protein containing the amino acid sequence represented by SEQ ID NO: 17 is represented by SEQ ID NO: 18. As DNA encoding a protein containing the amino acid sequence represented by SEQ ID NO: 19, such as DNA containing a base sequence The DNA containing the base sequence represented by SEQ ID NO: 20 and the DNA encoding the protein containing the amino acid sequence represented by SEQ ID NO: 21 include the base sequence represented by SEQ ID NO: 22. As a DNA encoding a protein containing the amino acid sequence represented by SEQ ID NO: 23, a DNA containing the base sequence represented by SEQ ID NO: 24 is represented by SEQ ID NO: 25. Examples of DNA encoding a protein containing an amino acid sequence include a DNA containing a base sequence represented by SEQ ID NO: 26, and a DNA encoding a protein containing an amino acid sequence represented by SEQ ID NO: 27. DNA containing the base sequence represented by SEQ ID NO: 28 is used.

The DNA encoding the partial peptide used in the present invention may be any DNA as long as it contains a base sequence encoding the partial peptide used in the present invention described above. Further, any of genomic DNA, genomic DNA library, cDNA derived from the cells / tissues described above, cDNA library derived from the cells / tissues described above, and synthetic DNA may be used.
Examples of the DNA encoding the partial peptide used in the present invention include SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, and SEQ ID NO: 14. SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26 or DNA having a part of the DNA containing the base sequence represented by SEQ ID NO: 28 Or SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, comprising a nucleotide sequence that hybridizes with the nucleotide sequence represented by SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26 or SEQ ID NO: 28 under highly stringent conditions, A DNA containing a portion of the DNA encoding a protein having a quality substantially the same quality of activity are used.
SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, The DNA that can hybridize with the base sequence represented by SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26 or SEQ ID NO: 28 has the same significance as described above.
Hybridization methods and highly stringent conditions are the same as those described above.

Means for cloning DNA that completely encodes the protein and partial peptide used in the present invention (hereinafter, in the description of cloning and expression of DNA encoding them, these may be simply abbreviated as the protein of the present invention) As described above, a synthetic DNA primer having a part of the base sequence encoding the protein of the present invention is used to amplify by PCR method, or DNA incorporated into an appropriate vector encodes a part or all region of the protein of the present invention. Can be selected by hybridization with a DNA fragment or synthetic DNA labeled. The hybridization method can be performed, for example, according to the method described in Molecular Cloning 2nd (J. Sambrook et al., Cold Spring Harbor Lab. Press, 1989). Moreover, when using a commercially available library, it can carry out according to the method as described in an attached instruction manual.
The DNA base sequence is converted by PCR, a known kit such as Mutan ^™ -super Express Km (Takara Shuzo), Mutan ^™ -K (Takara Shuzo), etc., using the ODA-LA PCR method, It can be carried out according to a method known per se such as the Gapped duplex method and the Kunkel method or a method analogous thereto.
The DNA encoding the cloned protein can be used as it is or after digestion with a restriction enzyme or addition of a linker if desired. The DNA may have ATG as a translation initiation codon on the 5 ′ end side, and may have TAA, TGA, or TAG as a translation termination codon on the 3 ′ end side. These translation initiation codon and translation termination codon can be added using an appropriate synthetic DNA adapter.
The protein expression vector of the present invention can be obtained by, for example, (i) cutting out a target DNA fragment from the DNA encoding the protein of the present invention, and (ii) linking the DNA fragment downstream of a promoter in an appropriate expression vector. Can be manufactured.

Examples of vectors include E. coli-derived plasmids (eg, pBR322, pBR325, pUC12, pUC13), Bacillus subtilis-derived plasmids (eg, pUB110, pTP5, pC194), yeast-derived plasmids (eg, pSH19, pSH15), λ phage, and the like. In addition to animal viruses such as bacteriophage, retrovirus, vaccinia virus and baculovirus, pA1-11, pXT1, pRc / CMV, pRc / RSV, pcDNAI / Neo and the like are used.
The promoter used in the present invention may be any promoter as long as it is suitable for the host used for gene expression. For example, when animal cells are used as the host, SRα promoter, SV40 promoter, LTR promoter, CMV promoter, HSV-TK promoter and the like can be mentioned.
Of these, it is preferable to use a CMV (cytomegalovirus) promoter, SRα promoter, or the like. When the host is Escherichia, trp promoter, lac promoter, recA promoter, .lambda.P _L promoter, lpp promoter, T7 promoter, etc. When the host is Bacillus, SPO1 promoter, SPO2 promoter, penP promoter, etc. When the host is yeast, the PHO5 promoter, PGK promoter, GAP promoter, ADH promoter and the like are preferable. When the host is an insect cell, a polyhedrin promoter, a P10 promoter and the like are preferable.
In addition to the above, an expression vector containing an enhancer, a splicing signal, a poly A addition signal, a selection marker, an SV40 replication origin (hereinafter sometimes abbreviated as SV40ori) and the like is used as desired. it can. Selectable markers include, for example, dihydrofolate reductase (hereinafter sometimes abbreviated as dhfr) gene [methotrexate (MTX) resistance], ampicillin resistance gene (hereinafter sometimes abbreviated as Amp ^r ), neomycin resistance gene (hereinafter sometimes abbreviated as Neo ^r, G418 resistance). In particular, when a dhfr gene-deficient Chinese hamster cell is used and the dhfr gene is used as a selection marker, the target gene can also be selected by a medium not containing thymidine.
If necessary, a signal sequence suitable for the host is added to the N-terminal side of the protein of the present invention. When the host is Escherichia, the PhoA signal sequence, OmpA, signal sequence, etc., and when the host is Bacillus, the α-amylase signal sequence, subtilisin signal sequence, etc. In some cases, MFα • signal sequence, SUC2 • signal sequence, etc., and when the host is an animal cell, insulin signal sequence, α-interferon signal sequence, antibody molecule / signal sequence, etc. can be used.
A transformant can be produced using a vector containing the DNA encoding the protein of the present invention thus constructed.

As the host, for example, Escherichia, Bacillus, yeast, insect cells, insects, animal cells and the like are used.
Specific examples of the genus Escherichia include, for example, Escherichia coli K12 · DH1 [Proc. Natl. Acad. Sci. USA, 60, 160 (1968)], JM103 [Nucleic Acids Research, 9, 309 (1981)], JA221 [Journal of Molecular Biology, 120, 517 (1978)], HB101 [Journal of Molecular Biology, 41, 459 (1969)], C600 [Genetics, 39, 440 (1954)] Etc. are used.
Examples of the Bacillus bacterium include Bacillus subtilis MI114 [Gene, 24, 255 (1983)], 207-21 [Journal of Biochemistry, 95, 87 (1984)].
Examples of yeast include Saccharomyces cerevisiae AH22, AH22R ⁻ , NA87-11A, DKD-5D, 20B-12, Schizosaccharomyces pombe NCYC1913, NCYC2036, Used.
As insect cells, for example, when the virus is AcNPV, larvae-derived cell lines (Spodoptera frugiperda cells; Sf cells), MG1 cells derived from the midgut of Trichoplusia ni, High Five ^™ derived from eggs of Trichoplusia ni Cells, cells derived from Mamestra brassicae or cells derived from Estigmena acrea are used. When the virus is BmNPV, sputum-derived cell lines (Bombyx mori N cells; BmN cells) and the like are used. Examples of the Sf cells include Sf9 cells (ATCC CRL 1711), Sf21 cells (Vaughn, JL et al., In Vivo, 13, 213-217, (1977)) and the like.
Examples of insects include silkworm larvae [Maeda et al., Nature, 315, 592 (1985)].
Examples of animal cells include monkey cells COS-7, Vero, Chinese hamster cells CHO (hereinafter abbreviated as CHO cells), dhfr gene-deficient Chinese hamster cells CHO (hereinafter abbreviated as CHO (dhfr ⁻ ) cells), mouse L Cells, mouse AtT-20, mouse myeloma cells, mouse ATDC5 cells, rat GH3, human FL cells and the like are used.
Transformation of Escherichia can be performed, for example, according to the method described in Proc. Natl. Acad. Sci. USA, 69, 2110 (1972), Gene, 17, 107 (1982).

Transformation of Bacillus can be performed, for example, according to the method described in Molecular & General Genetics, 168, 111 (1979).
In order to transform yeast, for example, the method described in Methods in Enzymology, 194, 182-287 (1991), Proc. Natl. Acad. Sci. USA, 75, 1929 (1978) and the like can be performed. it can.
Insect cells or insects can be transformed, for example, according to the method described in Bio / Technology, 6, 47-55 (1988).
In order to transform animal cells, for example, cell engineering separate volume 8 New Cell Engineering Experiment Protocol. 263-267 (1995) (published by Shujunsha), Virology, 52, 456 (1973) Can do.
In this way, a transformant transformed with an expression vector containing DNA encoding the protein can be obtained.
When culturing a transformant whose host is an Escherichia bacterium or Bacillus genus, a liquid medium is suitable as a medium used for the culture, and a carbon source necessary for the growth of the transformant, Nitrogen sources, inorganic substances, etc. are contained. Examples of the carbon source include glucose, dextrin, soluble starch, and sucrose. Examples of the nitrogen source include ammonium salts, nitrates, corn steep liquor, peptone, casein, meat extract, soybean cake, and potato extract. Examples of inorganic or organic substances and inorganic substances include calcium chloride, sodium dihydrogen phosphate, and magnesium chloride. In addition, yeast extract, vitamins, growth promoting factors and the like may be added. The pH of the medium is preferably about 5-8.
As a medium for culturing Escherichia, for example, M9 medium containing glucose and casamino acid (Miller, Journal of Experiments in Molecular Genetics, 431-433, Cold Spring Harbor Laboratory, New York 1972) is preferable. . In order to make the promoter work efficiently here, a drug such as 3β-indolylacrylic acid can be added.
When the host is Escherichia, the culture is usually performed at about 15 to 43 ° C. for about 3 to 24 hours, and if necessary, aeration or agitation can be added.
When the host is a genus Bacillus, the culture is usually performed at about 30 to 40 ° C. for about 6 to 24 hours, and if necessary, aeration or agitation can be added.
When cultivating a transformant whose host is yeast, examples of the medium include a Burkholder minimum medium [Bostian, KL et al., Proc. Natl. Acad. Sci. USA, vol. 77, 4505 (1980)]. And SD medium containing 0.5% casamino acid [Bitter, GA et al. Proc. Natl. Acad. Sci. USA, 81, 5330 (1984)]. The pH of the medium is preferably adjusted to about 5-8. The culture is usually performed at about 20 ° C. to 35 ° C. for about 24 to 72 hours, and aeration and agitation are added as necessary.
When cultivating a transformant whose host is an insect cell or an insect, an appropriate medium such as 10% bovine serum that has been immobilized on Grace's Insect Medium (Grace, TCC, Nature, 195,788 (1962)) is used as the medium. Additions etc. are used. The pH of the medium is preferably adjusted to about 6.2 to 6.4. The culture is usually performed at about 27 ° C. for about 3 to 5 days, and aeration and agitation are added as necessary.
When culturing a transformant whose host is an animal cell, examples of the medium include MEM medium (Science, Vol. 122, 501 (1952)) containing about 5 to 20% fetal calf serum, DMEM medium [Virology, 8, 396 (1959)], RPMI 1640 medium [The Journal of the American Medical Association 199, 519 (1967)], 199 medium [Proceeding of the Society for the Biological Medicine, 73, 1 (1950)], etc. Is used. The pH is preferably about 6-8.
Culture is usually performed at about 30 ° C. to 40 ° C. for about 15 to 60 hours, and aeration and agitation are added as necessary.
As described above, the protein of the present invention can be produced inside the cell, the cell membrane, or outside the cell of the transformant.

Separation and purification of the protein of the present invention from the culture can be performed, for example, by the following method.
When extracting the protein of the present invention from cultured cells or cells, after culturing, the cells or cells are collected by a known method, suspended in an appropriate buffer, and subjected to ultrasound, lysozyme and / or freeze-thaw, etc. A method of obtaining a crude protein extract by centrifugation or filtration after destroying cells or cells by the method is appropriately used. The buffer solution may contain a protein denaturant such as urea or guanidine hydrochloride, or a surfactant such as Triton X-100 ^™ . When the protein is secreted into the culture solution, after completion of the culture, the cells or cells and the supernatant are separated by a method known per se, and the supernatant is collected.
Purification of the protein contained in the thus obtained culture supernatant or extract can be performed by appropriately combining per se known separation and purification methods. These known separation and purification methods include mainly molecular weights such as methods utilizing solubility such as salting out and solvent precipitation, dialysis, ultrafiltration, gel filtration, and SDS-polyacrylamide gel electrophoresis. Using difference in charge, method using difference in charge such as ion exchange chromatography, method using specific affinity such as affinity chromatography, and difference in hydrophobicity such as reverse phase high performance liquid chromatography A method using a difference in isoelectric point, such as a method or isoelectric focusing method, is used.

When the protein thus obtained is obtained in a free form, it can be converted into a salt by a method known per se or a method analogous thereto, and conversely, when obtained as a salt, a method known per se or a method analogous thereto Can be converted to the free form or other salts.
The protein produced by the recombinant can be arbitrarily modified or the polypeptide can be partially removed by allowing an appropriate protein modifying enzyme to act before or after purification. Examples of the protein modifying enzyme include trypsin, chymotrypsin, arginyl endopeptidase, protein kinase, glycosidase and the like.
The presence of the protein of the present invention thus produced can be measured by enzyme immunoassay or Western blotting using a specific antibody.

The antibody to the protein or partial peptide or salt thereof used in the present invention may be either a polyclonal antibody or a monoclonal antibody as long as it can recognize the protein or partial peptide or salt thereof used in the present invention.
An antibody against the protein or partial peptide used in the present invention or a salt thereof (hereinafter, in the description of the antibody, these may be simply abbreviated as the protein of the present invention) is known per se using the protein of the present invention as an antigen The antibody or antiserum can be produced according to the production method.
[Production of monoclonal antibodies]
(A) Production of Monoclonal Antibody-Producing Cells The protein of the present invention is administered to a warm-blooded animal by itself, together with a carrier and a diluent, at a site where antibody production can be achieved. Complete Freund's adjuvant or incomplete Freund's adjuvant may be administered in order to enhance antibody production ability upon administration. The administration is usually performed once every 2 to 6 weeks, about 2 to 10 times in total. Examples of the warm-blooded animal used include monkeys, rabbits, dogs, guinea pigs, mice, rats, sheep, goats and chickens, and mice and rats are preferably used.
When producing monoclonal antibody-producing cells, select a warm-blooded animal immunized with an antigen, for example, an individual with a confirmed antibody titer from a mouse, and collect spleen or lymph nodes 2 to 5 days after the final immunization. Monoclonal antibody-producing hybridomas can be prepared by fusing the antibody-producing cells to be fused with allogeneic or xenogeneic myeloma cells. The antibody titer in the antiserum can be measured, for example, by reacting the labeled protein described below with the antiserum and then measuring the activity of the labeling agent bound to the antibody. The fusion operation can be carried out according to a known method, for example, the method of Kohler and Milstein [Nature, 256, 495 (1975)]. Examples of the fusion promoter include polyethylene glycol (PEG) and Sendai virus. Preferably, PEG is used.

Examples of myeloma cells include warm-blooded animal myeloma cells such as NS-1, P3U1, SP2 / 0, AP-1, and P3U1 is preferably used. The preferred ratio between the number of antibody-producing cells (spleen cells) and the number of myeloma cells used is about 1: 1 to 20: 1, and PEG (preferably PEG 1000 to PEG 6000) is added at a concentration of about 10 to 80%. Cell fusion can be efficiently carried out by incubating at 20 to 40 ° C., preferably 30 to 37 ° C. for 1 to 10 minutes.
Various methods can be used to screen for monoclonal antibody-producing hybridomas. For example, the hybridoma culture supernatant is added to a solid phase (eg, microplate) on which a protein antigen is adsorbed directly or together with a carrier, and then a radioactive substance or An anti-immunoglobulin antibody labeled with an enzyme or the like (when the cell used for cell fusion is a mouse, an anti-mouse immunoglobulin antibody is used) or protein A, and a method for detecting a monoclonal antibody bound to a solid phase; Examples include a method in which a hybridoma culture supernatant is added to a solid phase on which a globulin antibody or protein A is adsorbed, a protein labeled with a radioactive substance or an enzyme is added, and a monoclonal antibody bound to the solid phase is detected.
The selection of the monoclonal antibody can be performed according to a method known per se or a method analogous thereto. Usually, it can be performed in a medium for animal cells supplemented with HAT (hypoxanthine, aminopterin, thymidine). As the selection and breeding medium, any medium may be used as long as it can grow a hybridoma. For example, RPMI 1640 medium containing 1-20%, preferably 10-20% fetal bovine serum, GIT medium (Wako Pure Chemical Industries, Ltd.) containing 1-10% fetal bovine serum, or serum-free for hybridoma culture A culture medium (SFM-101, Nissui Pharmaceutical Co., Ltd.) etc. can be used. The culture temperature is usually 20 to 40 ° C, preferably about 37 ° C. The culture time is usually 5 days to 3 weeks, preferably 1 to 2 weeks. Culturing can usually be performed under 5% carbon dioxide gas. The antibody titer of the hybridoma culture supernatant can be measured in the same manner as the antibody titer in the above antiserum.

(B) Purification of monoclonal antibody Separation and purification of the monoclonal antibody can be performed by a method known per se, for example, an immunoglobulin separation and purification method [eg, salting out method, alcohol precipitation method, isoelectric point precipitation method, electrophoresis method, ion exchange method. Absorption / desorption method by body (eg, DEAE), ultracentrifugation method, gel filtration method, antigen-binding solid phase, or an antibody-active adsorbent such as protein A or protein G. Purification method].
[Preparation of polyclonal antibody]
The polyclonal antibody of the present invention can be produced according to a method known per se or a method analogous thereto. For example, an immune antigen (protein antigen) itself or a complex of it and a carrier protein is prepared, and a warm-blooded animal is immunized in the same manner as the monoclonal antibody production method described above, and the antibody against the protein of the present invention is contained from the immunized animal. It can be produced by collecting the product and performing separation and purification of the antibody.
Regarding the complex of immunizing antigen and carrier protein used to immunize warm-blooded animals, the type of carrier protein and the mixing ratio of carrier and hapten are effective for antibodies against hapten immunized by cross-linking to carrier. As long as it is possible, any substance may be cross-linked at any ratio. For example, bovine serum albumin, bovine thyroglobulin, hemocyanin and the like are about 0.1 to 20, preferably about 1 with respect to hapten 1 by weight ratio. A method of coupling at a rate of ˜5 is used.
Various coupling agents can be used for coupling of the hapten and the carrier, but active ester reagents containing glutaraldehyde, carbodiimide, maleimide active ester, thiol group, and dithiobilidyl group are used.
The condensation product is administered to warm-blooded animals at the site where antibody production is possible, or with a carrier and a diluent. Complete Freund's adjuvant or incomplete Freund's adjuvant may be administered in order to enhance antibody production ability upon administration. The administration is usually performed once every about 2 to 6 weeks and about 3 to 10 times in total.
Polyclonal antibodies can be collected from blood, ascites, etc., preferably from blood of warm-blooded animals immunized by the above method.
The polyclonal antibody titer in the antiserum can be measured in the same manner as the antibody titer in the antiserum described above. Separation and purification of the polyclonal antibody can be performed according to the same immunoglobulin separation and purification method as the above-described monoclonal antibody separation and purification.

In the base sequence of a polynucleotide (preferably DNA) encoding a protein or partial peptide used in the present invention (hereinafter, in the description of an antisense polynucleotide, these DNAs may be abbreviated as the DNA of the present invention). The antisense polynucleotide having a complementary or substantially complementary nucleotide sequence or a part thereof is a nucleotide sequence complementary to or substantially complementary to the nucleotide sequence of the DNA of the present invention. Any antisense polynucleotide may be used as long as it has a portion and has an action capable of suppressing the expression of the DNA, but antisense DNA is preferred.
The base sequence substantially complementary to the DNA of the present invention is, for example, about 70 of the total base sequence or partial base sequence of the base sequence complementary to the DNA of the present invention (that is, the complementary strand of the DNA of the present invention). % Or more, preferably about 80% or more, more preferably about 90% or more, and most preferably about 95% or more of a base sequence having homology. In particular, among the entire base sequences of the complementary strand of the DNA of the present invention, (i) in the case of an antisense polynucleotide directed to translation inhibition, the base sequence of the portion encoding the N-terminal site of the protein of the present invention (for example, the start An antisense polynucleotide having a homology of about 70% or more, preferably about 80% or more, more preferably about 90% or more, and most preferably about 95% or more with a complementary strand (such as a base sequence near a codon) ii) In the case of an antisense polynucleotide directed to RNA degradation by RNase H, it is about 70% or more, preferably about 80% or more, more preferably about 90% with the complementary strand of the entire base sequence of the DNA of the present invention including introns. As mentioned above, antisense polynucleotides having a homology of about 95% or more are most preferable.
Specifically, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, A nucleotide sequence complementary to or substantially complementary to the nucleotide sequence of DNA containing the nucleotide sequence represented by SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26 or SEQ ID NO: 28 Or an antisense polynucleotide having a portion thereof, preferably, for example, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26 or complementary to the base sequence of DNA containing the base sequence represented by SEQ ID NO: 28 An antisense polynucleotide having a base sequence or a part thereof (more preferably, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, complementary to the base sequence of DNA containing the base sequence represented by SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26 or SEQ ID NO: 28 An antisense polynucleotide having a simple nucleotide sequence or a part thereof.
The antisense polynucleotide is usually composed of about 10 to 40 bases, preferably about 15 to 30 bases.
In order to prevent degradation by a hydrolase such as nuclease, the phosphate residues (phosphates) of each nucleotide constituting the antisense DNA are changed to chemically modified phosphate residues such as phosphorothioate, methylphosphonate and phosphorodithionate. May be substituted. In addition, the sugar (deoxyribose) of each nucleotide may be substituted with a chemically modified sugar structure such as 2′-O-methylation, and the base portion (pyrimidine, purine) is also chemically modified. Any one may be used as long as it hybridizes to DNA containing the base sequence represented by SEQ ID NO: 2, SEQ ID NO: 6, SEQ ID NO: 8 or SEQ ID NO: 10. These antisense polynucleotides can be produced using a known DNA synthesizer.

According to the present invention, an antisense polynucleotide (nucleic acid) corresponding to the gene capable of inhibiting the replication or expression of the protein gene of the present invention is cloned, or the base of DNA encoding the determined protein is determined. Design and synthesis based on sequence information. Such antisense polynucleotides can hybridize with the RNA of the protein gene of the present invention, inhibit the synthesis or function of the RNA, or through interaction with the protein-related RNA of the present invention. The expression of the protein gene of the present invention can be regulated and controlled. The polynucleotide complementary to the selected sequence of the protein-related RNA of the present invention and the polynucleotide capable of specifically hybridizing with the protein-related RNA of the present invention are the protein gene of the present invention in vivo and in vitro. It is useful for regulating / controlling the expression of and for the treatment or diagnosis of diseases.
The term “corresponding” means homologous to or complementary to a specific sequence of nucleotides, base sequences or nucleic acids including genes. “Corresponding” between a nucleotide, base sequence or nucleic acid and protein usually refers to the amino acid of the protein (instructed) derived from the nucleotide (nucleic acid) sequence or its complement. 5 'end hairpin loop of protein gene, 5' end 6-base pair repeat, 5 'end untranslated region, polypeptide translation start codon, protein coding region, ORF translation stop codon, 3' end untranslated region, 3 ' An end palindromic region or a 3 ′ end hairpin loop or the like can be selected as a preferred region of interest, but any region within a protein gene can be selected as a target.
Regarding the relationship between the target nucleic acid and a polynucleotide complementary to at least a part of the target region, if the target nucleic acid can hybridize to the target region, the target nucleic acid It can be said that it is “antisense”. Antisense polynucleotides include polynucleotides containing 2-deoxy-D-ribose, polynucleotides containing D-ribose, other types of polynucleotides that are N-glycosides of purine or pyrimidine bases, non-polynucleotides, Other polymers with a nucleotide backbone (eg, commercially available protein nucleic acids and synthetic sequence-specific nucleic acid polymers) or other polymers containing special linkages (provided that the polymer is a base such as found in DNA or RNA) And a nucleotide having a configuration allowing the attachment of a pairing or base). They may be double-stranded DNA, single-stranded DNA, double-stranded RNA, single-stranded RNA, DNA: RNA hybrids, unmodified polynucleotides (or unmodified oligonucleotides), known modifications Additions, such as those with labels known in the art, capped, methylated, one or more natural nucleotides replaced with analogs, intramolecular nucleotide modifications Such as those having uncharged bonds (eg methylphosphonates, phosphotriesters, phosphoramidates, carbamates, etc.), charged or sulfur-containing bonds (eg phosphorothioates, phosphorodithioates, etc.) Things such as proteins (eg, nucleases, nuclease inhibitors, toxins, antibodies, Nalpeptides, poly-L-lysine, etc.) and sugars (eg, monosaccharides), etc., side chain groups, intercurrent compounds (eg, acridine, psoralen, etc.), chelate compounds (eg, , Metals, radioactive metals, boron, oxidizing metals, etc.), alkylating agents, and modified bonds (eg, alpha anomeric nucleic acids) Also good. Here, the “nucleoside”, “nucleotide” and “nucleic acid” may include not only purine and pyrimidine bases but also those having other modified heterocyclic bases. Such modifications may include methylated purines and pyrimidines, acylated purines and pyrimidines, or other heterocycles. Modified nucleotides and modified nucleotides may also be modified at the sugar moiety, eg, one or more hydroxyl groups are replaced by halogens, aliphatic groups, etc., or functional groups such as ethers, amines, etc. It may have been converted.
The antisense polynucleotide of the present invention is RNA, DNA or a modified nucleic acid (RNA, DNA). Specific examples of the modified nucleic acids include nucleic acid sulfur derivatives, thiophosphate derivatives, polynucleoside amides and oligonucleoside amides that are resistant to degradation. The antisense polynucleotide of the present invention can be designed, for example, as follows. That is, to make the antisense polynucleotide in the cell more stable, to increase the cell permeability of the antisense polynucleotide, to increase the affinity for the target sense strand, and to reduce the toxicity In some cases, the antisense polynucleotide is less toxic. Many such modifications have been reported in, for example, Pharm Tech Japan, 8, 247 or 395, 1992, Antisense Research and Applications, CRC Press, 1993, and the like.
The antisense polynucleotides of the present invention may be altered, contain modified sugars, bases, linkages, be provided in special forms such as liposomes, microspheres, or applied by gene therapy. Can be provided in an added form. In this way, the additional form can be used as a polycationic substance such as polylysine that works to neutralize the charge of the phosphate group skeleton, or a lipid that enhances the interaction with the cell membrane or increases the uptake of nucleic acids ( Examples include hydrophobic ones such as phospholipid and cholesterol. Preferred lipids for addition include cholesterol and derivatives thereof (eg, cholesteryl chloroformate, cholic acid, etc.). Such can be attached to the 3 'or 5' end of the nucleic acid and can be attached via a base, sugar, intramolecular nucleoside bond. Examples of the other group include a cap group specifically arranged at the 3 ′ end or 5 ′ end of a nucleic acid, which prevents degradation by a nuclease such as exonuclease or RNase. Such capping groups include, but are not limited to, hydroxyl protecting groups known in the art, including glycols such as polyethylene glycol and tetraethylene glycol.
The inhibitory activity of the antisense polynucleotide can be examined using the transformant of the present invention, the in vivo or in vitro gene expression system of the present invention, or the in vivo or in vitro translation system of the protein of the present invention. .

  The protein or partial peptide of the present invention or a salt thereof (hereinafter sometimes abbreviated as the protein of the present invention), a polynucleotide (eg, DNA) encoding the protein or partial peptide of the present invention (hereinafter referred to as the present invention). An antibody against the protein or partial peptide of the present invention or a salt thereof (hereinafter sometimes abbreviated as the antibody of the present invention), and the polynucleotide (eg, DNA) of the present invention. The use of a sense polynucleotide (hereinafter sometimes abbreviated as the antisense polynucleotide of the present invention) and the like will be described.

Since protein A of the present invention is highly expressed in a site-specific manner with the onset of colorectal cancer, it is useful as a marker for early diagnosis of colorectal cancer, determination of the severity of symptoms, and prediction of disease progression. . A compound which inhibits the activity of the protein A of the present invention or a salt thereof, an antibody against the protein A of the present invention, an antisense polynucleotide of the gene encoding the protein A of the present invention, an siRNA of the gene encoding the protein A of the present invention, shRNA can be used, for example, as a preventive / therapeutic agent for colon cancer, an apoptosis promoter for colon cancer cells, a growth inhibitor for colon cancer cells, a metastasis / recurrence inhibitor for colon cancer, and the like.
Protein B of the present invention is useful as a marker for early diagnosis of colorectal cancer, determination of symptom severity, and prediction of disease progression because expression decreases in a site-specific manner as colon cancer develops. The compound or its salt that promotes the activity of protein B of the present invention includes, for example, a preventive / therapeutic agent for colon cancer, an apoptosis promoter for colon cancer cells, a growth inhibitor for colon cancer cells, and a metastasis / recurrence inhibitor for colon cancer. It can be used as an agent.

(1) Screening of drug candidate compounds for diseases Protein A of the present invention has anti-apoptotic activity, cancer cell proliferation promoting activity and the like. Therefore, the compound or salt thereof that inhibits the activity of protein A of the present invention includes, for example, a low-toxic preventive / therapeutic agent for colorectal cancer, a cancer cell apoptosis promoter, a cancer cell growth inhibitor, a colorectal cancer metastasis / It can be used as a recurrence inhibitor. Therefore, the protein A of the present invention is useful as a reagent for screening a compound or a salt thereof that inhibits the activity of the protein A of the present invention.
On the other hand, protein B of the present invention has apoptotic activity, cancer cell growth inhibitory activity and the like. Therefore, the compound or its salt that promotes the activity of protein B of the present invention is, for example, a low-toxic preventive / therapeutic agent for colon cancer, an apoptosis promoter for cancer cells, a growth inhibitor for cancer cells, a metastasis / It can be used as a recurrence inhibitor. Therefore, the protein B of the present invention is useful as a reagent for screening a compound or a salt thereof that promotes the activity of the protein B of the present invention.
That is, the present invention has the activity of the protein of the present invention characterized by using the protein A of the present invention (eg, anti-apoptotic activity, cancer cell proliferation promoting activity, protease activity of CTSE, oxidase activity of NOX1). Provided are screening methods for inhibiting compounds or salts thereof. The present invention also provides a method for screening a compound or a salt thereof that promotes the activity (eg, apoptosis activity, cancer cell growth inhibitory activity, etc.) of the protein of the present invention, characterized by using the protein B of the present invention. .

Specific examples include the following methods.
A) (i) when cells having the ability to produce the protein of the present invention are cultured, and (ii) ability to produce the protein of the present invention (protein A or B of the present invention) in the presence of the test compound. Anti-apoptotic activity or apoptotic activity in the case of culturing cells having the same, respectively, and screening for a compound that inhibits the activity of protein A of the present invention or a salt thereof, or a compound that promotes the activity of protein B of the present invention .
Anti-apoptotic activity or apoptotic activity is measured according to a known method using Cell death detection ELISA (Roche), Apo-ONE Homogeneous Caspase-3 / 7 Assay (Promega) or the like.
B) (i) when cells having the ability to produce the protein of the present invention are cultured, and (ii) when cells having the ability to produce the protein of the present invention are cultured in the presence of the test compound, The cancer cell growth promoting activity or the cancer cell growth inhibiting activity is measured, respectively, and a compound that inhibits the activity of protein A of the present invention or a salt thereof, or a compound that promotes the activity of protein B of the present invention is screened.
Measurement of cancer cell growth promoting activity or cancer cell growth inhibitory activity is performed according to a known method using CellTiter-Glo (Promega), _BrdU ELISA (Roche) or the like.
C) a compound having the action of inhibiting or promoting the activity of the protein of the present invention by comparing (i) the protease activity of the protein of the present invention and (ii) the protease activity of the mixture of the protein of the present invention and the test compound, or a compound thereof Screen for salt.
This reaction is performed in an appropriate buffer. The protease activity is measured according to a known method, for example, the method described in Biological Chemistry, 386, 299-305, 2005 or a method analogous thereto.
D) A compound having an action of inhibiting or promoting the activity of the protein of the present invention by comparing (i) the oxidase activity of the protein of the present invention and (ii) the oxidase activity of the mixture of the protein of the present invention and the test compound Or screen its salts.
This reaction is performed in an appropriate buffer. The oxidase activity is measured according to a known method, for example, the method described in Biological & Pharmaceutical Bulletin, 27, pp. 147-155, 2004) or a method analogous thereto.
The above protein of the present invention is preferably produced by culturing a transformant containing DNA encoding the protein of the present invention. Furthermore, you may make it react similarly using the cell which can express the protein of this invention.
As the cell having the ability to produce the protein of the present invention, for example, a host (transformant) transformed with a vector containing the above-described DNA encoding the protein of the present invention is used. As the host, animal cells such as COS7 cells, CHO cells, HEK293 cells are preferably used. For the screening, for example, a transformant in which the protein of the present invention is expressed by culturing by the aforementioned method is preferably used. The method for culturing cells capable of expressing the protein of the present invention is the same as the method for culturing the transformant of the present invention described above.
Examples of the test compound include peptides, proteins, antibodies, non-peptide compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts, plasma and the like.
For example, a test compound that reduces (or increases) the activity in the above-mentioned case (ii) by about 20% or more, preferably 30% or more, more preferably about 50% or more compared to the above-mentioned case (i). It can be selected as a compound that inhibits (or promotes) the activity of the protein of the invention.

The compound having the activity of inhibiting the activity of protein A of the present invention is a safe and low-toxic drug for suppressing the physiological activity of the protein of the present invention, such as a preventive / therapeutic agent for colon cancer, apoptosis of colon cancer cells. It is useful as a promoter, a colon cancer cell growth inhibitor, a colon cancer metastasis / recurrence inhibitor, and the like.
The compound having the activity of promoting the activity of the protein B of the present invention is a safe and low-toxic drug for promoting the physiological activity of the protein B of the present invention, such as a preventive / therapeutic agent for colorectal cancer, colon cancer cells. It is useful as an apoptosis promoter, a colon cancer cell growth inhibitor, a colon cancer metastasis / relapse inhibitor, and the like. The compound or salt thereof obtained using the screening method or screening kit of the present invention is, for example, a peptide, protein, antibody, non-peptide compound, synthetic compound, fermentation product, cell extract, plant extract, animal tissue. It is a compound selected from extracts, plasma and the like. As the salt of the compound, the same salts as those of the peptide of the present invention described above can be used.

Furthermore, the gene encoding the protein A of the present invention produces a protein having anti-apoptotic activity, promotion of cancer cell growth, protease activity of CTSE, oxidase activity of NOX1, and the like. The compound that inhibits expression or a salt thereof can also be used as, for example, a preventive / therapeutic agent for colon cancer, an apoptosis promoter for colon cancer cells, a growth inhibitor for colon cancer cells, a metastasis / recurrence inhibitor for colon cancer, and the like. it can. Therefore, the DNA encoding the protein A of the present invention is useful as a reagent for screening for compounds or salts thereof that inhibit the expression of the gene encoding the protein of the present invention.
Furthermore, since the gene encoding protein B of the present invention produces a protein having apoptosis activity, cancer cell growth inhibitory activity, etc., a compound or a salt thereof that promotes the expression of the gene encoding protein B of the present invention, For example, it can be used as a prophylactic / therapeutic agent for colorectal cancer, a colorectal cancer cell apoptosis promoter, a colorectal cancer cell growth inhibitor, a colorectal cancer metastasis / recurrence inhibitor, and the like. Therefore, the DNA encoding the protein B of the present invention is useful as a reagent for screening a compound or a salt thereof that promotes the expression of the gene encoding the protein of the present invention.
As a screening method, (iii) when cells having the ability to produce the protein of the present invention were cultured, and (iv) cells having the ability to produce the protein used in the present invention were cultured in the presence of the test compound. There is a screening method characterized by comparison with the case.
In the above method, the expression level of the gene (specifically, the amount of the protein of the present invention or the amount of mRNA encoding the protein) in the cases of (iii) and (iv) is measured and compared.
Examples of the cell having the ability to produce the test compound and the protein of the present invention include those described above.
The amount of protein is measured by a known method, for example, by using an antibody that recognizes the protein of the present invention, the protein present in a cell extract or the like according to a method such as Western analysis, ELISA method or the like or a method equivalent thereto. can do.
Measurement of the amount of mRNA is a known method, for example, as a probe, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16 , SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26 or SEQ ID NO: 28, or Northern hybridization using a nucleic acid containing a part thereof, or SEQ ID NO: as a primer : 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, It can be measured according to a PCR method using a nucleic acid containing SEQ ID NO: 24, SEQ ID NO: 26 or SEQ ID NO: 28 or a part thereof, or a method analogous thereto. That.
For example, in the case of the gene encoding the protein A of the present invention, the gene expression level in the case (iv) is about 20% or more, preferably 30% or more, more preferably compared to the case (iii). A test compound that inhibits about 50% or more can be selected as a compound that suppresses (inhibits) the expression of the gene encoding protein A of the present invention. In the case of the gene encoding the protein B of the present invention, the gene expression level in the case (iv) is about 20% or more, preferably 30% or more, more preferably compared to the case (iii). A test compound that promotes about 50% or more can be selected as a compound that promotes the expression of the gene encoding protein B of the present invention.

The screening kit of the present invention contains cells having the ability to produce the protein or partial peptide used in the present invention or a salt thereof, or the protein or partial peptide used in the present invention.
The compound obtained by using the screening method or the screening kit of the present invention or a salt thereof is the above-described test compound, for example, peptide, protein, non-peptide compound, synthetic compound, fermentation product, cell extract, plant extract. (A) the activity of protein A of the present invention (eg, anti-apoptotic activity, cancer cell proliferation promoting activity, protease activity of CTSE, oxidation of NOX1) A compound or a salt thereof that inhibits the enzyme activity, etc., inhibits the expression of the protein A gene, or inhibits the expression of the protein A, (b) the activity of the protein B of the present invention (eg, apoptotic activity, cancer cell growth inhibitory activity) Etc.), the expression of the protein B gene, or the expression of the protein B or a salt thereof It is.
As the salt of the compound, the same salt as the protein salt of the present invention described above can be used.
A compound or salt thereof that inhibits the activity of protein A of the present invention, a compound or salt thereof that inhibits the expression of the gene encoding protein A of the present invention, a compound or salt thereof that inhibits the expression of protein A of the present invention, It has low toxicity and is useful, for example, as a medicament for preventing or treating colorectal cancer, colorectal cancer cell apoptosis promoter, colorectal cancer cell growth inhibitor, colorectal cancer metastasis / recurrence inhibitor, and the like.
A compound or a salt thereof that promotes the activity of the protein B of the present invention, a compound or a salt thereof that promotes the expression of the gene encoding the protein B of the present invention, a compound or a salt thereof that promotes the expression of the protein B of the present invention, It has low toxicity and is useful, for example, as a medicament for preventing or treating colorectal cancer, colorectal cancer cell apoptosis promoter, colorectal cancer cell growth inhibitor, colorectal cancer metastasis / recurrence inhibitor, and the like.
When the compound or salt thereof obtained using the screening method or screening kit of the present invention is used as the above-mentioned prophylactic / therapeutic agent, it can be formulated according to conventional means.

For example, compositions for oral administration include solid or liquid dosage forms, specifically tablets (including sugar-coated tablets and film-coated tablets), pills, granules, powders, capsules (including soft capsules). Syrup, emulsion, suspension and the like. Such a composition is produced by a method known per se, and contains a carrier, diluent or excipient usually used in the pharmaceutical field. For example, lactose, starch, sucrose, magnesium stearate and the like are used as carriers and excipients for tablets.
As a composition for parenteral administration, for example, injections, suppositories and the like are used, and injections are intravenous injections, subcutaneous injections, intradermal injections, intramuscular injections, intravenous injections, intraarticular injections. Includes dosage forms such as agents. Such an injection is prepared according to a method known per se, for example, by dissolving, suspending or emulsifying the above antibody or a salt thereof in a sterile aqueous or oily liquid usually used for injection. As an aqueous solution for injection, for example, isotonic solutions containing physiological saline, glucose and other adjuvants are used, and appropriate solubilizers such as alcohol (eg, ethanol), polyalcohol (eg, Propylene glycol, polyethylene glycol), nonionic surfactants (eg, polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)) and the like may be used in combination. As the oily liquid, for example, sesame oil, soybean oil and the like are used, and benzyl benzoate, benzyl alcohol and the like may be used in combination as a solubilizing agent. The prepared injection solution is usually filled in a suitable ampoule.
A suppository used for rectal administration is prepared by mixing the above-described antibody or a salt thereof with a normal suppository base.

The above oral or parenteral pharmaceutical compositions are conveniently prepared in dosage unit form to suit the dosage of the active ingredient. Examples of the dosage form of such a dosage unit include tablets, pills, capsules, injections (ampoules), suppositories, etc., and usually 5 to 500 mg, especially 5 to 100 mg for injections, Other dosage forms preferably contain 10 to 250 mg of the above antibody.
Each of the above-described compositions may contain other active ingredients as long as they do not cause an unfavorable interaction by blending with the antibody.
Since the preparation thus obtained is safe and has low toxicity, for example, humans or warm-blooded animals (eg, mice, rats, rabbits, sheep, pigs, cows, horses, birds, cats, dogs, monkeys, chimpanzees, etc. ) Orally or parenterally.
The dose of the compound or a salt thereof varies depending on its action, target disease, administration subject, administration route, etc. For example, a compound or salt thereof that inhibits the activity of the protein of the present invention for the purpose of treating colorectal cancer, for example. In general, in an adult (with a body weight of 60 kg), about 0.1 to 100 mg, preferably about 1.0 to 50 mg, more preferably about 1.0 Administer ~ 20 mg. When administered parenterally, the single dose of the compound or a salt thereof varies depending on the administration subject, target disease, etc., for example, a compound that inhibits the activity of the protein of the present invention for the purpose of treating colon cancer Or when the salt is administered to an adult (usually 60 kg body weight) in the form of an injection, the compound or salt thereof is about 0.01 to 30 mg, preferably about 0.1 to 20 mg, more preferably per day. It is convenient to administer about 0.1 to 10 mg to a cancer lesion site by injection. In the case of other animals, an amount converted per 60 kg body weight can be administered.
In addition, the above compound is an existing anticancer agent [eg, alkylating agent (eg, cyclophosphamide, busulfan, melphalan, etc.), antimetabolite (eg, cytarabine, 6-mercaptopurine, methotrexate, 5-fluorouracil, etc.) , Anticancer antibiotics (eg, daunorubicin, doxorubicin, pirarubicin, mitoxantrone, idarubicin, mitomycin, adriamycin, etc.), plant-derived anticancer agents (eg, vincristine, vindesine, taxol, etoposide, etc.), enzyme preparations (eg, L- Asparaginase), corticosteroids (eg, prednisolone, prednisone, dexamethasone, cortisone acetate), estrogen (eg, estradiol, ethinylestradiol, phosfestol, chlorotrianicene), Estrogen drugs (eg, epithiostanol, mepithiostane, tamoxifen, clomiphene, etc.), luteinizing hormone drugs (eg, hydroxyprogesterone caproate, didogesterone, medroxyprogesterone, norethisterone, norethindrone, etc.), LHRH derivatives (eg, leuprorelin acetate, etc.) ), Cisplatin, carboplatin, transretinoic acid, interferon α, imatinib, etc.]. In this case, the administration time is not limited, and these may be administered simultaneously to the administration subject or may be administered with a time difference. The dose can be appropriately selected based on the clinically used dose. Moreover, the compounding ratio of the said compound and anticancer agent can be suitably selected according to an administration subject, administration route, an object disease, a symptom, a combination, etc.

(2) Quantification of the protein of the present invention, a partial peptide thereof or a salt thereof The antibody against the protein of the present invention (hereinafter sometimes abbreviated as the antibody of the present invention) can specifically recognize the protein of the present invention. Therefore, it can be used for quantification of the protein of the present invention in a test solution, particularly quantification by sandwich immunoassay.
That is, the present invention
(I) The antibody of the present invention is competitively reacted with a test solution and the labeled protein of the present invention, and the ratio of the labeled protein of the present invention bound to the antibody is measured. And (ii) the test solution and the antibody of the present invention insolubilized on the carrier and the labeled another antibody of the present invention simultaneously or successively. Provided is a method for quantifying the protein of the present invention in a test solution, characterized by measuring the activity of a labeling agent on an insolubilized carrier after the reaction.
In the above quantification method (ii), it is desirable that one antibody is an antibody that recognizes the N-terminal part of the protein of the present invention and the other antibody is an antibody that reacts with the C-terminal part of the protein of the present invention.
In addition to quantification of the protein of the present invention using a monoclonal antibody against the protein of the present invention (hereinafter sometimes referred to as the monoclonal antibody of the present invention), detection by tissue staining or the like can also be performed. For these purposes, the antibody molecule itself may be used, or F (ab ′) ₂ , Fab ′ or Fab fraction of the antibody molecule may be used.
The method for quantifying the protein of the present invention using the antibody of the present invention is not particularly limited, and an antibody, antigen or antibody-antigen complex corresponding to the amount of antigen (for example, the amount of protein) in the solution to be measured is not limited. Any measurement method may be used as long as it is a measurement method in which the amount is detected by chemical or physical means and calculated from a standard curve prepared using a standard solution containing a known amount of antigen. For example, nephrometry, competition method, immunometric method and sandwich method are preferably used, but the sandwich method described later is particularly preferable in view of sensitivity and specificity.
Examples of the labeling agent used in the measurement method using the labeling substance include radioisotopes (eg,
^[125 I], ^[131 I], ^[3 H], ^[14 C], ^[32 P], ^[33 P] and ^[35 S]), a fluorescent substance [
Examples, cyanine fluorescent dyes (eg, Cy2, Cy3, Cy5, Cy5.5, Cy7 (manufactured by Amersham Biosciences)), fluorescamine, fluorescein isothiocyanate, NBD (7-nitrobenz-2-oxa-1, 3-diazol), enzymes (eg, β-galactosidase, β-glucosidase, alkaline phosphatase, peroxidase, malate dehydrogenase, etc.), luminescent substances (eg, luminol, luminol derivatives, luciferin, lucigenin, etc.), biotin Lanthanide elements are used. Furthermore, a biotin-avidin system can also be used for binding of an antibody or antigen to a labeling agent.

For insolubilization of an antigen or antibody, physical adsorption may be used, or a method using a chemical bond usually used to insolubilize or immobilize a protein or an enzyme may be used. Examples of the carrier include insoluble polysaccharides such as agarose, dextran, and cellulose, synthetic resins such as polystyrene, polyacrylamide, and silicon, or glass.
In the sandwich method, the test solution is reacted with the insolubilized monoclonal antibody of the present invention (primary reaction), and further labeled with another monoclonal antibody of the present invention (secondary reaction), and then on the insolubilized carrier. By measuring the activity of the labeling agent, the amount of the protein of the present invention in the test solution can be quantified. The primary reaction and the secondary reaction may be performed in the reverse order, or may be performed simultaneously or at different times. The labeling agent and the insolubilization method can be the same as those described above. In the immunoassay by the sandwich method, the antibody used for the solid phase antibody or the labeling antibody is not necessarily one type, and a mixture of two or more types of antibodies is used for the purpose of improving measurement sensitivity. May be.
In the method for measuring the protein of the present invention by the sandwich method of the present invention, the monoclonal antibody of the present invention used for the primary reaction and the secondary reaction is preferably an antibody having a different site to which the protein of the present invention binds. That is, the antibody used in the primary reaction and the secondary reaction, for example, when the antibody used in the secondary reaction recognizes the C-terminus of the protein of the present invention, the antibody used in the primary reaction is preferably For example, an antibody that recognizes the N end other than the C end is used.

The monoclonal antibody of the present invention can be used in measurement systems other than the sandwich method, for example, competitive method, immunometric method, nephrometry, and the like.
In the competition method, the antigen in the test solution and the labeled antigen are reacted competitively with the antibody, and then the unreacted labeled antigen (F) and the labeled antigen (B) bound to the antibody are separated. (B / F separation), the amount of labeling of either B or F is measured, and the amount of antigen in the test solution is quantified. In this reaction method, a soluble antibody is used as an antibody, B / F separation is performed using polyethylene glycol, a liquid phase method using a second antibody against the antibody, and a solid-phased antibody is used as the first antibody, or As the first antibody, a soluble method is used, and a solid phase method using a solid phase antibody as the second antibody is used.
In the immunometric method, the antigen in the test solution and the immobilized antigen are subjected to a competitive reaction with a certain amount of labeled antibody, and then the solid phase and the liquid phase are separated, or the antigen in the test solution is separated. And an excess amount of the labeled antibody are reacted, and then the solid phase antigen is added to bind the unreacted labeled antibody to the solid phase, and then the solid phase and the liquid phase are separated. Next, the labeled amount of any phase is measured to quantify the amount of antigen in the test solution.
In nephrometry, the amount of insoluble precipitate produced as a result of the antigen-antibody reaction in a gel or solution is measured. Laser nephrometry using laser scattering is preferably used even when the amount of antigen in the test solution is small and only a small amount of precipitate is obtained.

In applying these individual immunological measurement methods to the quantification method of the present invention, special conditions, operations and the like are not required to be set. What is necessary is just to construct | assemble the measurement system of the protein of this invention, adding the usual technical consideration of those skilled in the art to the usual conditions and operation methods in each method. For details of these general technical means, it is possible to refer to reviews, books and the like.
For example, Hiroshi Irie “Radioimmunoassay” (Kodansha, published in 1974), Hiroshi Irie “Sequel Radioimmunoassay” (published in Kodansha, 1979), “Enzyme Immunoassay” edited by Eiji Ishikawa et al. 53), "Enzyme Immunoassay" edited by Eiji Ishikawa et al. (2nd edition) (Medical Shoin, published in 1982), "Enzyme Immunoassay" edited by Eiji Ishikawa et al. (3rd edition) (Medical Shoin, Showa) 62), “Methods in ENZYMOLOGY” Vol. 70 (Immunochemical Techniques (Part A)), Id. Vol. 73 (Immunochemical Techniques (Part B)), Id. Vol. 74 (Immunochemical Techniques (Part C)), Id. 84 (Immunochemical Techniques (Part D: Selected Immunoassays)), ibid.Vol. 92 (Immunochemical Techniques (Part E: Monoclonal Antibodies and General Immunoassay Methods)), ibid. )) (From Academic Press ) And the like can be referred to.
As described above, the protein of the present invention can be quantified with high sensitivity by using the antibody of the present invention.
The antibody of the present invention can be used for detecting the protein of the present invention present in a subject such as a body fluid or tissue. In addition, for the production of an antibody column used for purifying the protein of the present invention, detection of the protein of the present invention in each fraction during purification, analysis of the behavior of the protein of the present invention in a test cell, etc. Can be used.

(3) Colorectal cancer diagnostic agent and diagnostic method DNA encoding the protein of the present invention (DNA of the present invention) can be used, for example, as a probe to allow humans or warm-blooded animals (eg, rats, mice, guinea pigs, (Abnormality of a DNA or mRNA encoding a protein of the present invention or a partial peptide thereof (gene abnormality) in a rabbit, bird, sheep, pig, cow, horse, cat, dog, monkey, chimpanzee, etc.) For example, it is useful as a genetic diagnostic agent for damaging, mutating or reducing expression of the DNA or mRNA, and increasing or overexpressing the DNA or mRNA.
The above gene diagnosis using the DNA of the present invention includes, for example, known Northern hybridization and PCR-SSCP method (Genomics, Vol. 5, pp. 874-879, 1989, Proceedings of the National Academy of Sciences of the United States). States of America, 86, 2766-2770, 1989).
For example, when an overexpression is detected by Northern hybridization or a DNA mutation is detected by the PCR-SSCP method, it can be diagnosed that there is a high possibility of colorectal cancer.

Specifically, LEFTB, FOXA2, S100P, CTSE, NOX1, CDH3, KLK10, DUOX2, S100A2, GABRP, MSLN, FAIM2, and TACSTD2 are upregulated in colon cancer tumor tissue, for example, Northern hybridization When mRNA that is a translation template for these proteins is detected or the expression level is recognized to be high, for example, there is a high possibility of having colorectal cancer, colorectal cancer, recurrence of colorectal cancer Can be diagnosed, determined, predicted or classified as having a high probability of metastasis, poor prognosis, high possibility of metastasis, high malignancy, and the like. In addition, when mRNA serving as a translation template for these proteins is not detected or its expression level is recognized to be low, for example, it is less likely to suffer from colorectal cancer, colorectal cancer, colon cancer Can be diagnosed, determined, predicted or classified as having a low probability of recurrence, good prognosis, low probability of metastasis, low grade of malignancy, etc.
On the other hand, since CEACAM1 has decreased expression in tumor tissue of colorectal cancer, for example, when mRNA serving as a translation template of these proteins is detected by Northern hybridization, or its expression level is recognized as high, For example, diagnosed as having a low likelihood of developing colorectal cancer, not having colorectal cancer, low likelihood of recurrence of colorectal cancer, good prognosis, low probability of metastasis, low malignancy, etc. Can be determined, predicted or classified. In addition, when mRNA serving as a translation template for these proteins is not detected or the expression level is recognized to be low, for example, it is highly probable to have colorectal cancer, colorectal cancer, colorectal cancer Can be diagnosed, determined, predicted, or classified as having a high probability of recurrence, poor prognosis, high possibility of metastasis, high malignancy, and the like.

  In addition, (i) mRNA serving as a translation template for LEFTB, mRNA serving as a translation template for FOXA2, mRNA serving as a translation template for S100P, mRNA serving as a translation template for CTSE, mRNA serving as a translation template for NOX1, and a translation template for CDH3 MRNA, KLK10 translation template mRNA, DUOX2 translation template mRNA, S100A2 translation template mRNA, GABRP translation template mRNA, MSLN translation template mRNA, FAIM2 translation template mRNA And / or (2) the presence / absence or level of expression of any two or more selected from mRNA serving as a translation template for TACSTD2 and / or (ii) the presence / absence or level of expression of mRNA serving as a translation template for CEACAM1 Analysis, for example, the possibility of developing colorectal cancer, the presence or absence of disease, the possibility of recurrence, the prognosis, the possibility of metastasis Height, diagnosis and level of malignancy, the determination may be predicted or classified. Known statistical analysis methods include, for example, (i) a method using Gene Spring (Silicon Genetics) Predict Parameter Value, and (ii) a weighted voting cross-validation algorithm (Science using leave-one-out cross-validation). 286, 531-537, 1999). The type and number of the DNA of the present invention used in the diagnosis, determination, prediction or classification method using such a statistical analysis method is not particularly limited, but usually 2 to 14 types, preferably 3 to 10 types. More preferably, 4 to 8 and particularly preferably 5 to 7 polynucleotides can be appropriately selected and used.

Similar to the above-described DNA of the present invention, the complementary DNA of the present invention, that is, the DNA containing the full-length complementary sequence of the base sequence of the mRNA used as the translation template of the protein used in the present invention or a partial base sequence thereof, for example, The protein used in the present invention in humans or warm-blooded animals (eg, rats, mice, guinea pigs, rabbits, birds, sheep, pigs, cows, horses, cats, dogs, monkeys, chimpanzees, etc.) Alternatively, since the presence / absence, expression level or abnormality (gene abnormality) of the polynucleotide (preferably DNA or mRNA) encoding the partial peptide can be detected, for example, the presence / absence or expression of the DNA or mRNA. Disease or condition related to the amount, or damage to the DNA or mRNA It is useful as a gene diagnostic agent, such as a mutation.
The complementary DNA of the present invention can be used to diagnose, determine, predict or predict the possibility of colorectal cancer, whether or not it is affected, the likelihood of recurrence, the prognosis, the possibility of metastasis, the degree of metastasis, and the like. When used for classification, it can be used in the same manner as the polynucleotide of the present invention described above.

Moreover, the DNA of the present invention, the complementary DNA of the present invention, and the like can be used for a microarray. A microarray (preferably a DNA microarray) can be prepared, used and analyzed by a known method or a method analogous thereto. Preparation, use, and analysis methods of microarrays are described in, for example, (i) DNA Microarrays: A Practical Approach (edited by M. Schena, Oxford University Press, London, 1999), (ii) Schena, M. et al., Proc. Natl. Acad. Sci. USA, 93, 10614-10619, 1996, (iii) Heller, RA et al., Proc. Natl. Acad. Sci. USA, 94, 2150-2155, 1997, etc. ing.
Furthermore, such a DNA microarray of the present invention can also be used in a diagnostic kit for diagnosing a disease or condition related to the presence or absence or expression level of a polynucleotide encoding the protein of the present invention.

In addition, since the antibody used in the present invention can detect or quantify the protein used in the present invention as described above, it is used for diagnosis of diseases or conditions related to the presence or absence of the expression of the protein of the present invention or the expression level. be able to.
That is, the present invention provides (i) a labeled present invention that binds to the antibody by competitively reacting an antibody used in the present invention with a test solution and a labeled protein used in the present invention. (Ii) the antibody used in the present invention insolubilized on the test solution and the carrier and the labeling method, characterized by measuring the ratio of the protein used in Wherein the activity of the labeling agent on the insolubilized carrier is measured after reacting with another antibody used in the present invention simultaneously or continuously, Provided are a quantification method, and (iii) a diagnostic method and a diagnostic agent for a disease or condition related to the presence or absence or expression level of the protein of the present invention using the quantification method of (i) or (ii) above. In the quantification method (ii) above, one antibody is an antibody that recognizes the N-terminal part of the protein used in the present invention, and the other antibody is an antibody that reacts with the C-terminal part of the protein used in the present invention. It is desirable.

The antibody used in the present invention can also be used in an antibody microarray, that is, an “antibody chip” formed of an array of a plurality of or one antibody immobilized on a substrate. Such an antibody microarray can be prepared, used and analyzed by a known method or a method analogous thereto. Preparation and use of such antibody microarrays are described in, for example, (i) MacBeath and Schriber, Science, 289, 1760-1763, 2000, (ii) Haab, BB et al., Genome Biology, 2 (2), It is described in 2001.
Furthermore, such an antibody microarray of the present invention can also be used in a diagnostic kit for diagnosing a disease or condition related to the presence or absence or expression level of the protein of the present invention.

Since the expression of LEFTB, FOXA2, S100P, CTSE, NOX1, CDH3, KLK10, DUOX2, S100A2, GABRP, MSLN, FAIM2 and TACSTD2 is increased in tumor tissues of colorectal cancer, these are used with the antibodies used in the present invention. By quantifying the protein concentration of these proteins, if these proteins are detected or the expression level is recognized to be high, for example, there is a high possibility of suffering from colon cancer, colon cancer, colon It can be diagnosed, determined, predicted or classified as having a high possibility of cancer recurrence, poor prognosis, high possibility of metastasis, high malignancy, and the like. In addition, when these proteins are not detected or are found to be low in expression level, for example, there is a low possibility of developing colorectal cancer, no colorectal cancer, or the possibility of recurrence of colorectal cancer. Diagnose, determine, predict or classify as low, good prognosis, low chance of metastasis, low malignancy, etc.
On the other hand, CEACAM1 has decreased expression in the tumor tissue of colorectal cancer, so by quantifying the concentration of these proteins using the antibody used in the present invention, these proteins can be detected and their expression levels Is found to be high, for example, low likelihood of developing colorectal cancer, no colorectal cancer, low likelihood of recurrence of colorectal cancer, good prognosis, low probability of metastasis It can be diagnosed, judged, predicted or classified as having a low malignancy.
In addition, (i) presence or absence of expression or expression level of any two or more selected from LEFTB, FOXA2, S100P, CTSE, NOX1, CDH3, KLK10, DUOX2, S100A2, GABRP, MSLN, FAIM2, and TACSTD, (ii ) Analyzing the presence or absence of CEACAM1 expression or the level of expression by known statistical analysis methods, for example, the possibility of developing colorectal cancer, the presence or absence of disease, the likelihood of recurrence, and the prognosis It is also possible to diagnose, determine, predict or classify the possibility of metastasis and the degree of malignancy. Known statistical analysis methods include, for example, (i) a method using Gene Spring (Silicon Genetics) Predict Parameter Value, and (ii) a weighted voting cross-validation algorithm (Science using leave-one-out cross-validation). 286, 531-537, 1999). The type and number of the protein of the present invention used in the diagnosis, determination, prediction or classification method using such a statistical analysis method is not particularly limited, but usually 2 to 14 types, preferably 3 to 10 types. More preferably, 4 to 8 proteins, particularly preferably 5 to 7 proteins can be appropriately selected and used.
In the present specification, “high expression level” or “low expression level” not only means absolute expression level, but also means relative expression level.

(4) Pharmaceutical containing antisense polynucleotide The antisense polynucleotide of the present invention capable of binding to DNA encoding the protein A of the present invention in a complementary manner and suppressing the expression of the DNA It has inhibitory action, cancer cell apoptosis promoting / promoting action, etc., has low toxicity, and functions of DNA encoding the protein of the present invention or protein A of the present invention in vivo (eg, anti-apoptotic activity, cancer cell proliferation) (Promoting activity) can be suppressed. For example, it can be used as a prophylactic / therapeutic agent for colon cancer, an apoptosis promoting agent for colon cancer cells, a growth inhibitor for colon cancer cells, a metastasis / recurrence inhibitor for colon cancer, etc. Can do.
When the antisense polynucleotide is used as the above-mentioned prophylactic / therapeutic agent, it can be formulated and administered according to a method known per se.
In addition, for example, after inserting the above-described antisense polynucleotide alone or into an appropriate vector such as a retrovirus vector, an adenovirus vector, an adenovirus associated virus vector, etc., a human or mammal (eg, rat, Rabbits, sheep, pigs, cows, cats, dogs, monkeys, etc.) orally or parenterally. The antisense polynucleotide can be formulated as it is or together with a physiologically recognized carrier such as an adjuvant for promoting ingestion, and can be administered by a gene gun or a catheter such as a hydrogel catheter. Alternatively, it can be aerosolized and locally administered into the trachea as an inhalant.
Furthermore, for the purpose of improving pharmacokinetics, prolonging the half-life, and improving the efficiency of cellular uptake, the above-mentioned antisense polynucleotides can be formulated (injection) alone or with a carrier such as liposome to produce veins, subcutaneouss, joint cavities Of these, it may be administered to cancerous lesions.
The dosage of the antisense polynucleotide varies depending on the target disease, administration subject, administration route, etc. For example, when the antisense polynucleotide of the present invention is administered for the purpose of treating colorectal cancer, In a body weight of 60 kg, about 0.1 to 100 mg of the antisense polynucleotide is administered per day.
Furthermore, the antisense polynucleotide can also be used as a diagnostic oligonucleotide probe for examining the presence and expression status of the DNA of the present invention in tissues and cells.

(4a) Pharmaceutical containing siRNA, shRNA, etc. Similar to the above-mentioned antisense polynucleotide, double-stranded RNA containing a part of RNA encoding protein A of the present invention (siRNA against the polynucleotide of the present invention (small ( short (interfering RNA), shRNA (small (short) hairpin RNA)), ribozymes containing a part of the RNA encoding the protein of the present invention can also suppress the expression of the gene of the present invention, Since the function of the protein used in the present invention or the DNA used in the present invention in the body can be suppressed, for example, a preventive / therapeutic agent for colon cancer, an apoptosis promoter for cancer cells, a growth inhibitor for cancer cells, a colon It can be used as a cancer metastasis / recurrence inhibitor.
Double-stranded RNA can be produced by designing based on the sequence of the polynucleotide of the present invention according to a known method (eg, Nature, 411, 494, 2001).
The ribozyme can be designed and produced based on the sequence of the polynucleotide of the present invention according to a known method (eg, TRENDS in Molecular Medicine, 7, 221 pages, 2001). For example, it can be produced by linking a known ribozyme to a part of the RNA encoding the protein of the present invention. Examples of the RNA encoding the protein of the present invention include a portion (RNA fragment) adjacent to the cleavage site on the RNA of the present invention that can be cleaved by a known ribozyme.
When the above double-stranded RNA or ribozyme is used as the prophylactic / therapeutic agent, it can be formulated and administered in the same manner as the antisense polynucleotide.

(5) Pharmaceutical containing the antibody of the present invention The antibody of the present invention, for example, an antibody having an action of neutralizing the activity of protein A of the present invention is, for example, a preventive / therapeutic agent for colorectal cancer, an apoptosis promoting agent for cancer cells. It can be used as a cancer cell growth inhibitor and a colon cancer metastasis / recurrence inhibitor.
The prophylactic / therapeutic agent for the above-mentioned diseases containing the antibody has low toxicity and can be used as a liquid or as a pharmaceutical composition of an appropriate dosage form as a human or mammal (eg, rat, rabbit, sheep, pig, bovine, Cats, dogs, monkeys, etc.) or can be administered parenterally (eg, intravenous injection). The dose varies depending on the administration subject, target disease, symptom, administration route, etc. For example, when used for treatment / prevention of colorectal cancer in adults, the antibody of the present invention is usually used as a single dose. About 0.01-20 mg / kg body weight, preferably about 0.1-10 mg / kg body weight, more preferably about 0.1-5 mg / kg body weight, about 1-5 times a day, preferably 1 It is convenient to administer about 3 times. In the case of other parenteral administration and oral administration, an equivalent amount can be administered. When symptoms are particularly severe, the dose may be increased according to the symptoms.
The antibody can be administered per se or as a suitable pharmaceutical composition. The pharmaceutical composition used for the administration comprises the antibody or a salt thereof and a pharmacologically acceptable carrier, diluent or excipient. Such compositions are provided as dosage forms suitable for oral or parenteral administration (eg, intravenous injection). Preferably it is provided as an inhalant.
Each of the above-described compositions may contain other active ingredients as long as they do not cause an unfavorable interaction by blending with the antibody.

(6) DNA-transferred animal In the present invention, a DNA encoding a foreign protein of the present invention (hereinafter abbreviated as a foreign DNA of the present invention) or a mutant DNA thereof (abbreviated as a foreign mutant DNA of the present invention) There is provided a non-human mammal having
That is, the present invention
(1) a non-human mammal having the exogenous DNA of the present invention or a mutant DNA thereof,
(2) the animal according to (1), wherein the non-human mammal is a rodent;
(3) An animal according to (2), wherein the rodent is a mouse or a rat, and (4) a recombinant vector containing the exogenous DNA of the present invention or a mutant DNA thereof and capable of being expressed in a mammal. Is.
A non-human mammal having the exogenous DNA of the present invention or a mutant DNA thereof (hereinafter abbreviated as the DNA-transferred animal of the present invention) is used for embryos including unfertilized eggs, fertilized eggs, spermatozoa and progenitor cells thereof. Preferably, at the stage of embryonic development in non-human mammal development (more preferably at the single cell or fertilized egg cell stage and generally prior to the 8-cell stage), the calcium phosphate method, electric pulse method, lipofection method, aggregation method, It can be produced by transferring the target DNA by the microinjection method, particle gun method, DEAE-dextran method or the like. Further, by the DNA transfer method, the target exogenous DNA of the present invention can be transferred to somatic cells, living organs, tissue cells, etc., and can be used for cell culture, tissue culture, etc. The DNA-transferred animal of the present invention can also be produced by fusing the above-mentioned embryo cells with a cell fusion method known per se.
Examples of non-human mammals include cows, pigs, sheep, goats, rabbits, dogs, cats, guinea pigs, hamsters, mice, rats and the like. Among them, rodents, especially mice (for example, C57BL / 6 strain, DBA2 strain, etc. as pure strains, etc.) that are relatively short in ontogenesis and biological cycle from the viewpoint of creating a disease animal model system and that are easy to reproduce. As the system, B6C3F ₁ system, BDF ₁ system, B6D2F ₁ system, BALB / c system, ICR system, etc.) or rats (for example, Wistar, SD, etc.) are preferable.
Examples of the “mammal” in the recombinant vector that can be expressed in the mammal include humans and the like in addition to the above non-human mammals.
The exogenous DNA of the present invention is not the DNA of the present invention inherently possessed by a non-human mammal but the DNA of the present invention once isolated and extracted from the mammal.
Examples of the mutant DNA of the present invention include those in which a mutation (for example, mutation or the like) has occurred in the base sequence of the original DNA of the present invention, specifically, addition of a base, deletion, substitution to another base, etc. The resulting DNA is used, and abnormal DNA is also included.
The abnormal DNA means DNA that expresses the abnormal protein of the present invention, and for example, DNA that expresses a protein that suppresses the function of the normal protein of the present invention is used.
The exogenous DNA of the present invention may be derived from mammals of the same or different species as the target animal. In transferring the DNA of the present invention to a target animal, it is generally advantageous to use the DNA as a DNA construct bound downstream of a promoter that can be expressed in animal cells. For example, when transferring the human DNA of the present invention, expression of DNA derived from various mammals (for example, rabbits, dogs, cats, guinea pigs, hamsters, rats, mice, etc.) having the DNA of the present invention having high homology with the human DNA of the present invention. The DNA of the present invention is highly expressed by microinjecting a DNA construct (eg, a vector, etc.) in which the human DNA of the present invention is bound downstream of various promoters into a fertilized egg of a target mammal, such as a mouse fertilized egg. DNA transfer mammals can be created.

Examples of the expression vector for the protein of the present invention include plasmids derived from Escherichia coli, plasmids derived from Bacillus subtilis, plasmids derived from yeast, bacteriophages such as λ phage, retroviruses such as Moloney leukemia virus, animal viruses such as vaccinia virus and baculovirus. Etc. are used. Of these, plasmids derived from Escherichia coli, plasmids derived from Bacillus subtilis or plasmids derived from yeast are preferably used.
Examples of promoters that regulate DNA expression include (i) promoters of DNA derived from viruses (eg, simian virus, cytomegalovirus, Moloney leukemia virus, JC virus, breast cancer virus, poliovirus, etc.) ) Promoters derived from various mammals (human, rabbit, dog, cat, guinea pig, hamster, rat, mouse, etc.), such as albumin, insulin II, uroplakin II, elastase, erythropoietin, endothelin, muscle creatine kinase, glial fibrillary acidity Protein, glutathione S-transferase, platelet-derived growth factor β, keratin K1, K10 and K14, collagen type I and type II, cyclic AMP-dependent protein kinase βI subunit, dystroph , Tartrate-resistant alkaline phosphatase, atrial natriuretic factor, endothelial receptor tyrosine kinase (generally abbreviated as Tie2), sodium potassium adenosine 3 kinase (Na, K-ATPase), neurofilament light chain, metallothionein I And IIA, metalloproteinase 1 tissue inhibitor, MHC class I antigen (H-2L), H-ras, renin, dopamine β-hydroxylase, thyroid peroxidase (TPO), peptide chain elongation factor 1α (EF-1α), β Actin, α and β myosin heavy chain, myosin light chain 1 and 2, myelin basic protein, thyroglobulin, Thy-1, immunoglobulin, heavy chain variable region (VNP), serum amyloid P component, myoglobin, troponin C, smooth muscle α act , Pre-pro-enkephalin A, such as a promoter, such as vasopressin is used. Among these, the cytomegalovirus promoter capable of being highly expressed throughout the body, the human peptide chain elongation factor 1α (EF-1α) promoter, the human and chicken β-actin promoters, and the like are suitable.
The vector preferably has a sequence (generally called a terminator) that terminates transcription of a target messenger RNA in a DNA-transferred mammal. For example, the sequence of each DNA derived from viruses and various mammals The SV40 terminator of simian virus or the like is preferably used.

In addition, the splicing signal of each DNA, enhancer region, a part of intron of eukaryotic DNA, etc. 5 ′ upstream of the promoter region, between the promoter region and the translation region or between the translation regions for the purpose of further expressing the target foreign DNA It is also possible to connect it 3 'downstream of the target.
The normal translation region of the protein of the present invention includes liver, kidney, thyroid cell, fibroblast-derived DNA derived from humans or various mammals (eg, rabbits, dogs, cats, guinea pigs, hamsters, rats, mice, etc.) and commercially available DNA. As a raw material, complementary DNA prepared by a known method as a whole or a part of genomic DNA or from RNA derived from liver, kidney, thyroid cells, or fibroblasts can be obtained. In addition, exogenous abnormal DNA can produce a translation region obtained by mutating a normal protein translation region obtained from the above cells or tissues by a point mutagenesis method.
The translation region can be prepared as a DNA construct that can be expressed in a metastasized animal by an ordinary DNA engineering technique in which it is linked downstream of the promoter and optionally upstream of the transcription termination site.
The transfer of the foreign DNA of the present invention at the fertilized egg cell stage is ensured to be present in all germ cells and somatic cells of the subject mammal. The presence of the exogenous DNA of the present invention in the embryo cells of the produced animal after the DNA transfer indicates that all the progeny of the produced animal retain the exogenous DNA of the present invention in all the germ cells and somatic cells. means. The offspring of this type of animal that has inherited the foreign DNA of the present invention has the foreign DNA of the present invention in all of its germ cells and somatic cells.
The non-human mammal to which the exogenous normal DNA of the present invention has been transferred can be subcultured in a normal breeding environment as the DNA-bearing animal after confirming that the exogenous DNA is stably retained by mating. .
The transfer of the exogenous DNA of the present invention at the fertilized egg cell stage is ensured to be excessively present in all germ cells and somatic cells of the target mammal. Excessive exogenous DNA of the present invention is present in the germ cells of the produced animal after DNA transfer. This means that all the offspring of the produced animal have the exogenous DNA of the present invention in all the germ cells and somatic cells. Means. The offspring of this type of animal that has inherited the foreign DNA of the present invention has an excess of the foreign DNA of the present invention in all germ cells and somatic cells.
By obtaining a homozygous animal having the introduced DNA in both homologous chromosomes and mating the male and female animals, all the offspring can be bred and passaged so as to have the DNA in excess.

In the non-human mammal having the normal DNA of the present invention, the normal DNA of the present invention is highly expressed, and finally the function of the endogenous normal DNA is promoted, thereby finally causing the hyperfunction of the protein of the present invention. It can develop and can be used as a disease model animal. For example, the normal DNA-transferred animal of the present invention can be used to elucidate the hyperfunction of the protein of the present invention, the pathological mechanism of the disease associated with the protein of the present invention, and the method of treatment for these diseases Is possible.
Further, since the mammal to which the foreign normal DNA of the present invention has been transferred has increased symptoms of the released protein of the present invention, a preventive / therapeutic agent for diseases related to the protein of the present invention, for example, colon cancer It can also be used in screening tests for prophylactic / therapeutic agents, colon cancer cell apoptosis promoters, colon cancer cell growth inhibitors, and colon cancer metastasis / relapse inhibitors.
On the other hand, the non-human mammal having the exogenous abnormal DNA of the present invention can be subcultured in a normal breeding environment as the DNA-bearing animal after confirming that the exogenous DNA is stably retained by mating. Furthermore, the target foreign DNA can be incorporated into the aforementioned plasmid and used as a raw material. A DNA construct with a promoter can be prepared by a normal DNA engineering technique. The abnormal DNA transfer of the present invention at the fertilized egg cell stage is ensured to be present in all germ cells and somatic cells of the target mammal. The presence of the abnormal DNA of the present invention in the embryo cell of the produced animal after DNA transfer means that all the offspring of the produced animal have the abnormal DNA of the present invention in all the germ cells and somatic cells. The offspring of this type of animal that has inherited the foreign DNA of the present invention has the abnormal DNA of the present invention in all of its germ cells and somatic cells. By obtaining a homozygous animal having the introduced DNA in both homologous chromosomes and mating the male and female animals, all the offspring can be bred and passaged so as to have the DNA.

In the non-human mammal having the abnormal DNA of the present invention, the abnormal DNA of the present invention is highly expressed, and finally the functional inactive form of the protein of the present invention is inhibited by inhibiting the function of the endogenous normal DNA. It may become refractory and can be used as a model animal for the disease state. For example, by using the abnormal DNA-transferred animal of the present invention, it is possible to elucidate the pathological mechanism of the functional inactive refractory of the protein of the present invention and to examine a method for treating this disease.
Further, as a specific applicability, the abnormal DNA high-expressing animal of the present invention exhibits the function inhibition (dominant negative action) of the normal protein by the abnormal protein of the present invention in the functional inactive refractory disease of the protein of the present invention. It becomes a model to elucidate.
Further, since the mammal to which the foreign abnormal DNA of the present invention has been transferred has an increased symptom of the released protein of the present invention, a preventive / therapeutic agent for the protein of the present invention or a functional inactive refractory disease, for example, the large intestine It can also be used in screening tests for cancer prevention / treatment agents, colon cancer cell apoptosis promoters, colon cancer cell growth inhibitors, and colon cancer metastasis / recurrence inhibitors.
Further, as other applicability of the above-mentioned two kinds of DNA transfer animals of the present invention, for example,
(I) use as a cell source for tissue culture;
(Ii) Peptides that are specifically expressed or activated by the protein of the present invention by directly analyzing DNA or RNA in the tissue of the DNA-transferred animal of the present invention or by analyzing peptide tissue expressed by the DNA Analysis of the relationship with
(Iii) culturing cells of a tissue having DNA by a standard tissue culture technique, and using them, studying the function of cells from tissues generally difficult to cultivate,
(Iv) Screening of drugs that enhance the function of cells by using the cells described in (iii) above, and (v) isolation and purification of the mutant protein of the present invention and production of antibodies thereof are conceivable.

Furthermore, using the DNA-transferred animal of the present invention, clinical symptoms of diseases related to the protein of the present invention, including functional inactive refractories of the protein of the present invention, can be examined, and the protein of the present invention More detailed pathological findings in each organ of the disease model related to the disease can be obtained, and it is possible to contribute to the development of a new treatment method and further to the study and treatment of secondary diseases caused by the disease.
Moreover, each organ can be taken out from the DNA-transferred animal of the present invention, and after minced, it is possible to obtain free DNA-transferred cells, culture them, or systematize the cultured cells with a proteolytic enzyme such as trypsin. . Furthermore, the protein of the present invention and its action can be investigated by identifying the protein-producing cells of the present invention, examining the relationship with apoptosis, differentiation or proliferation, or the signal transduction mechanism in them, and examining their abnormalities. It becomes an effective research material for.
Furthermore, in order to develop a therapeutic agent for a disease related to the protein of the present invention, including the functional inactive refractory of the protein of the present invention, using the DNA-transferred animal of the present invention, the above-described test method and quantification are performed. It is possible to provide an effective and rapid screening method for a therapeutic agent for the disease using a method or the like. Moreover, it is possible to examine and develop a DNA treatment method for diseases associated with the protein of the present invention using the DNA-transferred animal of the present invention or the exogenous DNA expression vector of the present invention.

(7) Knockout Animal The present invention provides a non-human mammal embryonic stem cell in which the DNA of the present invention is inactivated and the non-human mammal deficient in DNA expression of the present invention.
That is, the present invention
(1) a non-human mammalian embryonic stem cell in which the DNA of the present invention is inactivated,
(2) The embryonic stem cell according to (1), wherein the DNA is inactivated by introducing a reporter gene (eg, β-galactosidase gene derived from E. coli),
(3) The embryonic stem cell according to (1), which is neomycin-resistant,
(4) The embryonic stem cell according to item (1), wherein the non-human mammal is a rodent.
(5) The embryonic stem cell according to (4), wherein the rodent is a mouse,
(6) the DNA expression-deficient non-human mammal in which the DNA of the present invention is inactivated,
(7) The DNA can be inactivated by introducing a reporter gene (eg, β-galactosidase gene derived from E. coli), and the reporter gene can be expressed under the control of a promoter for the DNA of the present invention (6) The non-human mammal according to Item,
(8) The non-human mammal according to (6), wherein the non-human mammal is a rodent.
(9) administering a test compound to the non-human mammal according to (8) and (10) the animal according to (7), wherein the rodent is a mouse, and detecting the expression of the reporter gene The screening method of the compound or its salt which promotes or inhibits the promoter activity with respect to DNA of this invention characterized by these is provided.
The non-human mammal embryonic stem cell in which the DNA of the present invention is inactivated refers to suppressing the DNA expression ability by artificially adding mutation to the DNA of the present invention possessed by the non-human mammal, or By substantially losing the activity of the protein of the present invention encoded by the DNA, the DNA has substantially no ability to express the protein of the present invention (hereinafter sometimes referred to as the knockout DNA of the present invention). ) Non-human mammal embryonic stem cells (hereinafter abbreviated as ES cells).
As the non-human mammal, the same ones as described above are used.
As a method for artificially adding a mutation to the DNA of the present invention, for example, a part or all of the DNA sequence can be deleted or another DNA can be inserted or replaced by a genetic engineering technique. With these mutations, for example, the knockout DNA of the present invention may be prepared by shifting the reading frame of codons or destroying the function of the promoter or exon.

Specific examples of non-human mammalian embryonic stem cells in which the DNA of the present invention is inactivated (hereinafter abbreviated as the DNA inactivated ES cell of the present invention or the knockout ES cell of the present invention) A DNA of the present invention possessed by a non-human mammal is isolated and a neomycin resistance gene, a drug resistance gene typified by a hygromycin resistance gene, or lacZ (β-galactosidase gene), cat (chloramphenicol acetyl) Inserting a reporter gene or the like typified by a transferase gene), or inserting a DNA sequence (for example, a polyA addition signal) that terminates the transcription of the gene into an intron between exons, Preventing the synthesis of complete messenger RNA As a result, a DNA strand containing a DNA sequence constructed so as to disrupt the gene (hereinafter abbreviated as a targeting vector) is introduced into the chromosome of the animal by, for example, homologous recombination, and the resulting ES cell PCR using Southern hybridization analysis using the DNA sequence of or near the DNA of the present invention as a probe or the DNA sequence of the targeting vector and the DNA sequence of the neighboring region other than the DNA of the present invention used for the preparation of the targeting vector It can be obtained by analyzing by the method and selecting the knockout ES cell of the present invention.
In addition, as the original ES cell for inactivating the DNA of the present invention by homologous recombination method, for example, those already established as described above may be used, or according to the known Evans and Kaufma method. Newly established may be used. For example, in the case of mouse ES cells, currently 129 ES cells are generally used, but since the immunological background is unclear, an alternative purely immunologically genetic background For example, C57BL / 6 mice and BDF ₁ mice (C57BL / 6 and DBA / 2, which have improved the number of eggs collected from C57BL / 6 mice by crossing with DBA / 2) are obtained. Those established using F ₁ ) can be used well. BDF ₁ mice have the advantage of having a large number of eggs collected and strong eggs, and since C57BL / 6 mice are used as background, ES cells obtained using these mice can be used to produce pathological model mice. The genetic background can be advantageously replaced by C57BL / 6 mice by backcrossing with C57BL / 6 mice.
When establishing ES cells, blastocysts on the third day after fertilization are generally used, but in addition to this, many blastocysts can be efficiently obtained by collecting eggs and culturing them to blastocysts. Early embryos can be obtained.
Although both male and female ES cells may be used, male ES cells are usually more convenient for producing germline chimeras. In addition, it is desirable to discriminate between males and females as soon as possible in order to reduce troublesome culture work.

One example of a method for determining the sex of an ES cell is a method of amplifying and detecting a gene in the sex-determining region on the Y chromosome by PCR. If this method is used, the number of ES cells of about 1 colony (about 50) is required, whereas about 10 ⁶ cells were conventionally required for karyotype analysis. It is possible to perform primary selection of ES cells in the early stage by discriminating between males and females, and by allowing male cells to be selected at an early stage, labor in the initial stage of culture can be greatly reduced.
The secondary selection can be performed, for example, by confirming the number of chromosomes by the G-banding method. The number of chromosomes of the obtained ES cell is preferably 100% of the normal number. However, if it is difficult due to physical operations during establishment, the gene of the ES cell is knocked out, and then normal cells (for example, chromosomes in mice) It is desirable to clone again into cells where the number is 2n = 40.
The embryonic stem cell line obtained in this way is usually very proliferative, but it tends to lose its ontogenic ability, so it needs to be subcultured carefully. For example, in a carbon dioxide incubator (preferably 5% carbon dioxide, 95% air or 5% oxygen, 5%) in the presence of LIF (1-10000 U / ml) on a suitable feeder cell such as STO fibroblast. Culturing is carried out by a method such as culturing at about 37 ° C. with carbon dioxide gas and 90% air. Preferably, the cells are converted into single cells by treatment with about 0.1% trypsin / 1 mM EDTA and seeded on newly prepared feeder cells. Such passage is usually carried out every 1 to 3 days. At this time, the cells are observed, and if morphologically abnormal cells are observed, the cultured cells are preferably discarded.
ES cells can be differentiated into various types of cells such as parietal muscle, visceral muscle, and myocardium by monolayer culture to high density or suspension culture until a cell conglomerate is formed under appropriate conditions. [MJ Evans and MH Kaufman, Nature 292, 154, 1981; GR Martin Proceedings of National Academy of Sciences USA (Proc. Natl. Acad. Sci. USA) 78, 7634, 1981; TC Doetschman et al., Journal of Embrology and Experimental Morphology, 87, 27, 1985], ES cells of the present invention. The DNA expression-deficient cells of the present invention obtained by differentiation are useful for in vitro cell biology of the protein of the present invention.

The DNA expression-deficient non-human mammal of the present invention can be distinguished from normal animals by measuring the mRNA level of the animal using a known method and comparing the expression level indirectly.
As the non-human mammal, those similar to the above can be used.
The DNA expression-deficient non-human mammal of the present invention is a DNA in which, for example, a targeting vector prepared as described above is introduced into mouse embryonic stem cells or mouse egg cells, and the DNA of the present invention of the targeting vector is inactivated by the introduction. The DNA of the present invention can be knocked out by homologous recombination in which the sequence replaces the DNA of the present invention on the chromosome of mouse embryonic stem cells or mouse egg cells by gene homologous recombination.
A cell in which the DNA of the present invention is knocked out is obtained by analyzing a DNA sequence on a Southern hybridization analysis or targeting vector using the DNA sequence of or near the DNA of the present invention as a probe and the mouse derived from the mouse used for the targeting vector. It can be determined by analysis by a PCR method using a DNA sequence in a neighboring region other than DNA as a primer. When non-human mammalian embryonic stem cells are used, a cell line in which the DNA of the present invention has been inactivated by gene homologous recombination is cloned, and the cells are cultured at an appropriate time, for example, the 8-cell stage non-human mammal. It is injected into animal embryos or blastocysts, and the produced chimeric embryos are transplanted into the uterus of the non-human mammal that is pseudopregnant. The produced animal is a chimeric animal composed of both cells having the normal DNA locus of the present invention and cells having the artificially mutated DNA locus of the present invention.
When some of the germ cells of the chimeric animal have the mutated DNA locus of the present invention, all the tissues are artificially mutated from the population obtained by mating such a chimeric individual with a normal individual. It can be obtained by selecting an individual composed of cells having the added DNA locus of the present invention, for example, by determining the coat color. Individuals thus obtained are usually individuals with deficient hetero-expression of the protein of the present invention, and individuals with deficient hetero-expression of the protein of the present invention are crossed and homozygous expression of the protein of the present invention from their offspring. Failure individuals can be obtained.

In the case of using an egg cell, for example, a transgenic non-human mammal having a targeting vector introduced into a chromosome can be obtained by injecting a DNA solution into the nucleus of the egg cell by a microinjection method. Compared to mammals, it can be obtained by selecting those having mutations in the DNA locus of the present invention by gene homologous recombination.
Thus, an individual in which the DNA of the present invention is knocked out can be reared in a normal breeding environment after confirming that the animal individual obtained by mating has also been knocked out.
In addition, germline acquisition and retention may be followed in accordance with conventional methods. That is, a homozygous animal having the inactivated DNA in both homologous chromosomes can be obtained by mating male and female animals possessed by the inactivated DNA. The obtained homozygous animal can be efficiently obtained by rearing the mother animal in a state where there are 1 normal individual and multiple homozygous animals. By breeding male and female heterozygotes, homozygotes and heterozygotes having the inactivated DNA are bred and passaged.
The non-human mammal embryonic stem cell in which the DNA of the present invention is inactivated is very useful for producing the non-human mammal deficient in DNA expression of the present invention.
In addition, since the non-human mammal deficient in DNA expression of the present invention lacks various biological activities that can be induced by the protein of the present invention, it is a model for diseases caused by inactivation of the biological activity of the protein of the present invention. Therefore, it is useful for investigating the causes of these diseases and for examining therapeutic methods.

(7a) Method for screening compound having therapeutic / preventive effect on diseases caused by deficiency or damage of DNA of the present invention The non-human mammal deficient in the expression of DNA of the present invention may have deficiency or damage of the DNA of the present invention. Compounds that have therapeutic / preventive effects on diseases caused by cancer (specifically, colon cancer preventive / therapeutic effects, colon cancer cell apoptosis promoting effects, colon cancer cell growth inhibitory effects, colorectal cancer metastasis / relapse) It can be used for screening of compounds having an inhibitor effect.
That is, the present invention is caused by the deficiency or damage of the DNA of the present invention, which comprises administering a test compound to the non-human mammal deficient in DNA expression of the present invention and observing and measuring changes in the animal. Provided is a screening method for a compound having a therapeutic / prophylactic effect on a disease such as cancer, or a salt thereof.
Examples of the non-human mammal deficient in DNA expression of the present invention used in the screening method include those described above.
Examples of test compounds include peptides, proteins, non-peptidic compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts, plasma, and the like, and these compounds are novel compounds. It may be a known compound.
Specifically, the non-human mammal deficient in DNA expression of the present invention is treated with a test compound, compared with an untreated control animal, and tested using changes in each organ, tissue, disease symptom, etc. of the animal as an index. The therapeutic / prophylactic effect of the compound can be tested.
As a method for treating a test animal with a test compound, for example, oral administration, intravenous injection and the like are used, and can be appropriately selected according to the symptoms of the test animal, the properties of the test compound, and the like.
The dosage of the test compound can be appropriately selected according to the administration method, the properties of the test compound, and the like.

For example, when screening a compound having a therapeutic / preventive effect on colorectal cancer, a colorectal cancer cell apoptosis promoting effect, a colorectal cancer cell growth inhibitory effect, a colorectal cancer metastasis / relapse inhibitory effect, etc. A test compound is administered to a non-human mammal deficient in DNA expression of the invention, and the difference in the degree of onset of cancer and the degree of cure of cancer from the test compound non-administered group is observed over time in the tissue.
In the screening method, when a test compound is administered to a test animal, the test compound is improved when the disease symptoms of the test animal are improved by about 10% or more, preferably about 30% or more, more preferably about 50% or more. It can be selected as a compound having a therapeutic / prophylactic effect on the above-mentioned diseases.
The compound obtained by using the screening method is a compound selected from the test compounds described above, and has a therapeutic / prophylactic effect on diseases caused by deficiency or damage of the protein of the present invention. It can be used as a medicine such as a safe and low-toxic preventive / therapeutic agent. Furthermore, compounds derived from the compounds obtained by the above screening can be used as well.
The compound obtained by the screening method may form a salt. Examples of the salt of the compound include physiologically acceptable metal salts, ammonium salts, salts with organic bases, salts with inorganic acids, organic Examples include salts with acids, salts with basic or acidic amino acids, and the like. Preferable examples of the metal salt include alkali metal salts such as sodium salt and potassium salt; alkaline earth metal salts such as calcium salt, magnesium salt and barium salt; aluminum salt and the like. Preferable examples of the salt with an organic base include, for example, trimethylamine, triethylamine, pyridine, picoline, 2,6-lutidine, ethanolamine, diethanolamine, triethanolamine, cyclohexylamine, dicyclohexylamine, N, N′-dibenzylethylenediamine. And the like. Preferable examples of the salt with inorganic acid include salts with hydrochloric acid, hydrobromic acid, nitric acid, sulfuric acid, phosphoric acid and the like. Preferable examples of salts with organic acids include formic acid, acetic acid, trifluoroacetic acid, propionic acid, phthalic acid, fumaric acid, oxalic acid, tartaric acid, maleic acid, citric acid, succinic acid, malic acid, methanesulfonic acid And salts with benzoic acid, benzenesulfonic acid, p-toluenesulfonic acid and the like. Preferable examples of salts with basic amino acids include salts with arginine, lysine, ornithine and the like, and preferable examples of salts with acidic amino acids include salts with aspartic acid, glutamic acid and the like. Can be mentioned.
The medicament containing the compound obtained by the screening method or a salt thereof can be produced in the same manner as the aforementioned medicament containing the protein of the present invention.
Since the preparation thus obtained is safe and low toxic, it can be used, for example, in humans or mammals (eg, rats, mice, guinea pigs, rabbits, sheep, pigs, cows, horses, cats, dogs, monkeys, etc.). Can be administered.
Although the dose of the compound or a salt thereof varies depending on the target disease, administration subject, administration route, etc., for example, when the compound is administered orally, generally in an adult (with a body weight of 60 kg) colon cancer patients The compound is administered at about 0.1 to 100 mg, preferably about 1.0 to 50 mg, more preferably about 1.0 to 20 mg per day. When administered parenterally, the single dose of the compound varies depending on the administration subject, target disease, etc., but for example, the compound is usually administered to an adult (60 kg) colon cancer patient in the form of an injection. In this case, it is convenient to administer about 0.01 to 30 mg, preferably about 0.1 to 20 mg, more preferably about 0.1 to 10 mg of the compound per day by intravenous injection. In the case of other animals, an amount converted per 60 kg can be administered.

(7b) Method for screening a compound that promotes or inhibits the activity of a promoter for the DNA of the present invention In the present invention, a test compound is administered to a non-human mammal deficient in DNA expression of the present invention to detect the expression of a reporter gene. The screening method of the compound or its salt which promotes or inhibits the activity of the promoter with respect to DNA of this invention characterized by these is provided.
In the screening method described above, the DNA expression-deficient non-human mammal of the present invention is inactivated by introducing a reporter gene among the DNA expression-deficient non-human mammals of the present invention described above, A gene capable of expressing the reporter gene under the control of a promoter for the DNA of the present invention is used.
Examples of the test compound are the same as described above.
As the reporter gene, the same one as described above is used, and a β-galactosidase gene (lacZ), a soluble alkaline phosphatase gene, a luciferase gene or the like is preferable.
In the non-human mammal of the present invention in which the DNA of the present invention is replaced with a reporter gene, the expression of the substance encoded by the reporter gene is traced because the reporter gene exists under the control of the promoter for the DNA of the present invention. By doing so, the activity of the promoter can be detected.
For example, when a part of the DNA region encoding the protein of the present invention is replaced with the β-galactosidase gene (lacZ) derived from Escherichia coli, the protein of the present invention is originally replaced with the tissue expressing the protein of the present invention. Β-galactosidase is expressed. Therefore, for example, by staining with a reagent that is a substrate of β-galactosidase such as 5-bromo-4-chloro-3-indolyl-β-galactopyranoside (X-gal), the present invention can be easily performed. The expression state of the protein in the animal can be observed. Specifically, the protein-deficient mouse of the present invention or a tissue section thereof is fixed with glutaraldehyde or the like, washed with phosphate buffered saline (PBS), and then stained with X-gal at room temperature or around 37 ° C. Then, after reacting for about 30 minutes to 1 hour, the tissue specimen is washed with a 1 mM EDTA / PBS solution to stop the β-galactosidase reaction and observe the coloration. Moreover, you may detect mRNA which codes lacZ according to a conventional method.
The compound obtained by using the screening method or a salt thereof is a compound selected from the test compounds described above, and is a compound that promotes or inhibits the promoter activity for the DNA of the present invention.
The compound obtained by the screening method may form a salt, and examples of the salt of the compound include salts as described above.

Since the compound or its salt that promotes or inhibits the promoter activity for the DNA of the present invention can regulate the expression of the protein of the present invention and the function of the protein, for example, a preventive / therapeutic agent for colorectal cancer, colon It is useful as a cancer cell apoptosis promoter, a colon cancer cell growth inhibitor, a colon cancer metastasis / relapse inhibitor, and the like.
Furthermore, compounds derived from the compounds obtained by the above screening can be used as well.
The pharmaceutical containing the compound obtained by the screening method or a salt thereof can be produced in the same manner as the pharmaceutical containing the protein of the present invention or a salt thereof.
Since the preparation thus obtained is safe and low toxic, it can be used, for example, in humans or mammals (eg, rats, mice, guinea pigs, rabbits, sheep, pigs, cows, horses, cats, dogs, monkeys, etc.). Can be administered.
The dose of the compound or a salt thereof varies depending on the target disease, administration subject, administration route, and the like. For example, when a compound that inhibits the promoter activity against the DNA of the present invention is orally administered, generally an adult (body weight) In a colon cancer patient (as 60 kg), the compound is administered at about 0.1 to 100 mg, preferably about 1.0 to 50 mg, more preferably about 1.0 to 20 mg per day. When administered parenterally, the single dose of the compound varies depending on the administration subject, the target disease, etc. For example, a compound that inhibits the promoter activity against the DNA of the present invention is usually administered in the form of an injection (in adults) When administered to a colon cancer patient (with a body weight of 60 kg), about 0.01 to 30 mg, preferably about 0.1 to 20 mg, more preferably about 0.1 to 10 mg of the compound per day is intravenously injected. Are conveniently administered. In the case of other animals, an amount converted per 60 kg body weight can be administered.
Thus, the non-human mammal deficient in DNA expression of the present invention is extremely useful in screening for compounds or salts thereof that promote or inhibit the activity of the promoter for the DNA of the present invention. It can greatly contribute to the investigation of the causes of various diseases caused or the development of preventive / therapeutic agents.
In addition, using DNA containing the promoter region of the protein of the present invention, genes encoding various proteins are ligated downstream thereof, and this is injected into an egg cell of an animal to produce a so-called transgenic animal (gene transgenic animal). Once created, the protein can be specifically synthesized and its action in the living body can be studied. Furthermore, if a suitable reporter gene is bound to the above promoter portion and a cell line that expresses it is established, a low molecular weight compound that specifically promotes or suppresses the production ability of the protein itself of the present invention in the body. Can be used as a search system.

In the present specification and sequence listing, bases, amino acids and the like are indicated by abbreviations based on abbreviations by IUPAC-IUB Commission on Biochemical Nomenclature or conventional abbreviations in the field, examples of which are described below. In addition, when there is an optical isomer with respect to an amino acid, the L form is shown unless otherwise specified.
DNA: deoxyribonucleic acid cDNA: complementary deoxyribonucleic acid A: adenine T: thymine G: guanine C: cytosine RNA: ribonucleic acid mRNA: messenger ribonucleic acid dATP: deoxyadenosine triphosphate dTTP: deoxythymidine triphosphate dGTP: deoxyguanosine tri Phosphoric acid dCTP: Deoxycytidine triphosphate ATP: Adenosine triphosphate EDTA: Ethylenediaminetetraacetic acid SDS: Sodium dodecyl sulfate Gly: Glycine Ala: Alanine Val: Valine Leu: Leucine Ile: Isoleucine Ser: Threon Cys: Threon Cyny : Methionine Glu: Glutamic acid Asp: Aspartic acid Lys: Lysine Arg: Arginine His: Histidine Phe Phenylalanine Tyr: tyrosine Trp: tryptophan Pro: proline Asn: asparagine Gln: glutamine pGlu: pyroglutamic acid Sec: selenocysteine (selenocysteine)

In addition, substituents, protecting groups and reagents frequently used in the present specification are represented by the following symbols.
Me: methyl group Et: ethyl group Bu: butyl group Ph: phenyl group TC: thiazolidine-4 (R) -carboxamide group Tos: p-toluenesulfonyl CHO: formyl Bzl: benzyl
Cl ₂ -Bzl: 2,6-dichlorobenzyl Bom: benzyloxymethyl Z: benzyloxycarbonyl Cl-Z: 2-chlorobenzyloxycarbonyl Br-Z: 2-bromobenzyloxycarbonyl Boc: t-butoxycarbonyl DNP: dinitro Phenyl Trt: Trityl Bum: t-Butoxymethyl Fmoc: N-9-fluorenylmethoxycarbonyl HOBt: 1-hydroxybenztriazole HOOBt: 3,4-dihydro-3-hydroxy-4-oxo-
1,2,3-benzotriazine HONB: 1-hydroxy-5-norbornene-2,3-dicarboximide DCC: N, N'-dicyclohexylcarbodiimide

The sequence numbers in the sequence listing in the present specification indicate the following sequences.
[SEQ ID NO: 1]
The amino acid sequence of LEFTB is shown.
[SEQ ID NO: 2]
CDNA encoding a protein having the amino acid sequence represented by SEQ ID NO: 1 (LEFTB)
The base sequence is shown.
[SEQ ID NO: 3]
The amino acid sequence of FOXA2 is shown.
[SEQ ID NO: 4]
CDNA encoding a protein having the amino acid sequence represented by SEQ ID NO: 3 (FOXA2)
The base sequence is shown.
[SEQ ID NO: 5]
The amino acid sequence of S100P is shown.
[SEQ ID NO: 6]
CDNA encoding a protein having the amino acid sequence represented by SEQ ID NO: 5 (S100P)
The base sequence is shown.
[SEQ ID NO: 7]
Shows the amino acid sequence of CTSE.
[SEQ ID NO: 8]
This shows the base sequence of cDNA encoding the protein having the amino acid sequence represented by SEQ ID NO: 7 (CTSE).
[SEQ ID NO: 9]
The amino acid sequence of NOX1 is shown.
[SEQ ID NO: 10]
This shows the base sequence of cDNA encoding the protein (NOX1) having the amino acid sequence represented by SEQ ID NO: 9.
[SEQ ID NO: 11]
The amino acid sequence of CDH3 is shown.
[SEQ ID NO: 12]
This shows the base sequence of cDNA encoding the protein having the amino acid sequence represented by SEQ ID NO: 11 (CDH3).
[SEQ ID NO: 13]
The amino acid sequence of KLK10 is shown.
[SEQ ID NO: 14]
This shows the base sequence of cDNA encoding the protein having the amino acid sequence represented by SEQ ID NO: 13 (KLK10).
[SEQ ID NO: 15]
The amino acid sequence of DUOX2 is shown.
[SEQ ID NO: 16]
This shows the base sequence of cDNA encoding the protein having the amino acid sequence represented by SEQ ID NO: 15 (DUOX2).
[SEQ ID NO: 17]
The amino acid sequence of S100A2 is shown.
[SEQ ID NO: 18]
This shows the base sequence of cDNA encoding the protein having the amino acid sequence represented by SEQ ID NO: 17 (S100A2).
[SEQ ID NO: 19]
The amino acid sequence of GABRP is shown.
[SEQ ID NO: 20]
This shows the base sequence of cDNA encoding the protein having the amino acid sequence represented by SEQ ID NO: 19 (GABRP).
[SEQ ID NO: 21]
The amino acid sequence of MSLN is shown.
[SEQ ID NO: 22]
This shows the base sequence of cDNA encoding the protein having the amino acid sequence represented by SEQ ID NO: 21 (MSLN).
[SEQ ID NO: 23]
The amino acid sequence of FAIM2 is shown.
[SEQ ID NO: 24]
This shows the base sequence of cDNA encoding the protein having the amino acid sequence represented by SEQ ID NO: 23 (FAIM2).
[SEQ ID NO: 25]
The amino acid sequence of TACSTD2 is shown.
[SEQ ID NO: 26]
This shows the base sequence of cDNA encoding the protein having the amino acid sequence represented by SEQ ID NO: 25 (TACSTD2).
[SEQ ID NO: 27]
The amino acid sequence of CEACAM1 is shown.
[SEQ ID NO: 28]
This shows the base sequence of the cDNA encoding the protein having the amino acid sequence represented by SEQ ID NO: 27 (CEACAM1).
[SEQ ID NO: 29]
Shows the base sequence of HOXB13.
[SEQ ID NO: 30]
The amino acid sequence of HOXB13 is shown.
[SEQ ID NO: 31]
Shows the base sequence of PITX2.
[SEQ ID NO: 32]
The amino acid sequence of PITX2 is shown.

In the following, the present invention will be more specifically described with reference to examples, but the present invention is not limited thereto.

Example 1
The following experiment was conducted for the purpose of finding a gene whose expression changes specifically in the colorectal tumor onset site.
Total RNA extracted from colorectal tumor tissue and surrounding normal tissues was provided by Jichi Medical University (Table 1), and gene expression analysis was performed using an oligonucleotide microarray (Human Genome U133A; Affymetrix). The experiment method was in accordance with the Affymetrix experiment manual (Expression analysis technical manual). About the obtained experimental results, GeneSpring
(SiliconGenetics) was used for analysis. In this study, the rectum, sigmoid colon, and descending colon were classified as the left colon, transverse colon, ascending colon, and appendix as the right colon, and the right colon tumor group (patient number 1-5) was the CR group, the right colon Analysis was performed with the normal tissue group around the large intestine as the NR group, the tumor group in the left large intestine (patient number 6-12) as the CL group, and the normal tissue around the left large intestine as the NL group.
First, in order to compare the NR group with the NL group, genes that were significant (p <0.05) and showed an expression difference of 2.5 times or more in both groups were extracted. As a result, 225 genes were extracted.

Example 2
Among the genes obtained in Example 1, HOXB13 (SEQ ID NO: 29 [base sequence], SEQ ID NO: 30 [amino acid sequence]) and PITX2 (SEQ ID NO: 31 [base sequence]), SEQ ID NO: 32 [amino acid sequence] ]), Quantitative PCR was performed to confirm the GeneChip results. The cDNA used for quantitative PCR was prepared using TaqMan Reverse Transcription Reagents (Applied Biosystems) using the total RNA used in Example 1 as a template. Primers and probes for the 18s rRNA gene used to correct the expression level between each gene and specimen were purchased from Applied Biosystems.
As a result, it was found that the results of GeneChip and TaqMan correlated well. Table 2 shows the relative values when the expression of sample 1N is 1.

  Furthermore, in order to confirm that these genes are effective in distinguishing the left and right of the large intestine, quantitative PCR was performed by the same method as described above using the total RNA of the newly added sample (Table 3). The results are shown in Table 4. As a result, it was found that the left and right of the large intestine can be easily distinguished using these gene expression levels as an index.

Example 3
Analysis using GeneChip data obtained from Example 1 was performed. Among the gene groups of Example 1, S100P (SEQ ID NO: 6), FOXA2 (SEQ ID NO: 4), NOX1 (SEQ ID NO: 10) are genes whose expression is increased more than twice in the CR group compared to the NR group. ), LEFTB (SEQ ID NO: 2) and CTSE (SEQ ID NO: 8). In addition, among the gene group of Example 1, S100P and NOX1 were found as genes whose expression was enhanced twice or more in the CL group compared to the NL group. Quantitative PCR was performed on these genes. The method was in accordance with Example 2.
As a result, as shown in Table 5, it was found that the GeneChip result and the TaqMan result correlated well.

Example 4
In Example 1, the results obtained using the oligonucleotide microarray (Human Genome U133A; Affymetrix) were further analyzed using GeneSpring (SiliconGenetics). In this study, the normal tissue group around the right and left large intestine was analyzed as NR + NL, and the tumor tissue group in the right and left large intestine was analyzed as CR + CL. In order to compare the NR + NL group and the CR + CL group, genes that were significant (p <0.05) and showed an expression difference of 4 times or more were extracted in both groups.
As a result, 180 genes were extracted. As genes whose expression increases in the CR + CL group, CDH3 (SEQ ID NO: 12), KLK10 (SEQ ID NO: 14), DUOX2 (SEQ ID NO: 16), S100A2 (SEQ ID NO: 18), GABRP (SEQ ID NO: 20) ), MSLN (SEQ ID NO: 22), FAIM2 (SEQ ID NO: 24), TACSTD2 (SEQ ID NO: 26), and the like. As a gene whose expression decreases in the CR + CL group, CEACAM1 (SEQ ID NO: 28) and the like were found.

SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, A compound that inhibits the activity of a protein containing the same or substantially the same amino acid sequence represented by SEQ ID NO: 21, SEQ ID NO: 23 or SEQ ID NO: 25, or a salt thereof, and expression of the protein gene An inhibitory compound or salt thereof, an antisense polynucleotide containing a base sequence complementary to or substantially complementary to the base sequence of a polynucleotide encoding the protein or its partial peptide, or a part thereof, an antibody against the protein, etc. Preventive / therapeutic agents for colorectal cancer, colon cancer cell apoptosis promoter, colon cancer cell growth inhibitor, colon cancer metastasis / It can be used as such originating inhibitor.
In addition, a compound or a salt thereof that promotes the activity of a protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 27, a compound that promotes the expression of the gene of the protein, or a salt thereof, and the like Can be expected as a safe drug as a preventive / therapeutic agent for colorectal cancer, a metastasis / recurrence inhibitor for colorectal cancer, an apoptosis promoter for colorectal cancer cells, a proliferation inhibitor for colorectal cancer cell cells, and so on. It can be used for screening active ingredients of various pharmaceuticals.

Claims (33)

  SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, A compound or a salt thereof that inhibits the activity of a protein containing the same or substantially the same amino acid sequence represented by SEQ ID NO: 21, SEQ ID NO: 23 or SEQ ID NO: 25, or a partial peptide thereof, or a salt thereof A preventive / therapeutic agent or inhibitor for metastasis / recurrence of colorectal cancer, comprising:   SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, A compound that inhibits the expression of a gene of a protein or a partial peptide thereof or a salt thereof containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 21, SEQ ID NO: 23 or SEQ ID NO: 25, or A preventive / therapeutic agent or an inhibitor of metastasis / recurrence of colorectal cancer comprising the salt.   SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, Complementary to the nucleotide sequence of a polynucleotide encoding a protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 21, SEQ ID NO: 23 or SEQ ID NO: 25, or a partial peptide thereof, or An antisense polynucleotide containing a substantially complementary base sequence or a part thereof.   A medicament comprising the antisense polynucleotide according to claim 3.   The medicament according to claim 4, which is a preventive / therapeutic agent for colon cancer or an inhibitor for metastasis / recurrence.   SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SiRNA or shRNA against a polynucleotide encoding a protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 21, SEQ ID NO: 23 or SEQ ID NO: 25 or a partial peptide thereof, The medicine which becomes.   The medicament according to claim 6, which is a preventive / therapeutic agent for colon cancer or an inhibitor for metastasis / recurrence.   SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, Colorectal cancer comprising an antibody against a protein containing the same or substantially the same amino acid sequence represented by SEQ ID NO: 21, SEQ ID NO: 23 or SEQ ID NO: 25, or a partial peptide thereof or a salt thereof. Preventive / therapeutic or metastasis / recurrence inhibitor.   SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, Contains an antibody against a protein containing the same or substantially the same amino acid sequence represented by SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25 or SEQ ID NO: 27, or a partial peptide thereof or a salt thereof A diagnostic agent for colorectal cancer.   SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, A polynucleotide containing a protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25 or SEQ ID NO: 27, or a partial peptide thereof A diagnostic agent for colorectal cancer.   SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, An antibody against a protein containing the same or substantially the same amino acid sequence as shown in SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25 or SEQ ID NO: 27, or a partial peptide thereof, or a salt thereof; A method for diagnosing colorectal cancer, comprising using a polynucleotide encoding the protein or a partial peptide thereof.   SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, A protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25 or SEQ ID NO: 27, or a partial peptide thereof or a salt thereof Use as a diagnostic marker.   SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, Use of a protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25 or SEQ ID NO: 27, or a partial peptide thereof, or a salt thereof. A screening method for a drug for preventing / treating or suppressing metastasis / recurrence of a characteristic colorectal cancer.   SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, A protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25 or SEQ ID NO: 27, a partial peptide thereof, or a salt thereof A screening kit for a drug for preventing / treating or inhibiting metastasis / recurrence of colorectal cancer.   SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, A polynucleotide encoding a protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25 or SEQ ID NO: 27, or a partial peptide thereof is used. A screening method for a medicament for preventing / treating or inhibiting metastasis / recurrence of colorectal cancer.   SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, A polynucleotide containing a protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25 or SEQ ID NO: 27, or a partial peptide thereof A screening kit for a medicament for preventing / treating or inhibiting metastasis / recurrence of colorectal cancer.   SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, Measures the activity of a protein containing the same or substantially the same amino acid sequence represented by SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25 or SEQ ID NO: 27, or a partial peptide thereof or a salt thereof A screening method for a medicament for preventing / treating or inhibiting metastasis / recurrence of colorectal cancer.   SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, Measures the amount of a protein containing the same or substantially the same amino acid sequence represented by SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25 or SEQ ID NO: 27, or a partial peptide thereof or a salt thereof A screening method for a medicament for preventing / treating or inhibiting metastasis / recurrence of colorectal cancer.   SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, Inhibits the activity of a protein containing the same or substantially the same amino acid sequence as shown in SEQ ID NO: 21, SEQ ID NO: 23 or SEQ ID NO: 25, or a partial peptide thereof, or a salt thereof, or the above protein Alternatively, a method for preventing or treating colorectal cancer or a method for suppressing metastasis or recurrence, which comprises inhibiting the expression of a gene of a partial peptide or a salt thereof.   For mammals, (i) SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, sequence No. 17, SEQ ID No. 19, SEQ ID No. 21, SEQ ID No. 23, or a protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID No. 25 or a partial peptide thereof or a salt thereof (Ii) a compound or salt thereof that inhibits expression of the gene of the protein or partial peptide or salt thereof, (iii) an antibody to the protein or partial peptide or salt thereof, or (Iv) Complementary or substantially complementary to the nucleotide sequence of the polynucleotide encoding the protein or its partial peptide Nucleotide sequence or features that, colorectal cancer prevention and treatment or metastatic or recurrent suppression methods administering an effective amount of an antisense polynucleotide comprising a part thereof.   (I) SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: for producing a preventive / therapeutic agent for colon cancer or a metastasis / recurrence inhibitor 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, or the same or substantially the same as the amino acid sequence represented by SEQ ID NO: 25 A compound or a salt thereof that inhibits the activity of a protein containing the amino acid sequence or a partial peptide thereof or a salt thereof; (ii) a compound or a salt thereof which inhibits the expression of the gene of the protein or a partial peptide thereof or a salt thereof; An antibody against the protein or a partial peptide thereof or a salt thereof, or (iv) a polynucleotide encoding the protein or the partial peptide thereof Complementary or substantially complementary base sequence to the nucleotide sequence or the use of antisense polynucleotides containing part thereof.   SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, A compound or a salt thereof that inhibits the activity of a protein containing the same or substantially the same amino acid sequence represented by SEQ ID NO: 21, SEQ ID NO: 23 or SEQ ID NO: 25, or a partial peptide thereof, or a salt thereof A colon cancer cell apoptosis promoter or colon cancer cell growth inhibitor comprising   SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, A compound that inhibits the expression of a gene of a protein or a partial peptide thereof or a salt thereof containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 21, SEQ ID NO: 23 or SEQ ID NO: 25, or A colon cancer cell apoptosis promoter or colon cancer cell growth inhibitor comprising the salt.   The medicament according to claim 4, which is an agent for promoting apoptosis of colon cancer cells or an agent for suppressing growth of colon cancer cells. The medicament according to claim 6, which is a colon cancer cell apoptosis promoter or a colon cancer cell growth inhibitor.   SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, Colorectal cancer comprising an antibody against a protein containing the same or substantially the same amino acid sequence represented by SEQ ID NO: 21, SEQ ID NO: 23 or SEQ ID NO: 25, or a partial peptide thereof or a salt thereof. Cell apoptosis promoter or colon cancer cell growth inhibitor.   SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, Use of a protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25 or SEQ ID NO: 27, or a partial peptide thereof, or a salt thereof. A screening method for a pharmaceutical for promoting apoptosis of a characteristic colorectal cancer cell or for inhibiting proliferation of a colorectal cancer cell.   SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, A protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25 or SEQ ID NO: 27, a partial peptide thereof, or a salt thereof A screening kit for a drug for promoting apoptosis of colorectal cancer cells or suppressing proliferation of colorectal cancer cells.   SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, A polynucleotide encoding a protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25 or SEQ ID NO: 27, or a partial peptide thereof is used. A screening method for a medicament for promoting apoptosis of colorectal cancer cells or suppressing growth of colorectal cancer cells.   SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, A polynucleotide containing a protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25 or SEQ ID NO: 27, or a partial peptide thereof A screening kit for a drug for promoting apoptosis of colorectal cancer cells or suppressing proliferation of colorectal cancer cells.   SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, Inhibits the activity of a protein containing the same or substantially the same amino acid sequence as shown in SEQ ID NO: 21, SEQ ID NO: 23 or SEQ ID NO: 25, or a partial peptide thereof, or a salt thereof, or the above protein Alternatively, a method for promoting apoptosis of colorectal cancer cells or a method for suppressing growth of colorectal cancer cells, which comprises inhibiting expression of a gene of a partial peptide or a salt thereof.   For mammals, (i) SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, sequence No. 17, SEQ ID No. 19, SEQ ID No. 21, SEQ ID No. 23, or a protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID No. 25 or a partial peptide thereof or a salt thereof (Ii) a compound or salt thereof that inhibits expression of the gene of the protein or partial peptide or salt thereof, (iii) an antibody to the protein or partial peptide or salt thereof, or (Iv) Complementary or substantially complementary to the nucleotide sequence of the polynucleotide encoding the protein or its partial peptide Which comprises administering an effective amount of an antisense polynucleotide comprising a nucleotide sequence or a portion thereof, proapoptotic method or antiproliferative methods of colon cancer cells in colon cancer cells. (I) SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, sequence for producing a colon cancer cell apoptosis promoter or colon cancer cell growth inhibitor SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, or SEQ ID NO: 23. (Ii) a compound or salt thereof that inhibits the activity of a protein containing the same amino acid sequence, or a partial peptide thereof, or a salt thereof, (ii) a gene that inhibits the expression of the gene of the protein or the partial peptide or salt thereof, iii) an antibody against the protein or its partial peptide or salt thereof, or (iv) it encodes the protein or its partial peptide Use of an antisense polynucleotide comprising a complementary or substantially complementary base sequence or a portion thereof to the base sequence of the polynucleotide.