JP2006136319A - Marker gene of large intestine cancer - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a new human originated mannose transferase gene as a marker gene of large intestine cancer. <P>SOLUTION: Hmat-Xa gene coding either one of the following proteins (A) or (B) is provided; (A) a protein having a specific amino acid sequence, (B) a protein with an amino acid sequence containing substitution, deletion, insertion and/or addition of one or several amino acid substituents for/from/into/to the amino acid sequence having the specific amino acid sequence, and having sugar transferase activity. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

   The present invention relates to a novel tumor marker gene useful for diagnosis of colorectal cancer.

With the development of biotechnology, human hormones and blood coagulation factors have been produced as biopharmaceuticals. A considerable number of biopharmaceuticals are called glycoproteins in which a sugar chain is covalently bonded to a protein. In the process of establishing a production system in E. coli based on the cloning of these useful glycoprotein genes, it was found that the activity could not be expressed by recombination of the human gene into E. coli. The main reason for this is that in Escherichia coli, there is no sugar chain biosynthetic system to be added when it is produced in the human body, and therefore proteins lacking sugar chains are produced. For this reason, production systems using cultured animal cells such as Chinese hamster ovary cells (CHO cells) that have the same sugar chain biosynthesis system as humans have been developed as useful glycoprotein production systems. It has been put into practical use. The main sugar chains of glycoproteins include asparagine-linked sugar chains and mucin-type sugar chains. Asparagine-linked sugar chain of glycoprotein is a precursor of lipid intermediate [Glc ₃ Man ₉ GlcNAc ₂ -PP-Dolicol (Dol): Glc; glucose, Man; mannose, GlcNAc; N-acetylglucosamine, P: phosphate] The body. The biosynthesis of the lipid intermediate is initiated on the cytoplasmic side of the rough endoplasmic reticulum membrane, a seven-step reaction using sugar nucleotides (UDP-GlcNAc and GDP-Man) as sugar donors, and within the rough endoplasmic reticulum membrane The reaction is carried out by a seven-stage reaction using dolichol phosphate sugars (Dol-P-Man and Dol-P-Glc) after transfer to the cavity side as a sugar donor, for a total of 14 stages of glycosyltransferase reaction. The structure of the lipid intermediate and its biosynthesis process are common among eukaryotes such as yeast, plants and animals.

  One of the present inventors is a tunicamycin that specifically inhibits N-acetylglucosamine-1-phosphate transferase, which is the first reaction in the biosynthesis process of lipid intermediates in the culture of Raji cells derived from human Burkitt lymphoma. It was found that the cell proliferation was reversibly stopped in the G1 phase by administration of [Nishikawa, Y., Yamamoto, Y., Kaji, K. and Mitsui, H .: Biochem. Biophys. Res. Commun. 97 : 1296-1303 (1980)]. This is the first time that the biosynthesis of lipid intermediates and the biosynthesis of asparagine-linked glycans of glycoproteins are necessary for passing through the G1 phase of the eukaryotic cell cycle. This discovery provided the rationale for the isolation of a lipid intermediate biosynthetic mutant G258 (possibly a mannose transferase IV mutant) from mouse breast cancer-derived FM3A cells by one of the inventors [Nishikawa , Y .: J. Cell. Physiol. 119: 260-266 (1984)]. In addition, it was further tested in yeast and became a theoretical basis for the separation of lipid intermediate biosynthetic mutants in yeast. From the above research flow, the present inventors have focused on the first five steps of mannose transferase, which is performed on the cytoplasmic side of the rough endoplasmic reticulum membrane in the lipid intermediate biosynthesis process. Yes. Based on the amino acid sequence of the mannose transferase gene cloned in yeast, human genes have been homologated and cloned. That is, as human genes homologous to the ALG1 gene (mannose transferase I gene), ALG2 gene (which has been referred to as mannose transferase III gene), and ALG11 gene (which has been referred to as mannose transferase V gene) cloned in yeast The genes Hmat-1, Hmat-3 and Hmat-5 were cloned. And the base sequence was determined and the deduced amino acid sequence was obtained from it. They include a lycosyl transferase group 1 domain unique to glycosyltransferases using sugar nucleotides as sugar donors and a DXD (DDX) sequence that is considered to be a sugar nucleotide binding site (D: aspartic acid, X: any amino acid). had.

  The structural change of glycoprotein sugar chains accompanying canceration has been reported since around 1975. Here, as an asparagine-linked sugar chain branching structure of glycoprotein due to canceration, N-acetylglucosamine added by β1-6 bond to Manα1-6 of α1-6 arm, followed by β1- The expression promotion of 6N-acetylglucosamine transferase (N-acetylglucosamine transferase VI) is described.

The β1-6 branch of the asparagine-linked sugar chain of the glycoprotein is formed by N-acetylglucosamine transferase V, which is followed by the subsequent generation of polylactosamine structure and sialic acid addition. The formation of this branch affects the adhesion of cells related to glycoproteins present in the cell surface layer or extracellular matrix to the substrate. According to Yamashita et al. [J. Biol. Chem. 260: 3893-3969 (1985); Non-Patent Document 1], transformation of BHK cells with polyoma virus increases β1-6 branching, and N-acetylglucosaminyltransferase. Increased V activity was reported. With this report, N-acetylglucosamine transferase V activity (expression) and transformation, tumor metastasis [Dennis et al. Science 236: 582-585 (1987); Non-Patent Document 2, Taniguchi et al. Clin. Cancer Res. 6: 1772-1776 (2000); non-patent document 3] etc. were focused on. According to Dennis et al. [J. Biol. Chem. 266: 1772-1782 (1991)]; Non-patent document 4], β1-6 branching is increased in human lung cancer and the like, and N-acetylglucosaminyltransferase V is added. It has been shown that the activity corresponds. Furthermore, there are many reports that this increase in N-acetylglucosamine transferase V activity is parallel to the transcriptional activity of N-acetylglucosamine transferase V gene (Mgat-5) [Petretti et al. Biochim. Biophys. Acta 1428: 209-218 (1999); Non-Patent Document 5]. This transcription is known to be involved in Ets-1, src, erbB2, etc., and promotedly involved Ets-1 protein binding to the ets-1 binding site in the 5 'upstream region of the gene. This is shown by Taniguchi et al. [J. Biol. Chem. 271: 26706-26712 (1966); Non-Patent Document 6]. Regarding the relationship between N-acetylglucosaminyltransferase V activity and tumor metastasis, Taniguchi et al. [J. Biol. Chem. 277: 16960-16966 (2001); As a result, matriptase increases its β1-6 branch, becomes resistant to proteolysis, and is proteinically stabilized, which is a urokinase-type plasminogen activator and HGF (liver) that act on tumor metastasis. It is explained by promoting the activation of both proteins (cell growth factor). Further, in the process of malignant transformation, the beta ₁ integrin asparagine-linked oligosaccharide fibronectin receptor alpha ₅ beta ₁ is integrin cell surface, an increase in β1-6 branch due N- acetylglucosaminyltransferase V activity, alpha _{It has also been shown} that it inhibits the compact formation of both ₅ and β ₁ subunits, resulting in weaker adhesion of cancer cells to the substrate and easier migration [Guo et al. Cancer Res. 62: 6837- 6845 (2002); Non-Patent Document 8]. Furthermore, in analysis using N-acetylglucosamine transferase V gene knockout mice, N-acetylglucosamine transferase V is essential for tumor growth and tumor metastasis [Dennis et al. Nature Med. 6: 306-312 (2000). Non-Patent Document 9] is shown and is consistent with the current research flow.
In the future, it will be necessary to conduct a detailed analysis of the regulation of Hmat-Xa expression that we found in the same way.

Yamashita, K., Tachibana, Y., Ohkura, T., and Kobata, A. J. Biol. Chem. 260: 3893-3969 (1985) Dennis, J.W., Laferte, S., Waghorne, C., Breitman, M.L., Kerbel, R.S.Science 236: 582-585 (1987) Murata, K., Miyoshi, E., Kameyama, M., Ishikawa, O., Kabuto, T., Sasaki, Y., Hiratsuka, M., Ohigashi, H., Ishiguro, S., Ito, S., Honda, H., Takemura, F., Taniguchi, N., and Imaoka, S. Clin. Cancer Res. 6: 1772-1776 (2000) Yousefi, S., Higgins, E., Daoling, Z., Pollex-Kruger, A., Hindsgaul, O., and Dennis, J.W. J. Biol. Chem. 266: 1772-1782 (1991) Petretti, T., Schulze, B., Schlag, P.M., and Kemmner, W. Biochim. Biophys. Acta 1428: 209-218 (1999) Kang, R., Saito, H., Ihara, Y., Miyoshi, E., Koyama, N., Sheng, Y., and Taniguchi, N. J. Biol. Chem. 271: 26706-26712 (1966) Ihara, S., Miyoshi, E., Ko, JH, Murata, K., Nakahara, S., Honke, K., Dickson, RB, Lin, CY, and Taniguchi, NJ Biol. Chem. 277: 16960-16966 (2001) Guo, H.B., Lee, I., Kamar, M., Akiyama, S.K., and Pierce, M. Cancer Res. 62: 6837-6845 (2002) Granovsky, M., Fata, J., Pawling, J., Muller, W.J., Khokha, R., and Dennis, J.W.Nature Med. 6: 306-312 (2000)

As described above, among mannose transferases that act on lipid intermediate biosynthesis, some mannose transferases have been cloned, but not all mannose transferases have been cloned. In the first half of the mannose transferase for lipid intermediate biosynthesis, GDP-Man is a sugar donor, and at least two of these processes are isolated from yeast as temperature-sensitive mutants. Are we further mannose transferase IV from mouse breast cancer-derived FM3A cells? Since the G258 mutant strain has been isolated as a mutant strain, it is considered essential for the growth of eukaryotic cells. In the latter half, the four steps using Dol-P-Man as a sugar donor consisted of a Dol-P-Man synthase-deficient mutant strain [Thy-1 ^- class E (dpm1), Lec15 (dpm2)] is not essential because it can grow normally. However, as a subtype of Human Congenital Disorders of Glycosylation (CDG) type I, which exhibits severe cranial nerve symptoms and liver dysfunction, these mannose transferase activities are defective. There are cases of human genetic diseases around the world where the supply of lipid intermediates in size is not in time, and the number of asparagine-linked glycans per polypeptide in the glycoprotein is reduced, resulting in the above dysfunction. It has been found one after another in recent years.
As mannose transferase, in addition to lipid intermediate biosynthesis, enzymes that catalyze mannosylation of mannose transferase and O-mannosylated protein involved in the biosynthesis of sugar chains in GPI anchor proteins have also been found. And also in these, the human genetic disease which shows a serious symptom by the defect | deletion has been found. However, the existence of mannose transferase other than these is not known.
As described above, knowledge regarding the relationship with diseases such as cancer as a role of mannose transferase in vivo is still being accumulated. The present invention aims to solve the cloning of novel mannose transferase genes derived from humans (especially, mannose transferase II and mannose transferase IV using GDP-mannose as a sugar donor, which is unknown throughout all living organisms). It was. Furthermore, the present invention has a problem to be solved, for example, to clone a mannose transferase gene such that the defect becomes a causative gene of congenital human glycosylation and a marker gene for cancer. did. Furthermore, another object of the present invention is to provide a genetic diagnosis for congenital human glycosylation and a cancer diagnosis method using the obtained mannose transferase gene.

In order to solve the above-mentioned problems, the present inventors attempted to clone an unknown gene by a bioinformatics technique using the following parameters. The parameters used in the search were (1) human mannose transferase I, mannose transferase III, mannose transferase V using CaZY, a database of classification based on homology of known glycosyltransferases. Amino acid sequences of glycosyltransferases and glycosyltransferase candidates that have a domain common to glycosyltransferases that use sugar nucleotides such as GDP-mannose as sugar donors, including (2) all known organisms (3) DXD or DDX sequence common to glycosyltransferases using sugar nucleotides as donors, and (4) α-glycosidic bonds in sugar nucleotides are maintained and retained. EX ₇ E sequence common to all types of glycosyltransferases, (5) glycosyltransferases acting on yeast lipid intermediate biosynthesis, yeast dolichol kinase (Sec59 protein), These are five of the hypothetical dolichol recognition sequences PDRS proposed by the canine oligosaccharide transferase ribophorin I subunit.

First, as a result of searching a human EST database based on the amino acid sequence of the Drosophila Q9VXN0 protein having the Glycosyltransferase group 1 domain, BI770600 and AU119344 were identified as EST clones encoding a human protein homologous to the Q9VXN0 protein.
Next, based on these nucleotide and amino acid sequence information, human fetal brain cDNA pools were constructed using primers designed based on the nucleotide sequence including the region considered to be all ORFs obtained by further searching. PCR was performed on the template. Furthermore, as a result of searching the human fetal brain cDNA library using the obtained PCR product, the inventors succeeded in identifying an ORF consisting of 1371 bases encoding the amino acid 457 residue and completed the present invention. .

That is, according to the present invention, the following protein (A) or (B) is provided.
(A) A protein having the amino acid sequence set forth in SEQ ID NO: 2.
(B) A protein having an amino acid sequence containing substitution, deletion, insertion and / or addition of one or several amino acids and having glycosyltransferase activity in the amino acid sequence shown in SEQ ID NO: 2.

According to another aspect of the present invention, DNA encoding any of the following proteins (A) or (B) is provided.
(A) A protein having the amino acid sequence set forth in SEQ ID NO: 2.
(B) A protein having an amino acid sequence containing substitution, deletion, insertion and / or addition of one or several amino acids and having glycosyltransferase activity in the amino acid sequence shown in SEQ ID NO: 2.

According to still another aspect of the present invention, the following DNA (a), (b) or (c) is provided.
(A) DNA having the base sequence set forth in SEQ ID NO: 1.
(B) The base sequence described in SEQ ID NO: 1 has a base sequence including substitution, deletion, insertion and / or addition of one or several bases, and encodes a protein having glycosyltransferase activity DNA.
(C) A DNA that hybridizes with a DNA having the nucleotide sequence set forth in SEQ ID NO: 1 under a stringent condition and encodes a protein having glycosyltransferase activity.

According to still another aspect of the present invention, there is provided a recombinant vector containing the above-described DNA of the present invention.
According to still another aspect of the present invention, a transformant having the above-described DNA or recombinant vector of the present invention is provided.

  According to still another aspect of the present invention, an antibody against the protein of the present invention described above, a diagnostic agent for colorectal cancer containing an antibody against the protein of the present invention described above, and expression of the protein in a biological sample using the antibody. And a method for diagnosing colorectal cancer comprising detecting or measuring the same.

According to still another aspect of the present invention, the oligonucleotide comprises a partial sequence of the base sequence of the DNA of the present invention described above or a complementary sequence thereof, and comprises a partial sequence of the base sequence of the DNA of the present invention described above or a complementary sequence thereof. A diagnostic agent for colorectal cancer comprising an oligonucleotide and a method for diagnosing colorectal cancer comprising detecting or measuring expression of the DNA in a biological sample using the oligonucleotide are provided.
According to still another aspect of the present invention, there is provided a method of using the above-described protein of the present invention as a marker for colorectal cancer.

  According to the present invention, the Hmat-Xa gene is provided as a novel gene containing Glycosyltransferase group 1 domain. The gene of the present invention is useful as a tumor marker for colorectal adenocarcinoma because its expression is low in the colon gland epithelial tissue of healthy individuals but is specifically promoted in colorectal adenocarcinoma tissue.

Hereinafter, the present invention will be described more specifically.
(1) Protein of the present invention The protein of the present invention is one of the following proteins (A) or (B).
(A) A protein having the amino acid sequence set forth in SEQ ID NO: 2.
(B) A protein having an amino acid sequence containing substitution, deletion, insertion and / or addition of one or several amino acids and having glycosyltransferase activity in the amino acid sequence shown in SEQ ID NO: 2.

  The protein having glycosyltransferase activity referred to in the present specification is not particularly limited as long as it has an enzyme activity to transfer sugar, but preferably has a Glycosyltransferase group 1 domain, more preferably a sugar nucleotide as a sugar donor. Means a protein having a DXD (X is arbitrary: for example, DKD) or DDX (X is arbitrary: for example, DDY) sequence unique to glycosyltransferases.

The range of “one or several” in the above-mentioned “amino acid sequence including substitution, deletion, insertion and / or addition of one or several amino acids in the amino acid sequence shown in SEQ ID NO: 2” is not particularly limited. Is, for example, 1 to 20, preferably 1 to 10, more preferably 1 to 7, further preferably 1 to 5, and particularly preferably about 1 to 3.

  The method for obtaining and producing the protein of the present invention is not particularly limited, and may be a naturally derived protein, a chemically synthesized protein, or a recombinant protein produced by a gene recombination technique. Recombinant proteins are preferred in that they can be produced in large quantities with relatively easy operations.

  When a naturally derived protein is obtained, it can be isolated from cells or tissues expressing the protein by an appropriate combination of protein isolation and purification methods. When obtaining a chemically synthesized protein, for example, the protein of the present invention can be synthesized according to a chemical synthesis method such as the Fmoc method (fluorenylmethyloxycarbonyl method) or the tBoc method (t-butyloxycarbonyl method). . Moreover, the protein of the present invention can be synthesized using various commercially available peptide synthesizers.

  In order to produce the protein of the present invention as a recombinant protein, a DNA having a base sequence encoding the protein (for example, the base sequence described in SEQ ID NO: 1) or a variant or homologue thereof is obtained, and this is preferably used. The protein of the present invention can be produced by introducing it into an expression system.

   The expression vector is preferably any one that can replicate autonomously in the host cell or can be integrated into the host cell chromosome, and contains a promoter at a position where the gene of the present invention can be expressed. Is used. Moreover, the transformant which has the gene which codes the protein of this invention can be produced by introduce | transducing said expression vector into a host. The host may be any of bacteria, yeast, animal cells, and insect cells, and the expression vector may be introduced into the host by a known method according to each host.

  In the present invention, the transformant having the gene of the present invention produced as described above is cultured, the protein of the present invention is produced and accumulated in the culture, and the protein of the present invention is collected from the culture. The recombinant protein can be isolated by

  When the transformant of the present invention is a prokaryote such as Escherichia coli or a eukaryote such as yeast, the medium for culturing these microorganisms contains a carbon source, a nitrogen source, inorganic salts and the like that can be assimilated by the microorganism. As long as the medium can efficiently culture the transformant, either a natural medium or a synthetic medium may be used. Moreover, the culture conditions may be the conditions usually used for culturing the microorganism. In order to isolate and purify the protein of the present invention from the culture of the transformant after culturing, ordinary protein isolation and purification methods may be used.

  A protein having an amino acid sequence containing substitution, deletion, insertion and / or addition of one or several amino acids in the amino acid sequence shown in SEQ ID NO: 2 is a DNA encoding the amino acid sequence shown in SEQ ID NO: 2. Those skilled in the art can appropriately produce or obtain the information based on the information of the base sequence described in SEQ ID NO: 1 showing an example of the sequence.

  That is, in the amino acid sequence shown in SEQ ID NO: 2, a gene (mutant gene) having a base sequence encoding a protein having an amino acid sequence containing substitution, deletion, insertion and / or addition of one or several amino acids, It can also be produced by any method known to those skilled in the art, such as chemical synthesis, genetic engineering techniques or mutagenesis. Specifically, mutant DNA can be obtained by using DNA having the base sequence described in SEQ ID NO: 1 and introducing mutation into these DNAs.

For example, the DNA having the base sequence described in SEQ ID NO: 1 can be contacted with a mutagen agent, a method of irradiating with ultraviolet light, a genetic engineering method, or the like. Site-directed mutagenesis, which is one of the genetic engineering methods, is useful because it can introduce a specific mutation at a specific position. Molecular Cloning: A laboratory Mannual, 3 ^rd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY., 2001 (hereinafter abbreviated as Molecular Cloning 3rd Edition), Current Protocols in Molecular Biology, Supplement 1-38, John Wiley & Sons (1987-1997) (hereinafter referred to as Current Protocols (Abbreviated as in-molecular biology) and the like.

(2) DNA of the present invention
The DNA of the present invention is a DNA encoding the protein of the present invention described in (1) above, and is preferably any one of the following DNAs (a), (b) or (c).
(A) DNA having the base sequence set forth in SEQ ID NO: 1.
(B) The base sequence described in SEQ ID NO: 1 has a base sequence including substitution, deletion, insertion and / or addition of one or several bases, and encodes a protein having glycosyltransferase activity DNA.
(C) A DNA that hybridizes with a DNA having the nucleotide sequence set forth in SEQ ID NO: 1 under a stringent condition and encodes a protein having glycosyltransferase activity.

  The range of “one or several” in the above-described “base sequence including substitution, deletion, insertion and / or addition of one or several bases in the base sequence described in SEQ ID NO: 1” is not particularly limited. For example, 1 to 60, preferably 1 to 30, more preferably 1 to 20, more preferably 1 to 15, further preferably 1 to 10, further preferably 1 to 5, particularly preferably It means about 1 to 3 pieces.

  The above-mentioned “hybridizes under stringent conditions” means a DNA base obtained by using DNA as a probe and using a colony hybridization method, a plaque hybridization method, a Southern blot hybridization method, or the like. Means a sequence, for example, after hybridization at 65 ° C. in the presence of 0.7 to 1.0 M NaCl using a filter on which DNA derived from colonies or plaques or a fragment of the DNA is immobilized, 0 .1 to 2 × SSC solution (1 × SSC solution is 150 mM sodium chloride, 15 mM sodium citrate), and DNA that can be identified by washing the filter under 65 ° C. can be used. Hybridization can be performed according to the method described in Molecular Cloning 3rd edition and the like.

  Examples of DNA that hybridizes under stringent conditions include DNA having a certain degree of homology with the base sequence of DNA used as a probe, for example, 70% or more, preferably 80% or more, more preferably 90%. More preferably, DNA having homology of 93% or more, particularly preferably 95% or more, and most preferably 98% or more is mentioned.

  The method for obtaining the DNA of the present invention is not particularly limited. Appropriate probes and primers are prepared based on the nucleotide sequence and amino acid sequence information described in SEQ ID NO: 1 and SEQ ID NO: 2 in the sequence listing in this specification, and a human or other cDNA library is screened using them. Thus, the DNA of the present invention can be isolated. The cDNA library is preferably prepared from cells, organs or tissues expressing the DNA of the present invention.

  DNA having the base sequence described in SEQ ID NO: 1 can also be obtained by PCR. Using human chromosomal DNA or a human cDNA library as a template, PCR is performed using a pair of primers designed to amplify the base sequence described in SEQ ID NO: 1. PCR reaction conditions can be set as appropriate. For example, one cycle of a reaction step consisting of 94 ° C. for 30 seconds (denaturation), 55 ° C. for 30 seconds to 1 minute (annealing), and 72 ° C. for 2 minutes (extension) For example, after 30 cycles, a condition of reacting at 72 ° C. for 7 minutes can be exemplified. The amplified DNA fragment can then be cloned into a suitable vector that can be amplified in a host such as E. coli.

  The above-described procedures such as probe or primer preparation, cDNA library construction, cDNA library screening, and target gene cloning are known to those skilled in the art. For example, Molecular Cloning 3rd Edition, Current Protocols In -It can be performed according to the method described in Molecular Biology etc.

(3) Antibody of the present invention The present invention relates to an antibody that recognizes the above-described protein of the present invention as an epitope (antigen).

  The antibody of the present invention may be a polyclonal antibody or a monoclonal antibody, and can be produced by a conventional method.

  For example, a polyclonal antibody can be obtained by immunizing a mammal with the protein of the present invention as an antigen, collecting blood from the mammal, and separating and purifying the antibody from the collected blood. For example, mammals such as mice, hamsters, guinea pigs, chickens, rats, rabbits, dogs, goats, sheep and cows can be immunized. Methods of immunization are known to those skilled in the art. For example, the antigen may be administered 2 to 3 times at intervals of 7 to 30 days, for example. The dose can be about 0.05 to 2 mg of antigen per dose, for example. The administration route is not particularly limited, and subcutaneous administration, intradermal administration, intraperitoneal administration, intravenous administration, intramuscular administration, and the like can be appropriately selected. The antigen can be used after being dissolved in an appropriate buffer, for example, a complete buffer containing a commonly used adjuvant such as Freund's complete adjuvant or aluminum hydroxide.

  After the immunized mammal has been bred for a certain period of time, if the antibody titer increases, booster immunization can be performed using, for example, 100 μg to 1000 μg of antigen. Blood is collected from the immunized mammal 1 to 2 months after the last administration, and the blood is collected, for example, by centrifugation, precipitation using ammonium sulfate or polyethylene glycol, gel filtration chromatography, ion exchange chromatography, affinity. A polyclonal antibody that recognizes the protein of the present invention can be obtained as a polyclonal antiserum by separation and purification by a conventional method such as chromatography.

   On the other hand, a monoclonal antibody can be obtained by preparing a hybridoma. For example, a hybridoma can be obtained by cell fusion between an antibody-producing cell and a myeloma cell line. The hybridoma producing the monoclonal antibody of the present invention can be obtained by the following cell fusion method.

  As antibody-producing cells, spleen cells, lymph node cells, B lymphocytes and the like from immunized animals are used. As the antigen, the protein of the present invention or a partial peptide thereof is used. Mice, rats and the like can be used as immunized animals, and antigens are administered to these animals by conventional methods. For example, an animal is administered by administering a suspension or emulsion of an adjuvant such as complete Freund's adjuvant or incomplete Freund's adjuvant and the protein of the present invention as an antigen several times into the vein, subcutaneous, intradermal, intraperitoneal, etc. of the animal. Immunize. For example, spleen cells are obtained as antibody-producing cells from the immunized animal and fused with myeloma cells by a known method (G. Kohler et al., Nature, 256 495 (1975)) to produce a hybridoma. Can do.

Examples of myeloma cell strains used for cell fusion include P3X63Ag8, P3U1 strain, Sp2 / 0 strain and the like in mice. When cell fusion is performed, a fusion promoter such as polyethylene glycol or Sendai virus is used, and hypoxanthine / aminopterin / thymidine (HAT) medium is used in accordance with a conventional method for selection of hybridomas after cell fusion. Hybridomas obtained by cell fusion are cloned by limiting dilution or the like. Furthermore, if necessary, a cell line producing a monoclonal antibody that specifically recognizes the protein of the present invention can be obtained by screening by an enzyme immunoassay using the protein of the present invention.

  In order to produce the desired monoclonal antibody from the hybridoma thus obtained, the hybridoma is cultured by a normal cell culture method or ascites formation method, and the monoclonal antibody is purified from the culture supernatant or ascites. . Purification of the monoclonal antibody from the culture supernatant or ascites can be performed by a conventional method. For example, ammonium sulfate fractionation, gel filtration, ion exchange chromatography, affinity chromatography and the like can be used in appropriate combination.

   The above-mentioned antibody fragments are also within the scope of the present invention. Examples of the antibody fragment include F (ab ′) 2 fragment, Fab ′ fragment and the like.

Furthermore, labeled antibodies of the above-mentioned antibodies are also within the scope of the present invention. That is, the antibody of the present invention produced as described above can be used after being labeled. The types of antibody labeling and labeling methods are known to those skilled in the art. For example, enzyme labels such as horseradish peroxidase or alkaline phosphatase, fluorescent labels such as FITC (fluorescein isothiocyanate) or TRITC (tetramethylrhodamine B isothiocyanate), labels with colored substances such as colloidal metals and colored latex, biotin, etc. An affinity label or an isotope label such as ¹²⁵ I can be mentioned.

(4) Diagnostic Agent and Diagnostic Method for Colorectal Cancer Using the Antibody of the Present Invention The present invention relates to a diagnostic agent for colorectal cancer containing an antibody against the protein of the present invention described above, and the expression of the protein of the present invention in a biological sample. The present invention relates to a method for diagnosing colorectal cancer, which comprises detecting or measuring the above using the antibody of the present invention. As the biological sample used here, biological samples collected from humans or animals (for example, organs, biological tissues, blood, urine, etc.) or primary cultured cells or cell lines established from these biological samples are used.

  As a method for immunologically detecting the protein of the present invention using the antibody of the present invention described in (3) above, enzyme immunization such as immunohistochemical staining, flow cytometry, Western blotting, sandwich ELISA, etc. There are assay methods (EIA) or radioimmunoassay methods (RIA), and these techniques are well known to those skilled in the art.

  In the immunohistochemical staining method, biological tissue or cells are immobilized, the antibody of the present invention is reacted, and an anti-immunoglobulin antibody or antibody fragment labeled with a fluorescent substance, enzyme, biotin, colloidal gold, radioactive substance or the like is further reacted. Thereafter, the labeled antibody is visualized as necessary, and observed using a microscope or the like to detect or measure the presence of the protein of the present invention in tissues or cells. As the fluorescent substance label, fluorescein isothiocyanate (FITC), tetramethylrhodamine isothiocyanate, or the like is used. In the case of enzyme labeling, peroxidase, alkaline phosphatase, or the like is used, and a color developing reaction can be performed by adding a substrate that develops color with an enzyme.

  In flow cytometry, cells are reacted with the antibody of the present invention, further reacted with a fluorescent substance-labeled anti-immunoglobulin antibody or antibody fragment, and then the fluorescent dye is measured with a flow cytometer. Expression of the protein of the present invention can be detected.

In the Western blotting method, a biological tissue or cell lysate is fractionated by SDS-PAGE, the gel is blotted on a membrane, the antibody of the present invention is reacted with this membrane, and an enzyme such as peroxidase or alkaline phosphatase, or ¹²⁵ This is a method for detecting a protein band of the present invention after reacting with an anti-immunoglobulin antibody labeled with a radioactive substance such as I.

  In sandwich ELISA, which is a kind of enzyme immunoassay, two types of antibodies of the present invention having different antigen recognition sites are prepared, and one antibody is adsorbed on a plate. Anti-immunoglobulin labeled with an enzyme such as peroxidase or alkaline phosphatase, which is reacted by contacting a cell disruption solution prepared from biological tissue or cells with a test sample as a test sample, and reacting with another antibody with a different antigen recognition site. The antibody is reacted. After adding a substrate that develops color with an enzyme and causing a color reaction, the protein of the present invention in the sample can be detected or measured by measuring the color intensity with a spectrophotometer.

The radioimmunoassay is a method in which radiation is measured with a scintillation counter by performing the same operation as the enzyme immunoassay using an antibody labeled with a radioactive substance such as ¹²⁵ I instead of an enzyme.

  By utilizing the above-described method, the expression of the protein of the present invention in a biological sample can be detected or measured using the antibody of the present invention, whereby colorectal cancer can be diagnosed. That is, when the expression level of the protein of the present invention in the biological sample is increased compared to the normal case, it can be diagnosed that there is a suspicion of colorectal cancer.

(5) Oligonucleotide of the present invention The present invention relates to an oligonucleotide comprising a partial sequence of the base sequence of the DNA of the present invention or a complementary sequence thereof.
Specific examples of the oligonucleotide of the present invention include a sense oligonucleotide having the same sequence as 5 to 100 consecutive bases in the base sequence of the DNA of the present invention, and an antisense oligonucleotide having a sequence complementary to the sense oligonucleotide. And oligonucleotide derivatives of the sense or antisense oligonucleotide. More specifically, a DNA having the same sequence as 5 to 100 consecutive bases in the base sequence of SEQ ID NO: 1 or a DNA having a sequence complementary to the DNA can be mentioned. The length of the sequence is generally 5 to 100 bases, preferably 10 to 60 bases, and more preferably 15 to 50 bases.

  The oligonucleotide of the present invention can be produced by a conventional method using a nucleic acid synthesizer or the like based on the base sequence information of the DNA of the present invention or a fragment thereof.

  In addition, derivatives of the above oligonucleotides can also be used as the oligonucleotide of the present invention. Oligonucleotide derivatives include oligonucleotide derivatives in which phosphodiester bonds in oligonucleotides are converted to phosphorothioate bonds, oligonucleotides in which phosphodiester bonds in oligonucleotides are converted to N3′-P5 ′ phosphoramidate bonds Derivatives, oligonucleotide derivatives in which ribose and phosphodiester bonds in oligonucleotides are converted to peptide nucleic acid bonds, oligonucleotide derivatives in which uracil in oligonucleotides is replaced with C-5 propynyluracil, uracil in oligonucleotides is C Oligonucleotide derivatives substituted with -5 thiazoleuracil, cytosines in oligonucleotides substituted with C-5 propynylcytosine, cytosines in oligonucleotides Is an oligonucleotide derivative substituted with phenoxazine modified cytosine, an oligonucleotide derivative in which the ribose in the oligonucleotide is substituted with 2'-O-propylribose, or the ribose in the oligonucleotide is substituted with 2'-methoxyethoxyribose And oligonucleotide derivatives.

(6) Diagnostic Agent and Diagnostic Method for Colorectal Cancer Using Oligonucleotide of the Present Invention The present invention relates to a diagnostic agent for colorectal cancer containing the oligonucleotide of the present invention described above, and the expression of the DNA of the present invention in a biological sample. The present invention relates to a method for diagnosing colorectal cancer, which comprises detecting or measuring the above using the oligonucleotide of the present invention.

  Specifically, the expression of the DNA of the present invention can be detected or measured by detecting the mRNA of the DNA of the present invention using the oligonucleotide of the present invention described in (5) above.

  Examples of the method for detecting mRNA of DNA of the present invention include Northern blotting, in situ hybridization, quantitative PCR, differential hybridization, DNA chip method, ribonuclease protection assay and the like. By these methods, the expression level of the DNA of the present invention at the mRNA level in various samples can be examined. By these methods, it is possible to obtain information on in which tissues and cells the DNA of the present invention is expressed. Examples of the sample used here include biological samples collected from humans and animals (for example, organs, biological tissues, blood, urine, etc.) or primary cultured cells or cell lines established from these biological samples.

  Northern blotting is a method in which total RNA or mRNA extracted from a sample is separated by gel electrophoresis, transferred to a membrane, and subjected to hybridization and washing using a labeled probe prepared by labeling the oligonucleotide of the present invention with a label. This is a method for detecting mRNA of the DNA of the present invention as a band bound to a labeled probe. Since the band intensity reflects the mRNA level of the DNA of the present invention, the expression level at the mRNA level of the DNA of the present invention can also be quantified.

The in situ hybridization method prepares a paraffin-embedded section or a cryostat section from a sample organ or biological tissue, and performs hybridization and washing on the section using a labeled probe prepared from the oligonucleotide of the present invention. This is a method for detecting in detail the type and expression site of cells expressing the DNA of the present invention in the tissue.

  In the quantitative PCR method, PCR is performed using a cDNA synthesized from a sample-derived RNA using an oligo dT primer and a reverse transcriptase, using the oligonucleotide primer of the present invention as a template, and a DNA fragment derived from the mRNA of the DNA of the present invention. Is a method of specifically amplifying Since the amount of the amplified DNA fragment reflects the amount of mRNA of the DNA of the present invention in a sample, it is possible to quantify the mRNA of the DNA of the present invention.

  The differential hybridization method and the DNA chip method are performed by performing hybridization and washing using a labeled cDNA synthesized from a sample-derived RNA as a probe on a substrate on which the oligonucleotide of the present invention is immobilized. It is a method for detecting the expression of the DNA of the invention at the mRNA level.

  In the ribonuclease protection assay, first, a promoter sequence such as a T7 promoter is bound to the 3 ′ end of the DNA of the present invention, and a labeled antisense RNA is synthesized by an in vitro transcription system using labeled NTP and a promoter-specific RNA polymerase. . This labeled antisense RNA was hybridized with RNA prepared from a sample to form an RNA-RNA hybrid with the mRNA of the present invention in a specimen, and then digested with ribonuclease, and protected from digestion by ribonuclease by the formation of the hybrid. Bands are detected by gel electrophoresis. By quantifying the protected band, the expression of the DNA of the present invention at the mRNA level can be quantified.

  Using the above-described method, a biological sample such as an organ, biological tissue, blood or the like collected from a subject and a healthy person by biopsy or the like, or a primary cultured cell cultured from now on, is used as a specimen. By quantifying the expression at the mRNA level and comparing it, it is possible to diagnose whether the expression level of the mRNA level of the DNA of the present invention is increased compared with that of a healthy person. Then, when the expression level of the DNA of the present invention in the sample is increased as compared with that of a healthy person, it can be diagnosed that there is a suspicion of colorectal cancer.

  The following examples further illustrate the present invention, but the present invention is not limited to the examples.

Example 1: Search for novel gene and identification of Hmat-Xa gene As described in paragraph 0008, mannose transferase II and mannose transferase IV in which the gene has not yet been cloned are also expressed in the above-described mannose. Similar to transferase I, mannose transferase III, and mannose transferase V, the sugar nucleotide GDP-mannose is used as a mannose donor. Therefore, we attempted to clone an unknown gene using bioinformatics techniques using the following parameters. The parameters used in the search are (1) common to glycosyltransferases using sugar nucleotides as sugar donors, such as human mannose transferase I, mannose transferase III, and mannose transferase V using CaZY. Glycosyltransferase group 1 domain glycosyltransferase and amino acid sequences of glycosyltransferase candidates, (2) amino acid sequences of mannose transferases of all known organisms, (3) common to glycosyltransferases using sugar nucleotides as donors DX4 or DDX sequence, (4) EX ₇ E sequence common to glycosyltransferases that maintain α-glycoside bonds in sugar nucleotides, (5) Glycosyltransferases acting on yeast lipid intermediate biosynthesis, yeast This is a hypothetical dolichol recognition sequence PDRS proposed by dolichol kinase (Sec59 protein), canine oligosaccharide transferase ribophorin I subunit and the like.
From the above, first of all, about 1000 proteins belonging to the glycosyltransferase group having Glycosyltransferase group 1 domain registered in Pfam, those that are derived from eukaryotes and whose functions are unknown are listed.

  As a result, Drosophila Q9VXN0, Arabidopsis Q9LPN6, rice Q9AXB1, Trypanosoma Q9U532, Arabidopsis Q9M147, etc. were listed. Then, tDBLASTn search was performed on the human EST database of DDBJ using those amino acid sequences as parameters.

  Among them, the result of the gene of the present invention was obtained by tBLASTn search of the human EST database with the amino acid sequence of Q9VXN0. In other words, two human unknown ESTs were hit with high bits. One group was homologous to the Glycosyltransferase group 1 domain portion located on the C-terminal side of the amino acid sequence of Q9VXN0, and the other was homologous to the N-terminus of the amino acid sequence of Q9VXN0. Examples of the human EST clone belonging to the former include BI770600 and AU119344, and examples of the human EST clone belonging to the latter include BI488854. In addition, as a known human gene showing homology to the Glycosyltransferase group 1 domain part of Q9VXN0, mannose transferase III gene Hmat-3 and N-acetylglucosaminyltransferase gene PIG- acting in the first stage of sugar chain biosynthesis in GPI anchor A was detected.

  Therefore, when a human EST homologous to the human EST clone BI770600 was further searched, a group showing high homology and three unknown genes were hit. It was also found at this stage that BI770600 was derived from chromosome 2. Next, a partial amino acid sequence (SEQ ID NO: 5) consisting of 272 amino acids was created based on AU119344, AI082171, and BG682274 detected as human ESTs homologous to BI770600 and BI770600.

Next, based on this sequence, Prodom, an Internet site that searches the domain structure of proteins, searched for a domain based on the same program. As a result, this sequence was found to contain the mannose transferase domain (K150-W253). It turns out to have. Furthermore, the Prodom search revealed that there was Q9HE5 as a human protein having these sequences (however, the function was unknown and it was not included in the list of those having the Glycosyltransferase group 1 domain). Based on the above, it was considered that this was a novel glycosyltransferase gene, and this gene was named Hmat-Xa and cloning of the gene was started. A sequence homologous to the Hmat-Xa gene was not found in the yeast gene.
In addition, when searching for the glycosyltransferase family of CAZY, there is an unknown human EST clone AK021815 in glycosyltransferase family 4 to which the Hmat-3 and Hmat-5 genes belong, which is homologous to the Hmat-Xa gene we found. I found out.

Example 2: Cloning of the Hmat-Xa gene:
Based on this AK021815, the following searches were performed. Comparing EST clone AK021815 with EST clone AK091684 that was hit by BLAST, we found that AK091684 was extended 637 bp to the 5 'side of AK021815 (at the same time AK091684 skipped one exon) (Corresponding to the exon lacking the black capital sequence in the figure below). The genomic clone AC010681 containing the most upstream exon of AK021815 did not contain the 637 bp sequence of AK091684. Therefore, when BLAST search was performed again with the sequence of AK091684 (full length 2353 bp), a genomic clone AC009957 adjacent to AC010681 was detected, and this included the 2nd to 613rd nucleotides of AK091684 divided into 4 parts ( The nucleotides 614 to 967 were contained in AC010681 detected at the same time). In addition, when the EST database was searched based on the 2nd to 400th nucleotides of AK091684, multiple EST clones extended further 5 'than AK091684 were detected (BG027230, BG427368, etc.). The consensus of the extended portion was taken, and the Hmat-Xa cDNA sequence was further extended by 59 bp to the 5 ′ side (this 59 bp sequence is contained in the genomic clone AC010130 further adjacent to the genomic clone AC009957 by 5 ′). On the 5 'side of this 59 bp sequence, there is still a possibility that an exon is linked by splicing, but since there is a stop codon ( TAG ) in frame in the 59 bp sequence, the start codon It was concluded that this is the 93rd to 95th position ( ATG ) of AK091684.

From the above results, it was considered that the full length of the Hmat-Xa gene ORF was included in the sequence (SEQ ID NO: 6) in the figure below. / (Line feed) indicates an exon boundary. Splicing of the AK091684 pattern, which lacks the exon in the black uppercase part, terminates the ORF (ORF2) in the uppercase TAA downstream, whereas the splicing of the AK021815 pattern that contains this exon causes further lowercase capitalization ORF (ORF1) continues to TGA, and shows significant homology to the Glycosyltransferase group 1 domain.

/ gtatctctgcat TAG ccagattggacatatgtaaagggacatcagtatatctggatcatcag /
attgtgcttcaagaagaccatagggaagaacgcattttcatcttgctgcaaaagttgcaatagaa /
gatctgaaggtttgaatgggcca ATG agtatcctcatcattgaagcattctatggaggctcccataaacagctggtggat
cttcttcaagaagagttaggagactgtgtcgtttatacccttcctgcaaagaaatggcattggagagcccggacatctgc
tttatatttctctcagaccattcccatcagtgagcattacag /
gaccctctttgcaagttcagtgcttaacctgaccgaactggctgcccttcggcctgaccttgggaaactgaaaaagattc
tgtattttcacgagaaccagttgatatatcctgtcaagaaatgtcaggagagggatttccaatatggatacaaccaaatt
ctttcatg /
cctggtggctgatgtggttgtattcaactcagtttttaatatggaatcatttctcacttccatgggaaaatttatgaagc
tgattcctgatcacagacccaaggatctggaaagcatcatcagacccaagtgccaagttatttactttcccatcaggttt
cctgatgtgagcag /
attcatgcccaagcacaaaacaacccatttaaagaagatgctcggccttaaaggaaatggcggtgcggttctgtccatgg
cccttccttttcagccagagcagagagattcagaggatttattgaagaattttaattcagagtgtgatacacactgtggc
cttgatactgcacgacaagaatatttgggtaactcattaagacaggaatcagacttgaaaaaatccacctcgtcagataa
ttcaagctctcatcatggtgaaaataaacaaaatctgactgttgatccctgtgacattttgggtggagttgataatcagc
aaagactgctacacattgtctggcctcacaggtg /
ggagcatgataaagatccagaaagcttttttaaggtattaatgcatcttaaagacttag gactcaatttccacgtgtctg
tacttggag aaaccttcacagatgtcccag
ATATTTTTTCAGAGGCCAAAAAGGCATTGGGATCTTCTGTCTTACACTGGGGCTACTTACCCAGCAAAGATGACTACTTC
CAAGTACTGTGCATGGCTGATGTTGTCATCTCAACAGCTAAGCATGAATTCTTTGGAGTGGCAAT /
gttggaagctgtgtactgtgggtgttacccactttgtcctaaagatttggtttatcccgaaatatttccag /
ctgaatatctgtattctacacctgaacagctttcaaaaaggctccagaatttctgcaagagaccagatatta TAA gaaaa
catctctataag /
ggtgaaatcgctccgttttcttgggcagccctacatggtaaattcaggtctctgcttacaacagaacctagggaagattt
g TGA cagatggggctaagtcacaaacttgcagcctaaggcagagtctgaagaactttccagagtgtgcccatatttacct
gatcagagagaaaagaaaatct gcagaggaagctgagcctggctgcttgtca tagctgacacagagccatctgccacaaa
cctgtggcggcttcagatctccaatccctgccaccaccccaactcaaattaaatacagattcctagagacgttatgatgg
ttacacatgtcctcggcatcacatgtaggagactgttcaaaaaaaatatgtggcctgttgtataaccgcactcatgtatc
ccatatgtggtgccacattgaatttccggttgaatccgtttttatcctttgtactggatgacatggtgcctgaattcttt
cttttcgccgacacgatggcagccaaactgcagcttcaaacgctcacacttggctgggtttctacctaggttgccaggtt
atcatcggagccttcttgtgtcctcaaagggccacgaggcctgaaaggaggatcagaatgctttgggattaattgggcag
ccatcgcagaattgtttgtgggcaaagggctgctttagcacttttcttttagcaaattaatgactctcaggcacaggggg
ttttaagtgaaggtattaataagaggtctggcaggtattcccatgattcacagagttacatttgcatttaattaatctta
aagttgcaagataaacagctgtaattcggacaaacatgacaaacacagtgaagccaactatcccataaaatgaacactga
catacttgttttaatttttttcccagcgtaaaaatagaaaaatcaaaatactcctaacaaaaccagtgattttgatagaa
atatttctccaatatacttgcatccacctacaaatataaccttttcaagataaatcgcttatgatttcaatagtcaaact
gctgtgtttgttgatgtaaagatgttttgaatggctagatggtaaaataaattcttaataaagtacccactgc

Based on this deduced full-length sequence, as a PCR primer for cloning into the SmaI site of pUC118 or pUC119 vector,
XA-9F: ATCAGATTGTGCTTCAAGAAGACCATAGGG (SEQ ID NO: 7)
XA-10R: TGACAAGCAGCCAGGCTCAGCTTCCTCTGC (SEQ ID NO: 8)
Was designed (consigned synthesis to Greiner Japan), and PCR was performed using the brain and liver cDNA pools (MTC Panel Human I; Clontech) as a template under the following conditions.

Reaction solution composition:
Human MTC Panel cDNA (brain or liver) 5 μl
dH ₂ O 36.25μl
10 x buffer (enzyme attached, No.1) 5μl
10 mM dNTP mix 1 μl
1 μl of 10 μM each primer
Expand Long Template PCR System (Roche Diagnostics) enzyme mix 0.75μl
Total 50 μl of reaction solution

Reaction conditions:
After 94 ℃ 1 min, (94 ℃ 30 sec ・ 68 ℃ 3 min) × 50 cycles, 68 ℃ 2 min

As a result of 1% agarose gel electrophoresis, amplified fragments of two sizes (about 1.6 kb and 1.45 kb) expected from human brain and liver, both tissues, were confirmed. This PCR reaction solution was purified using the PCR Product Purification Kit (Roche Diagnostics) according to the manual, concentrated by EtOH precipitation, and then ligated to the Sma I cleaved pUC118 or pUC119 vector (Takara Ligation Kit ver. E. coli JM109 was transformed and cloned.
Thus, after cloning into the pUC vector, the base sequences of these two size positive clones were determined.

The following primers were used as primers for sequencing after cloning into the pUC vector.
puCF: CGCCAGGGTTTTCCCAGTCACGAC (SEQ ID NO: 9)
pUCR: CGGATAACAATTTCACACAGGAAAC (SEQ ID NO: 10)
XA-5R: TCTGCTCTGGCTGAAAAGGAAGGGCCATGG (SEQ ID NO: 11)
XA-7F: GAAGATGCTCGGCCTTAAAGGAAATGGCGG (SEQ ID NO: 12)

  As described above, there are two types of ORFs. The first (ORF1) has 1374 bases (encoding amino acid 458 residues), and the second (ORF2) has 1128 bases ORF (coding amino acid 376 residues). there were. Of these two ORFs, only ORF1 contained Glycosyltransferase group 1 domain. That is, this ORF1 is the Hmat-Xa gene.

SEQ ID NO: 1: Base sequence containing Hmat-Xa gene (ORF1) The start and stop codons are shown in capital letters.
gtatctctgcatTAGccagattggacatatgtaaagggacatcagtatatctggatcatcagattgtgcttcaagaagac
catagggaagaacgcattttcatcttgctgcaaaagttgcaatagaagatctgaaggtttgaatgggcca ATG agtatcc
tcatcattgaagcattctatggaggctcccataaacagctggtggatcttcttcaagaagagttaggagactgtgtcgtt
tatacccttcctgcaaagaaatggcattggagagcccggacatctgctttatatttctctcagaccattcccatcagtga
gcattacaggaccctctttgcaagttcagtgcttaacctgaccgaactggctgcccttcggcctgaccttgggaaactga
aaaagattctgtattttcacgagaaccagttgatatatcctgtcaagaaatgtcaggagagggatttccaatatggatac
aaccaaattctttcatgcctggtggctgatgtggttgtattcaactcagtttttaatatggaatcatttctcacttccat
gggaaaatttatgaagctgattcctgatcacagacccaaggatctggaaagcatcatcagacccaagtgccaagttattt
actttcccatcaggtttcctgatgtgagcagattcatgcccaagcacaaaacaacccatttaaagaagatgctcggcctt
aaaggaaatggcggtgcggttctgtccatggcccttccttttcagccagagcagagagattcagaggatttattgaagaa
ttttaattcagagtgtgatacacactgtggccttgatactgcacgacaagaatatttgggtaactcattaagacaggaat
cagacttgaaaaaatccacctcgtcagataattcaagctctcatcatggtgaaaataaacaaaatctgactgttgatccc
tgtgacattttgggtggagttgataatcagcaaagactgctacacattgtctggcctcacaggtgggagcatgataaaga
tccagaaagcttttttaaggtattaatgcatcttaaagacttag gactcaatttccacgtgtctgtacttggag aaacct
tcacagatgtcccagATATTTTTTCAGAGGCCAAAAAGGCATTGGGATCTTCTGTCTTACACTGGGGCTACTTACCCAGC
AAAGATGACTACTTCCAAGTACTGTGCATGGCTGATGTTGTCATCTCAACAGCTAAGCATGAATTCTTTGGAGTGGCAAT
gttggaagctgtgtactgtgggtgttacccactttgtcctaaagatttggtttatcccgaaatatttccagctgaatatc
tgtattctacacctgaacagctttcaaaaaggctccagaatttctgcaagagaccagatattaTAAgaaaacatctctat
aagggtgaaatcgctccgttttcttgggcagccctacatggtaaattcaggtctctgcttacaacagaacctagggaaga
tttg TGA cagatggggctaagtcacaaacttgcagcctaaggcagagtctgaagaactttccagagtgtgcccatattta
cctgatcagagagaaaagaaaatct gcagaggaagctgagcctggctgcttgtca tagctgacacagagccatctgccac
aaacctgtggcggcttcagatctccaatccctgccaccaccccaactcaaattaaatacagattcctagagacgttatga
tggttacacatgtcctcggcatcacatgtaggagactgttcaaaaaaaatatgtggcctgttgtataaccgcactcatgt
atcccatatgtggtgccacattgaatttccggttgaatccgtttttatcctttgtactggatgacatggtgcctgaattc
tttcttttcgccgacacgatggcagccaaactgcagcttcaaacgctcacacttggctgggtttctacctaggttgccag
gttatcatcggagccttcttgtgtcctcaaagggccacgaggcctgaaaggaggatcagaatgctttgggattaattggg
cagccatcgcagaattgtttgtgggcaaagggctgctttagcacttttcttttagcaaattaatgactctcaggcacagg
gggttttaagtgaaggtattaataagaggtctggcaggtattcccatgattcacagagttacatttgcatttaattaatc
ttaaagttgcaagataaacagctgtaattcggacaaacatgacaaacacagtgaagccaactatcccataaaatgaacac
tgacatacttgttttaatttttttcccagcgtaaaaatagaaaaatcaaaatactcctaacaaaaccagtgattttgata
gaaatatttctccaatatacttgcatccacctacaaatataaccttttcaagataaatcgcttatgatttcaatagtcaa
actgctgtgtttgttgatgtaaagatgttttgaatggctagatggtaaaataaattcttaataaagtacccactgc

SEQ ID NO: 2: amino acid sequence of ORF1 The underlined portion indicates a sequence different from the amino acid sequence of ORF2.
MSILIIEAFYGGSHKQLVDLLQEELGDCVVYTLPAKKWHWRARTSALYFSQTIPISEHYRTLFASSVLNLTELAALRPDL
GKLKKILYFHENQLIYPVKKCQERDFQYGYNQILSCLVADVVVFNSVFNMESFLTSMGKFMKLIPDHRPKDLESIIRPKC
QVIYFPIRFPDVSRFMPKHKTTHLKKMLGLKGNGGAVLSMALPFQPEQRDSEDLLKNFNSECDTHCGLDTARQEYLGNSL
RQESDLKKSTSSDNSSSHHGENKQNLTVDPCDILGGVDNQQRLLHIVWPHRWEHDKDPESFFKVLMHLKDLGLNFHVSVL
GETFTDVP DIFSEAKKALGSSVLHWGYLPSKDDYFQVLCMADVVISTAKHEFFGVAMLEAVYCGCYPLCPKDLVYPEIFP
AEYLYSTPEQLSKRLQNFCKRPDIIRKHLYKGEIAPFSWAALHGKFRSLLTTEPREDL (term)

SEQ ID NO: 3: Base sequence including ORF2 Start codon and end codon are shown in capital letters.
gtatctctgcatTAGccagattggacatatgtaaagggacatcagtatatctggatcatcagattgtgcttcaagaagac
catagggaagaacgcattttcatcttgctgcaaaagttgcaatagaagatctgaaggtttgaatgggcca ATG agtatcc
tcatcattgaagcattctatggaggctcccataaacagctggtggatcttcttcaagaagagttaggagactgtgtcgtt
tatacccttcctgcaaagaaatggcattggagagcccggacatctgctttatatttctctcagaccattcccatcagtga
gcattacaggaccctctttgcaagttcagtgcttaacctgaccgaactggctgcccttcggcctgaccttgggaaactga
aaaagattctgtattttcacgagaaccagttgatatatcctgtcaagaaatgtcaggagagggatttccaatatggatac
aaccaaattctttcatgcctggtggctgatgtggttgtattcaactcagtttttaatatggaatcatttctcacttccat
gggaaaatttatgaagctgattcctgatcacagacccaaggatctggaaagcatcatcagacccaagtgccaagttattt
actttcccatcaggtttcctgatgtgagcagattcatgcccaagcacaaaacaacccatttaaagaagatgctcggcctt
aaaggaaatggcggtgcggttctgtccatggcccttccttttcagccagagcagagagattcagaggatttattgaagaa
ttttaattcagagtgtgatacacactgtggccttgatactgcacgacaagaatatttgggtaactcattaagacaggaat
cagacttgaaaaaatccacctcgtcagataattcaagctctcatcatggtgaaaataaacaaaatctgactgttgatccc
tgtgacattttgggtggagttgataatcagcaaagactgctacacattgtctggcctcacaggtgggagcatgataaaga
tccagaaagcttttttaaggtattaatgcatcttaaagacttag gactcaatttccacgtgtctgtacttggag aaacct
tcacagatgtcccaggttggaagctgtgtactgtgggtgttacccactttgtcctaaagatttggtttatcccgaaatat
ttccagctgaatatctgtattctacacctgaacagctttcaaaaaggctccagaatttctgcaagagaccagatatta TA
A gaaaacatctctataagggtgaaatcgctccgttttcttgggcagccctacatggtaaattcaggtctctgcttacaac
agaacctagggaagatttgTGAcagatggggctaagtcacaaacttgcagcctaaggcagagtctgaagaactttccaga
gtgtgcccatatttacctgatcagagagaaaagaaaatct gcagaggaagctgagcctggctgcttgtca tagctgacac
agagccatctgccacaaacctgtggcggcttcagatctccaatccctgccaccaccccaactcaaattaaatacagattc
ctagagacgttatgatggttacacatgtcctcggcatcacatgtaggagactgttcaaaaaaaatatgtggcctgttgta
taaccgcactcatgtatcccatatgtggtgccacattgaatttccggttgaatccgtttttatcctttgtactggatgac
atggtgcctgaattctttcttttcgccgacacgatggcagccaaactgcagcttcaaacgctcacacttggctgggtttc
tacctaggttgccaggttatcatcggagccttcttgtgtcctcaaagggccacgaggcctgaaaggaggatcagaatgct
ttgggattaattgggcagccatcgcagaattgtttgtgggcaaagggctgctttagcacttttcttttagcaaattaatg
actctcaggcacagggggttttaagtgaaggtattaataagaggtctggcaggtattcccatgattcacagagttacatt
tgcatttaattaatcttaaagttgcaagataaacagctgtaattcggacaaacatgacaaacacagtgaagccaactatc
ccataaaatgaacactgacatacttgttttaatttttttcccagcgtaaaaatagaaaaatcaaaatactcctaacaaaa
ccagtgattttgatagaaatatttctccaatatacttgcatccacctacaaatataaccttttcaagataaatcgcttat
gatttcaatagtcaaactgctgtgtttgttgatgtaaagatgttttgaatggctagatggtaaaataaattcttaataaa
gtacccactgc

SEQ ID NO: 4: Amino acid sequence of ORF2 The underlined portion indicates a sequence different from the amino acid sequence of ORF1.
MSILIIEAFYGGSHKQLVDLLQEELGDCVVYTLPAKKWHWRARTSALYFSQTIPISEHYRTLFASSVLNLTELAALRPDL
GKLKKILYFHENQLIYPVKKCQERDFQYGYNQILSCLVADVVVFNSVFNMESFLTSMGKFMKLIPDHRPKDLESIIRPKC
QVIYFPIRFPDVSRFMPKHKTTHLKKMLGLKGNGGAVLSMALPFQPEQRDSEDLLKNFNSECDTHCGLDTARQEYLGNSL
RQESDLKKSTSSDNSSSHHGENKQNLTVDPCDILGGVDNQQRLLHIVWPHRWEHDKDPESFFKVLMHLKDLGLNFHVSVL
GETFTDVP GWKLCTVGVTHFVLKIWFIPKYFQLNICILHLNSFQKGSRISARDQIL (term)

Example 3: Homology search In parallel with the cloning of the Hmat-Xa gene by the above method, a novel glycosyltransferase gene candidate was obtained from a human EST database based on the amino acid sequence of a mannose transferase derived from microorganisms. As a result of the search, the hypothetical mannose transferase (Q9RLP4) and Methanosarcina mazei mannose transferase (Q8PZ44), which are gene products of the PimC gene of Mycobacterium tuberculosis, were detected as proteins with homology to the Hmat-Xa gene. It was. This suggested that the Hmat-Xa gene may be a gene encoding mannose transferase.

Example 4: Expression of Hmat-Xa gene (mRNA expression)
The expression of Hmat-Xa gene (ORF1) in human tissues and in normal glandular epithelium and adenocarcinoma was semi-quantitatively examined by RT-PCR. Specifically, it was performed as follows.

  XA-17F: RT-PCR was performed using GACTCAATTTCCACGTGTCTGTACTTGGAG (SEQ ID NO: 13) and XA-10R (SEQ ID NO: 8). The tissues examined were 14 tissues included in MTC Panel Human I and MTC Panel Human II (both normal tissue origin, Clontech) and MTC Panel Human Tumor (cancer tissue origin, Clontech).

Reaction solution composition:
5 μl of each MTC Panel cDNA
dH ₂ O 36.25μl
10 x buffer (enzyme attached, No.1) 5μl
10 mM dNTP mix 1 μl
Primer mix (XA-17F and XA-10R; 10 μM each) 2 μl
Expand Long Template PCR System (Roche Diagnostics) enzyme mix 0.75μl
Total 50 μl of reaction solution

Reaction conditions:
After 94 ° C for 30 sec, (94 ° C for 30 sec (denaturation), 68 ° C for 2 min (annealing)) x 30 or 34 cycles, 68 ° C for 2 min (extension)

  After PCR, 1% agarose gel electrophoresis was performed using 10 μl of the reaction mixture. From all tissues, two predicted sizes of ORF1 partial sequence (about 570 bp) and ORF2 partial sequence (420 bp) were obtained. Amplified fragments were detected.

The results are shown in FIGS. 1 (a) and (b). As can be seen from the results in FIG. 1A, the expression of the Hmat-Xa gene was high in the placenta, ovary, kidney, liver and pancreas, and low in the large intestine, heart and skeletal muscle. As a control, glyceraldehyde-3-phosphate dehydrogenase (G3PDH) was also subjected to RT-PCR (22 cycles) using Clontech's human G3PDH control primer set. As a result, G3PDH showed the same level of expression in all tissues examined.
As described above, the Hmat-Xa gene does not have a homologous sequence in yeast, and as shown in this section, the expression level differs between human tissues. On the other hand, genes that act on lipid intermediate biosynthesis are genes that are ubiquitous (conserved) among eukaryotes, and are so-called housekeeping genes that are always expressed. It is a gene that is expressed at the same level in each tissue of living organisms (we investigated the expression of human mannose transferase I gene and human mannose transferase III gene by tissue, Not much difference). Therefore, it is expected that the Hmat-Xa gene is not a mannose transferase that acts on lipid intermediate biosynthesis.

  The result shown in FIG. 1 (b) is a comparison of the transcription of the Hmat-Xa gene in normal glandular epithelium and adenocarcinoma by RT-PCR for five tissues: lung, large intestine, prostate, ovary, and pancreas. . As a control, RT-PCR (22 cycles of PCR) was also performed for glyceraldehyde-3-phosphate dehydrogenase (G3PDH). As a result, almost the same level of G3PDH was observed in 5 tissues regardless of normal or cancer. On the other hand, the following results were obtained for the Hmat-Xa gene. The expression was low in normal and adenocarcinoma tissues in the lung. In the large intestine, the expression was low in normal tissues, but the expression was significantly increased in adenocarcinoma. On the other hand, in the prostate, ovary, and pancreas, significant expression was observed in normal tissues, but in adenocarcinoma, the expression was significantly decreased. This indicates that the transcriptional progression of the Hmat-Xa gene can be a tumor marker in colorectal adenocarcinoma.

Example 5: Expression of Hmat-Xa gene (expression at the protein level)
For this purpose, an HA-tagged Hmat-Xa gene was first prepared. That is, a sequence (SEQ ID NO: 1) containing ORF1 already cloned into the pUC vector was amplified by PCR and inserted between KpnI-NotI sites of an animal cell expression vector (pMH; Roche Diagnostics).
PCR conditions and reaction composition are as described in Example 2. However, the template DNA is derived from the transformant and the primers are as follows.

PCR primers used:
XA-11FK aaggtaccaccATGAGTATCCTCATCATTG (SEQ ID NO: 14)
XA-12RN gggcggccgCAAATCTTCCCTAGGCTCTGT (SEQ ID NO: 15)

In addition, for yeast expression, the Hmat-Xa gene was constructed by incorporating the Hmat-Xa gene into the pMH vector by the above method, and then the constructed HA tag-containing Hmat-Xa gene portion was amplified by PCR, and the yeast expression vector (pESC-URA; Stratagene) was integrated between the SalI and XhoI sites.
PCR conditions and reaction solution composition are as described in Example 2. However, the template DNA is derived from the transformant and the primers are as follows.

Primer
HA-RX: ggctcgagTTAGACGTAGTCTGGGACGTCG (SEQ ID NO: 16)
XA-11FS: gggtcgacATGAGTATCCTCATCATTGAAG (SEQ ID NO: 17)

  As described above, transformation of HeLa cells or yeast was performed using the constructed HA tag-added Hmat-Xa gene-containing pMH vector and HA-tag-added Hmat-Xa gene-containing pESC-URA vector.

A. Preparation of HA-tagged Hmat-Xa transgenic HeLa cells
After containing 6 μl of Fugene 6 (Roche) and 2 μg of the HA-tagged Hmat-Xa gene-containing construct, the total volume was adjusted to 100 μl with serum-free DMEM medium, and then the total volume of the HeLa cells was expanded to the logarithmic growth phase (3 × 10 ⁵ ml / ml) was added to a petri dish cultured in a volume of 4 ml. After 3 days of culture, the cell suspension was diluted 1/64 and 1/128 and colonized in 4 ml of DMEM medium containing geneticin (400 μg / ml) for colony formation. After further culturing for 10 days, colony-formed cells were collected and cultured in the order of 96-well plate, 24-well plate, and 100 mm petri dish to increase the number of cells derived from one colony. A transformant was obtained from the above.

B. Preparation of H-tagged Hmat-Xa transgenic yeast
<Method>
The yeast YPH499 strain is shaken and cultured in 1 ml of YPDA medium for 9 hours in a 30 ° C constant temperature bath, then collected by centrifugation, and 100 μl of One Step Buffer (0.2 M lithium acetate, 40% PEG4000, 100 mM DTT) is added thereto. Suspended. Add HA-tagged Hmat-Xa gene-containing pESC-URA vector solution 3 μl (2 μg), mix, heat shock for 30 minutes in a 45 ° C constant temperature bath, apply to SD-URA agar medium, and apply 30 The cells were cultured for 3 days at 0 ° C.

C. Confirmation of transgene and its transcription to mRNA in transformed cells
The presence of the HA-tagged Hmat-Xa gene in HeLa cells was confirmed by PCR, and the expression (transcription) of the gene to RNA was confirmed by RT-PCR. The presence of HA-tagged Hmat-Xa gene in yeast was confirmed by PCR.

Primers used for PCR and RT-PCR in Hmat-Xa transgenic HeLa cells with HA tag sequence
Presence and transcription confirmation of HLa-introduced Htag-added Hmat-Xa gene
Forward: XR-17F (described above)
Reverse: H1-CHR (HA tag sequence-containing primer)
GACGTAGTCTGGGACGTCGTATGGGTACACTACCCATACGACGTCCCAGACTACGTCTAA (SEQ ID NO: 18)

Presence and transcription confirmation of HeLa endogenous Hmat-Xa gene
Forward: XR-17F (described above)
Reverse: XR-10R (described above)

A. PCR
(1) PCR reaction solution composition:
0.2 mM dNTPmix
0.2 μM F primer
0.2 μM R primer
0.75μg template DNA
2.6U Expand High Fidelity PCR System Enzyme (Roche Diagnostics)
10 X Buffer (attached to enzyme solution)
15 mM MgCl ₂
Total 50μl

(2) PCR reaction conditions:
1, 94 ℃ 1min (denaturation)
2, 94 ℃ 1min (denaturation)
3, 55 ℃ 1min (annealing)
4, 72 ℃ 1min (extension)
5, 4 ℃ 30min
Repeat steps 2 to 4 35 times.

B. RT-PCR
(1) RT-PCR reaction solution composition:
Ready-To-Go RT-PCR Beads (Amersham Pharmacia Biotech)
200nM F, R primer
RNA 1ng
Sterilized water containing 0.1% DEPC
Total 50μl

(2) RT-PCR reaction conditions:
1, 42 ℃ 45min (reverse transcriptase reaction)
2, 94 ℃ 5min (denaturation)
3, 94 ℃ 1min (denaturation)
4, 55 ℃ 1min (annealing)
5, 72 ℃ 1min (extension)
6, 4 ℃ 30min
Repeat steps 3-5 45 times.

In the HA tag sequence-added Hmat-Xa gene-introduced HeLa yeast, the primers used for PCR for confirming the presence of the HA tag sequence-added Hmat-Xa gene are:
Forward: XR-17F (described above)
Reverse: H1-CHR (HA tag sequence-containing primer, mentioned above)

The results are shown in FIG.
FIG. 2 (a) shows the results of PCR confirming the presence of the same gene in the HA-tagged Hmat-Xa gene transformed cells. As a result, as shown in the figure, a band of about 980 bP in length predicted from the primer was detected in the HeLa cell system (lane 2) and the yeast system (lane 3). It was confirmed that it was introduced inside.

  FIG. 2 (b) shows the results of RT-PCR using HA-tagged Hmat-Xa specific primers after extraction of total RNA from HA-tagged Hmat-Xa-introduced HeLa cells. As a result, as shown in lane 2, a band of about 980 bp in length expected from the primer was detected, and it was found that the Hmat-Xa gene was transcribed into mRNA. The 520 bp band seen in lane 2 (HA-tagged Hmat-Xa gene transformed HeLa) and lane 3 (non-transformed HeLa) is due to the expression of the endogenous Hmat-Xa gene in HeLa cells. .

Moreover, the expression as a protein was examined in Western using an anti-HA tag antibody.
Western method Preparation of SDS-PAGE samples from HA-tagged Hmat-Xa gene-transformed HeLa cells
After culturing HA-tagged Hmat-Xa gene-transformed HeLa cells in 12 100 mm tissue culture dishes containing DMEM medium + 10% FCS, the medium was removed, and the cells were further washed with ice-cold PBS (-). Washed once. Next, the cells were removed with a 2% EDTA-PBS (−) solution and collected by centrifugation. The precipitated cells were washed with 200 μl of PBS (−) and wet weight was measured. Sample buffer [2% SDS, 100 mM DTT, 0.36 M Tris-HCl (pH 6.8), 1 mM PMSF] 10 times the wet cell weight was added and pipetting, followed by boiling at 100 ° C. for 5 minutes. After cooling the sample on ice, the sample was aspirated and discharged for 30 seconds with a syringe with a 20G needle, followed by 30 seconds with a syringe with a 26G needle, and then centrifuged at 15000 rpm, 4 ° C for 15 minutes, and the supernatant (SDS The solubilized fraction) was collected. Then, the amount of protein in the supernatant was measured.

2. SDS-PAGE of sample and transfer to PDF membrane (blotting)
A 1% SDS-10% polyacrylamide gel having a length of 9 cm, a width of 10 cm, and a thickness of 1 mm was prepared using a gel plate, and a sample solution with the same protein content was added to the well of the gel. Electrophoresis was performed at a constant current of 30 mA / gel. The gel was transferred to a plastic container, washed with 100 ml of transfer buffer (this washing was repeated 5 times), and transferred to a PVDF membrane with a blotting apparatus. Further, the PVDF membrane after blotting was blocked with a 10% skim milk solution and then washed with TBS-T.

3. Antibody treatment
The PVDF membrane was reacted in a hybridization pack with 5 ml of a 1000-fold diluted solution of rabbit anti-HA antibody (Bethyl laboratories, Inc: A190-108A) in 10% skim milk at room temperature for 1 hour. After the PVDF membrane was washed with TBS-T (this wash was repeated 5 times), a 10-fold diluted solution of peroxidase-labeled donkey anti-rabbit antibody (Amersham Biosciences NIF824) in a 10% skim milk solution and 1 hour at room temperature Reacted. PVDF membrane was washed with TBS-T (this wash was repeated 5 times)

4). detection
The PVDF membrane was placed on the wrap, and 0.1 ml of detection solution (Amersham Biosciences: RPN2132) was placed on the PVDF membrane area (cm ² ) and left for 5 minutes. After removing the detection solution, wrap the PVDF membrane with a wrap, place an X-ray film [Hyper film ECL (Amersham: Model No. RPN2104K)] on it, set it in the Hyper film cassette, 30 seconds, 1 minute, 5 minutes, 30 Exposed for 1 minute. Subsequently, the exposed X-ray film was developed.

  As described above, the HA-tagged Hmat-Xa-introduced HeLa cells and Mock system and the HA-tagged Hmat-Xa-introduced yeast (transgene-induced system and non-inducible system) were solubilized with surfactants and SDS- After PAGE, Western blot analysis using anti-HA antibody was performed. As a result, as shown in FIG. 3, in the Hmat-Xa expression induction system of the HA-tagged Hmat-Xa-introduced HeLa cells and the HA-tagged Hmat-Xa-introduced yeast, the HA-tagged Hmat at the expected 52.07 kDa position. -Xa protein band was detected. That is, it was confirmed that the Hmat-Xa gene was expressed (translated) as a protein.

FIG. 1 shows the results of examining the expression of the Hmat-Xa gene (ORF-1) for each human tissue by RT-PCR. FIG. 2 shows the results of examining the transcription of the HA-tagged Hmat-Xa gene in HA-tagged Hmat-Xa gene-transformed HeLa cells. (A) Presence of HA-tagged Hmat-Xa gene in transformed cells Lane 1: Marker, Lane 2: HA-tagged Hmat-Xa-introduced HeLa, Lane 3: HA-tagged Hmat-Xa-introduced yeast (b) Transformed cell Transcription of HA-tagged Hmat-Xa gene in lane 1: marker, lane 2: HA-tagged Hmat-Xa introduced HeLa, lane 3: non-introduced HeLa FIG. 3 shows the results of Western analysis using anti-HA antibody.

SEQUENCE LISTING
<110> Tokai University
<120> A marker gene for colon cancer
<130> A51782A
<160> 18
<210> 1
<211> 2556
<212> DNA
<213> Human
<400> 1
gtatctctgc attagccaga ttggacatat gtaaagggac atcagtatat ctggatcatc 60
agattgtgct tcaagaagac catagggaag aacgcatttt catcttgctg caaaagttgc 120
aatagaagat ctgaaggttt gaatgggcca atgagtatcc tcatcattga agcattctat 180
ggaggctccc ataaacagct ggtggatctt cttcaagaag agttaggaga ctgtgtcgtt 240
tatacccttc ctgcaaagaa atggcattgg agagcccgga catctgcttt atatttctct 300
cagaccattc ccatcagtga gcattacagg accctctttg caagttcagt gcttaacctg 360
accgaactgg ctgcccttcg gcctgacctt gggaaactga aaaagattct gtattttcac 420
gagaaccagt tgatatatcc tgtcaagaaa tgtcaggaga gggatttcca atatggatac 480
aaccaaattc tttcatgcct ggtggctgat gtggttgtat tcaactcagt ttttaatatg 540
gaatcatttc tcacttccat gggaaaattt atgaagctga ttcctgatca cagacccaag 600
gatctggaaa gcatcatcag acccaagtgc caagttattt actttcccat caggtttcct 660
gatgtgagca gattcatgcc caagcacaaa acaacccatt taaagaagat gctcggcctt 720
aaaggaaatg gcggtgcggt tctgtccatg gcccttcctt ttcagccaga gcagagagat 780
tcagaggatt tattgaagaa ttttaattca gagtgtgata cacactgtgg ccttgatact 840
gcacgacaag aatatttggg taactcatta agacaggaat cagacttgaa aaaatccacc 900
tcgtcagata attcaagctc tcatcatggt gaaaataaac aaaatctgac tgttgatccc 960
tgtgacattt tgggtggagt tgataatcag caaagactgc tacacattgt ctggcctcac 1020
aggtgggagc atgataaaga tccagaaagc ttttttaagg tattaatgca tcttaaagac 1080
ttaggactca atttccacgt gtctgtactt ggagaaacct tcacagatgt cccagatatt 1140
ttttcagagg ccaaaaaggc attgggatct tctgtcttac actggggcta cttacccagc 1200
aaagatgact acttccaagt actgtgcatg gctgatgttg tcatctcaac agctaagcat 1260
gaattctttg gagtggcaat gttggaagct gtgtactgtg ggtgttaccc actttgtcct 1320
aaagatttgg tttatcccga aatatttcca gctgaatatc tgtattctac acctgaacag 1380
ctttcaaaaa ggctccagaa tttctgcaag agaccagata ttataagaaa acatctctat 1440
aagggtgaaa tcgctccgtt ttcttgggca gccctacatg gtaaattcag gtctctgctt 1500
acaacagaac ctagggaaga tttgtgacag atggggctaa gtcacaaact tgcagcctaa 1560
ggcagagtct gaagaacttt ccagagtgtg cccatattta cctgatcaga gagaaaagaa 1620
aatctgcaga ggaagctgag cctggctgct tgtcatagct gacacagagc catctgccac 1680
aaacctgtgg cggcttcaga tctccaatcc ctgccaccac cccaactcaa attaaataca 1740
gattcctaga gacgttatga tggttacaca tgtcctcggc atcacatgta ggagactgtt 1800
caaaaaaaat atgtggcctg ttgtataacc gcactcatgt atcccatatg tggtgccaca 1860
ttgaatttcc ggttgaatcc gtttttatcc tttgtactgg atgacatggt gcctgaattc 1920
tttcttttcg ccgacacgat ggcagccaaa ctgcagcttc aaacgctcac acttggctgg 1980
gtttctacct aggttgccag gttatcatcg gagccttctt gtgtcctcaa agggccacga 2040
ggcctgaaag gaggatcaga atgctttggg attaattggg cagccatcgc agaattgttt 2100
gtgggcaaag ggctgcttta gcacttttct tttagcaaat taatgactct caggcacagg 2160
gggttttaag tgaaggtatt aataagaggt ctggcaggta ttcccatgat tcacagagtt 2220
acatttgcat ttaattaatc ttaaagttgc aagataaaca gctgtaattc ggacaaacat 2280
gacaaacaca gtgaagccaa ctatcccata aaatgaacac tgacatactt gttttaattt 2340
ttttcccagc gtaaaaatag aaaaatcaaa atactcctaa caaaaccagt gattttgata 2400
gaaatatttc tccaatatac ttgcatccac ctacaaatat aaccttttca agataaatcg 2460
cttatgattt caatagtcaa actgctgtgt ttgttgatgt aaagatgttt tgaatggcta 2520
gatggtaaaa taaattctta ataaagtacc cactgc 2556
<210> 2
<211> 458
<212> PRT
<213> Human
<400> 2
Met Ser Ile Leu Ile Ile Glu Ala Phe Tyr Gly Gly Ser His Lys Gln
1 5 10 15
Leu Val Asp Leu Leu Gln Glu Glu Leu Gly Asp Cys Val Val Tyr Thr
20 25 30
Leu Pro Ala Lys Lys Trp His Trp Arg Ala Arg Thr Ser Ala Leu Tyr
35 40 45
Phe Ser Gln Thr Ile Pro Ile Ser Glu His Tyr Arg Thr Leu Phe Ala
50 55 60
Ser Ser Val Leu Asn Leu Thr Glu Leu Ala Ala Leu Arg Pro Asp Leu
65 70 75 80
Gly Lys Leu Lys Lys Ile Leu Tyr Phe His Glu Asn Gln Leu Ile Tyr
85 90 96
Pro Val Lys Lys Cys Gln Glu Arg Asp Phe Gln Tyr Gly Tyr Asn Gln
100 105 110
Ile Leu Ser Cys Leu Val Ala Asp Val Val Val Phe Asn Ser Val Phe
115 120 125
Asn Met Glu Ser Phe Leu Thr Ser Met Gly Lys Phe Met Lys Leu Ile
130 135 140
Pro Asp His Arg Pro Lys Asp Leu Glu Ser Ile Ile Arg Pro Lys Cys
145 150 155 160
Gln Val Ile Tyr Phe Pro Ile Arg Phe Pro Asp Val Ser Arg Phe Met
165 170 175
Pro Lys His Lys Thr Thr His Leu Lys Lys Met Leu Gly Leu Lys Gly
180 185 190
Asn Gly Gly Ala Val Leu Ser Met Ala Leu Pro Phe Gln Pro Glu Gln
195 200 205
Arg Asp Ser Glu Asp Leu Leu Lys Asn Phe Asn Ser Glu Cys Asp Thr
210 215 220
His Cys Gly Leu Asp Thr Ala Arg Gln Glu Tyr Leu Gly Asn Ser Leu
225 230 235 240
Arg Gln Glu Ser Asp Leu Lys Lys Ser Thr Ser Ser Asp Asn Ser Ser
245 250 255
Ser His His Gly Glu Asn Lys Gln Asn Leu Thr Val Asp Pro Cys Asp
260 265 270
Ile Leu Gly Gly Val Asp Asn Gln Gln Arg Leu Leu His Ile Val Trp
275 280 285
Pro His Arg Trp Glu His Asp Lys Asp Pro Glu Ser Phe Phe Lys Val
290 295 300
Leu Met His Leu Lys Asp Leu Gly Leu Asn Phe His Val Ser Val Leu
305 310 315 320
Gly Glu Thr Phe Thr Asp Val Pro Asp Ile Phe Ser Glu Ala Lys Lys
325 330 335
Ala Leu Gly Ser Ser Val Leu His Trp Gly Tyr Leu Pro Ser Lys Asp
340 345 350
Asp Tyr Phe Gln Val Leu Cys Met Ala Asp Val Val Ile Ser Thr Ala
355 360 365
Lys His Glu Phe Phe Gly Val Ala Met Leu Glu Ala Val Tyr Cys Gly
370 375 380
Cys Tyr Pro Leu Cys Pro Lys Asp Leu Val Tyr Pro Glu Ile Phe Pro
385 390 395 400
Ala Glu Tyr Leu Tyr Ser Thr Pro Glu Gln Leu Ser Lys Arg Leu Gln
405 410 415
Asn Phe Cys Lys Arg Pro Asp Ile Ile Arg Lys His Leu Tyr Lys Gly
420 425 430
Glu Ile Ala Pro Phe Ser Trp Ala Ala Leu His Gly Lys Phe Arg Ser
435 440 445
Leu Leu Thr Thr Glu Pro Arg Glu Asp Leu
450 455
<210> 3
<211> 2411
<212> DNA
<213> Human
<400> 3
gtatctctgc attagccaga ttggacatat gtaaagggac atcagtatat ctggatcatc 60
agattgtgct tcaagaagac catagggaag aacgcatttt catcttgctg caaaagttgc 120
aatagaagat ctgaaggttt gaatgggcca atgagtatcc tcatcattga agcattctat 180
ggaggctccc ataaacagct ggtggatctt cttcaagaag agttaggaga ctgtgtcgtt 240
tatacccttc ctgcaaagaa atggcattgg agagcccgga catctgcttt atatttctct 300
cagaccattc ccatcagtga gcattacagg accctctttg caagttcagt gcttaacctg 360
accgaactgg ctgcccttcg gcctgacctt gggaaactga aaaagattct gtattttcac 420
gagaaccagt tgatatatcc tgtcaagaaa tgtcaggaga gggatttcca atatggatac 480
aaccaaattc tttcatgcct ggtggctgat gtggttgtat tcaactcagt ttttaatatg 540
gaatcatttc tcacttccat gggaaaattt atgaagctga ttcctgatca cagacccaag 600
gatctggaaa gcatcatcag acccaagtgc caagttattt actttcccat caggtttcct 660
gatgtgagca gattcatgcc caagcacaaa acaacccatt taaagaagat gctcggcctt 720
aaaggaaatg gcggtgcggt tctgtccatg gcccttcctt ttcagccaga gcagagagat 780
tcagaggatt tattgaagaa ttttaattca gagtgtgata cacactgtgg ccttgatact 840
gcacgacaag aatatttggg taactcatta agacaggaat cagacttgaa aaaatccacc 900
tcgtcagata attcaagctc tcatcatggt gaaaataaac aaaatctgac tgttgatccc 960
tgtgacattt tgggtggagt tgataatcag caaagactgc tacacattgt ctggcctcac 1020
aggtgggagc atgataaaga tccagaaagc ttttttaagg tattaatgca tcttaaagac 1080
ttaggactca atttccacgt gtctgtactt ggagaaacct tcacagatgt cccaggttgg 1140
aagctgtgta ctgtgggtgt tacccacttt gtcctaaaga tttggtttat cccgaaatat 1200
ttccagctga atatctgtat tctacacctg aacagctttc aaaaaggctc cagaatttct 1260
gcaagagacc agatattata agaaaacatc tctataaggg tgaaatcgct ccgttttctt 1320
gggcagccct acatggtaaa ttcaggtctc tgcttacaac agaacctagg gaagatttgt 1380
gacagatggg gctaagtcac aaacttgcag cctaaggcag agtctgaaga actttccaga 1440
gtgtgcccat atttacctga tcagagagaa aagaaaatct gcagaggaag ctgagcctgg 1500
ctgcttgtca tagctgacac agagccatct gccacaaacc tgtggcggct tcagatctcc 1560
aatccctgcc accaccccaa ctcaaattaa atacagattc ctagagacgt tatgatggtt 1620
acacatgtcc tcggcatcac atgtaggaga ctgttcaaaa aaaatatgtg gcctgttgta 1680
taaccgcact catgtatccc atatgtggtg ccacattgaa tttccggttg aatccgtttt 1740
tatcctttgt actggatgac atggtgcctg aattctttct tttcgccgac acgatggcag 1800
ccaaactgca gcttcaaacg ctcacacttg gctgggtttc tacctaggtt gccaggttat 1860
catcggagcc ttcttgtgtc ctcaaagggc cacgaggcct gaaaggagga tcagaatgct 1920
ttgggattaa ttgggcagcc atcgcagaat tgtttgtggg caaagggctg ctttagcact 1980
tttcttttag caaattaatg actctcaggc acagggggtt ttaagtgaag gtattaataa 2040
gaggtctggc aggtattccc atgattcaca gagttacatt tgcatttaat taatcttaaa 2100
gttgcaagat aaacagctgt aattcggaca aacatgacaa acacagtgaa gccaactatc 2160
ccataaaatg aacactgaca tacttgtttt aatttttttc ccagcgtaaa aatagaaaaa 2220
tcaaaatact cctaacaaaa ccagtgattt tgatagaaat atttctccaa tatacttgca 2280
tccacctaca aatataacct tttcaagata aatcgcttat gatttcaata gtcaaactgc 2340
tgtgtttgtt gatgtaaaga tgttttgaat ggctagatgg taaaataaat tcttaataaa 2400
gtacccactg c 2411
<210> 4
<211> 376
<212> PRT
<213> Human
<400> 4
Met Ser Ile Leu Ile Ile Glu Ala Phe Tyr Gly Gly Ser His Lys Gln
1 5 10 15
Leu Val Asp Leu Leu Gln Glu Glu Leu Gly Asp Cys Val Val Tyr Thr
20 25 30
Leu Pro Ala Lys Lys Trp His Trp Arg Ala Arg Thr Ser Ala Leu Tyr
35 40 45
Phe Ser Gln Thr Ile Pro Ile Ser Glu His Tyr Arg Thr Leu Phe Ala
50 55 60
Ser Ser Val Leu Asn Leu Thr Glu Leu Ala Ala Leu Arg Pro Asp Leu
65 70 75 80
Gly Lys Leu Lys Lys Ile Leu Tyr Phe His Glu Asn Gln Leu Ile Tyr
85 90 96
Pro Val Lys Lys Cys Gln Glu Arg Asp Phe Gln Tyr Gly Tyr Asn Gln
100 105 110
Ile Leu Ser Cys Leu Val Ala Asp Val Val Val Phe Asn Ser Val Phe
115 120 125
Asn Met Glu Ser Phe Leu Thr Ser Met Gly Lys Phe Met Lys Leu Ile
130 135 140
Pro Asp His Arg Pro Lys Asp Leu Glu Ser Ile Ile Arg Pro Lys Cys
145 150 155 160
Gln Val Ile Tyr Phe Pro Ile Arg Phe Pro Asp Val Ser Arg Phe Met
165 170 175
Pro Lys His Lys Thr Thr His Leu Lys Lys Met Leu Gly Leu Lys Gly
180 185 190
Asn Gly Gly Ala Val Leu Ser Met Ala Leu Pro Phe Gln Pro Glu Gln
195 200 205
Arg Asp Ser Glu Asp Leu Leu Lys Asn Phe Asn Ser Glu Cys Asp Thr
210 215 220
His Cys Gly Leu Asp Thr Ala Arg Gln Glu Tyr Leu Gly Asn Ser Leu
225 230 235 240
Arg Gln Glu Ser Asp Leu Lys Lys Ser Thr Ser Ser Asp Asn Ser Ser
245 250 255
Ser His His Gly Glu Asn Lys Gln Asn Leu Thr Val Asp Pro Cys Asp
260 265 270
Ile Leu Gly Gly Val Asp Asn Gln Gln Arg Leu Leu His Ile Val Trp
275 280 285
Pro His Arg Trp Glu His Asp Lys Asp Pro Glu Ser Phe Phe Lys Val
290 295 300
Leu Met His Leu Lys Asp Leu Gly Leu Asn Phe His Val Ser Val Leu
305 310 315 320
Gly Glu Thr Phe Thr Asp Val Pro Gly Trp Lys Leu Cys Thr Val Gly
325 330 335
Val Thr His Phe Val Leu Lys Ile Trp Phe Ile Pro Lys Tyr Phe Gln
340 345 350
Leu Asn Ile Cys Ile Leu His Leu Asn Ser Phe Gln Lys Gly Ser Arg
355 360 365
Ile Ser Ala Arg Asp Gln Ile Leu
370 375
<210> 5
<211> 272
<212> PRT
<213> Human
<400> 5
Met Leu Gly Leu Lys Gly Asn Gly Gly Ala Val Leu Ser Met Ala Leu
1 5 10 15
Pro Phe Gln Pro Glu Gln Arg Asp Ser Glu Asp Leu Leu Lys Asn Phe
20 25 30
Asn Ser Glu Cys Asp Thr His Cys Gly Leu Asp Thr Ala Arg Gln Glu
35 40 45
Tyr Leu Gly Asn Ser Leu Arg Gln Glu Ser Asp Leu Lys Lys Ser Thr
50 55 60
Ser Ser Asp Asn Ser Ser Ser His His Gly Glu Asn Lys Gln Asn Leu
65 70 75 80
Thr Val Asp Pro Cys Asp Ile Leu Gly Gly Val Asp Asn Gln Gln Arg
85 90 95
Leu Leu His Ile Val Trp Pro His Arg Trp Glu His Asp Lys Asp Pro
100 105 110
Glu Ser Phe Phe Lys Val Leu Met His Leu Lys Asp Leu Gly Leu Asn
115 120 125
Phe His Val Ser Val Leu Gly Glu Thr Phe Thr Asp Val Pro Asp Ile
130 135 140
Phe Ser Glu Ala Lys Lys Ala Leu Gly Ser Ser Val Leu His Trp Gly
145 150 155 160
Tyr Leu Pro Ser Lys Asp Asp Tyr Phe Gln Val Leu Cys Met Ala Asp
165 170 175
Val Val Ile Ser Thr Ala Lys His Glu Phe Phe Gly Val Ala Met Leu
180 185 190
Glu Ala Val Tyr Cys Gly Cys Tyr Pro Leu Cys Pro Lys Asp Leu Val
195 200 205
Tyr Pro Glu Ile Phe Pro Ala Glu Tyr Leu Tyr Ser Thr Pro Glu Gln
210 215 220
Leu Ser Lys Arg Leu Gln Asn Phe Cys Lys Arg Pro Asp Ile Ile Arg
225 230 235 240
Lys His Leu Tyr Lys Gly Glu Ile Ala Pro Phe Ser Trp Ala Ala Leu
245 250 255
His Gly Lys Phe Arg Ser Leu Leu Thr Thr Glu Pro Arg Glu Asp Leu
260 265 270
<210> 6
<211> 2556
<212> DNA
<213> Human
<400> 6
gtatctctgc attagccaga ttggacatat gtaaagggac atcagtatat ctggatcatc 60
agattgtgct tcaagaagac catagggaag aacgcatttt catcttgctg caaaagttgc 120
aatagaagat ctgaaggttt gaatgggcca atgagtatcc tcatcattga agcattctatv180
ggaggctccc ataaacagct ggtggatctt cttcaagaag agttaggaga ctgtgtcgtt 240
tatacccttc ctgcaaagaa atggcattgg agagcccgga catctgcttt atatttctct 300
cagaccattc ccatcagtga gcattacagg accctctttg caagttcagt gcttaacctg 360
accgaactgg ctgcccttcg gcctgacctt gggaaactga aaaagattct gtattttcac 420
gagaaccagt tgatatatcc tgtcaagaaa tgtcaggaga gggatttcca atatggatac 480
aaccaaattc tttcatgcct ggtggctgat gtggttgtat tcaactcagt ttttaatatg 540
gaatcatttc tcacttccat gggaaaattt atgaagctga ttcctgatca cagacccaag 600
gatctggaaa gcatcatcag acccaagtgc caagttattt actttcccat caggtttcct 660
gatgtgagca gattcatgcc caagcacaaa acaacccatt taaagaagat gctcggcctt 720
aaaggaaatg gcggtgcggt tctgtccatg gcccttcctt ttcagccaga gcagagagat 780
tcagaggatt tattgaagaa ttttaattca gagtgtgata cacactgtgg ccttgatact 840
gcacgacaag aatatttggg taactcatta agacaggaat cagacttgaa aaaatccacc 900
tcgtcagata attcaagctc tcatcatggt gaaaataaac aaaatctgac tgttgatccc 960
tgtgacattt tgggtggagt tgataatcag caaagactgc tacacattgt ctggcctcac 1020
aggtgggagc atgataaaga tccagaaagc ttttttaagg tattaatgca tcttaaagac 1080
ttaggactca atttccacgt gtctgtactt ggagaaacct tcacagatgt cccagatatt 1140
ttttcagagg ccaaaaaggc attgggatct tctgtcttac actggggcta cttacccagc 1200
aaagatgact acttccaagt actgtgcatg gctgatgttg tcatctcaac agctaagcat 1260
gaattctttg gagtggcaat gttggaagct gtgtactgtg ggtgttaccc actttgtcct 1320
aaagatttgg tttatcccga aatatttcca gctgaatatc tgtattctac acctgaacag 1380
ctttcaaaaa ggctccagaa tttctgcaag agaccagata ttataagaaa acatctctat 1440
aagggtgaaa tcgctccgtt ttcttgggca gccctacatg gtaaattcag gtctctgctt 1500
acaacagaac ctagggaaga tttgtgacag atggggctaa gtcacaaact tgcagcctaa 1560
ggcagagtct gaagaacttt ccagagtgtg cccatattta cctgatcaga gagaaaagaa 1620
aatctgcaga ggaagctgag cctggctgct tgtcatagct gacacagagc catctgccac 1680
aaacctgtgg cggcttcaga tctccaatcc ctgccaccac cccaactcaa attaaataca 1740
gattcctaga gacgttatga tggttacaca tgtcctcggc atcacatgta ggagactgtt 1800
caaaaaaaat atgtggcctg ttgtataacc gcactcatgt atcccatatg tggtgccaca 1860
ttgaatttcc ggttgaatcc gtttttatcc tttgtactgg atgacatggt gcctgaattc 1920
tttcttttcg ccgacacgat ggcagccaaa ctgcagcttc aaacgctcac acttggctgg 1980
gtttctacct aggttgccag gttatcatcg gagccttctt gtgtcctcaa agggccacga 2040
ggcctgaaag gaggatcaga atgctttggg attaattggg cagccatcgc agaattgttt 2100
gtgggcaaag ggctgcttta gcacttttct tttagcaaat taatgactct caggcacagg 2160
gggttttaag tgaaggtatt aataagaggt ctggcaggta ttcccatgat tcacagagtt 2220
acatttgcat ttaattaatc ttaaagttgc aagataaaca gctgtaattc ggacaaacat 2280
gacaaacaca gtgaagccaa ctatcccata aaatgaacac tgacatactt gttttaattt 2340
ttttcccagc gtaaaaatag aaaaatcaaa atactcctaa caaaaccagt gattttgata 2400
gaaatatttc tccaatatac ttgcatccac ctacaaatat aaccttttca agataaatcg 2460
cttatgattt caatagtcaa actgctgtgt ttgttgatgt aaagatgttt tgaatggcta 2520
gatggtaaaa taaattctta ataaagtacc cactgc 2556
<210> 7
<211> 30
<212> DNA
<213> Artificial
<220>
<223> primer
<400> 7
atcagattgt gcttcaagaa gaccataggg 30
<210> 8
<211> 30
<212> DNA
<213> Artificial
<220>
<223> primer
<400> 8
tgacaagcag ccaggctcag cttcctctgc 30
<210> 9
<211> 24
<212> DNA
<213> Artificial
<220>
<223> primer
<400> 9
cgccagggtt ttcccagtca cgac 24
<210> 10
<211> 24
<212> DNA
<213> Artificial
<220>
<223> primer
<400> 10
cggataacaa tttcacacag gaaac 24
<210> 11
<211> 30
<212> DNA
<213> Artificial
<220>
<223> primer
<400> 11
tctgctctgg ctgaaaagga agggccatgg 30
<210> 12
<211> 30
<212> DNA
<213> Artificial
<220>
<223> primer
<400> 12
gaagatgctc ggccttaaag gaaatggcgg 30
<210> 13
<211> 30
<212> DNA
<213> Artificial
<220>
<223> primer
<400> 13
gactcaattt ccacgtgtct gtacttggag 30
<210> 14
<211> 30
<212> DNA
<213> Artificial
<220>
<223> primer
<400> 14
aaggtaccac catgagtatc ctcatcattg 30
<210> 15
<211> 30
<212> DNA
<213> Artificial
<220>
<223> primer
<400> 15
gggcggccgc aaatcttccc taggctctgt 30
<210> 16
<211> 30
<212> DNA
<213> Artificial
<220>
<223> primer
<400> 16
ggctcgagtt agacgtagtc tgggacgtcg 30
<210> 17
<211> 30
<212> DNA
<213> Artificial
<220>
<223> primer
<400> 17
gggtcgacat gagtatcctc atcattgaag 30
<210> 18
<211> 60
<212> DNA
<213> Artificial
<220>
<223> primer
<400> 18
gacgtagtct gggacgtcgt atgggtacac tacccatacg acgtcccaga ctacgtctaa 60

Claims (12)

The following protein (A) or (B).
(A) A protein having the amino acid sequence set forth in SEQ ID NO: 2.
(B) A protein having an amino acid sequence containing substitution, deletion, insertion and / or addition of one or several amino acids and having glycosyltransferase activity in the amino acid sequence shown in SEQ ID NO: 2. DNA encoding any of the following proteins (A) or (B).
(A) A protein having the amino acid sequence set forth in SEQ ID NO: 2.
(B) A protein having an amino acid sequence containing substitution, deletion, insertion and / or addition of one or several amino acids and having glycosyltransferase activity in the amino acid sequence shown in SEQ ID NO: 2. DNA of any of the following (a), (b) or (c).
(A) DNA having the base sequence set forth in SEQ ID NO: 1.
(B) The base sequence described in SEQ ID NO: 1 has a base sequence including substitution, deletion, insertion and / or addition of one or several bases, and encodes a protein having glycosyltransferase activity DNA.
(C) A DNA that hybridizes with a DNA having the nucleotide sequence set forth in SEQ ID NO: 1 under a stringent condition and encodes a protein having glycosyltransferase activity. A recombinant vector comprising the DNA according to claim 2 or 3. A transformant comprising the DNA according to claim 2 or 3 or the recombinant vector according to claim 4. An antibody against the protein according to claim 1. A diagnostic agent for colorectal cancer, comprising an antibody against the protein according to claim 1. A method for diagnosing colorectal cancer, comprising detecting or measuring the expression of the protein of claim 1 in a biological sample using the antibody of claim 6. An oligonucleotide comprising a partial sequence of the base sequence of the DNA according to claim 2 or 3 or a complementary sequence thereof. A diagnostic agent for colorectal cancer comprising an oligonucleotide consisting of a partial sequence of the base sequence of the DNA according to claim 2 or 3 or a complementary sequence thereof. A method for diagnosing colorectal cancer, comprising detecting or measuring the expression of the DNA according to claim 2 or 3 in a biological sample using the oligonucleotide according to claim 9. A method of using the protein of claim 1 as a marker for colorectal cancer.