JP2004357702A - New protein and dna encoding the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To analyze base sequences of cDNA included in a full-length cDNA library and specifying the bioactivities of a protein encoded by the cDNA having a new sequence in them and to provide a method for using a protein based on the bioactivity and DNA encoding the same. <P>SOLUTION: The protein is either one of the following (a)-(c). (a) a protein comprising a specific amino acid sequence derived from human and having EGF (epidermal growth factor) activity. (b) a protein comprising amino acid sequence having one or more amino acids deleted, substituted and/or added from/for/to the above amino acid sequence and having increased expression in cancer cells or cancer tissues. (c) a protein comprising a partial amino acid sequence of the amino acid sequence of the above protein and having increased expression in cancer cells or cancer tissues. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to a novel cancer-related protein, a DNA encoding the protein, a full-length cDNA encoding the protein, a recombinant vector having the DNA, an oligonucleotide comprising a partial sequence of the DNA, and a gene into which the DNA has been introduced. The present invention relates to an introduced cell, an antibody that specifically binds to the protein, and the like.

  At present, genomic sequences of various organisms are being elucidated and analyzed on a global level. The entire genome sequence of about one hundred and several dozen prokaryotic microorganisms, budding yeast of lower eukaryotes, and nematodes that are multicellular eukaryotes have already been determined. A draft of the nucleotide sequence of a human genome called 3 billion base pairs was published in February 2001, but the complete sequence was decoded and published in April 2003. The purpose of elucidating the genomic sequence is to understand the complex biological phenomena as a network of interactions and functions between all genes, between genes, between proteins, between cells, and even between individuals. Elucidating life phenomena from the genome information of various species is not only important as a research topic in the academic field, but also how to develop the research results obtained therefrom into industrial applications. In that respect, its social significance is also great.

  However, simply determining the genome sequence cannot clarify the functions of all genes. For example, in yeast, only about half of the about 6,000 genes estimated from the genomic sequence could estimate its function. On the other hand, it is said that about 100,000 kinds of proteins exist in humans. Therefore, there is a strong demand for the establishment of a “high-throughput gene function analysis system” for quickly and efficiently elucidating the functions of an enormous amount of new genes revealed from genome sequences.

  In eukaryotic genomic sequences, one gene is often divided into multiple exons by introns. Therefore, there are many problems in accurately predicting the structure of the protein encoded therefrom only from the genome sequence information. On the other hand, in cDNA prepared from mRNA from which introns have been removed, the amino acid sequence information of the protein can be obtained as one continuous sequence information, so that the primary structure can be easily clarified. In human cDNA research, more than 5 million EST (Expressed Sequence Tags) data have been published in public databases so far.

  Such information is used from various angles such as elucidation of human gene structure, prediction of exon region in genomic sequence, and estimation of its expression profile. However, since most of these human EST information is concentrated near the 3 'end of cDNA, the information particularly near the 5' end of mRNA is extremely insufficient. In addition, 40,000 cDNAs have been revealed as a result of analyzes conducted at research institutions around the world (Helix Research Institute, Kazusa DNA Research Institute, The University of Tokyo Medical Research Institute, German Cancer Research Center, MGC Project, etc.) It seems that it covers most of the 30,000 loci in number, but the ratio of cDNA obtained as a full-length clone is about 80%, Considering that variants are included, it is considered that there are many cDNAs that have not yet been obtained.

  If a full-length cDNA can be obtained, the mRNA transcription start point on the genomic sequence can be estimated from the 5 'end sequence, and the factors involved in the stability of mRNA contained in the sequence and the regulation of expression at the translation stage are analyzed. Is possible. In addition, since the translation initiation codon atg is included on the 5 'side, translation into a protein can be performed in the correct frame. Therefore, by applying an appropriate gene expression system, it becomes possible to mass-produce the protein encoded by the cDNA or to express the protein and analyze its biological activity. Thus, the analysis of full-length cDNA provides important information that complements genomic sequence analysis. In addition, an expressible full-length cDNA clone is extremely important in empirical analysis of the function of the gene and in application to industrial applications.

  On the other hand, even when mRNA is transcription-modified from the same gene, some exons in the gene sequence are inserted and deleted and joined when undergoing splicing in the process of producing mature mRNA from the precursor RNA immediately after transcription. There are isomers (hereinafter sometimes referred to as “splicing variant mRNA”). In fact, a plurality of similar proteins (hereinafter sometimes referred to as “splicing variants”) produced by translating these mRNAs have been identified in vivo. Splicing variants are expressed in a tissue-specific, developmental stage-specific, or disease-specific manner, and are thought to have different functions.

  For example, for estrogen receptor β, ten exon deletion splicing variant mRNAs have been identified from various human tissues. Nine of them are expressed in normal breast tissue, among which mRNA expression lacking exons 5 and 6 has been shown to be significantly reduced in most cancer tissues. I have. In addition, it has been revealed that the expression level of mRNA lacking exon 5 significantly varies depending on the degree of progression of cancer (for example, see Non-Patent Document 1). Thus, the expression level of the splicing variant may show a significant correlation with the disease state.

  Such splicing variant mRNA or cDNA is also difficult to obtain from a conventional cDNA library or EST, and is a clone highly likely to be obtained from a full-length cDNA library including a transcription start site.

  Among them, cancer-related proteins have been discovered in various ways since α-fetoprotein (AFP) was reported in 1963, followed by carcino embryonic antigen (CEA) in 1963, and “tumor (cancer) A substance produced by cells or a substance produced by non-tumor cells in response to the presence of a tumor, the measurement of which indicates the presence of a tumor and is useful for assisting clinical diagnosis and early detection of metastasis / recurrence. '' It is utilized as a biological marker of a malignant tumor different from morphological information obtained by X-ray CT, ultrasound, MRI, or the like.

  Pre-cancerous cells and cancer cells are characterized by producing cancer-related marker proteins. These proteins are often composed of aberrant (modified) forms of the wild-type protein, which are those produced by cancer cells as a result of genetic mutation or altered post-translational processing. Alternatively, the cancer marker may be a protein that is overexpressed in tumor cells, usually as a result of gene amplification or aberrant transcriptional regulation. In some cases, these two phenomena occur simultaneously and continue to accumulate modified proteins throughout the course of the disease. For example, modified forms such as Ras, p53, c-myc, MUC-1, c-erbβ2 have been found to be associated with various cancers.

  Cancer-associated proteins are found in both tissues and body fluids of individuals who have precancerous or cancer cells. Their levels are very low during the early stages of the carcinogenesis process, but increase as the condition progresses. Although the detection of these proteins has been effectively used for diagnosing cancer in daily tests, unfortunately, these measurement reagents have various limitations. In particular, commercially available antibodies for standard testing are usually sensitive to the very early stages of the pathology where treatment is most effective, for example, to detect low levels of cancer-associated proteins as found in asymptomatic patients. Is insufficient. In addition, most commercially available antibodies are not specific for modified forms of the cancer-associated marker and cross-react with the wild-type of these proteins. Therefore, these commercially available antibodies were only useful in detecting substantial increases in serum levels of cancer marker proteins that typically occur at the stage of cancer progression.

  For example, a reagent for measuring a breast cancer marker, CA15-3 (carbohydrate antigen 15-3), detects both modified and unmodified forms of MUC1, and is characterized by elevated serum levels of MUC1. Useful for diagnosis. However, this measurement reagent cannot be used for screening for neoplastic or early-stage breast cancer, because the serum levels of MUC1 at these stages are not significantly different from those in healthy subjects (see, for example, Non-Patent Document 2). . Other cancer markers, such as CEA and CA 19.9 (carbohydrate antigen 19.9), have been reported to be elevated in the serum of patients with metastatic breast and colorectal cancer, but not in patients with early stage cancer (eg, , Non-Patent Documents 3 and 4). In addition, in the case of these cancer markers, commercially available measurement reagents cannot be used to discriminate between a modified protein and a wild-type protein, and thus have a limitation in use. Furthermore, commercially available antibodies may lead to false positive results by cross-reacting with normal forms of cancer-associated proteins. Thus, there is a need in the art for more sensitive and specific antibodies for use in these measurements to detect precancerous and early oncogenic changes, while at the same time raising the above-mentioned problems. It is desired to provide a new cancer-related protein that does not allow the cancer to be caused.

  In particular, breast cancer is a major cancer in women, and in Europe and the United States, it is said that one in ten women suffers from breast cancer. Although the number of breast cancer patients in Japan tended to be smaller than in the United States and Europe, the incidence of breast cancer in the Japanese population has rapidly increased due to the recent westernization of lifestyles, and since 1995 it has become the number one female cancer incidence. Currently, about 30,000 women suffer from breast cancer annually, and about 8,000 more die.

  For the diagnosis of breast cancer, palpation, ultrasonography, mammography (X-ray (radiography) dedicated to the mammary gland), cytology or histology have been used. However, the early detection of breast cancer has limitations in palpation and ultrasonography, while radiography requires expensive equipment and highly trained specialists to operate it and interpret contrast diagrams. In addition to this, there is a problem that the chance of X-ray exposure is increased. In addition, cytology and histology also require the collection of a sample at a specific facility, and have the problem that they are invasive methods that require specialized histological examination and interpretation.

As described above, in particular, the detection of cancer using a serum marker in serum is an excellent method that can be easily performed, and a novel cancer marker protein that can detect cancer with higher accuracy is required.
Poola I et al., J. Steroid Biochem. Mol. Biol., 82: 169-179 (2002). Robertson, et al., Eur. J. Cancer, 26: 1127-1132 (1990). Robertson et al., Cancer Immunol.Immunother., 133: 403-410 (1991) Thomas et al., Br. J. Cancer 63: 975-976 (1991) Payne, et al., Clin. Chem., 46: 175-182 (2000).

  An object of the present invention is to provide a novel protein, a DNA encoding the protein, an antibody that specifically binds to the protein, and the use thereof. In particular, the present invention relates to a novel cancer-related protein, a DNA encoding the protein, an antibody that specifically binds to the protein, an antisense oligonucleotide against a transcript of the DNA, and a diagnostic agent using the same. And other new applications.

  The present inventors have proposed the oligocap method (Maruyama, K., et al., Gene, 138: 171-174 (1994); Suzuki, Y. et al., Gene, 200: 149-156 (1997)). A database was searched for a full-length cDNA containing a splicing variant obtained using the novel splicing variant based on the homology of the nucleotide sequence of the cDNA clone, and a full-length cDNA presumed to encode a cancer-associated protein was selected. Among the cDNAs presumed to encode these cancer-related proteins, it was found that TBAES2007379 (SEQ ID NOs: 1 and 2) was specifically expressed in cancer, and the protein encoded by TBAES2007379 was It was identified as a protein. The present invention has been achieved based on these findings.

That is, according to the present invention, the following inventions 1 to 21 are provided.
1. Any one of the following proteins (a) to (f):
(A) a protein consisting of the amino acid sequence of SEQ ID NO: 2,
(B) a protein comprising an amino acid sequence in which one or several amino acids are deleted, substituted and / or added in the amino acid sequence of SEQ ID NO: 2, and whose expression is increased in cancer cells or cancer tissues,
(C) a protein consisting of the partial amino acid sequence of the amino acid sequence set forth in SEQ ID NO: 2 and having increased expression in cancer cells or cancer tissues;
(D) a protein comprising an amino acid sequence in which one or several amino acids have been deleted, substituted and / or added in the amino acid sequence of SEQ ID NO: 2, and having an EGF-like physiological activity,
(E) a protein comprising a partial amino acid sequence in the amino acid sequence of SEQ ID NO: 2 and having an EGF-like physiological activity,
(F) an amino acid sequence represented by SEQ ID NO: 2 in which one or several amino acids have been deleted, substituted and / or added, and at least the amino acid sequence represented by any of SEQ ID NOs: 10 to 12 Contains one protein.
2. 2. The protein according to item 1, wherein the cancer is breast cancer, colon cancer, lung cancer or pancreatic cancer.
3. 3. A DNA encoding the protein according to the above 1 or 2.
4. 3. A full-length cDNA encoding the protein according to the above 1 or 2.
5. Any one of the following DNAs (a) to (f);
(A) a DNA having the nucleotide sequence of SEQ ID NO: 1,
(B) encoding a protein having a nucleotide sequence in which one or several nucleotides are deleted, substituted and / or added in the nucleotide sequence of SEQ ID NO: 1, and whose expression is increased in cancer cells or cancer tissues DNA
(C) a DNA comprising a partial nucleotide sequence in the nucleotide sequence of SEQ ID NO: 1 and encoding a protein whose expression is increased in cancer cells or cancer tissues;
(D) a DNA having a base sequence in which one or several bases are deleted, substituted and / or added in the base sequence of SEQ ID NO: 1, and encoding a protein having an EGF-like physiological activity;
(E) a DNA consisting of a partial nucleotide sequence in the nucleotide sequence of SEQ ID NO: 1 and encoding a protein having an EGF-like physiological activity;
(F) the base sequence of SEQ ID NO: 1 having a base sequence in which one or several bases have been deleted, substituted and / or added, and the amino acid sequence of any of SEQ ID NOs: 10 to 12; DNA comprising at least one nucleotide sequence encoding
6. 6. The DNA according to the above item 5, wherein the cancer is breast cancer, colon cancer, lung cancer or pancreatic cancer.
7. A recombinant vector containing the DNA according to any one of the above items 3 to 6.
8. 7. A transgenic cell into which the DNA according to any one of the above items 3 to 6 or the recombinant vector according to the above item 7 has been introduced, or an individual comprising the cell.
9. 9. A protein produced by the cell according to the above item 8.
10. 7. A sense oligonucleotide having the same sequence as 5 to 100 consecutive nucleotides in the base sequence of the DNA according to any one of the above 3 to 6, an antisense oligonucleotide having a sequence complementary to the sense oligonucleotide, and Oligonucleotide derivatives of sense or antisense oligonucleotides, double-stranded oligonucleotides consisting of sense or antisense oligonucleotides having the same sequence as 15 to 30 consecutive bases in the base sequence, and the double-stranded oligonucleotides An oligonucleotide selected from the group consisting of oligonucleotide derivatives of oligonucleotides.
11. An antibody or a partial fragment thereof that specifically binds to the protein according to any one of the above items 1, 2 or 9.
12. 12. The antibody according to the above item 11, wherein the antibody has an action of neutralizing the activity of the protein according to any one of the above items 1, 2 or 9.
13. 10. A screening method for an activity-regulating substance for a protein, which comprises bringing the protein according to any one of the above items 1, 2 or 9 into contact with a test substance, and measuring a change in the activity of the protein caused by the test substance. Method.
14． Contacting a test substance with a cell that expresses the DNA encoding the protein according to the above 1 or 2 or the gene-transfected cell according to the above 8 and detecting a change in the expression level of the DNA introduced into the cell. A method for screening for a substance that regulates expression of said DNA, characterized in that:
15. At least one or more amino acid sequence information selected from the amino acid sequence of the protein according to the above 1 or 2 and / or at least one or more nucleotide sequence information selected from the DNA base sequence according to any of the above 3 to 6 A computer-readable recording medium storing a computer.
16. A carrier to which the protein according to any one of the above items 1, 2 or 9, the DNA according to any one of the above items 3 to 6, and / or the antibody according to the above item 11 or 12 are bound.
17. 7. A method for detecting cancer, comprising measuring the expression state of the DNA according to any one of Items 3 to 6 in a biological sample.
18. A method for detecting cancer, comprising immunologically measuring the protein according to the above 1 or 2 in a biological sample using the antibody or the partial fragment thereof according to the above 11 or 12.
19. A method for detecting cancer, which comprises immunologically measuring an autoantibody against the protein according to the above 1 or 2 in a biological sample using the protein according to any one of the above items 1, 2 or 9.
20. 20. The detection method according to any one of the above items 17 to 19, wherein the cancer is breast cancer, colon cancer, lung cancer or pancreatic cancer.
21. 17. Detection of cancer comprising at least one selected from the group consisting of the protein according to any one of the above items 1, 2 or 9, the antibody according to the above item 11 or 12, or the carrier according to the above item 16. Reagent kit for performing

  Since the protein encoded by the DNA of the present invention has increased expression in cancer cells or cancer tissues, various cancers using the protein, an antibody against the protein, DNA or RNA encoding the protein or a part thereof are used. For example, it is useful for developing diagnostic agents for breast cancer, colon cancer, lung cancer, pancreatic cancer and the like. In addition, it is useful for the development of pharmaceuticals that can act on diseases related to the protein. Further, gene therapy using a DNA encoding the protein, and in vivo using the siRNA, antisense RNA or DNA obtained from the DNA sequence encoding the protein or various aptamers, or the mRNA encoding the protein in vivo It can also be used as an expression inhibitor for.

  More specifically, the gene of the present invention selected and isolated from breast cancer tissue has a significantly increased expression in breast cancer cells, precancerous cells, or tissues containing these cells. Can be used for prevention, treatment or diagnosis of various cancers, for example, breast cancer.

  For example, when the protein encoded by the gene of the present invention is involved in the development and / or metastasis of breast cancer, a protein (eg, a ligand) that binds to the present protein in a metastatic tissue is useful for prevention or treatment of metastasis. It can be used. As will be described later, it is possible to isolate the ligand using the protein encoded by the gene of the present invention, and competitively inhibit the binding between the protein encoded by the gene of the present invention and the protein that binds thereto. The compound is a drug candidate compound for preventing the development and metastasis of breast cancer.

  The gene consisting of the nucleotide sequence provided by the present invention can be said to be particularly closely related to the development of breast cancer. Therefore, it is considered that breast cancer diagnosis and treatment can be achieved by regulating the expression of this gene and the action of the protein encoded by this gene.

  That is, by inhibiting the expression of the gene of the present invention in a living body, the occurrence, progress and metastasis of breast cancer can be effectively suppressed. Alternatively, suppression of breast cancer is also achieved by inhibiting the function of the protein of the present invention. In order to inhibit the expression of the gene, the expression can be inhibited by a decoy nucleic acid after elucidating an antisense nucleic acid drug or a transcription regulatory region thereof. In order to inhibit the function of the protein itself, it is effective to change the tertiary structure of the active site by administering a compound that binds to the protein, or to prevent the protein from binding to its target compound.

  Furthermore, a cancer vaccine can be developed using the protein of the present invention. That is, if an immune response to the protein encoded by the gene of the present invention or a fragment thereof can be induced, the immunological elimination mechanism for breast cancer can be strengthened. Such an immune response is caused by administering the protein of the present invention or a fragment thereof. Administration of a protein into a living body can be achieved by administering the protein and introducing and expressing a gene encoding the protein. The necessary gene can be introduced using an adenovirus vector or a retrovirus vector based on a known method.

  Furthermore, the gene of the present invention may play a similar role in cancers other than breast cancer, and can be used for prevention or treatment of cancer or prediction of malignancy, similarly to breast cancer. That is, the gene “TBAES2007379” having the sequence represented by SEQ ID NO: 1 (the amino acid sequence is SEQ ID NO: 2) is not only used in breast cancer cells, but also in colon cancer cells, lung cancer cells, pancreatic cancer cells, or tissues containing these cancer cells. Expression was found to be significantly increased.

  Therefore, it is of great significance to provide a full-length cDNA of a novel cancer-associated protein containing a splicing variant that has not been isolated in humans, and can be used for the development of a diagnostic or pharmaceutical for various diseases in which this gene is involved. In addition, the protein encoded by this gene or an antibody against the protein can be expected to be useful as a diagnostic or pharmaceutical. Therefore, obtaining the full length of the cDNA encoding the novel human protein is of great significance.

Hereinafter, the present invention will be described in more detail. However, these descriptions are merely examples (representative examples) of the embodiments of the present invention, and do not limit the scope of the present invention.
(1) Acquisition of full-length cDNA The DNA of the present invention comprises a protein consisting of the amino acid sequence of SEQ ID NO: 2 or one or several amino acids in the amino acid sequence of SEQ ID NO: 2 (here, several means, for example, 5 or less, preferably 3 or less, more preferably 2 or less) amino acid residue substitution, deletion and / or addition, and increased expression in cancer cells or cancer tissues. Any protein can be used as long as it can encode a protein to be expressed. Specifically, it may be only the translation region encoding the amino acid sequence (hereinafter sometimes referred to as “open reading frame” or “ORF”), or may include the full length of the cDNA.

  More specifically, examples of the DNA containing the full-length cDNA include a DNA having the nucleotide sequence of SEQ ID NO: 1. Examples of the translation region include those having a sequence represented by base numbers 70 to 2118 (including a stop codon) of SEQ ID NO: 1. Further, the DNA of the present invention includes not only the full length of the above-mentioned cDNA but also those containing the above-mentioned translation region and a portion necessary for the expression of the translation region adjacent to the 3 ′ and / or 5 ′ end thereof. .

  The DNA of the present invention may be obtained by any method as long as it can be obtained, and specifically, for example, can be obtained by the method described below. First, mRNA is prepared from a human tissue or a cultured cell or the like by a method known per se and generally used. Next, cDNA is obtained by oligo cap method (Maruyama, K., et al., Gene, 138: 171-174 (1994)) using this mRNA as a template. Specifically, the 5 'cap is removed from the obtained mRNA by acid pyrophosphatase, and then an oligocap linker is ligated to the exposed 5' terminal phosphate group using RNA ligase. Here, for an RNA molecule not having a cap structure at the 5 ′ end, a phosphate group existing at the 5 ′ end is not removed in advance but the 5 ′ end is removed so that the oligocap linker does not bind. It is effective to remove by using a phosphatase having the activity of removing only the phosphate group of the above. Using this RNA molecule as a template, reverse transcription is performed with a reverse transcriptase using an oligo dT primer as a 3'-side primer, and then the RNA strand is degraded and removed.

  Furthermore, using the obtained single-stranded DNA as a template, an oligonucleotide having a partial sequence of the above oligocap linker as a 5 ′ primer, and using a primer specific to the 3 ′ end (oligo dT primer or the like) for polymerase chain reaction ( PCR), a full-length cDNA library can be prepared. Here, the 5 'primer and the 3' primer are not complementary to the full length of the synthetic oligonucleotide and the reverse transcription primer, and it is preferable to use sequences shifted by 3 to 10 bases to the 3 'side. The chain length of the primer is usually 15 to 100 bases, preferably 15 to 30 bases. When the cDNA to be amplified has a long chain length, the length is preferably 25 to 35 bases. and Accurate PCR (LA PCR: Takeshi Hayashi, Experimental Medicine Separate Volume / Latest PCR Technology, Yodosha; Cheng, S. et al., Nature 369: 684-685 (1994)).

  The thus obtained cDNA is cloned by inserting it into an appropriate cloning vector. As the vector used herein, a protein expression vector that can introduce the obtained cDNA clone into cells and express the protein encoded by the cDNA is preferably used. Specifically, for example, when the host is a mammalian cell or the like, pME18SFL3 (Genbank AB009864) or the like is preferable. In the case of yeast, pESP-I expression vector (manufactured by Stratagene) and the like are used. In the case of insect cells, BacPAK6 (manufactured by Clontech) is used. When the host is an animal cell, examples include ZAP Express (manufactured by Stratagene) and pSVK3 (manufactured by Amersham Pharmacia Biotech).

  The nucleotide sequence of the thus obtained cDNA library is analyzed by a commonly used method known per se. The DNA of the present invention analyzes the base sequence at the 5 'end or 3' end of the obtained cDNA and converts it to NCBI (National Center for Biotechnology Information; http://www.ncbi.nlm.nih.gov/). A base sequence database such as Genbank, EMBL, DDBJ, dbEST, etc., used in BLAST (Basic local alignment search tool; Altschul, SF, et al., J. Mol. Biol., 215, 403-410 (1990)) , And when a sequence completely matching the full length was not found, it was decided to newly provide the following analysis.

  The clone whose cDNA sequence is considered to be novel as described above, that is, a DNA having the nucleotide sequence of full-length cDNA includes, for example, a DNA having the nucleotide sequence of SEQ ID NO: 1, and the like. The amino acid sequence shown is that shown in SEQ ID NO: 2. Furthermore, specific examples of the translation region include those having a sequence represented by base numbers 70 to 2118 (including a termination codon) of SEQ ID NO: 1. Further, the DNA of the present invention includes not only the full length of the above-mentioned cDNA but also those containing the above-mentioned translation region and a portion necessary for the expression of the translation region adjacent to the 3 ′ and / or 5 ′ end thereof. .

  Further, the DNA of the present invention may be obtained by the above-described method or may be synthesized. The DNA base sequence can be easily replaced with a commercially available kit such as a site-directed mutagenesis kit (Takara Shuzo) or a quick change site-directed mutagenesis kit (Stratagene).

  Thus obtained, the base sequence is determined, and the DNA of the present invention whose function is estimated is not limited to the base sequence described in SEQ ID NO: 1 or the one having the base sequence shown above as a translation region thereof, In these base sequences, one or several bases (here, several means, for example, 15 or less, preferably 9 or less, more preferably 6 or less) bases are deleted, substituted and / or substituted. Alternatively, it also includes a DNA having an added base sequence and encoding a protein whose expression is increased in cancer cells or cancer tissues. These DNAs include a protein comprising an amino acid sequence in which one or several amino acid sequences are deleted, substituted, and / or added to the amino acid sequence of SEQ ID NO: 2, and which further increases the expression in cancer cells or cancer tissues. Includes code.

(2) Analysis of base sequence and amino acid sequence of full-length cDNA Genbank (http://www.ncbi.nlm.nih.gov/web/GenBank), EMBL (http://www.ebi.ac.uk/embl) /index.html), base sequence databases such as DDBJ (http://www.ddbj.nig.ac.jp/), dbEST (http://www.ncbi.nlm.nih.gov/dbEST/) A new nucleotide sequence as a full length cDNA obtained by performing a homology search (homology search) using BLAST was further analyzed as a detailed analysis of the sequence by using the NRDB protein database (SWISS-PROT (http: //www.ebi). .ac.uk / ebi_docsSwissProt_db / swisshome.html), PIR (http://pir.georgetown.edu/pirwww/pirhome.shtml), TREMBL (http://kr.expasy.org/sprot/sprot-search.html) ), GENPEPT (ftp://ftp.ncbi.nih.gov/genbank), PDB (http://www.rcsb.org/pdb/index.html), PRF (http: //www.prf.or. jp / en /)) By performing BLAST by homologous search like the amino acid sequence database were included in the database) such as the sequence, it is possible to estimate the function of the protein which the base sequence codes. In the homology search by BLAST, the function of the clone to be analyzed can be estimated from various kinds of annotation information associated with hit sequences having sufficiently significant homology obtained as a result of the search. A specific example of the function prediction is described below.

  The DNA having the nucleotide sequence of SEQ ID NO: 1 (TBAES2007379) consists of 3119 bases, of which base numbers 70 to 2118 are an open reading frame (including a stop codon). The amino acid sequence predicted from the open reading frame consists of 682 amino acid residues (SEQ ID NO: 2). In TBAES2007379, four exons among 22 exons covering the ORF region of the registration symbol NM020974 (SCUBE2 (Signal peptide-CUB-EGF-like domain containing protein 2)) of the sequence database (RefSeq) are deleted. It is a splicing variant and the sequence is novel (see FIG. 2). Although NM020974 was a variant called “SCUBE2a” of SCUBE2 in detail, it is known that SCUBE2a exists most widely among SCUBE2, and is often abbreviated as SCUBE2. , This is referred to as SCUBE2.

  In the case of preliminary homology search prior to detailed analysis of the sequence, GenBank, Swiss-Prot, UniGene, NRDB, RefSeq (http://www.ncbi.nlm.nih.gov/LocusLink/refseq.html) Estimating the function of the protein encoded by the cDNA of the present invention by performing a homology search on each database using BLAST, FASTA, or the like, and referring to the functions of the hit gene and the protein encoded thereby. Can be. In the prediction from the structure, the amino acid sequence deduced from the entire nucleotide sequence is compared with the PSORT (K. Nakai & M. Kanehisa, Genomics, 14: 897-911 (1992)) for the prediction of the signal sequence and the transmembrane region. )), SOSUI (T. Hirokawa et.al. Bioimformatics, 14: 378-379 (1998), sold by Mitsui Information & Development Co., Ltd.), MEMSAT (DTJones, WRTaylor & JMThornton, Biochemistry, 33: 3038-3049 ( 1994)), and for the prediction of motifs and domains, search for Pfam and PROSITE (http://www.expasy.ch/prosite/), etc., to find out more about the proteins encoded in the clones. Function prediction is possible.

  In this way, for TBAES2007379 (SEQ ID NO: 1), which has been found to have a novel nucleotide sequence, homology search can be performed for each database of GenBank, Swiss-Prot, NRDB, and RefSeq (implementation). Examples 3 and 5). In addition, a signal sequence using PSORT and SOSUI and a transmembrane region can be searched for the amino acid sequence deduced from the full-length nucleotide sequence (see Example 4).

If the Swiss-Prot hit data and the NRDB and RefSeq hit data are genes and proteins registered in the Online Mendelian Inheritance in Man (OMIM), which is a database of human genes and diseases, it is estimated to be disease-related proteins. (See Example 5). In this manner, the function prediction based on the annotation (for the hit data of Swiss-Prot, refer to the keyword; for the hit data of NRDB, RefSeq, refer to Definition and Reference information), and the PSORT for the estimated ORF In addition to the results of the signal sequence search using, the transmembrane region search using SOSUI, and the OMIM registration, 14 functional categories (secretion / membrane proteins, glycoprotein-related proteins, signal transduction-related proteins, transcription-related proteins, Disease-related proteins, enzymes / metabolism-related proteins, cell division / proliferation-related proteins, cytoskeleton-related proteins, nucleoprotein / RNA synthesis-related proteins, protein synthesis / transport-related proteins, cell defense-related proteins, development / differentiation-related proteins, DNA / RNA binding protein, ATP / GTP binding protein) Can be classified.
Specifically, it can be estimated that the protein having the amino acid sequence of SEQ ID NO: 2 belongs to secretory / membrane proteins.

(3) Analysis of the amino acid sequence motif and domain of the protein encoded by the novel cDNA The entire structure of the protein is composed of a collection of minimal structures such as motifs and domains. It is conceivable that the present invention focuses on the common motif and domain structure of the minimum unit of various proteins with respect to the amino acid sequence of the protein encoded by the novel full-length cDNA whose entire nucleotide sequence has been revealed in the present invention. The protein of the present invention can be analyzed to analyze common motifs, domain structures or common motifs, and domain structure functions. Specifically, a protein feature search (profile search: http: // pfam) using HMMPFAM, which is one of the functions of HMMER (sequence analysis method using hidden Markov model; Eddy, SR, Bioinformatics 14: 755-763 (1998)) By performing the analysis of domains and motifs, it is possible to predict, at the molecular level, what kind of function the protein plays in the cell as a whole (Example 4). And 6). HMMPFAM is an analysis by a method in which the characteristics of the amino acid sequence of an entry in a database in which a protein profile called Pfam is accumulated are checked to determine whether or not the amino acid sequence encoded by the base sequence to be analyzed has. The profile is extracted from a series of proteins having the same characteristic. Even if the function cannot be clarified by comparing the entire sequence of one sequence to one sequence, if the characteristic region is present in the sequence, the function can be identified and the function can be predicted. As described above, a cDNA whose protein is predicted to have a specific activity can be confirmed for the specific activity by a biochemical experiment described later.

  For example, by finding a signal sequence, a transmembrane region, a nuclear translocation signal, a glycosylation signal, a phosphorylation site, a Zinc finger motif, an SH3 domain, and the like in the amino acid sequence, the protein encoded by the cDNA of the present invention can be identified. A common function estimation can be attempted. In particular, the structure of motifs, domains, etc. is a partial sequence structure commonly found in several proteins, which is the minimum functional structure of the protein. In Pfam (http://www.sanger.ac.uk/Software/Pfam/index.shtml), 4832 types have been identified and stored in a database.

  Further, with respect to a clone having a novel full-length cDNA whose entire nucleotide sequence has been revealed in the present invention, the Pfam of the deduced amino acid sequence (http://www.sanger.ac.uk/Software/Pfam/index.shtml) ), Using the domain, motif name, and accession number of the hit data obtained from the result of the domain search (see Example 6) on the Pfam site or InterPro (http://www.ebi.ac.uk/interpro /), Detailed description of each domain and motif at each link such as PROSITE (http://www.expasy.ch/cgi-bin/prosite-list.pl), and unique functional category classification especially for PROSITE Can be referred to. In this way, the functions of the proteins encoded in the clones hit by Pfam are predicted, and 14 functional categories (secretion / membrane proteins, glycoprotein-related proteins, signal transduction-related proteins, transcription-related proteins, disease-related proteins) , Enzymes / metabolism-related proteins, cell division / proliferation-related proteins, cytoskeleton-related proteins, nucleoprotein / RNA synthesis-related proteins, protein synthesis / transport-related proteins, cell defense-related proteins, development / differentiation-related proteins, DNA / RNA binding proteins , ATP / GTP binding protein).

  The amino acid sequence of SEQ ID NO: 2 has a domain called EGF-like (EGF-like domain) and a repeat of the EGF-like domain (EGF-like repeat). Based on the properties of the domain and the repeat, it is considered that the protein having the amino acid sequence of SEQ ID NO: 2 has an EGF-like physiological activity, that is, the same physiological activity as the protein having the EGF-like domain or the EGF-like repeat. . The amino acid sequence set forth in SEQ ID NO: 2 has a cysteine-rich motif and domain characteristic of a membrane protein, such as a trypsin inhibitor-like cysteine-rich domain, a keratin (high sulfur B2 protein) motif, and a granulin motif.

  In addition, since the function of a motif or a domain does not necessarily belong to only one of the function categories described above, the function may correspond to any of the predicted function categories. In addition, even if there is no hit data in Pfam, if a new domain or motif is found together with accumulation of protein data in the future, the deduced amino acid sequence of the clone is again analyzed by a new database to obtain a new one. Domains and motifs with functions may be discovered and categorized.

(4) The protein encoded by the cDNA of the present invention The translation region of the protein encoded by the DNA of the present invention is, for example, the nucleotide sequence of the DNA which is converted into amino acids in three reading frames and the longest polypeptide Can be deduced as the translation region of the protein of the present invention using the range encoding Examples of such an amino acid sequence include those described in SEQ ID NO: 2. Further, the protein of the present invention is not limited to the above-mentioned amino acid sequence, but consists of an amino acid sequence in which one or several amino acids have been substituted, deleted, and / or added in the amino acid sequence, and has a cancer cell or Also included are those whose expression is increased in cancer tissues.

  As a method for obtaining the protein of the present invention, the method of transcription / translation of the DNA of the present invention described in (1) by an appropriate method is preferably used. Specifically, a recombinant vector inserted with a suitable expression vector or a suitable vector together with a suitable promoter is prepared, and a suitable host microorganism is transformed with the recombinant vector or introduced into a suitable cultured cell. Thus, it can be obtained by purifying this.

  The protein of the present invention also includes a protein which is tagged with a tag which is inserted into an N-terminus or C-terminus of a suitable tag and the like. Specific examples of the tag include glutathione-S-transferase, polyhistidine, Flag, and Fc region of human IgG.

  The protein produced by the above transformant can be modified by incorporating amino acids substituted or modified with heavy atoms or the like during protein synthesis. In addition, the protein can be converted into a modified protein by arbitrarily modifying the protein or partially removing the polypeptide by applying an appropriate protein modifying enzyme before or after purification. For example, N-terminal acetylation, terminal modification such as C-terminal amidation, sugar chain addition, lipid addition, acylation, methylation, sulfonation, carboxylation, hydroxylation, phosphorylation, ADP-ribosylation, etc. It is not necessarily limited to these. These modified proteins are also included in the scope of the present invention as long as they are functionally equivalent to the protein of the present invention. Whether a protein is functionally equivalent to the protein of the present invention is determined by examining whether or not the protein has the activity by using the biological activity of the protein of the present invention, such as antigenicity, as an index. Can be confirmed by

  In addition, the protein produced by the transformant may be arbitrarily modified or partially removed by treating with a suitable protein modifying enzyme before or after purification to obtain a modified protein. Can be. As described above, these modified proteins are also included in the scope of the present invention as long as they are functionally equivalent to the protein of the present invention.

  When producing the protein of the present invention, the vector used for the production of the recombinant vector containing the DNA of the present invention is not particularly limited as long as the DNA is expressed in the transformant. Any of vectors may be used. Usually, a commercially available protein expression vector into which an expression control region DNA such as a promoter suitable for a host into which the DNA is introduced has already been inserted is used. Specific examples of such protein expression vectors include pET3, pET11 (manufactured by Stratagene) and pGEX (manufactured by Amersham Pharmacia Biotech) when the host is Escherichia coli, and pESP when the host is yeast. -I expression vector (Stratagene) and the like. In the case of insect cells, BacPAK6 (Clontech) and the like are used. When the host is an animal cell, examples include ZAP Express (manufactured by Stratagene) and pSVK3 (manufactured by Amersham Pharmacia Biotech). When the host is a mammalian cell or the like, pME18SFL3 (Genbank AB009864) is preferable.

  When using a vector into which the expression control region has not been inserted, it is necessary to insert at least a promoter as the expression control region. Here, as the promoter, a promoter possessed by a host microorganism or a cultured cell can be used, but is not limited thereto. Specifically, for example, when the host is E. coli, T3, T7, tac, lac A promoter and the like can be used. In the case of yeast, an nmt1 promoter, a Gal1 promoter and the like can be used. In the case of insect cells, a polyhedrin promoter or the like can be used. When the host is an animal cell, SV40 promoter, CMV promoter and the like are preferably used.

  When a host capable of functioning with a mammalian-derived promoter is used, a promoter specific to the gene of the present invention can also be used. Insertion of the DNA of the present invention into these vectors may be carried out by connecting the DNA or a DNA fragment containing the DNA to the amino acid sequence of the protein encoded by the gene DNA downstream of the promoter in the vector.

  The recombinant vector thus prepared can be used to transform a host described below by a method known per se to prepare a DNA transductant. As a method for introducing the vector into a host, specifically, a heat shock method (J. Mol. Biol., 53: 154 (1970)), a calcium phosphate method (Science, 221: 551 (1983)), a DEAE dextran method (Science, 215: 166 (1982)), in vitro packaging method (Proc. Natl. Acad. Sci. USA, 72: 581 (1975)), viral vector method (Cell, 37: 1053 (1984)), and electrical Pulse method (Chu. Et al., Nuc. Acids Res., 15: 1331 (1987)) and the like.

  The host for preparing the DNA transfectant is not particularly limited as long as the DNA of the present invention is expressed in the body. For example, Escherichia coli, yeast, baculovirus (arthropod polyhedrosis virus) -insect cells, or Animal cells and the like can be mentioned. Specifically, BL21 and XL-2Blue (manufactured by Stratagene) for Escherichia coli, SP-Q01 (manufactured by Stratagene) and the like for yeast, and AcNPV (J. Biol. Chem., 263: 7406 (1988) for baculovirus )) And its host Sf9 (ATCC CRL-1711; J. Biol. Chem., 263: 7406 (1988)). As animal cells, mouse fibroblast cell line C127 (ATCC CRL-1804; J. Viol., 26: 291 (1978)) and Chinese hamster ovary cell line CHO-K1 (ATCC CCL-61; Proc. Natl. Acad. Sci. USA, 77: 4216 (1980)), etc., and preferably African green monkey kidney-derived cell line COS-7 (ATCC CRL1651: American Type Culture Collection preserved cell), human adeno, in terms of expression level and simplicity of screening. A human embryonic kidney-derived cell line HEK293 (ATCC CRL1573; hereinafter sometimes referred to as “HEK293 cell”) transformed with the virus type 5 or a human cervical cancer-derived cell line HeLa (ATCC CCL-2; hereinafter, “ HeLa cells ").

  In addition to the above-described expression method using a protein expression vector, a homologous recombination technique (Vertes, AA et al., Biosci. Biotechnol) in which a DNA fragment of the present invention linked to a promoter is directly inserted into a chromosome of a host microorganism. Biochem., 57: 2036 (1993)), or a transposon or an insertion sequence (Vertes, AA et al., Molecular Microbiol., 11: 739 (1994)) can be used to prepare a DNA transductant.

  The culture obtained above is obtained by collecting cells or cells by a method such as centrifugation, suspending the cells or cells in a suitable buffer, and sonication, lysozyme, and / or freeze-thaw. After disruption by the method, a crude protein solution is obtained by centrifugation, filtration, or the like, and can be further purified by combining an appropriate purification method.

Thus, the protein of the present invention is obtained. In addition to the above-mentioned expression method using the protein expression recombinant vector, the DNA of the present invention obtained in the above (1) is subjected to a cell-free transcription / translation system to induce protein expression to obtain the protein of the present invention. be able to. The cell-free transcription / translation system (or also referred to as "cell-free protein synthesis system") used in the present invention is a system containing all elements necessary for transcription from DNA to mRNA and translation from mRNA to protein. And any system in which the protein encoded by the DNA is synthesized by adding the DNA thereto. Specific examples of the cell-free transcription / translation system include transcription / translation systems prepared based on extracts from eukaryotic cells and bacterial cells, or a part thereof. Specific examples of the cell-free protein synthesis system include known ones such as Escherichia coli, plant seed germ, cell extract of rabbit reticulocytes and the like. These can be used commercially, or a method known per se, specifically, an E. coli extract can be obtained from Pratt, JM et al., Transcription and Translation, Hames, 179-209, BD & Higgins, SJ, eds , IRL Press, Oxford (1984). As a commercially available cell-free protein synthesis system or cell extract, those derived from Escherichia coli include E. coli S30 extract system (Promega) and RTS500 Rapid Translation System (Roche), and rabbit reticulocytes. Those derived from Rabbit Reticulocyte Lysate System (Promega) and those derived from wheat germ include PROTEIOS ^™ (TOYOBO).

  Separation and purification of the protein of the present invention from the obtained transcription-translation product of the cell-free transcription / translation system can be carried out by a commonly used method known per se. Specifically, for example, a DNA region encoding an epitope peptide, a polyhistidine peptide, glutathione-S-transferase (GST), a maltose binding protein, or the like is introduced into the DNA to be transcribed and translated, and expressed as described above. The protein can be purified by utilizing the affinity of the protein with a substance having an affinity.

  The expression of the target protein is separated by SDS-polyacrylamide gel electrophoresis or the like, and stained with Coomassie brilliant blue (manufactured by Sigma) or detected by an antibody that specifically binds to the protein of the present invention described later. It can be confirmed by the method of performing. In general, it is known that an expressed protein is cleaved (processed) by a proteolytic enzyme present in a living body. Naturally, even the partial fragment of the amino acid sequence thus cleaved is included in the protein of the present invention as long as it is functionally equivalent to the protein of the present invention.

(5) Confirmation of the activity possessed by the protein of the present invention The protein of the present invention is prepared as a recombinant protein as described in (4) above, and is analyzed to analyze the protein to be estimated in (2) or (3). Can be confirmed. Further, as described in the following (6), it can be confirmed by introducing the DNA of the present invention into a certain kind of cell and analyzing a change in its phenotype. Furthermore, analysis can also be performed in combination with the antibody or the like described in (9) below.

The fact that the protein of the present invention has, for example, an EGF-like physiological activity can be confirmed by a conventional method known per se. For example, cell proliferation action, gastric acid secretion suppression action, anti-ulcer action, gastrointestinal mucosa protection action, DNA synthesis promotion action, corneal repair action, calcium release promotion action, wound healing promotion action, anti-inflammatory action, analgesic action, hepatocellular injury Physiological activity of EGF such as inhibitory action and hair follicle retraction action (edited by the Japanese Society for Tissue Culture, “Cell Growth Factor”, Asakura Shoten, 1984) can be used as an index. More specifically, there is a method of culturing cells by adding the protein to a medium and measuring cell proliferation by absorbance with a spectrophotometer or the like or uptake of ³ H-thymidine. The cells may be germline or somatic, totipotent or pluripotent, divided or undivided, parenchymal or epithelial, immortalized or transformed, etc., with epithelial cells being preferred. Here, epithelial cells are cells that cover the free surface of an organ (keratinous, mucosal or membranous), or that are located in a cellular, non-vascular layer that lines a duct or body cavity of the animal body. Means Specific examples include human mammary epithelial cells, normal human skin epithelial cells (such as NHEK cells (Normal human epidermal keratinocytes)), human vascular smooth muscle cells, and human retinal pigment epithelial cells.

  Such an analysis system for EGF-like bioactivity can also be used for evaluating agonists and antagonists of the protein having EGF-like bioactivity of the present invention. Note that confirmation of the activity of the protein of the present invention is not limited to the method described above.

(6) Functional analysis of the protein of the present invention The protein of the present invention, which is encoded by the full-length cDNA thus obtained and has an EGF-like physiological activity or the like, has its function further determined by the method described below. The analysis provides a new use for it. In particular, since the protein of the present invention is a splicing variant of a known protein, it is important to identify what functions this variant has with the known variant.

  Specific methods for analyzing functions include, for example, (i) a method for comparing and analyzing the expression state in each tissue, disease, or developmental stage, (ii) a method for analyzing the interaction with other proteins and DNA, ( iii) a method of analyzing a phenotypic change by introducing into a suitable cell or individual, and (iv) a method of analyzing a phenotypic change by inhibiting the expression of the protein in a suitable cell or individual.

  In the method (i), the expression of the protein of the present invention can be analyzed at the mRNA level or the protein level. When the expression level is analyzed at the mRNA level, for example, in situ hybridization (Application to Developmental Biology & Medicine., Ed. by Harris, N. and Wilkinson, DG), Cambridge University Press (1990) ), A hybridization method using a DNA chip, a quantitative PCR method, and the like. Here, in the case of a splicing variant in which a known variant exists in the protein to be analyzed, a probe that is present only in the cDNA encoding the protein to be analyzed and does not hybridize with the cDNA encoding the known variant should be used. Is preferred. In the case of the quantitative PCR method, there is a method of selecting a primer capable of producing an amplified fragment having a different length between the target variant and the known variant (Wong, YW, Neuroscience Lett., 320: 141-145 (2002)) and the like. No. In the case of analyzing at the protein level, a tissue staining method using an antibody that specifically binds to the protein of the present invention, which will be described later, may be mentioned. In this case, it is preferable to use an antibody that reacts only with the target protein and does not react with a known variant.

  As a method for analyzing the interaction (ii), a conventional method known per se can be used. Specifically, for example, yeast two-hybrid method, fluorescence depolarization method, surface plasmon method, phage display method, Ribosomal display method and the like can be mentioned. Also in this method, when the protein to be analyzed is a splicing variant in which a known variant is present, the known variant is similarly analyzed for interacting substances, and a substance that specifically interacts with the target protein is identified. Is preferred.

  In the method (iii), the cells into which the cDNA of the present invention is introduced are not particularly limited, but human cultured cells and the like are particularly preferably used. Methods for introducing DNA into cells include those described in (2) above. In addition, the phenotype of the introduced cells can be observed under a microscope, such as cell viability, cell growth rate, cell differentiation, neurite outgrowth when cells are neurons, localization and migration of intracellular proteins, etc. And those that can be analyzed by biochemical experiments, such as changes in the expression of specific proteins in cells. In the case of a splicing variant in which a known variant exists, these phenotypes can be similarly introduced into cells, and a phenotype related to the variant to be analyzed can be identified by comparative analysis. In addition, since the protein of the present invention is known to have increased expression in cancer cells or cancer tissues and / or to have an EGF-like physiological activity, it has a phenotype such as that found in cancer and EGF-related diseases. It is also preferable to focus attention on analysis.

  In the present invention, the fact that a certain gene causes canceration can be confirmed by observing the canceration of a host cell due to the transformation of the gene. Alternatively, the occurrence of malignancy can be confirmed by using, as an index, the fact that cells acquire metastatic potential when the gene is transformed into a cancer cell line having no metastatic potential.

  Since the protein encoded by the DNA of the present invention has a full-length amino acid sequence, as described above, the protein can be expressed as a recombinant by applying an appropriate expression system, or injected into cells, or By preparing and using an antibody that specifically recognizes, it is possible to analyze its biological activity and its effect on cell state changes such as cell proliferation and differentiation.

Each protein can be analyzed for the biological activity of each protein based on the method described in the following literature.
Secreted proteins, membrane proteins:
`` The Practical Approach Series '' (IRL PRESS), `` Ion Channels '' (ed by RHAshley, 1995),
Growth Factors (I. McKay, I. Leigh, 1993), Extracellular Matrix (MAHaralson, JRHassell, 1995)
Glycoprotein-related proteins:
`` Glycobiology '' (edited by M. Fukuda, A. Kobata, 1993) in `` The Practical Approach Series '' (IRL PRESS),
`` Glycoprotein Analysis in Biomedicine '' of the `` Method in Molecular Biology '' (Humana Press) series (Elizabeth F. Hounsell, 1993),
Signaling-related proteins:
`` Signal Transduction '' of The Practical Approach Series (IRL PRESS) (edited by G. Milligan, 1992),
`` Protein Phosphorylation '' (DG Hardie ed., 1993), or `` Method in Molecular Biology '' (Humana Press) series `` Signal Transduction Protocols '' (David A. Kendall, Stephen J. Hill ed., 1995),
Transcription-related proteins:
`` Gene Transcription '' (BD Hames, SJ Higgins, 1993) of `` The Practical Approach Series '' (IRL PRESS),
Transcription Factors (DSLatchman, 1993),
Enzyme and metabolism-related proteins:
`` The Practical Approach Series '' (IRL PRESS), `` Enzyme Assays '' (ROBERT EISENTHAL and MICHAEL J. DANSON, 1992),
Cell division / proliferation-related proteins:
`` Cell Growth, Differentiation and Senescence '' of `` The Practical Approach Series '' (IRL PRESS) (GEORGE STUDZINSKI, 2000),
Cytoskeleton-related proteins:
`` Cytoskeleton: Signaling and Cell Regulation '' of `` The Practical Approach Series '' (IRL PRESS) (KERMIT L. CARRAWAY and CAROLIE A. CAROTHERS CARRAWAY, 2000),
`` Cytoskeleton Methods and Protocols '' in the `` Method in Molecular Biology '' (Humana Press) series (Gavin, Ray H. ed., 2000),
Nucleoprotein / RNA synthesis-related proteins:
`` Nuclear Receptors '' (DIDIER PICARD, 1999) of `` The Practical Approach Series '' (IRL PRESS),
`` RNA Processing '' (STEPHEN J. HIGGINS and B. DAVID HAMES, 1994),
Protein synthesis / transport-related proteins:
"The Practical Approach Series" (IRL PRESS) "Membrane Transport" (STEPHEN A. BALDWIN, 2000),
`` Protein Synthesis Methods and Protocols '' of the `` Method in Molecular Biology '' (Humana Press) series (Martin, Robin ed., 1998),
Cell defense-related proteins:
`` Method in Molecular Biology '' (Humana Press) series `` DNA Repair Protocols '' (Henderson, Daryl S., 1999),
`` Chaperonin Protocols '' (Schneider, Christine ed., 2000),
Developmental / differentiation related proteins:
`` Developmental Biology Protocols '' of the `` Method in Molecular Biology '' (Humana Press) series (ROBERT EISENTHAL and MICHAEL J. DANSON, 1992),
DNA / RNA binding proteins:
`` Method in Molecular Biology '' (Humana Press) series `` DNA-Protein Interactions Principles and Protocols '' (Kneale, G. Geoff ed., 1994),
`` RNA-Protein Interaction Protocols '' (Haynes, Susan R. ed., 1999),
ATP / GTP binding protein:
"Signal Transduction Protocols" in the "Method in Molecular Biology" (Humana Press) series (ed. David A. Kendall, Stephen J. Hill, 1995).

  For other methods, protein activity can be analyzed with reference to Methods in Enzymology (Academic Press).

  The method (iv) can be efficiently performed by a method using an oligonucleotide described later or an RNA interference method. In this method, when the target protein to be analyzed is a splicing variant in which a known variant is present, a similar analysis is performed for the known variant and other variants, and the target protein-specific protein is analyzed by comparative analysis. Function can be identified.

(7) Analysis of association between DNA and disease of the present invention (7-1) Analysis using OMIM As described above, disease-related proteins can be analyzed for each function, and can express disease-related proteins. Using the specific recognition antibody thus obtained, the correlation between a specific disease and the expression level or activity of the protein can be known. Alternatively, analysis can be performed using Online Mendelian Inheritance in Man (OMIM), which is a database of human genes and diseases (see Example 5). Note that new information is always added to the OMIM. Therefore, those skilled in the art can find new relationships between specific diseases and the genes of the present invention from the latest databases. The disease-related protein is useful for the development of pharmaceuticals, such as a diagnostic marker, a drug for controlling the increase or decrease of expression or activity, or a target for gene therapy.

  OMIM is also used for not only proteins in the above 14 categories, but also proteins having various functions, including secretory proteins, membrane proteins, signal transduction-related proteins, glycoprotein-related proteins, and transcription-related proteins. When searching by keywords, results related to many diseases are obtained for each keyword. Alternatively, for example, regarding transcription-related proteins and signal transduction-related proteins, the association with disease is respectively related to Experimental Medicine, edited by Fujii, Tamura, Morohashi, Kageyama, Satake, `` Transcription Factor Research 1999 '' Vol.17, No.3, ( 1999) and Gene Medicine Vol. 3, No. 2 (1999). For example, in the case of cancer, as described in Shokabo Life Science Series, "Cancelology of Biology" (Satoshi Matsubara, 1992), cancer includes secreted proteins, membrane proteins, signal transduction-related proteins, glycoproteins, etc. It has been shown that not only related proteins and transcription-related proteins but also many proteins such as enzymes / metabolism-related proteins, cytoskeleton-related proteins, and cell division / proliferation-related proteins are involved. In this way, not only disease-related proteins but also secretory proteins, membrane proteins, signal transduction-related proteins, glycoprotein-related proteins, transcription-related proteins, etc. are often involved in diseases, and are useful as targets in the medical industry. I understand.

(7-2) Prediction of function and association with disease by expression analysis of DNA of the present invention Using the nucleotide sequence of the cDNA of the present invention, the expression frequency of a gene having the nucleotide sequence of the cDNA can be analyzed. Further, the function of the gene can be predicted based on the expression frequency information thus analyzed (see Examples 8 and 9).

  As a method for examining a gene associated with a disease, there is expression frequency analysis for examining a difference in gene expression level between a diseased tissue and a normal tissue. Expression frequency analysis includes Northern blotting, RT-PCR, and expression frequency analysis using a DNA macroarray or DNA microarray (Experimental Medicine Vol. 17, No. 8, 980-1056 (1999), Muramatsu).・ Supervised by Nami, Separate Volume on Cell Engineering, "DNA Microarray and the Latest PCR Method" (Shujunsha, 2000). In addition to the above analysis methods, the expression frequency can also be analyzed by comparing the nucleotide sequences of expressed genes by computer-assisted analysis. For example, a database called BODYMAP is a database in which gene clones are randomly extracted from cDNA libraries of various tissues and cells and homologous based on the homology information of the base sequence of the 3 ′ terminal region. Collects into clusters, classifies genes in cluster units, and obtains gene expression frequency information by comparing the number of clones contained in each cluster (http: //bodymap.ims. u-tokyo.ac.jp/).

  A gene whose expression level is apparent from the result of examining the difference in the level of gene expression between a diseased tissue and a normal tissue by such an analysis method can be said to be a gene related to the disease. In addition, even if it is not a diseased tissue, a gene whose expression level is clear from the result of examining the difference in gene expression level between cultured cells and normal cells that reproduces specific phenomena related to the disease state is related to the disease It can be said that it was a gene.

  With regard to TBAES2007379, which is a clone having the DNA of the present invention, a gene associated with a specific disease state or function can be selected using the following database (see Example 8). The database used for the analysis of the present invention is a database in which the nucleotide sequences of 1,402,069 clones are prepared, and is a sufficient database as an analysis parameter. The sequence information constituting this database is obtained by randomly selecting a cDNA clone from a cDNA library derived from various tissues and cells shown in Example 1 and determining the sequence of its 5 ′ terminal region. Obtainable.

  Next, the base sequence of each clone in this database is categorized (clustered) between homologous sequences by a base sequence homology search program, and the number of clones belonging to each cluster is totalized and normalized for each library. This makes it possible to analyze the abundance ratio of a certain gene in a cDNA library. By this analysis, it is possible to obtain information on the expression frequency of a certain gene in the tissue or cell that is the source of the cDNA library.

  Next, in order to analyze the expression of the gene having the nucleotide sequence of the cDNA of the present invention between tissues and cells, a library derived from tissues or cells obtained by analyzing a large amount of cDNA clones was used to express the amount of expression between tissues and cells. Can be compared. That is, for tissues and cells in which the base sequences of 600 or more cDNA clones have been analyzed, previously normalized numerical values can be compared between tissues and cells to analyze changes in gene expression frequencies. This analysis indicates that the gene is related to the following pathological conditions and functions.

(8) Preparation of Oligonucleotide and Functional Analysis Using the Oligonucleotide Using the DNA of the present invention or a fragment thereof obtained by the method described in (1) above, the DNA of the present invention Oligonucleotides such as antisense oligonucleotides and sense oligonucleotides having a partial sequence of DNA can be prepared. Examples of the oligonucleotide include a DNA having the same sequence as 5 to 100 consecutive bases in the base sequence of the DNA or a DNA having a sequence complementary to the DNA. Specific examples include a DNA having the same sequence as 5 to 100 consecutive nucleotides in the base sequence represented by SEQ ID NO: 1 or a DNA having a sequence complementary to the DNA. Here, when the target protein is a splicing variant in which a known variant DNA is present, it is preferable to select a base sequence different from that of the known variant. When used as a sense primer and an antisense primer, the above-mentioned oligonucleotides in which the melting temperature (Tm) and the number of bases of both do not extremely change are preferable. The length of the sequence is generally 5 to 100 bases, preferably 10 to 60 bases, and more preferably 15 to 50 bases.

  In addition, derivatives of these oligonucleotides can also be used as the oligonucleotide of the present invention. Examples of the oligonucleotide derivative include an oligonucleotide derivative in which a phosphoric diester bond in an oligonucleotide is converted to a phosphorothioate bond, and an oligonucleotide in which a phosphoric diester bond in an oligonucleotide is converted to an N3′-P5 ′ phosphoramidate bond. Nucleotide derivative, oligonucleotide derivative in which ribose and phosphodiester bond in oligonucleotide are converted to peptide nucleic acid bond, oligonucleotide derivative in which uracil in oligonucleotide is substituted with C-5 propynyluracil, uracil in oligonucleotide is Oligonucleotide derivatives substituted with C-5 thiazole uracil, oligonucleotide derivatives substituted with cytosine in the oligonucleotide with C-5 propynylcytosine, oligonucleotides Is an oligonucleotide derivative in which cytosine is replaced by phenoxazine-modified cytosine, an oligonucleotide derivative in which ribose in the oligonucleotide is replaced by 2′-O-propyl ribose, or ribose in the oligonucleotide is 2 Oligonucleotide derivatives substituted with '-methoxyethoxyribose can be mentioned.

  The oligonucleotide of the present invention can be applied to the RNA interference method (RNAi method) by preparing it as double-stranded RNA (dsRNA). Among them, those having a short chain length, particularly those having about 21 bases, may be referred to as "siRNA" (small interfering RNAs). For the method for preparing double-stranded RNA and the RNA interference method, for example, the method described in Elbashir, S., et al., Nature, 411: 494-498 (2001) can be used. The double-stranded RNA does not need to be all RNA. Specifically, as a part of which is DNA, those described in WO 02/10374 can be used.

  The gene to be targeted in this RNA interference method (hereinafter sometimes referred to as “target gene”) may be any gene as long as it is the DNA of the present invention. In addition, a gene predicted to be an ortholog of the gene DNA can also be a target gene. A double-stranded oligonucleotide consisting of RNA having a sequence substantially the same as at least a part of the base sequence of these DNAs (hereinafter sometimes referred to as “double-stranded oligonucleotide”) is a target gene. And a sequence substantially identical to a sequence of 15 bp or more, which may be any part of the base sequence. Here, “substantially the same” means that it has 80% or more homology with the sequence of the target gene.

  When the protein to be analyzed is an insertion type or substitution type variant as compared with a known protein, the double-stranded oligonucleotide sequence can be set at the insertion or substitution site. Further, when the protein to be analyzed is a deletion type variant of a known protein, a sequence that spans the deletion portion may be a double-stranded oligonucleotide sequence to select a sequence that is specifically effective for the protein. it can. Furthermore, by comparing the nucleotide sequence of the DNA encoding the protein to be analyzed and the nucleotide sequence of the DNA encoding the known protein and selecting a nucleotide sequence specific to the DNA encoding the protein to be analyzed, Expression can be inhibited.

  The nucleotide length may be any length from 15 bp to the entire length of the open reading frame (ORF) of the target gene, but a length of about 15 to 500 bp is preferably used. However, it is known that mammalian-derived cells have a signal transduction system that activates in response to a long double-stranded RNA of 30 bp or more. This is called an interferon reaction (Mareus, PI, et al., Interferon, 5: 115-180 (1983)), and when the double-stranded RNA enters a cell, PKR (dsRNA-responsive protein kinase: Bass, BL, Nature, 411: 428-429 (2001)), the translation initiation of many genes is non-specifically inhibited, and at the same time, 2'-5'oligoadenylate synthetase (Bass, BL, Nature, 411: 428-429 (2001)), RNase L is activated, and nonspecific degradation of intracellular RNA is caused. These non-specific reactions mask the specific response of the target gene. Therefore, when a mammal or a cell or tissue derived from the animal is used as a recipient, a double-stranded oligonucleotide of 15 to 30 bp, preferably 19 to 24 bp, more preferably 21 bp is preferably used. The double-stranded oligonucleotide does not need to be entirely double-stranded, and includes those in which the 5 'or 3' end is partially protruded, but those in which the 3 'end is protruded by two bases are preferably used. The double-stranded oligonucleotide means a double-stranded oligonucleotide having complementarity, but may be a self-annealed single-stranded oligonucleotide having self-complementarity. Single-stranded oligonucleotides having self-complementarity include, for example, those having an inverted repeat sequence.

  The method for preparing the double-stranded oligonucleotide is not particularly limited, but a known chemical synthesis method is preferably used. In chemical synthesis, a single-stranded oligonucleotide having complementarity is separately synthesized, and can be converted into a double-stranded oligonucleotide by associating the oligonucleotides by an appropriate method. Examples of the method of association include a method in which the oligonucleotides are mixed, heated to a temperature at which the double strand is dissociated, and then gradually cooled. The associated double-stranded oligonucleotide is confirmed using an agarose gel or the like, and the remaining single-stranded oligonucleotide is removed by, for example, decomposing it with a suitable enzyme.

  The transfectant into which the double-stranded oligonucleotide prepared in this way is introduced may be any as long as the target gene can be transcribed into RNA or translated into protein in the cell. Specific examples include those belonging to plants and animals. The plant may be a monocotyledonous, dicotyledonous or gymnosperm, and the animal may be a vertebrate or invertebrate. Examples of vertebrates include mammals, including fish, cows, goats, pigs, sheep, hamsters, mice, rats, and humans, and invertebrates include nematodes, Drosophila, and other vertebrates. Insects are included. Preferably, the cells are vertebrate cells.

  The transductant means a cell, tissue or individual. Here, the cell may be from germline or somatic, totipotent, or pluripotent, split or undivided, parenchymal tissue or epithelium, immortalized or transformed, and the like. The cells may be gametes or embryos, in the case of embryos, single-cell or constitutive cells, or cells from multi-cell embryos, including fetal tissue. Furthermore, they may be undifferentiated cells, such as stem cells, or differentiated cells, such as from cells of an organ or tissue, including fetal tissue, or any other cells present in an organism. Differentiating cell types include adipocytes, fibroblasts, muscle cells, cardiomyocytes, endothelial cells, nerve cells, glial cells, blood cells, megakaryocytes, lymphocytes, macrophages, neutrophils, eosinophils, Includes basophils, mast cells, leukocytes, granulocytes, keratinocytes, chondrocytes, osteoblasts, osteoclasts, hepatocytes and cells of the endocrine or exocrine glands.

  As a method for introducing the double-stranded oligonucleotide into the transfectant, when the transfectant is a cell or tissue, calcium phosphate method, electroporation method, lipofection method, virus infection, double-stranded oligonucleotide solution Immersion, transformation, or the like. Examples of the method of introducing the gene into an embryo include microinjection, electroporation, and viral infection. When the recipient is a plant, a method of injecting or perfusing the plant into a body cavity or stromal cells, or spraying is used. In the case of an animal individual, it is introduced systemically by oral, topical, parenteral (including subcutaneous, intramuscular and intravenous administration), vaginal, rectal, nasal, ophthalmic and intraperitoneal administration. A method, an electroporation method, a virus infection, or the like is used. For methods for oral introduction, the double-stranded oligonucleotide can be mixed directly with the food of the organism. Further, when introduced into an individual, it can be administered, for example, by administration as an implanted long-term release preparation or the like, or by ingesting an introduced body into which a double-stranded oligonucleotide has been introduced.

  The amount of the double-stranded oligonucleotide to be introduced can be appropriately selected depending on the transductant and the target gene, but it is preferable to introduce an amount sufficient to introduce at least one copy per cell. Specifically, for example, when the recipient is a cultured human cell and the double-stranded oligonucleotide is introduced by the calcium phosphate method, the concentration is preferably 0.1 to 1000 nM.

  By suppressing the expression of the DNA of the present invention in the introduced body by RNA interference, it is possible to confirm the function of the protein encoded by the DNA of the present invention or to analyze a new function.

  When the oligonucleotide of the present invention is applied to clinical use, for example, application to gene therapy can be considered. Since the protein of the present invention has increased expression in cancer cells or cancer tissues and / or has EGF-like physiological activity, it is considered that, for example, cancer is suitable as a target disease for gene therapy. When the oligonucleotide is used for gene therapy, for example, a retrovirus vector, an adenovirus vector, a virus vector such as an adeno-associated virus vector, a non-viral vector such as a liposome, or the like, using an ex vivo method or an in vivo method For example, it may be administered to a patient and used as an anticancer agent.

(9) Antibody that specifically binds to the protein of the present invention As a method for preparing an antibody that specifically binds to the protein of the present invention, a commonly used known method can be used. For example, when preparing a polyclonal antibody, a suitable condensing agent such as carbodiimide or maleimide is used for a carrier protein such as BSA (bovine serum albumin), porcine thyroid globulin, or KLH (keyhole limpet hemocyanin). The antigen polypeptide is bound to prepare an antigen (immunogen) for immunization. Here, the binding of the antigen polypeptide to the carrier protein may be carried out by a commonly used method known per se. For example, using KLH as a carrier protein, maleimide is used to bind the antigen polypeptide. In the case of this method, KLH is preferably reacted with a bifunctional condensing agent such as Sulfo-SMCC (Sulfosuccimidyl 4- (N-maleimidomethyl) cyclohexane-1-carboxylate) to produce maleimide, which is then converted to an amino terminal or By reacting an antigen polypeptide having a cysteine added to the end of the carboxyl terminal at which a bond is to be formed, an antigen for immunization can be prepared by easily binding via a thiol. When carbodiimide is used, KLH and the antigen polypeptide can be bound by forming a peptide bond by dehydration condensation.

  Also for polypeptides used as antigens, sequences having high antigenicity and suitable as epitopes (antigenic determinants) can be selected and used according to known methods. As a method for selecting an epitope, for example, commercially available software such as Epitope Adviser (manufactured by Fujitsu Kyushu System Engineering Co., Ltd.) can be used. When the target protein is a splicing variant in which a known variant is present, the antibody specific to the target protein is used by using an antibody that reacts only with the target protein and does not react with a known or other variant. Functions can be identified. As an epitope of such an antibody, for example, when there is an amino acid sequence in which the target protein is deleted as compared with a known variant, an amino acid sequence (junction portion) before and after the deleted portion is preferable. In addition, when the target protein has an amino acid sequence to which an N-terminal or C-terminal of a known variant is added, it is preferable that the added amino acid sequence is used as an epitope.

  Specifically, as the amino acid sequence specific to TBAES2007379 which is the DNA of the present invention, for example, the amino acid at the junction of the amino acid sequence encoded by the 11th exon and the amino acid sequence encoded by the 12th exon of TBAES2007379 Sequences (for example, amino acid numbers 443 to 444 of SEQ ID NO: 2) and the like. As the antigen polypeptide, for example, it is preferable to use a polypeptide having 5 or more amino acid residues including the amino acid sequence of the connecting portion (amino acids of 443 (Val) and 444 (Ala)), more preferably 8 residues As described above, a polypeptide having 10 residues or more is most preferably used. The upper limit of the length of the antigen polypeptide is not particularly limited as long as an antibody specific to TBAES2007379 can be obtained, but is usually 20 residues or less, preferably 15 residues or less. The preferred sequence of the antigen polypeptide used in the present invention includes at least the amino acid sequence of the exon connecting portion (Val Ala), and the sequence added to both ends thereof is not particularly limited, but the sequence itself of TBAES2007379 shown in SEQ ID NO: 2 can be used. Preferably, it is used. The position of the amino acid sequence of the exon junction is not particularly limited, but it is preferable that the sequence is located at the center. That is, the antigen polypeptide preferably includes a polypeptide having the sequence itself of TBAES2007379 of 1 to 8 residues, preferably about 3 to 6 residues at both ends of the amino acid sequence (Val Ala) of the exon connecting portion. . Specific examples of preferable sequences thereof include SEQ ID NOs: 10 and 13 as amino acid sequences of antigenic peptides including amino acid numbers 443 to 444 of SEQ ID NO: 2.

  In addition, in TBAES2007379, due to the deletion of the exon, a stop codon is generated due to a frame shift in the subsequent 16th exon. As a result, the amino acid sequence of TBAES2007379 has a C-terminal sequence (amino acids 678 to 682 of SEQ ID NO: 2) specific to TBAES2007379. Specifically, SEQ ID NOs: 11 and 12 and the like are given as the amino acid sequences of antigenic peptides containing amino acids 678 to 682 of SEQ ID NO: 2.

  As a method other than the above, an antibody that reacts only with the target protein can be obtained by removing an antibody that reacts with a known or other variant from the polyclonal antibody obtained with respect to the target protein. As a removing method, affinity chromatography in which a known or other variant is immobilized as a ligand, or immunoprecipitation using a known or other variant is used.

  As the polypeptide used as the antigen, a synthetic peptide synthesized according to a known method, or the protein itself of the present invention can be used. The polypeptide serving as the antigen may be prepared in an appropriate solution or the like according to a known method, and immunization may be performed on mammals, for example, rabbits, mice, rats, and the like, and birds, for example, chickens. In order to increase the antibody titer, it is preferable to use an antigen peptide in the form of a conjugate with an appropriate carrier protein, or to add an adjuvant or the like for immunization.

  The route of administration of the antigen upon immunization is not particularly limited, and any route such as subcutaneous, intraperitoneal, intravenous, or intramuscular may be used. Specifically, for example, a method of inoculating BALB / c mice several times every several days to several weeks with an antigen polypeptide is used. The amount of the antigen to be taken is preferably about 0.3 to 0.5 mg / time when the antigen is a polypeptide, but is appropriately adjusted depending on the type of the polypeptide and the animal species to be immunized.

  Alternatively, an antibody may be prepared by directly administering DNA encoding TBAES2007379 or cells transfected with TBAES2007379 gene to an animal for immunization (Katherin E. et al., HYBRIDOMA, 19: 297-302 (2000)). Keiichi Enjoji, Journal of the Japanese Society of Thrombosis and Hemostasis., 11, 265-267 (2000)).

  After immunization, blood is appropriately collected as a test, and an increase in the antibody titer is confirmed by a method such as Ouchterlony method, enzyme-linked immunosorbent assay (hereinafter sometimes referred to as “ELISA method”) or Western blotting. Blood is collected from animals with sufficiently raised antibody titers. A polyclonal antibody can be obtained by subjecting this to an appropriate treatment used for antibody preparation. Specifically, for example, there is a method of obtaining a purified antibody obtained by purifying an antibody component from serum according to a known method. For purification of the antibody component, methods such as salting out, ion exchange chromatography, and affinity chromatography can be used.

  Alternatively, a monoclonal antibody can be prepared by using a hybridoma fused with spleen cells and myeloma cells of the animal according to a known method (Milstein, et al., Nature, 256: 495 (1975)). . The monoclonal antibody can be obtained, for example, by the following method.

  First, antibody-producing cells are obtained from an animal whose antibody titer has increased due to immunization with the above-described antigen. The antibody-producing cells are plasma cells and lymphocytes which are precursor cells thereof, which may be obtained from any of the individuals, but is preferably obtained from spleen, lymph nodes, peripheral blood and the like. As a myeloma to be fused with these cells, generally, a cell line obtained from a mouse, for example, a P3X63-Ag8.653 (ATCC: CRL) which is an 8-azaguanine-resistant mouse (derived from BALB / c) myeloma cell line is used. -1580), P3-NS1 / 1Ag4.1 (RIKEN cell bank: RCB0095) and the like are preferably used. For cell fusion, antibody-producing cells and myeloma cells are mixed at an appropriate ratio, and 50% polyethylene is added to an appropriate cell fusion medium such as RPMI1640, Iskov's modified Dulbecco's medium (IMDM), or Dulbecco's modified Eagle's medium (DMEM). It can be carried out by using a solution in which glycol (PEG) is dissolved. It can also be performed by the electrofusion method (Zimmermann, U. et al., Naturwissenschaften, 68: 577 (1981)).

The hybridoma was prepared by using the myeloma cell line used as an 8-azaguanine resistant strain in a normal medium (HAT medium) containing an appropriate amount of a hypoxanthine / aminopterin / thymidine (HAT) solution at 5% CO ₂ , 37%. It can be selected by culturing at an appropriate temperature for an appropriate time. This selection method can be appropriately selected and used depending on the myeloma cell line to be used. The antibody titer of the antibody produced by the selected hybridoma is analyzed by the above-described method, the hybridoma producing the antibody having a high antibody titer is separated by a limiting dilution method or the like, and the separated fused cells are cultured in an appropriate medium. The monoclonal supernatant can be obtained by purifying the resulting culture supernatant by an appropriate method such as ammonium sulfate fractionation and affinity chromatography. For purification, a commercially available monoclonal antibody purification kit can also be used. Furthermore, ascites containing a large amount of the monoclonal antibody of the present invention can be obtained by growing the antibody-producing hybridoma obtained above in the abdominal cavity of an animal of the same strain as the immunized animal or a nude mouse.

  Further, when a human-derived protein is obtained as the protein of the present invention, the above-described method is applied to a Severe combined immune deficiency (SCID) mouse transplanted with human peripheral blood lymphocytes using the polypeptide or a partial peptide thereof as an antigen. A humanized antibody can also be prepared by immunization in the same manner as described above and preparing a hybridoma between the antibody-producing cells of the immunized animal and human myeloma cells (Mosier, DE, et al., Nature, 335: 256-259 (1988); Duchosal, MA, et al., Nature, 355: 258-262 (1992)).

  Further, RNA is extracted from the obtained hybridoma producing the human antibody, a gene encoding the target human antibody is cloned, this gene is inserted into an appropriate vector, and this is introduced into an appropriate host. By expression, human antibodies can be produced in larger quantities. Here, an antibody with low binding to an antigen can be obtained as an antibody with even higher binding by using an evolutionary engineering technique known per se. A partial fragment such as a monovalent antibody can be prepared by cleaving the Fab and Fc portions using, for example, papain or the like, and collecting the Fab portion using an affinity column or the like.

  The thus-obtained antibody that specifically binds to the protein of the present invention can also be used as a neutralizing antibody that specifically binds to the protein of the present invention and thereby inhibits the EGF-like physiological activity of the protein. There is no particular limitation on the method for selecting a substance that inhibits the activity of the protein. For example, the antibody may be brought into contact with the DNA transfectant prepared in (4) above to determine whether the function of the target protein in the transfectant is inhibited. And a method of analyzing the above.

  Such a neutralizing antibody may be used alone when the clinical application is performed, or may be used as a pharmaceutical composition by mixing with a pharmaceutically acceptable carrier. At this time, the ratio of the active ingredient to the carrier can be varied between 1 and 90% by weight. Such drugs can be administered in various forms, such as tablets, capsules, granules, powders, orally administered by syrup or the like, or injections, drops, liposomes, Parenteral administration with suppositories and the like can be mentioned. In addition, the dose can be appropriately selected depending on symptoms, age, body weight, and the like.

(10) Screening and Screening Kit for Molecules that Regulate the Activity of the Protein of the Present Invention The action of specifically binding to the protein of the present invention and inhibiting, antagonizing or enhancing the function (activity) of the protein of the present invention. By screening for a substance having the function, a function modulator of the protein of the present invention (hereinafter, this may be referred to as a "modulator") can be obtained.

  This method of screening for a regulatory substance may be any method as long as it can obtain a substance that specifically binds to the protein of the present invention and has an activity of inhibiting, antagonizing or enhancing the activity of the protein. For example, first, the protein of the present invention is brought into contact with a substance to be subjected to screening (hereinafter, this may be referred to as “test substance”), and selection is performed using the binding property to the protein as an index. A method of selecting a test substance using the change in the activity of the protein as an index can be used.

  The test substance may be any substance that can interact with the protein of the present invention and affect the activity of the protein, and specifically, for example, a peptide , Proteins, non-peptidic compounds, low molecular compounds, synthetic compounds, fermentation products, cell extracts, animal tissue extracts, and the like. These substances may be novel substances or known substances. As a method for analyzing the interaction between the test substance and the protein of the present invention, a conventional method known per se can be used. Specifically, for example, yeast two-hybrid method, fluorescence depolarization method, surface plasmon method Phage display method, ribosomal display method, or the competition analysis method with the antibody described in the above (9). By such a method, a substance found to bind to the protein of the present invention is then analyzed by analyzing how the activity of the protein of the present invention is affected in the presence of the substance. Whether it is used as a modulator is identified. Here, when the target protein is a splicing variant in which a known variant is present, the effect of a substance that binds only to the target protein and does not bind to a known or other variant is analyzed, or a known protein is analyzed. Alternatively, it is possible to analyze the effect of the substance on the target protein by identifying whether or not the same binds to other variants and analyzing the difference in the effect of the same when the two bind. In addition, by examining the distribution of the substance in an individual, it is possible to analyze the effect on the target protein and known or other variants.

  As a specific analysis method, for example, when analyzing a substance that regulates EGF-like physiological activity, a protein (eg, a receptor protein) targeted to the DNA transfectant described in (4) is also subjected to the same method. Introduce. The activity of the target protein in the presence / absence of the selected substance with respect to the introduced substance is analyzed by a known method which is known per se. Specifically, it can be performed using the method described in (5) above. When the activity or the like of the target protein is increased as compared with the absence of the substance, the substance may function as an activator of the EGF-like protein, and may be reduced or inhibited. In that case, the substance can be identified as potentially functioning as an inhibitor of the EGF-like protein.

  Here, when using the DNA of the present invention or a recombinant protein used for screening for a regulatory substance for the purpose of obtaining a pharmaceutically active ingredient, it is preferable to use human DNA or protein. Further, the substance screened by the above method may be further selected as a drug candidate by in vivo screening. The evaluation of the protein function regulating substance of the present invention is not limited to the above method.

  Examples of the EGF-like physiological activity which is one of the activities of the protein of the present invention include cell proliferation, gastric acid secretion, anti-ulcer, gastrointestinal mucosa protection, DNA synthesis promotion, corneal repair, calcium It is a physiological activity similar to that of a protein having an EGF-like domain or an EGF-like repeat, such as a release promoting action, a wound healing promoting action, an anti-inflammatory action, an analgesic action, a hepatocellular injury suppressing action, and a hair follicle retraction action. Therefore, modulators that can be identified by the present screening method can be used as anticancer agents, therapeutic agents for digestive tract diseases, regenerating tissue inducers, therapeutic agents for immune / inflammatory diseases, and the like.

  The DNA encoding the protein of the present invention has been cloned from a cDNA library constructed from RNA derived from tissues, organs or cells of breast cancer or the like. Since the protein may have a specific function, the protein function modulator of the present invention can be used as a therapeutic agent for diseases specific to the tissue or organ.

  Such modulators can be used alone for the clinical application, but can also be used as pharmaceutical compositions by mixing with a pharmaceutically acceptable carrier. At this time, the ratio of the active ingredient to the carrier can be varied between 1 and 90% by weight. Such drugs can be administered in various forms, such as tablets, capsules, granules, powders, orally administered by syrup or the like, or injections, drops, liposomes, Parenteral administration with suppositories and the like can be mentioned. In addition, the dose can be appropriately selected depending on symptoms, age, body weight, and the like.

  In addition, the screening kit for such a regulatory substance may be any kit containing the protein of the present invention and the like.

(11) Screening and screening kit for a DNA expression regulator of the present invention As a screening method, a method of analyzing the expression level of the protein of the present invention or the mRNA encoding the same in the presence of a test substance, and the like are described. No. Specifically, for example, cells expressing the protein of the present invention described in (4) are cultured in an appropriate medium containing a test substance, and the amount of the protein of the present invention expressed in the cells is determined by ELISA. Or by analyzing the amount of mRNA encoding the protein of the present invention in the cells by quantitative reverse transcription PCR, Northern blot, or the like.

  As the test substance, those described in (10) can be used. According to this analysis, if the amount of the test substance increases in comparison with the amount of the protein or mRNA expressed in the cells cultured in the absence of the test substance, the test substance is a substance for promoting the expression of the DNA of the present invention. When the test substance decreases, it can be determined that this test substance can be used as the DNA expression inhibitor of the present invention.

  The above-mentioned active ingredient can be used alone for clinical application, but can also be used as a pharmaceutical composition by blending it with a pharmaceutically acceptable carrier. At this time, the ratio of the active ingredient to the carrier can be varied between 1 and 90% by weight. Such drugs can be administered in various forms, such as tablets, capsules, granules, powders, orally administered by syrup or the like, or injections, drops, liposomes, Parenteral administration with suppositories and the like can be mentioned. In addition, the dose can be appropriately selected depending on symptoms, age, body weight, and the like.

  Further, the screening kit for such an expression regulator may be any as long as it contains the DNA of the present invention, a recombinant vector containing the DNA, or a transgenic cell into which the recombinant vector has been introduced.

(12) The DNA-transfected animal of the present invention The transfected DNA containing the DNA of the present invention described in the above (1) is constructed, introduced into a fertilized egg of a mammal other than a human, and transplanted into a female individual oviduct. Thus, a non-human mammal into which the DNA of the present invention has been introduced can be produced. More specifically, for example, after superovulation of a female individual by hormone administration, it is mated with a male, a fertilized egg is extracted from an oviduct on the first day after mating, and the introduced DNA is microinjected into the fertilized egg. Introduce by the method of After culturing by an appropriate method, the surviving fertilized eggs are transplanted into the oviduct of a pseudopregnant female individual (foster parent) to give birth. Whether or not the target DNA has been introduced into the newborn can be identified by performing Southern blot analysis on DNA extracted from cells of the individual. Examples of mammals other than humans include mice, rats, guinea pigs, hamsters, rabbits, goats, pigs, dogs, cats, and the like.

  The thus-obtained DNA-introduced animal of the present invention obtains its offspring by crossing this individual and subculturing it in a normal breeding environment while confirming that the introduced DNA is stably retained. be able to. In addition, the offspring can be obtained by repeating in vitro fertilization, and the strain can be maintained.

  The non-human mammal into which the DNA of the present invention has been introduced can be used as an analysis of the function of the DNA of the present invention in a living body, or as a screening system for a substance regulating the function.

(13) Other uses of the protein of the present invention, an antibody that specifically recognizes the same, and a DNA containing a base sequence encoding the protein of the present invention. It can be used as a carrier having it bound on a substrate. In addition, a nucleotide sequence encoding the protein of the present invention, for example, a DNA having the nucleotide sequence of SEQ ID NO: 1 or a partial fragment thereof can be used as a carrier having them bound on a substrate. These may be hereinafter referred to as “protein chips”, “antibody chips”, “DNA chips” or “DNA arrays” (DNA microarrays and DNA macroarrays). These protein chips, antibody chips, or DNA chips or arrays may contain other proteins, antibodies, or DNAs in addition to the proteins, antibodies, or DNAs of the present invention. Here, when the target protein is a splicing variant in which a known variant is present, a target protein-specific amino acid sequence partial fragment can be used as the protein chip, but the protein chip has a different three-dimensional structure from other variants. The full length can be used because there is a possibility. In addition, it is preferable to use an antibody that recognizes an amino acid sequence or a three-dimensional structure that is different from other variants in the amino acid sequence of the target protein for the antibody chip. Similarly, it is preferable to select, from the DNA sequence encoding the target protein, a sequence different from other variant DNAs for the DNA array.

  Further, as a substrate for binding proteins, antibodies, and DNA, a resin substrate such as a nylon film or a polypropylene film, a nitrocellulose film, a glass plate, a silicon plate, a resin substrate coated with a metal thin film, particularly a gold thin film, and the like are used. When the detection of hybridization is performed non-RI, for example, using a fluorescent substance or the like, a glass plate or a silicon plate containing no fluorescent substance is preferably used. The binding of a protein, antibody or DNA to the substrate can be easily carried out by a commonly used method known per se. These protein chips, antibody chips, DNA chips, or DNA arrays are also included in the scope of the present invention.

  Further, the amino acid sequence of the protein of the present invention and the nucleotide sequence of DNA can also be used as sequence information. The base sequence of this DNA includes the base sequence of the corresponding RNA. That is, by storing the obtained amino acid sequence or base sequence in an appropriate recording medium in a predetermined format readable by a computer, a database of the amino acid sequence or base sequence can be constructed. This database may contain other types of proteins and the base sequences of DNAs encoding the same. Further, in the present invention, the database also means a computer system that writes the above-mentioned sequence on an appropriate recording medium and performs a search according to a predetermined program. Suitable recording media include, for example, magnetic media such as a flexible disk, a hard disk, and a magnetic tape; optical disks such as a CD-ROM, MO, CD-R, CD-RW, DVD-R, and DVD-RAM; And the like.

(14) Preparation of biological sample to be analyzed The biological sample to be analyzed in the present invention may be any biological sample that may contain the protein of the present invention. For example, blood, cerebrospinal fluid, ascites, urine, milk, sweat, body fluid such as saliva, etc. obtained from an individual suspected of having cancer may be mentioned, with blood being particularly preferred. For example, in the case of blood, a sample collected from an individual suspected of having cancer may contain an enzyme inhibitor in order to prevent a change (decomposition, etc.) of the protein of the present invention in the sample and blood coagulation. It is preferably added at the time of collection or after collection of the sample. Examples of the enzyme inhibitor include protease inhibitors such as aprotinin, antipine, pepstatin, leupeptin, EGTA, PMSF (phenylmethanesulfonyl fluoride), TLCK (tricyllysine chloromethyl ketone), and anticoagulant. As the agent, for example, heparin or the like is used. Among them, most preferably as the sample of the present invention, blood samples are collected using a blood collection tube or the like to which aprotinin, heparin, etc. are added, and serum components or plasma obtained by separating the obtained blood by centrifugation or the like are obtained. Used. After collection, these samples are preferably stored at, for example, 4 ° C. or lower, and more preferably frozen at −20 ° C. or lower, until subjected to analysis of the protein of the present invention.

  Further, if desired, the sample is preferably denatured in the presence of a protein solubilizing agent to remove contaminating proteins, and then used as a sample. In particular, when blood is used as a sample, it is preferable to remove the protein, for example, because the antigen-antibody reaction is hindered by a high concentration of the protein. Further, a sample which has been further concentrated as necessary can be used as a sample in the method of the present invention.

  The sample thus obtained can be further concentrated, if necessary, according to a known method to increase the detection sensitivity of the protein of the present invention.

(15) Cancer Detection Method of the Present Invention As described in the above (14), the presence of the protein of the present invention is analyzed in a biological sample solution obtained from an individual suspected of having cancer to detect cancer. The analysis of the presence of the protein of the present invention is not particularly limited as long as it is performed using an antibody specific to the protein of the present invention, and the reactivity between the sample and the antibody specific to the protein of the present invention, that is, Any method can be used as long as the immunoreactivity between the protein contained in the sample and the antibody can be compared with the immunoreactivity between the positive control and the antibody. The results obtained using a positive control of known concentration and the results obtained using the sample are compared to determine the amount of the protein of the present invention in the sample, and by relating the measurement result to the presence of cancer, Cancer can be detected.

  The analysis of the antibody of the present invention and the protein in a biological sample obtained from an individual suspected of having cancer by immunological reaction is described in Biochemical Experimental Method 11 "Enzyme Immunoassay" (Tijssen P., Tokyo Chemical Dojin), "Antibodies: A LABORATORY MANUAL "(Ed Harlow et al., Cold Spring Harbor Laboratory (1988)) and other commonly used methods known per se. Specifically, for example, a sandwich method such as an immunoblot method and an enzyme immunoassay (ELISA method), a competition method and the like can be mentioned.

  Here, a method for detecting the presence of the protein of the present invention in a sample by the dot blot method will be described below as an example. The sample obtained by the method described in the above (14) is transferred to a film usually used for transferring proteins, such as a PVDF (Polyvinyliden difluoride) film or a nitrocellulose film. After blocking the obtained membrane with a protein solution such as skim milk, an antibody specific to the protein of the present invention is reacted with the resulting membrane. After removing the unreacted antibody by washing the membrane, a secondary antibody to which a labeling substance has been previously bound is reacted. When the labeling substance is a substance that does not itself emit a signal, such as an enzyme, the membrane after the reaction is washed to remove unreacted secondary antibodies, and then the specific substance is labeled. A substance, such as a substrate, which reacts and reacts to generate a signal is reacted, and a signal emitted from the substance is detected. By comparing the detection result with the result obtained using a positive control with a known concentration, the amount of the protein of the present invention in the sample can be known. Examples of the enzyme used as the labeling substance include alkaline phosphatase and Horseradish peroxidase (hereinafter, this may be referred to as “HRP”).

  When the labeling substance is a substance having a property of emitting a signal itself, such as a fluorescent substance or a radioactive substance, the membrane after the reaction is washed to remove unreacted secondary antibodies, and then emitted from the labeling substance. By detecting the signal obtained and comparing the detection result with the result obtained using a positive control with a known concentration, the amount of the protein of the present invention in the sample can be known.

  In such detection, a reaction by a substance having specific binding ability, which is not an antigen-antibody reaction, can be used in order to remove the influence of an antibody brought in from a sample or the like. As the substance having specific binding ability, for example, a combination of biotin and streptavidin, a receptor protein and its ligand substance, and the like are used, and a combination of biotin and streptavidin is particularly preferable.

  Next, as another example of the analysis method, a case where the presence of the protein of the present invention in a sample is analyzed by an ELISA method will be described below. First, the antibody of the present invention is immobilized on a 96-well ELISA plate as an antibody for immobilization. The antibody-immobilized plate is preferably subjected to a blocking treatment with a protein solution such as skim milk or bovine serum albumin (hereinafter, this may be referred to as “BSA”). Then, the biological sample obtained by the method described in the above (14) is added to bind the protein of the present invention in the sample to the solid phase. Subsequently, as the detection antibody for detecting the bound protein of the present invention, another antibody of the present invention and the immobilized antibody is added and incubated.

  When a polyclonal antibody against the protein of the present invention is used, two types of polyclonal antibodies obtained by immunizing the protein of the present invention with different animal species are combined, and an antibody for immobilization and an antibody for detection are respectively used. Should be used as For example, a rabbit polyclonal antibody can be used as the antibody for immobilization, and a mouse polyclonal antibody can be used as the detection antibody, but the combination is not limited thereto.

  After adding the detection antibody and incubating, wash with a washing solution, recognize the detection antibody, and label the antibody with a labeling substance, for example, when the detection antibody is a mouse antibody, use an enzyme-labeled antibody. A mouse IgG antibody or the like is reacted. Alternatively, biotin or the like is bound in advance to the detection antibody, and the resultant is reacted with an enzyme-labeled streptavidin or the like. Measuring the amount of the protein of the present invention in a sample, such as by measuring the activity of a labeled substance bound to a solid phase after washing with a washing solution and comparing the result with a result obtained using a positive control having a known concentration. Can be.

  The positive control used in the detection method of the present invention may be any positive control as long as it specifically binds to the antibody of the present invention. For example, the amino acid of SEQ ID NO: 2 prepared by genetic recombination may be used. A protein consisting of

  Also, cancer markers are often recognized as heterologous molecules by the individual's immune system and elicit an autoimmune response. This immune response is humoral and can generate autoantibodies to the cancer marker protein. Autoantibodies are antibodies that naturally occur against an antigen that the individual's immune system recognizes as heterologous, even though the antigen is actually derived from the individual. Since the protein of the present invention is a protein whose expression is increased in cancer cells or cancer tissues, it may induce the production of autoantibodies. Therefore, the presence of an autoantibody against the protein of the present invention in a biological sample can be an indicator of cancer. Such an autoantibody can be immunologically measured using the protein of the present invention, for example, according to the method described above. Further, by measuring such autoantibodies, it is possible to detect autoimmune diseases.

  Furthermore, cancer can also be detected by examining the expression state of the DNA of the present invention in cells, tissues, and biological samples. As described in the above (6), the expression state of the DNA can be analyzed at the mRNA level or the protein level. When the expression level is analyzed at the mRNA level, for example, an in situ hybridization method, a hybridization method using a DNA chip, a quantitative PCR method, or the like is used. When the expression level is analyzed at the protein level, for example, the amount of the protein of the present invention may be immunologically measured as described above.

(16) Reagent kit containing protein of the present invention or antibody of the present invention The reagent kit of the present invention contains at least the protein encoded by TBAES2007379 or an antibody specific to the protein, and utilizes a normal immune reaction. Provided in a configuration according to the kit. Further optional components include a washing solution, a labeled antibody for detection, a pretreatment solution containing a protein solubilizing agent, a positive control, a diluting solution, a protein denaturing agent, an instrument or reagent for concentration, and the like.

  Specifically, for example, in the case of a kit applied to the ELISA method, the kit contains at least an antibody specific to the protein encoded by TBAES2007379, and further contains an immobilized anti-TBAES2007379 protein antibody and a labeled anti-IgG antibody. And a pretreatment liquid containing a protein solubilizing agent as an optional element. In the case of a kit applied to the competition method, the kit contains a labeled TBAES2007379 protein, a TBAES2007379 protein-specific antibody, and the like, and a pretreatment solution containing a protein solubilizing agent as an optional element. By using such a reagent kit, the method for detecting cancer of the present invention can be performed more quickly and easily.

  Hereinafter, the present invention will be described in detail with reference to examples, but the scope of the present invention is not limited to these examples. The preparation of various vectors, the expression of proteins, and the like are performed by known methods described in Molecular Cloning, A Laboratory Manual, 3rd edition (by Sambrook and Russell, published by Cold Spring Harbor Laboratory Press (2001)) unless otherwise specified. Performed according to the procedure. In the following examples, the DNA or protein of the present invention may be simply referred to as a clone name and described as “TBAES2007379”.

Production of cDNA Library by Oligocap Method Commercially available mRNA (Clontech: breast cancer (# 64015-1)) extracted as total RNA from each human tissue was subjected to oligocap method (Maruyama, K., et al., Gene , 138: 171-174 (1994)).

  First, after treating the above RNA with BAP (Bacterial Alkaline Phosphatase) and TAP (Tobacco Acid Pyrophosphatase), an oligocap linker (SEQ ID NO: 3) was ligated using RNA ligase. Using this RNA strand as a template, a first-strand cDNA was synthesized by reverse transcription using an oligo dT primer (SEQ ID NO: 4), and then the RNA strand was degraded and removed (Suzuki et al., Protein Nucleic Acid Enzyme, 41: 603-607). (1996); Suzuki, Y. et al., Gene, 200: 149-156 (1997)). Next, a double-stranded cDNA was amplified by PCR (polymerase chain reaction) using a 5 ′ PCR primer (SEQ ID NO: 5) and a 3 ′ PCR primer (SEQ ID NO: 6), and the amplified DNA strand was cut with SfiI. .

  Next, the SfiI-cut fragment obtained above was cloned into the DraIII site of pME18SFL3 (GenBank AB009864), which is an expression vector, to prepare a cDNA library. The pME18SFL3 vector used above has an SRα promoter and an SV40 small t intron incorporated upstream of the cloning site, and an SV40 poly (A) addition signal sequence inserted downstream thereof. The cloning site of pME18SFL3 is an asymmetric DraIII site, and a complementary SfiI site is added to the end of the cDNA fragment, so that the cloned cDNA fragment is unidirectional downstream of the SRα promoter. Is inserted into Therefore, in a clone containing the full-length cDNA, the gene can be transiently expressed by directly introducing the obtained plasmid into COS cells. That is, it is very easy to experimentally analyze the protein as a gene product or its biological activity.

  For the plasmid DNA of the clone obtained therefrom, the nucleotide sequence at the 5 'end or 3' end of the cDNA was converted to a DNA sequencing reagent (Dye Terminator Cycle Sequencing FS Ready Reaction Kit, dRhodamine Terminator Cycle Sequencing FS Ready Reaction Kit or BigDye Terminator Cycle Sequencing). Using a FS Ready Reaction Kit (manufactured by PE Biosystems) and a sequencing reaction according to the manual, the DNA base sequence was analyzed using a DNA sequencer (ABI PRISM 3700: manufactured by PE Biosystems).

CDNA libraries containing the clones used in the databases used for the expression frequency analysis in Examples 8 and 9 were also prepared in the same manner as described above. The relationship between the cDNA library name and its origin is shown below. The library name is enclosed in brackets, and the type and origin of the library source are indicated in parentheses, separated by /.
"3NB69" (cultured cells / NB69 cells (RCB # RCB0480))
"ADIPS" (Tissue / Adipose (Adipose) (Invitrogen # D6005-01))
"ADRGL" (Tissue / Adrenal gland) (CLONTECH # 64016-1)
“AHMSC” (cultured cells / HMSC cells (Human mesenchymal cells))
"ASTRO" (primary cultured cells / Normal Human Astrocytes) NHA5732 (Takara Shuzo # CC2565)
"BEAST" (Tissue / Adult Breast (STARATAGENE # 735044))
"BLADE" (Tissue / Bladder (Invitrogen # D6020-01))
"BRALZ" (Tissue / Alzheimer patient cerebral cortex (Brain, cortex, Alzheimer) (Invitrogen # D6830-01))
"BRCAN" (Tissue / Caudate nucleus (CLONTECH # 6575-1))
"BRCOC" (Tissue / Brain, corpus callosum) (CLONTECH # 6577-1)
"BRSSN" (tissue / substantia nigra) (CLONTECH # 6580-1)
"BRSTN" (Tissue / Brain, subthalamic nucleus) (CLONTECH # 6581-1)
"CERVX" (Tissue / Cervix (Invitrogen # D6047-01))
"CHONS" (cultured cells / chondrocytes)
"COLON" (Tissue / Colon (Invitrogen # D6050-0))
"CTONG" (Tissue, Tongue, Cancer)
"D9OST" (primary cultured cells / CD34 + cells (ODF induction 9 days))
"DFNES" (Normal Human Dermal Fibroblasts (Neonatal Skin); NHDF-Neo) NHDF2564 (Takara Shuzo # CC2509)
“ERLTF” (cultured cells / TF-1 cells (erythroleukemia cells; erythroleukemia))
"FEBRA" (Tissue / Fetal Brain (Brain, Fetal) (CLONTECH # 64019-1))
"FEHRT" (tissue / fetal heart (Heart, Fetal) (STARATAGENE # 738012))
"FEKID" (Tissue / Fetal Kidney (Kidney; Fetal))
"FELNG" (tissue, fetal lung (Lung, Fetal) (STARATAGENE # 738020))
"HCASM" (Primary cultured cells / normal coronary artery smooth muscle cells HCASMC (Human coronary artery smooth muscle cells) (Toyobo # T305K-05))
"HCHON" (primary cultured cells / normal chondrocytes HC (Human Chondrocytes) (Toyobo # T402K-05))
"HEART" (Tissue / Heart) (CLONTECH # 64025-1)
"HHDPC" (Primary cultured cells / Human dermal papilla cells (HDPC) (Toyobo # THPCK-001))
"HLUNG" (Tissue / Lung (CLONTECH # 64023-1))
"HSYRA" (Human synoviocytes from rheumatioid arthritis) (Toyobo # T404K-05)
"JCMLC" (cultured cells / leukemia cells (Leukemia, myelogenous))
"KIDNE" (Tissue / Kidney (CLONTECH # 64030-1))
"LIVER" (Tissue / Liver (Liver) (CLONTECH # 64022-1))
"LYMPB" (Lymphoblast, EB virus transferred B cell)
"MESAN" (Primary cultured cells / Normal human mesangial cells) NHMC56046-2 (Takara Shuzo # CC2559)
"MESTC" (Cultured cells / Mesenchyme stem cells)
"N1ESE" (Cultured cells / Mesenchymal stem cells)
"NETRP" (primary cultured cells / neutrophils (Neutrophil))
"NOVAR" (Tissue / Adult Ovary (STARATAGENE # 735260))
"NT2NE" (cultured cells / NT2 cells concentrated after neuronal differentiation (NT2 Neuron))
"NT2RI" (cultured cells / NT2 cells 5 weeks after RA induction, 2 weeks with growth inhibitor treatment)
"NT2RP" (cultured cell / NT2 cell RA induction 5 weeks)
"NTONG" (tissue / Tongue)
"OCBBF" (tissue / fetal brain (Brain, Fetal))
"PEBLM" (Primary cultured cells / Human peripheral blood mononuclear cells HPBMC5939 (Takara Shuzo # CC2702))
"PERIC" (Tissue / Pericardium (Invitrogen # D6105-01))
"PLACE" (tissue / placenta)
"PROST" (Tissue / Prostate (CLONTECH # 64038-1))
"PUAEN" (Primary cultured cells / Human pulmonary artery endothelial cells (Toyobo # T302K-05))
“RECTM” (Tissue / Rectum (Rectum) (Invitrogen # D6110-01))
"SKMUS" (Tissue / Skeletal Muscle (CLONTECH # 64033-1))
"SKNMC" (cultured cells / SK-N-MC cells (ATCC # HTB-10))
"SKNSH" (cultured cells / SK-N-SH cells (RCB # RCB0426))
"SMINT" (Tissue / Small Intestine (CLONTECH # 64039-1))
"SPLEN" (Tissue / Spleen (CLONTECH # 64034-1))
"STOMA" (Tissue / Stomach (CLONTECH # 64090-1))
"SYNOV" (Synovial membrane tissue from rheumatioid arthritis)
"T1ESE" (cultured cells / Mesenchymal stem cells (Trichostatin and 5-azacytidine treatment))
"TBAES" (tissue / breast cancer (Breast, Tumor) (CLONTECH # 64015-1))
"TESOP" (Tissue / Esophageal, Tumor) (Invitrogen # D6860-01)
“TKIDN” (tissue / kidney cancer (Kidney, Tumor) (Invitrogen # D6870-01))
"TLIVE" (Tissue / Liver cancer (Liver, Tumor) (Invitrogen # D6880-01))
"TLUNG" (Tissue / Lung Cancer (Lung; Tumor))
"TRACH" (Tissue / Trachea (CLONTECH # 64091-1))
"TSTOM" (Tissue / Stomach cancer (Stomach, Tumor) (Invitrogen # D6920-01))
"TUTER" (Tissue / Uterus cancer (Uterus, Tumor) (CLONTECH # 64008-1))
"UTERU" (Tissue / Uterus (CLONTECH # 64029-1))

Evaluation of the full length of the 5 'end of the clone from the cDNA library prepared by the oligocap method The nucleotide sequence of the 5' end of the human cDNA library prepared in Example 1 For all clones that match the 5 'end sequence compared to the sequence, when the 5' end is longer than the known mRNA sequence in the public database, or when the 5 'end is shorter but has a translation initiation codon Was determined to be "full length", and the case not including the translation initiation codon was determined to be "non-full length".

  Next, the clones were evaluated by ESTiMateFL. ESTiMateFL is a method developed by Nishikawa and Ota et al. Of the Helix Research Institute to select clones having a high possibility of full-length cDNA by comparing with the 5'-terminal sequence or 3'-terminal sequence of EST in a public database. is there. The 5'-end and 3'-end sequences of the cDNA clone analyzed in Example 1 were compared with the base sequences registered in the EST database, and extended to the 5'-side or 3'-side from the sequence of the obtained cDNA clone. If any EST exists, the clone was determined to be "possibly not full length". If the 5 'end is longer than the EST sequence in the public database, or even if the clone has a shorter 5' end, the difference is less than 50 bases for convenience and the shorter is the non-full length. .

  As a result, TBAES2007379 (SEQ ID NOS: 1 and 2) was found to have a novel full-length cDNA.

Analysis by homology search For TBAES2007379 (SEQ ID NOS: 1 and 2), which was found to have a novel cDNA in Example 2, the nucleotide sequence determined and the amino acid sequence of the ORF portion presumed to encode the protein were determined by SwissProt. , RefSeq, and BLAST search for NRDB. In the analysis for the amino acid database in which the P value or the E value is 10 ⁻⁴ or less, among the BLAST search hit data of consensus length × homology = 30 or more, the homology is higher, the base sequence and the estimated Representative data was selected from hit data whose function was relatively easy to predict with respect to the amino acid sequence, and is shown below as homology search result data. Therefore, the data shown are representative only, and molecules showing homology to clones are not limited thereto.

The homology search result data for the full-length nucleotide sequence and the deduced amino acid sequence are shown below. Each data is described by // in order of sequence name (sequence number), Definition of hit data, P value, length of comparison sequence, homology, Accession No. of hit data.
TBAES2007379 (SEQ ID NOS: 1 and 2) //// SCUBE2 (CEGP1) protein [Homo sapiens] // 0 // 545aa // 67% // NM_020974.

Search for signal sequence, transmembrane region and functional domain for deduced amino acid sequence Presence of amino-terminal signal sequence and transmembrane region for amino acid sequence deduced from full-length nucleotide sequence, and functional domain of protein ( Motif) search. PSORT (K. Nakai, M. Kanehisa, Genomics, 14: 897-911 (1992)) for the amino-terminal signal sequence and SOSUI (T. Hirokawa et. Al., Bioinformatics, 14: 378) for the transmembrane region. -379 (1998), sold by Mitsui Information & Development Co., Ltd.). For search of functional domains, Pfam (http://www.sanger.ac.uk/Software/Pfam/index.shtml) was used. According to PSORT and SOSUI, the amino acid sequence whose amino-terminal signal sequence and transmembrane region were predicted was predicted to be secreted and membrane proteins. In a functional domain search by Pfam, an amino acid sequence that hits a certain functional domain is found in, for example, PROSITE (http://www.expasy.ch/cgi-bin/prosite-list.pl) based on the hit data. The function of the protein can be predicted with reference to the functional category classification. It is also possible to search for a functional domain in PROSITE. The search results by each software are shown below.

  For the protein having the amino acid sequence of SEQ ID NO: 2, a signal sequence was detected in the deduced amino acid sequence by PSORT. SOSUI detected one transmembrane region in the deduced amino acid sequence.

A functional domain is detected in the deduced amino acid sequence by Pfam, and the search result is shown as a clone name (sequence number) // functional domain name, and when multiple functional domains are hit, they are separated by // and listed. did. It should be noted that the same functional domain is described without omission even when a plurality of hits occur.
TBAES2007379 (SEQ ID NO: 2) // EGF-like domain // EGF-like domain // Trypsin Inhibitor like cysteine rich domain // EGF-like domain // EGF-like domain // Keratin, high sulfur B2 protein // EGF- like domain // EGF-like domain // Granulins // EGF-like domain // EGF-like domain // EGF-like domain.

Functional category classification by homology search of deduced amino acid sequence For TBAES2007379, which was found to have a novel cDNA in Example 2, homology was determined for each database of Swiss-Prot, NRDB, and RefSeq of deduced amino acid sequence. From the results of the search (see Example 3), the function prediction and category classification of the protein encoded in the clone were performed.

  The clones estimated to belong to the secretory / membrane protein category are the growth data, cytokine, hormone, signal, transmembrane, membrane, extracellular matrix, receptor, G-protein coupled receptor, ionic channel, voltage-gated There was a description that was estimated to be a secretory / membrane protein such as channel, calcium channel, cell adhesion, collagen, connective tissue, etc., or as a result of analysis of the estimated ORF by Psort and SOSUI, the clone had a signal sequence or transmembrane region. is there.

  A clone presumed to belong to the category of glycoprotein-related proteins is a clone whose hit data has a description of a glycoprotein-related protein such as glycoprotein.

  The clones presumed to belong to the category of signal transduction-related proteins are described in the hit data as serine / threonine-protein kinase, tyrosine-protein kinase, SH3 domain, SH2 domain, etc. Clone.

  Clones that are presumed to belong to the category of transcription-related proteins are clones that have been described in the hit data, such as transcription regulation, zinc finger, and homeobox, as being presumed to be transcription-related proteins.

  The clones presumed to belong to the category of the disease-related protein include those described in the hit data as disease-related proteins such as disease mutation and syndrome, or Swiss-Prot hit data for the full-length nucleotide sequence, and NRDB. , RefSeq hit data are clones whose genes and proteins are registered in Online Mendelian Inheritance in Man (OMIM), which is a database of human genes and diseases.

  Clones presumed to belong to the category of enzyme / metabolism-related proteins are clones whose metabolism, oxidoreductase, ECNo. is there.

  Clones presumed to belong to the category of cell division / proliferation-related proteins are clones that have been described as being cell division / proliferation-related proteins, such as cell division, cell cycle, mitosis, chromosomal protein, cell growth, and apoptosis. It is.

  The clones presumed to belong to the category of cytoskeleton-related proteins are clones described in the hit data, such as structural proteins, cytoskeletons, actin-binding, and microtubles, which are presumed to be cytoskeleton-related proteins.

  The clones presumed to belong to the category of nuclear protein / RNA synthesis-related protein are described in the hit data as nuclear protein / RNA synthesis-related proteins such as nuclear protein, RNA splicing, RNA processing, RNA helicase, and polyadenylation. There was a clone that had.

  The clones that are presumed to belong to the category of protein synthesis / transport-related proteins are those described in the hit data, such as translation regulation, protein biosynthesis, amino-acid biosynthesis, ribosomal protein, protein transport, signal recognition particles, etc. This is a clone that is presumed to be described.

  The clones presumed to belong to the category of cell defense-related proteins are clones in which hit data, such as heat shock, DNA repair, and DNA damage, are described in the hit data.

  A clone presumed to belong to the category of development / differentiation-related proteins is a clone described as a development / differentiation-related protein, such as a developmental protein.

  A clone presumed to belong to the category of DNA / RNA binding protein is a clone in which hit data is described as DNA-binding, RNA-binding, or the like.

  A clone presumed to belong to the ATP / GTP binding protein category is a clone whose hit data has been described as ATP-binding, GTP-binding, or the like.

  In this functional category classification, when one clone corresponds to the plurality of categories described above, the clone was directly classified into the plurality of categories. However, the functions of proteins are not necessarily limited to the classified functional categories, and other functions may be revealed in the future.

  The protein having the amino acid sequence of SEQ ID NO: 2 could be estimated to belong to secretory / membrane proteins.

  In addition, even if information for estimating the function cannot be obtained from the homology search information at present, the function may be clarified by updating the database in the future.

Functional category classification domains and motifs by searching for functional domains for the deduced amino acid sequence are the minimum functional structures of proteins. The structure of a protein is composed of this minimum structure, and as a result, the function of the whole protein is determined. Therefore, it is possible to relatively accurately predict the function of the entire protein from the analysis of the domain and motif structure. In addition, creating a database of the results by function means that a clone having a specific function can be easily selected, which is very useful in analyzing the function of each clone.

  From the results of domain search for Pfam of the amino acid sequence deduced from the full-length nucleotide sequence (see Example 4), the domain of the hit data, the motif name and the accession number, and Pfam (http://www.sanger.ac.uk/ Software / Pfam / index.shtml) and detailed description data in PROSITE (http://www.expasy.ch/cgi-bin/prosite-list.pl) Function prediction and category classification of the protein encoded by.

  The clones presumed to belong to the category of secretion / membrane proteins include, for example, receptors, ion channels, hormones, and growth factors, such as 7 transmembrane receptor, Pancreatic hormone peptides, Ion transport protein, Fibroblast growth factor, etc. This clone has a domain and motif.

  Clones that are presumed to belong to the category of glycoprotein-related proteins are clones that have domains and motifs such as the immunoglobulin domain and Glycosyl transferases group 1 that are presumed to be involved in Glycobiology such as glycoproteins and glycosyltransferases. .

  The clones presumed to belong to the category of signal transduction-related proteins include, for example, Eukaryotic protein kinase domain, Protein phosphatase 2C, Ras, which are presumed to be protein kinases, phosphatases, SH2 domains, Small G proteins, and the like. It is a clone with domains and motifs such as family.

  The clones presumed to belong to the category of transcription-related proteins are those having domains and motifs such as bZIP transcription factor, Zinc finger, and C2H2 type that are presumed to be transcription factors and proteins involved in transcription regulation. .

  Clones that are presumed to belong to the category of disease-related proteins include proteins that are expressed in a specific disease, and those that are presumed to have increased or decreased expression in a disease, such as Wilm's tumour protein, von This clone has domains and motifs such as Hippel-Lindau disease tumor suppressor protein.

  Clones presumed to belong to the category of enzymes and metabolism-related proteins are domains such as Aldehyde dehydrogenase family, Chitin synthase, Glucose-6-phosphate dehydrogenase, which are presumed to be transferase, synthase, hydrolase, etc. , A clone with a motif.

  Clones that are presumed to belong to the category of cell division / proliferation-related proteins are clones that have domains and motifs such as Cyclin and Cell division protein, which are presumed to be cyclins, cell growth control proteins, and the like.

  The clones presumed to belong to the category of cytoskeleton-related proteins are clones having domains and motifs such as Actin, Fibronectin type I domain, and Kinesin motor domain, which are presumed to be actin, kinesin, fibronectin, and the like.

  The clones presumed to belong to the category of nucleoprotein / RNA synthesis-related proteins include splicing factors, RNA synthases, and helicases such as Hepatitis C virus RNA dependent RNA polymerase, DEAD / DEAH box helicase, etc. This clone has a domain and motif.

  Clones that are presumed to belong to the category of protein synthesis / transport-related proteins are translation-related proteins, ubiquitin-related proteins, domains such as the translation initiation factor SUI1, Ubiquitin family, and Ribosomal protein L16 that are presumed to be Ribosomal proteins. , A clone with a motif.

  The clones presumed to belong to the category of cell defense-related proteins are clones having domains and motifs such as Hsp90 protein and DNA mismatch repair protein, which are presumed to be molecular chaperones, DNA repair proteins, and the like.

  The clones presumed to belong to the category of development / differentiation-related proteins are clones having domains and motifs such as Floricaula / Leafy protein, which are presumed to be organogenesis-related proteins.

  The clones presumed to belong to the category of DNA / RNA binding protein include transcription factors, DNA / RNA-related enzymes including DNA ligase, and Zinc-finger-related proteins, for example, Transcription factor WhiB, This clone has domains and motifs such as B-box zinc finger and tRNA synthetases class I (C).

  The clones presumed to belong to the category of ATP / GTP-binding protein include ATP / GTP-related enzymes such as ATPase and domains such as E1-E2 ATPase and Ras family which are presumed to be G proteins. , A clone with a motif.

  In addition, in this functional category classification, when one clone corresponds to the above-mentioned plurality of categories, it was classified into a plurality of categories as it is. However, the functions of proteins are not necessarily limited to the classified functional categories.

  The protein having the amino acid sequence of SEQ ID NO: 2 was a clone that did not clearly belong to any of the above categories, although hit data was found for Pfam (see Example 4). However, it has a domain called EGF-like (EGF-like domain) and a repeat of the EGF-like domain (EGF-like repeat). From the properties of the domain and the repeat, it can be estimated that the protein having the amino acid sequence of SEQ ID NO: 2 has an EGF-like physiological activity, that is, the same physiological activity as the protein having the EGF-like domain or the EGF-like repeat. Was. In addition, the amino acid sequence of SEQ ID NO: 2 has a cysteine-rich motif and domain characteristic of membrane proteins, such as a trypsin inhibitor-like cysteine-rich domain, a keratin (high sulfur B2 protein) motif, and a granulin motif. It could be estimated to belong to secretory and membrane proteins.

  In the future, with the accumulation of data on proteins with similar domains and motifs, the functions will be elucidated in more detail, and it may be possible to categorize them into the above categories.

  In addition, even if there is no hit data in Pfam, if a new domain or motif is found together with accumulation of protein data in the future, the deduced amino acid sequence of the clone is again analyzed by a new database to obtain a new one. Domains and motifs with functions may be discovered and categorized.

Detailed Analysis of Sequence of cDNA Clone TBAES2007379 (SEQ ID NOS: 1 and 2) For the entire base sequence of the cDNA clone analyzed in Examples 3 to 6, BLAST (Basic local alignment search tool; Altschul, SF, et al., J. Am. Mol. Biol., 215: 403-410 (1990)), and HMMER (sequence analysis method using hidden Markov model; Eddy, SR, Bioinformatics, 14: 755-763 (1998)). In addition to protein feature search (profile search: http://pfam.wustl.edu) by HMMPFAM, which is one of the functional groups, PROSITE (a database of amino acid patterns that classify domain structures and families according to similarity of protein functions; Nucleic Acids Res., 30: 235-8 (2002)), and the function of the protein encoded by each cDNA clone was estimated. In addition, for a clone presumed to be a splicing variant in which a known clone in which a part of the nucleotide sequence is completely identical exists, any exon is deleted and spliced if its genome sequence can be analyzed. Was analyzed. Further, detailed analysis was performed using PSORTII (Trends Biochem. Sci., 24: 34-6 (1999)), a program for predicting the intracellular localization of proteins. The results are shown below.

  TBAES2007379, which is a clone presumed to be a secretory / membrane protein in Examples 3 to 6, is composed of 3119 bases, as shown in SEQ ID NO: 1, of which base numbers 70 to 2118 have an open reading frame (terminated). Including codons). The amino acid sequence predicted from the open reading frame consists of 682 amino acid residues (SEQ ID NO: 2). A homology search was performed on the sequence of SEQ ID NO: 1 with respect to the public DB and GeneSeq using BLAST, and a plurality of sequences having high homology to the amino acid sequence of SEQ ID NO: 2 were found. Among these, the protein encoded by the RefSeq registration code NM020974 was a known protein called SCUBE2 (Signal peptide-CUB-EGF-like domain containing protein 2) (J. Biol. Chem., 277: 46364). -46373 (2002)). As described above, what was found here was a variant called “SCUBE2a” of SCUBE2, but it is known that this SCUBE2a is the most widespread among SCUBE2, and is abbreviated as SCUBE2. This is called SCUBE2 in the present embodiment because there are many such cases.

  In addition, GeneSeq registration numbers ABK92209 (WO02 / 30268), ABN86363 (WO02 / 55988), ABT07724 (WO02 / 59377), ABT17101 (WO02 / 98358), ABZ11546 (WO02 / 70539). ), ACC50098 (WO03 / 004989) and ADG89346 (WO03 / 078662). These sequences were completely identical to NM020974 (SCUBE2) in amino acid sequence although a difference in nucleotide sequence was recognized. . FIG. 1 shows a structural comparison between TBAES2007379 and NM020974 (SCUBE2).

  The sequences of GeneSeq registration numbers ABT31918 (WO03 / 000012), AAL51944 (WO03 / 002610), ADD78281 (WO03 / 077875), and RefSeq registration numbers AK123039 are NM020974 (SCUBE2), respectively. In comparison, similar to TBAES2007379, partial deletion, amino acid substitution, and partial sequence addition were observed.

  Among them, particularly in TBAES2007379, a relatively long deletion was observed in the central part and C-terminal part of NM020974 (SCUBE2) (FIG. 1). These deletions were considered to be due to deletion of specific exons from the results of genome analysis. A comparison of the genomic structures of TBAES2007379 and NM020974 (SCUBE2) is shown in FIG. As shown in FIG. 2, NM020974 (SCUBE2) has a total of 22 exons, but TBAES2007379 lacks the 12th to 14th exons and the 19th exon, and the 19th exon Deletion results in a subsequent frame shift within the exon, resulting in a stop codon. As a result, the amino acid sequence of TBAES2007379 contained a specific linking sequence (amino acids 443 to 444 of SEQ ID NO: 2) and a C-terminal sequence (amino acids 678 to 682 of SEQ ID NO: 2) as compared to NM020974 (SCUBE2). Turn) was found to exist. Further, arginine at amino acid number 280 in NM020974 (SCUBE2) is glutamine at number 280 in TBAES2007379. It was presumed that the use of these TBAES2007379-specific sequences would enable the production of TBAES2007379-specific antibodies and the like.

Expression frequency analysis in silico cDNA libraries derived from various tissues and cells shown in Example 1 were prepared, cDNA clones were randomly selected from each library, and the sequence of the 5 ′ terminal region was determined. , Made into a database. This database is a database of base sequences of 1,402,069 clones, and is a sufficient database for analysis parameters. Although cDNA clones were randomly selected, TBAES2007379 was included in the database, so the analysis was proceeded as follows.

  The base sequence of each clone in this database is categorized (clustered) by the base sequence homology search program, and the number of clones belonging to each cluster is totalized and normalized for each library. The abundance ratio of a certain gene in a cDNA library was analyzed. Through this analysis, information on the expression frequency of a certain gene in the tissue or cell that is the source of the cDNA library was obtained.

  Next, in order to analyze the expression of the gene having the nucleotide sequence of the cDNA of the present invention between tissues and cells, a library derived from tissues or cells obtained by analyzing a large amount of cDNA clones was used to express the amount of expression between tissues and cells. Was included in the comparison. That is, with respect to tissues and cells obtained by analyzing the base sequences of 600 or more cDNA clones, previously normalized values were compared between tissues and cells, and changes in gene expression frequencies were analyzed.

  In cancer tissues, it is thought that a set of genes different from normal tissues is expressed and contributes to canceration of tissues and cells. Therefore, genes that are expressed differently from normal tissues are cancer-related genes. Genes whose expression was altered in cancer tissues compared to normal tissues were searched.

Among them, TBAES2007379 (SEQ ID NO: 1) is remarkable in both cases as a result of analyzing and comparing cDNAs of a breast cancer-derived library (TBAES) and a normal breast-derived library (BEAST) (Table 1). An expression change was indicated. Therefore, TBAES2007379 was presumed to be a gene related to breast cancer. In addition, each numerical value in Table 1 shows a relative expression frequency, and the larger the numerical value, the greater the expression level.

[Table 1]
-----------------------------
Clone ID BEAST TBAES
-----------------------------
TBAES2007379 0.000 100.000
-----------------------------

  The expression frequency of TBAES2007379 was analyzed for cDNAs of libraries derived from various other cancer tissues, various normal tissues, various adult tissues, various fetal tissues, and various cells, and various comparisons were made. There was no.

Preparation of protein
(1) Construction of TBAES2007379 CHO expression system TBAES2007379 gene was amplified by PCR using TBAES2007379 which is a clone which was found to have a novel cDNA in Example 2 as a template. In the PCR method, a synthetic forward primer (SEQ ID NO: 7) and a synthetic reverse primer (SEQ ID NO: 8) were used.

  The amplified PCR product was treated with the restriction enzyme EcoRI (manufactured by TAKARA), partially digested with PstI (manufactured by TAKARA), a band of about 2 kb was cut out by agarose gel electrophoresis, and a DNA fragment was recovered. . This DNA fragment was introduced into the EcoRI (890) and PstI (1319) sites of pME-sigXaIg (SEQ ID NO: 9) as a target gene to prepare an expression vector. The number shown in parentheses after the name of the restriction enzyme is the base number on the sequence of the plasmid pME-sigXaIg described in SEQ ID NO: 9, and indicates the restriction enzyme site. Escherichia coli DH5α strain (manufactured by TOYOBO) was transformed with the obtained plasmid, and plasmid DNA was extracted and purified from the colonies, and the DNA sequence was confirmed.

The expression vector prepared here contains the signal sequence of the HAI-2 gene (FEBS Letters, 436: 111-114 (1998)) upstream of the inserted target gene, and the Fc region of human IgG1 downstream of the inserted target gene. (For tag addition), and a vector having a protease recognition sequence (for Factor Xa) between the target gene and the human Fc region. When this is expressed, a fusion protein in which a human Fc tag is linked to the C-terminal side of the full-length protein of TBAES2007379 via a protease recognition sequence is expressed.
(2) Expression of TBAES2007379 by CHO expression system

The CHO cell line was transduced with the expression vector prepared in the above (1) together with pBSpacΔp (Gene, 62 (1): 121-126 (1988)) which is a vector for imparting puromycin resistance. Transfectam ^R (manufactured by BIOSEPRA) was used for transduction, and the experimental protocol was in accordance with the instructions attached to the reagent. Puromycin resistance was imparted, and transduced strains that had been grown on an eRDF medium (manufactured by Kyokuto Pharmaceutical Co., Ltd.) containing puromycin (10 μg / ml) and FBS (10%: GIBCO) were selected, and each was used for 96 wells. On a cell culture plate (Corning).

  Next, a clone expressing the desired fusion protein was selected by performing ELISA (enzyme-linked immunosorbent assay) for detecting human Fc on the culture supernatant cultured on the 96-well cell culture plate. In ELISA for detecting human Fc, a goat polyclonal antibody (manufactured by ICN) that reacts with human IgG is immobilized on a 96-well cell culture plate in advance, and the culture supernatant of each clone described above is added thereto. After reacting at 37 ° C. for 1 hour, the plate was washed, reacted with an HRP (horseradish peroxidase) -labeled anti-human IgG goat polyclonal antibody (ICN), washed, and subjected to a color development reaction using OPD (o-Phenylenediamine) as a substrate. The measurement was performed by measuring the intensity of color development with an absorptiometer.

  The clones selected as described above were cultured on culture plates (Corning) each having a diameter of 10 cm, and the cells were collected. 100 μl of PBS buffer was added to the collected cells, and the cells were disrupted by ultrasonic waves on ice. Western blot analysis was performed on the cell lysates.

  Antibodies for detection used in western blotting analysis include (a) a goat polyclonal antibody (ICN) that reacts with HRP-labeled human Fc, and (b) immunization with a peptide described later in Example 10. Two kinds of rabbit antisera described later in Example 11 were used. That is, a rabbit antiserum obtained by immunizing partial peptide 4 and a rabbit antiserum obtained by immunizing a mixture of partial peptides 2 to 4 are used, and a mixture of these two types of antisera is also used. Using. In the case of (b), in order to reduce the non-specific reaction of the antiserum, it was used after preliminarily reacting at 37 ° C. for 1 hour with a crushed CHO cell into which no gene had been introduced. In the case of (b), an HRP-labeled anti-rabbit IgG goat polyclonal antibody (manufactured by DAKO) was used as the labeled antibody, and the detection was performed in both cases (a) and (b) using the ECL plus chemiluminescence kit (Amersham Bioscience) ).

  FIG. 3 shows the results of the above protein expression confirmation. In FIG. 3, when any antibody was reacted, a band was observed in the vicinity of the molecular weight (98.4 kDa) expected as a fusion protein of TBAES2007379 full length and human Fc, confirming that TBAES2007379 was expressed. .

  In addition, it was shown that the two kinds of rabbit antisera described later in Example 11 both specifically bind to the expressed protein.

Preparation of partial peptide of TBAES2007379 and antigen for immunization A partial peptide of TBAES2007379 was synthesized using a peptide synthesizer (Applied Bio). As the sequence to be synthesized, the amino acid sequences of TBAES2007379 and SCUBE2 were compared, and different sequences (sequences specific to TBAES2007379) were used. That is, partial peptides 2 and 3 are sequences containing specific linking sequences (amino acid numbers 443 to 444) generated by deletion of exons 12 to 14 of TBAES2007379. Since partial peptide 1 lacks exon 19, it causes a frame shift within the subsequent exon to generate a stop codon. Therefore, the C-terminal sequence (amino acids 678 to 682) Number).

For antibody production, partial peptide 1 (SEQ ID NO: 10: corresponding to amino acid numbers 666 to 682 of SEQ ID NO: 2) was obtained by introducing partial peptide 4 (SEQ ID NO: 13) into which a functional group necessary for binding to a carrier protein and a linker were introduced. ). Partial peptide 2 (SEQ ID NO: 11: corresponding to amino acids 439 to 458 of SEQ ID NO: 2) and partial peptide 3 (SEQ ID NO: 12: corresponding to amino acids 431 to 450 of SEQ ID NO: 2) are amino acids having a functional group at the terminal Was synthesized using the sequence as it was.
Partial peptide 1: TTDFDGSTNITQFCDNI (SEQ ID NO: 10)
Partial peptide 2: KKDCVASCDLSCIVKRTEKR (SEQ ID NO: 11)
Partial peptide 3: PGYKLHWNKKDCVASCDLSC (SEQ ID NO: 12)
Partial peptide 4: CGTTDFDGSTNITQFCDNI (SEQ ID NO: 13)

  For each of the prepared peptides, a peptide protein conjugate for immunization (peptide KLH conjugate) was prepared using KLH (Keyhole Limpet Hemocyanin: PIERCE) as a carrier protein.

  Two methods were used to bind each partial peptide to KLH. In the first method (carbodiimide method), a solution prepared by dissolving each peptide and KLH in a MES buffer pH 6.0 at a molar ratio of 10: 1 is prepared. Carbodiimide (Dojinsha) was added so as to be twice equivalent, and reacted at room temperature for 1 hour. Thereafter, dialysis was carried out overnight using a phosphate buffer pH 7.0 to remove unreacted substances. The second method (maleimide method) was performed according to the method described in this kit using Imject Maleimide Activated Carrier Proteins (PIERCE) in which a maleimide group was introduced into KLH.

Specifically, first, three kinds of partial peptides 2, 3, and 4 were respectively bound to KLH by a carbodiimide method or a maleimide method to prepare a total of six kinds of conjugates. Next, three types of conjugates prepared by two methods were mixed to prepare two types of mixtures. Table 2 shows the combinations of the prepared conjugates and the immunized animals.

Preparation of Polyclonal Antibody against TBAES2007379 Each solution containing the peptide KLH conjugate prepared in Example 10 was subcutaneously and intraperitoneally administered twice a week to Balb / c mice together with the same volume of Freund's complete adjuvant. Thereafter, it was confirmed that the antibody titer was increased in the mouse serum. Blood was collected from the tail vein of the mouse, and the serum was separated by centrifugation to obtain a mouse polyclonal antibody against TBAES2007379.

  Rabbit polyclonal antibodies were also made. The solution containing the peptide KLH conjugate prepared in Example 10 was subcutaneously administered to rabbits together with the same volume of Freund's complete adjuvant. Eight weeks later, blood was collected from the ear, and serum was separated by centrifugation to obtain a rabbit polyclonal antibody against TBAES2007379.

  Combinations of the conjugate and the immunized animal used for immunization are as shown in Table 2 of Example 10 above.

Preparation of ELISA Plate for Analysis of Antibody to TBAES2007379 The six peptide KLH conjugates prepared in Example 10 were used as antigens for antibody analysis, and each peptide KLH conjugate was mixed to a final concentration of 2 μg / ml and physiologically mixed. After dilution in a hydrogen phosphate buffer (PBS (-)), 50 μl was added to each well of a 96-well plate (Coaster). Thereafter, the cells were stored at 4 ° C. for 24 hours, and each antigen was adsorbed to a 96-well plate. The solution was removed from the antigen-adhered plate, and 250 μl of PBS (−) containing 5% BSA was added to each well, and the blocking operation was carried out at 4 ° C. for 24 hours (about 12 hours) or at 37 ° C. for 2 hours or more. And stored at 4 ° C. as an ELISA plate for specificity analysis. A 96-well plate on which only KLH was adsorbed was prepared in the same manner as a control. These ELISA plates were used immediately before use except for the blocking solution in the plates.

Analysis of Specificity and Affinity of Antibody to TBAES2007379 Among the ELISA plates for analyzing the antibody to TBAES2007379 prepared in Example 12, the six types of mice obtained in Example 11 were compared with the plate to which the peptide KLH conjugate was adsorbed. Antiserum and two kinds of rabbit antisera were diluted and added, and the reactivity of the polyclonal antibody contained in the antiserum was analyzed.

  To two types of ELISA plates for specificity analysis of antibodies to TBAES2007379 (peptide KLH conjugate adsorption plate and plate to which only KLH was adsorbed), diluted antisera were added at 50 μl / well, and then added at 4 ° C. The reaction was allowed to proceed for more than an hour. PBS (-) was used as the diluent, and the dilution ratio was 20250, 60750, 182250, 546750 times.

Thereafter, a sufficient washing was performed using a PBS (−) solution containing 0.05% Tween 20 (hereinafter, this may be referred to as “PBST solution”), and the mouse antiserum was used as a secondary antibody. In this case, HRP-labeled sheep anti-mouse IgG polyclonal antibody (DAKO) (1 μg / ml) and PBS (-) containing 1% BSA were added to each well in an amount of 100 μl. 100 μl of PBS (−) containing 1 μg / ml and 1% BSA was added to each well, and the mixture was further reacted at room temperature for 1 hour. After sufficiently washing with a PBST solution, a citrate-phosphate buffer containing 0.4 mg / ml orthophenylenediamine (OPD, Sigma P-9029) and a 0.015 to 0.03% hydrogen peroxide solution. A liquid (pH 5.0) was added and reacted at room temperature to perform a color development reaction. Thereafter, a 1N H ₂ SO ₄ solution was added to stop the reaction, and the absorbance was measured at a measurement wavelength of 490 nm and a reference wavelength of 650 nm. FIG. 4 shows the obtained results. FIG. 5 shows the difference from the reaction with the plate on which only KLH was adsorbed.

  The antibody prepared in Example 11 reacted with the peptide KLH conjugate plate prepared in Example 12. In addition, it was confirmed that an antibody specifically reacting with TBAES2007379 was produced, having higher reactivity than the plate on which only the same amount of KLH as the peptide KLH conjugate was adsorbed. In addition, since there was a reaction even after dilution at a high magnification of 546750 times, it was also confirmed that a high-affinity antibody could be produced.

Analysis using antibody against TBAES2007379 Using the antibody obtained in Example 11, the presence of TBAES2007379 in various cancer cell lines and human normal tissues was analyzed by dot blot.

The names of the cancer cell lines used are shown in Table 3. As each cancer cell line, a commercially available product available from ATCC or the like was used. Each cancer cell line was cultured on a culture plate (Corning) having a diameter of 10 cm, and the cells were collected. 100 μl of PBS buffer was added to the collected cells, and the cells were disrupted by ultrasonic waves on ice. 1 μl of the obtained cell lysate was blotted on a nitrocellulose membrane and analyzed.

[Table 3]
---------------------------------
Name organ
---------------------------------
1 MKN1 stomach
2 MKN28 stomach
3 MKN45 stomach
4 HSC-3 stomach
5 Lic liver
6 HepG2 liver
7 MCF7 breast
8 SW387 Large intestine
9 DLD-1 Large intestine
10 Colo205 large intestine
11 HLC lung
12 LKAZ Lung
13 PC-3 Lung
14 PC-9 Lung
15 PANC-1 pancreas
16 H62 Lung
17 Control (recombinantly expressed TBAES2007379)
---------------------------------

Experiments on human normal tissues were performed according to the experimental protocol attached to commercially available normal tissues (Protein Medley, Cat. No. 1602-1) purchased from Clontech. Table 4 shows the types of normal tissues used. In each case, 1 μl of a 10 mg / ml solution was used for blotting.

[Table 4]
---------------------------------
Organ
---------------------------------
1 brain
2 Thyroid
3 heart
4 lungs
5 lymph nodes
6 spleen
7 Liver
8 stomach
9 small intestine
10 kidney
11 ovaries
12 uterus
13 placenta
14 testes
15 bladder
16 controls (recombinantly expressed TBAES2007379)
---------------------------------

  As a control, the recombinant TBAES2007379 expressed in Example 9 was used, and an appropriate amount of a CHO cell expressing the recombinant TBAES2007379 was blotted as a positive control.

  The antibody prepared in Example 11 was used as a primary antibody. In order to reduce non-specific reactions, the cells were used after preliminarily reacting at 37 ° C. for 1 hour with crushed CHO cells into which no gene was introduced. As a secondary antibody, an HRP-labeled anti-rabbit IgG goat polyclonal antibody (DAKO) was used. Detection was performed using an ECL plus chemiluminescence kit (Amersham Bioscience). FIG. 6 shows the results of dot blots of various cancer cell lines, and FIG. 7 shows the results of dot blots of normal tissues.

  As a result, in the cancer cell line, a clear reaction similar to that of the control was obtained in breast-derived MCF7, large intestine-derived DLD-1 and Colo205, and lung-derived HLC. No response was observed in normal human tissues, and only the control reacted. Therefore, it was found that TBAES2007379 specifically expresses a protein in cancer cells, and particularly expresses a strong protein in breast cancer cells, colon cancer cells, and lung cancer cells. From these results, it was considered that TBAES2007379 could be applied to the diagnosis and treatment of cancer, and was particularly highly useful for the diagnosis of cancer.

Construction of TBAES2007379 measurement system and measurement of TBAES2007379 in human blood The rabbit antibody obtained in Example 11 was used as a primary antibody. This antibody was dissolved in a 0.05 M carbonate-bicarbonate buffer (pH 9.6) to a concentration of 30 μg / ml, added to a 96-well plate at 100 μl / well, and incubated at 4 ° C. overnight (12 hours). Or more). After removing the primary antibody solution from the primary antibody-attached plate, PBS (-) containing 1% BSA was added at 250 to 300 μl / well, and the whole solution was added at 4 ° C. overnight (about 12 hours) or at 37 ° C. for 2 hours. Blocking was performed as described above. The blocking solution was removed from this plate, and 100 μl of human serum of various cancer patients was added thereto, or 100 μl of the recombinant TBAES2007379 expressed in Example 9 as a control was added as a CHO cell culture supernatant at a room temperature. For about 1 hour. Human sera from various cancer patients were purchased from Veritas. 8 sera from pancreatic cancer patients, 6 sera from liver cancer patients, 5 sera from colorectal cancer patients, 1 sera from gastric cancer patients, 1 sera from small intestine cancer patients, and healthy subjects (normal) Two serum samples were used.

After the reaction, a sufficient washing was performed using a washing solution composed of 500 mM NaCl, 0.05% Tween 20, and 20 mM Tris-HCl, pH 7.5, and then the mouse antibody prepared in Example 11 at 1 μg / ml and 1 μg / ml. 100 μl of PBS (−) containing% BSA was added to each well and reacted at room temperature for about 1 hour. Next, sufficient washing is performed using the above-mentioned washing solution, and 100 μl of PBS (−) containing 1% BSA diluted 2000-fold with an HRP-labeled sheep anti-mouse IgG polyclonal antibody (DAKO) as a secondary antibody is added to each well. The mixture was added and reacted at room temperature for 1 hour. After sufficiently performing a washing operation with a washing solution, a citrate-phosphate buffer solution containing 0.4 mg / ml orthophenylenediamine (OPD, Sigma P-9029) and a 0.015 to 0.03% hydrogen peroxide solution (PH 5.0) was added in an amount of 100 μl / well, and reacted at room temperature to develop color. Thereafter, a 1N H ₂ SO ₄ solution was added at 100 μl / well to stop the reaction, and the absorbance was measured at a measurement wavelength of 490 nm and a reference wavelength of 650 nm. FIG. 8 shows the results.

  As a result, 15 out of 21 sera of cancer patients showed a higher value than normal. In particular, some patients showed high levels in pancreatic cancer patients. Some specimens also showed high values in liver cancer and small intestine cancer. The results showed that the measurement of TBAES2007379 in blood was useful for diagnosing cancer.

Preparation of Monoclonal Antibody Against TBAES2007379 Each solution containing the peptide KLH conjugate prepared in Example 10 is subcutaneously and intraperitoneally administered to Balb / c mice three times at 4 week intervals together with the same volume of complete Freund's adjuvant. After confirming that the antibody is produced in the serum of the mouse, a solution containing 10 μg of the peptide KLH conjugate is administered into the tail vein. Three days later, the spleen was removed, and the spleen cells were fused with myeloma cells P3U1 using Polyethylene Glycol 1500 according to the "Monoclonal Antibody Experiment Manual" (published by Kodansha Scientific 1987) and injected into a 96-well plate. Thereafter, a HAT medium is added and culturing is performed for 14 days.

  Thereafter, hybridomas producing a monoclonal antibody specific to TBAES2007379 in the medium are selected. That is, the culture supernatant of the selected hybridoma was added to the plate to which the peptide KLH conjugate was adsorbed among the ELISA plates for analysis of the antibody against TBAES2007379 prepared in Example 12, and the monoclonal antibody present in the culture supernatant was Analyze reactivity. The culture supernatant of the hybridoma to be selected is added to an ELISA plate for analyzing the specificity of the antibody to TBAES2007379 at 100 μl / well, and then reacted at 4 ° C. for 2 hours or more.

Thereafter, the plate was sufficiently washed with a PBS (−) solution containing 0.05% Tween 20 (PBST solution), and 1 μg / ml of HRP-labeled sheep anti-mouse IgG / Fc polyclonal antibody (ICN) and 1% BSA were added. 100 μl of PBS (−) is added to each well, and the mixture is further reacted at room temperature for 1 hour. After washing thoroughly with a PBST solution, a citrate-phosphate buffer containing 0.4 mg / ml orthophenylenediamine (OPD, Sigma P-9029) and a 0.015 to 0.03% hydrogen peroxide solution. A solution (pH 5.0) is added and reacted at room temperature to develop color. Thereafter, a 1N H ₂ SO ₄ solution is added to stop the reaction, and the absorbance is measured at a measurement wavelength of 490 nm and a reference wavelength of 650 nm.

  Next, using the culture supernatant of the monoclonal antibody-producing hybridoma having reactivity with the peptide KLH conjugate obtained here, screening was carried out in the same manner on an ELISA plate prepared in Example 12 to which only KLH was adsorbed. A hybridoma showing no reactivity to KLH is selected. By this screening, a hybridoma producing a monoclonal antibody specific to TBAES2007379 is obtained.

  Each of the obtained hybridomas is subjected to cloning operation four times by the limiting dilution method, and then the supernatant is recovered, and the monoclonal antibody is purified by affinity chromatography (Amersham Pharmacia Biotech) to which protein A is bound. Thus, a monoclonal antibody having specific reactivity to the protein of the present invention (TBAES2007379) can be obtained.

This application is based on Japanese Patent Application (No. 2003-131452) filed on May 9, 2003, the contents of which are incorporated herein by reference. Also, all prior art documents cited herein are incorporated herein by reference.

FIG. 1 is a diagram comparing the structure of a protein having the amino acid sequence of SEQ ID NO: 2 (TBAES2007379 protein) with the structure of a known protein NM020974 (SCUBE2). FIG. 2 is a diagram comparing the genomic structures of a DNA having the nucleotide sequence of SEQ ID NO: 1 (TBAES2007379) and a cDNA encoding the known protein NM020974 (SCUBE2). FIG. 3 shows the results of confirming the expression of the TBAES2007379 protein by the CHO expression system by Western blotting. FIG. 4 shows the results of measuring the reactivity of a polyclonal antibody obtained by using a conjugate of KLH with a conjugate of partial peptides 2 to 4 (including a mixture) and KLH as an immunogen. FIG. 5 shows the difference between the reactivity of a polyclonal antibody obtained using a peptide conjugate as an immunogen to a peptide KLH plate and the reactivity of a plate to which only KLH was adsorbed. FIG. 6 shows the results of dot blot on various cancer cells using an antibody specific to TBAES2007379. FIG. 7 shows the results of dot blot on normal tissues using an antibody specific to TBAES2007379. FIG. 8 shows the results of measuring TBAES2007379 in the serum of various cancer patients by ELISA.

Claims (21)

Any one of the following proteins (a) to (f):
(A) a protein consisting of the amino acid sequence of SEQ ID NO: 2,
(B) a protein comprising an amino acid sequence in which one or several amino acids are deleted, substituted and / or added in the amino acid sequence of SEQ ID NO: 2, and whose expression is increased in cancer cells or cancer tissues,
(C) a protein consisting of the partial amino acid sequence of the amino acid sequence set forth in SEQ ID NO: 2 and having increased expression in cancer cells or cancer tissues;
(D) a protein comprising an amino acid sequence in which one or several amino acids have been deleted, substituted and / or added in the amino acid sequence of SEQ ID NO: 2, and having an EGF-like physiological activity,
(E) a protein comprising a partial amino acid sequence in the amino acid sequence of SEQ ID NO: 2 and having an EGF-like physiological activity,
(F) an amino acid sequence represented by SEQ ID NO: 2 in which one or several amino acids have been deleted, substituted and / or added, and at least the amino acid sequence represented by any of SEQ ID NOs: 10 to 12 Contains one protein. The protein according to claim 1, wherein the cancer is breast cancer, colon cancer, lung cancer or pancreatic cancer. A DNA encoding the protein according to claim 1. A full-length cDNA encoding the protein according to claim 1. Any one of the following DNAs (a) to (f);
(A) a DNA having the nucleotide sequence of SEQ ID NO: 1,
(B) encoding a protein having a nucleotide sequence in which one or several nucleotides are deleted, substituted and / or added in the nucleotide sequence of SEQ ID NO: 1, and whose expression is increased in cancer cells or cancer tissues DNA
(C) a DNA comprising a partial nucleotide sequence in the nucleotide sequence of SEQ ID NO: 1 and encoding a protein whose expression is increased in cancer cells or cancer tissues;
(D) a DNA having a base sequence in which one or several bases are deleted, substituted and / or added in the base sequence of SEQ ID NO: 1, and encoding a protein having an EGF-like physiological activity;
(E) a DNA consisting of a partial nucleotide sequence in the nucleotide sequence of SEQ ID NO: 1 and encoding a protein having an EGF-like physiological activity;
(F) the base sequence of SEQ ID NO: 1 having a base sequence in which one or several bases have been deleted, substituted and / or added, and the amino acid sequence of any of SEQ ID NOs: 10 to 12; DNA comprising at least one nucleotide sequence encoding The DNA according to claim 5, wherein the cancer is breast cancer, colon cancer, lung cancer or pancreatic cancer. A recombinant vector comprising the DNA according to claim 3. A transgenic cell into which the DNA according to any one of claims 3 to 6 or the recombinant vector according to claim 7 has been introduced, or an individual comprising the cell. A protein produced by the cell according to claim 8. A sense oligonucleotide having the same sequence as 5 to 100 consecutive nucleotides in the base sequence of the DNA according to any one of claims 3 to 6, an antisense oligonucleotide having a sequence complementary to the sense oligonucleotide, and An oligonucleotide derivative of the sense or antisense oligonucleotide, a double-stranded oligonucleotide consisting of a sense oligonucleotide or an antisense oligonucleotide having the same sequence as 15 to 30 consecutive bases in the base sequence, and the two Oligonucleotides selected from the group consisting of oligonucleotide derivatives of strand oligonucleotides. An antibody or a partial fragment thereof which specifically binds to the protein according to any one of claims 1, 2 and 9. The antibody according to claim 11, wherein the antibody has an action of neutralizing the activity of the protein according to any one of claims 1, 2 and 9. 10. A method for controlling the activity of a protein according to claim 1, which comprises bringing the protein according to claim 1, 2 or 9 into contact with a test substance, and measuring a change in the activity of the protein caused by the test substance. Screening method. A test substance is brought into contact with a cell that expresses the DNA encoding the protein according to claim 1 or 2 or the gene-introduced cell according to claim 8, and a change in the expression level of the DNA introduced into the cell is detected. A method for screening for a substance regulating the expression of said DNA. 7. At least one or more amino acid sequence information selected from the amino acid sequence of the protein according to claim 1 and / or at least one or more bases selected from the DNA base sequence according to any one of claims 3 to 6. A computer-readable recording medium that stores sequence information. A carrier to which the protein according to any one of claims 1, 2 and 9, the DNA according to any one of claims 3 to 6, and / or the antibody according to claim 11 or 12 is bound. A method for detecting cancer, comprising measuring the expression state of the DNA according to any one of claims 3 to 6 in a biological sample. A method for detecting cancer, comprising immunologically measuring the protein according to claim 1 or 2 in a biological sample using the antibody or a partial fragment thereof according to claim 11 or 12. 10. Detection of cancer, characterized in that an autoantibody against the protein according to claim 1 or 2 in a biological sample is immunologically measured using the protein according to claim 1, 2 or 9. Method. The detection method according to any one of claims 17 to 19, wherein the cancer is breast cancer, colon cancer, lung cancer, or pancreatic cancer. A cancer comprising at least one selected from the group consisting of the protein according to any one of claims 1, 2 and 9, the antibody according to claim 11 or 12, or the carrier according to claim 16. Reagent kit for detecting DNA.