JP2006238757A - Marker for detecting cancer - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for detecting cancer, to provide a pharmaceutical composition for treating the cancer, and to provide a method for screening a substance inhibiting the cancer. <P>SOLUTION: This method for detecting the cancer includes measuring expression of an Egf17 gene. An antibody specifically combines with an Egf17 polypeptide or a fragment thereof. The pharmaceutical composition for treating the cancer contains an Egf17 polynucleotide and/or a derivative thereof or the Egf17 polypeptide and/or a derivative thereof. The method for screening the substance inhibiting the cancer includes selecting a substance inhibiting the expression of the Egf17 gene or a substance accelerating the expression. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a method for detecting cancer, a marker for detecting cancer, a method for screening for a substance that inhibits cancer, and a pharmaceutical composition for treating cancer.

  Egfl7 expressed and secreted in endothelial cells is not involved in endothelial cell proliferation in angiogenesis, but is known as an essential protein for the formation of vascular luminal structures. Therefore, the expression of Egfl7 continues to be maintained at the stage of development such as embryonic fetus and neonatal period. On the other hand, in many organs of grown organisms, the expression of Egfl7 decreases and is not recognized. However, expression of Egfl7 is observed in organs such as lungs with many blood vessels, heart and kidney, vascular regeneration parts caused by vascular wounds, proliferating tissues such as tumors, reproductive organs in pregnancy, and inflamed tissues. This suggests that Egfl7 expression is involved in angiogenesis and regeneration. As described above, Egfl7 is known to be involved in the formation of the luminal structure of blood vessels in angiogenesis, but functions other than this finding have not been elucidated.

Leon H. Parker et al. (2004), Nature, 428, 754-758.

  An object of the present invention is to provide a method for detecting cancer, a pharmaceutical composition for treating cancer, and a method for screening a substance that inhibits cancer.

  The present inventors have found that expression of Egfl7 increases with canceration in organs such as the liver but decreases with canceration in other organs, and the present invention has been completed. .

That is, the present invention includes the following inventions.
(1) A method for detecting cancer, comprising measuring expression of an Egfl7 gene.
(2) The method according to (1), wherein the Egfl7 gene is a gene specified by the nucleotide sequence of SEQ ID NO: 1 or 2.
(3) A marker for detecting cancer, comprising Egfl7 polynucleotide.
(4) The marker according to (3), wherein the Egfl7 polynucleotide is a polynucleotide comprising all or part of the base sequence of SEQ ID NO: 1 or 2.

(5) A marker for detecting cancer, comprising Egfl7 polypeptide or a fragment thereof.
(6) The marker according to (5), wherein the Egfl7 polypeptide is a polypeptide comprising all or part of the amino acid sequence of SEQ ID NO: 3.
(7) A kit for detecting cancer, comprising an antibody that specifically binds to Egfl7 polypeptide or a fragment thereof.
(8) To detect cancer comprising a continuous primer having a length of at least 15 bases for specifically amplifying Egfl7 polynucleotide or a continuous probe having a length of at least 15 bases specifically hybridizing to Egfl7 polynucleotide Kit.
(9) A pharmaceutical composition for treating cancer, comprising an antibody that specifically binds to an Egfl7 polypeptide or a fragment thereof.

(10) A pharmaceutical composition for treating cancer, comprising Egfl7 polynucleotide and / or a derivative thereof, or Egfl7 polypeptide and / or a derivative thereof.
(11) Screening for a substance that inhibits cancer, comprising contacting a test sample with a cell expressing the Egfl7 gene and selecting a substance that inhibits or promotes the expression of the Egfl7 gene from the test sample. how to.

  According to the present invention, cancer can be detected. In addition, a substance that inhibits cancer can be screened.

  The present inventors have found that the expression of the Egfl7 gene, which is a protein essential for the formation of the luminal structure of blood vessels, is increased in a specific cancer tissue and decreased in another cancer tissue. Then, it was found that cancer can be detected by measuring the expression of the Egfl7 gene. In the present invention, measuring the expression of a gene includes measuring the expression level and detecting the presence or absence of expression.

  In the present invention, the cancer includes various malignant tumors such as solid cancer, hemangioma, hemangioendothelioma, sarcoma, Kaposi sarcoma, hematopoietic tumor, adenocarcinoma and squamous cell carcinoma, such as liver cancer, lung cancer, Examples include kidney cancer, small intestine cancer, colon cancer, ovarian cancer, stomach cancer, tongue cancer, esophageal cancer, breast cancer, uterine cancer, and pharyngeal cancer. Furthermore, these cancer metastases are also included. The detection method of the present invention is effective for detection of liver cancer, lung cancer, kidney cancer, colon cancer, small intestine cancer, and ovarian cancer, and particularly effective for detection of kidney cancer, lung cancer, and colon cancer.

  In the present invention, known base sequences of the Egfl7 gene have been disclosed. For example, the base sequence of the human Egfl7 gene can be found in Accession No. 5 in public databases (GenBank, EMBL, DDBJ). BC088371 (SEQ ID NO: 1) and No. It is registered as BC012377 (SEQ ID NO: 2). In addition, the amino acid sequence (SEQ ID NO: 3) of the polypeptide encoded by the human Egfl7 gene is disclosed in Accession No. in the published database (GenBank, EMBL, DDBJ). AAH88371.1 and no. It is registered as AAH12377.1. In the base sequence of SEQ ID NO: 2, the first to 265th bases of the base sequence of SEQ ID NO: 1 are replaced with the 1st to 25th bases of SEQ ID NO: 2, and the 1539th to 25th bases This is a base sequence from which the 1562th base is deleted. Each sequence is described, for example, at http: // www. ncbi. nlm. nih. available from gov /.

  In the present invention, the Egfl7 gene whose expression is to be measured can be appropriately selected depending on the subject to be detected, and is not particularly limited, but is usually a vertebrate-derived Egfl7 gene, preferably a mammal-derived Egfl7 gene, preferably It is a human-derived Egfl7 gene.

  In one embodiment of the present invention, the Egfl7 gene is a gene specified by the nucleotide sequence of SEQ ID NO: 1 or 2. In the present invention, the gene specified by SEQ ID NO: 1 or 2 includes a gene consisting of the base sequence represented by SEQ ID NO: 1 or 2, and a gene functionally equivalent to the gene. Here, “functionally equivalent” means that the polypeptide encoded by the gene of interest has the same biological function as the polypeptide encoded by the gene consisting of the nucleotide sequence represented by SEQ ID NO: 1 or 2. , Refers to having a biochemical function.

  Methods well known to those skilled in the art for preparing a gene encoding a polypeptide functionally equivalent to a certain polypeptide include hybridization techniques (Sambrook, J et al., Molecular Cloning 2nd ed., 9.47-). 9.58, Cold Spring Harbor Lab.press (1989)). In the present specification, the term “gene” includes not only DNA but also mRNA and cDNA thereof. Moreover, not only a full-length gene but EST shall be included.

  Therefore, the gene specified by the base sequence represented by SEQ ID NO: 1 or 2 includes a gene comprising a base sequence including all or part of the base sequence represented by SEQ ID NO: 1 or 2. The base length of a gene consisting of a base sequence containing all or part of the base sequence represented by SEQ ID NO: 1 or 2 is not particularly limited as long as it encodes functionally equivalent polypeptides. The part of the base sequence is a base sequence of a part of each base sequence and has a length of the base sequence sufficient to hybridize under stringent conditions, for example, at least 15 bases, The sequence is preferably at least 100 bases, more preferably at least 200 bases, and most preferably at least 1000 bases. Preferably, each base sequence is a sequence of at least 15 bases, preferably at least 100 bases, more preferably at least 200 bases, and most preferably at least 1000 bases. Here, “continuous” means that a continuous base sequence is included in the reference base sequence.

  More specifically, the gene identified by SEQ ID NO: 1 hybridizes with a polynucleotide comprising a base sequence complementary to the base sequence consisting of SEQ ID NO: 1 under stringent conditions and is equivalent to Egfl7 A gene consisting of a base sequence encoding a polypeptide having the biological function of, and the gene specified by SEQ ID NO: 2 is a polynucleotide consisting of a base sequence complementary to the base sequence consisting of SEQ ID NO: 2 And a gene consisting of a base sequence encoding a polypeptide that hybridizes under stringent conditions and has a biological function equivalent to that of Egfl7. In the present specification, the polynucleotide includes DNA and RNA.

  In the present specification, stringent conditions refer to conditions in which a specific hybrid is formed and a non-specific hybrid is not formed, that is, high homology to each base sequence (homology is 60% or more, 70% or more, more preferably 80% or more, still more preferably 90% or more, and most preferably 95% or more). More specifically, such conditions can be determined by conventional techniques well known in the art, such as colony hybridization, plaque hybridization, microarray method, or Southern blot hybridization. Hybridization was performed at 65 ° C. in the presence of 0.7 to 1.0 M NaCl using a membrane on which plaque-derived polynucleotide was immobilized, and then 0.1 to 2 times the concentration of SSC (Saline Sodium Citrate; 150mM sodium chloride, 15mM sodium citrate) solution and washing the membrane at 65 ° C.

  Under these conditions, it can be expected that a polynucleotide having high homology can be efficiently obtained as the temperature is increased. However, a plurality of factors such as temperature and salt concentration can be considered as factors affecting the stringency of hybridization, and those skilled in the art can realize the same stringency by appropriately selecting these factors. .

  It is also possible to isolate genes functionally equivalent to each gene using gene amplification methods using primers synthesized based on the nucleotide sequence information, such as polymerase chain reaction (PCR) method, LAMP method, etc. Is possible.

  Functionally equivalent genes isolated by these hybridization techniques and gene amplification techniques usually have high homology or identity at the amino acid sequence level. High homology or identity generally refers to homology or identity of at least 50% or more, preferably 75% or more, more preferably 85% or more, more preferably 95% or more at the amino acid level.

  The homology or identity of amino acid sequences and base sequences can be determined by the algorithm BLAST (Proc. Natl. Acad. Sci. USA 90: 5873-5877, 1993) by Karlin and Altschul. Based on this algorithm, programs called BLASTN and BLASTX have been developed (Altschul et al. J. Mol. Biol. 215: 403-410, 1990). Specific techniques for these analysis methods are known (http://www.ncbi.nlm.nih.gov.).

Method for Detecting Cancer The method for detecting cancer in the present invention is characterized by measuring the expression of the Egfl7 gene. As a method for detecting the expression of the Egfl7 gene in a sample derived from a subject, a method for detecting a polypeptide encoded by the Egfl7 gene in a sample derived from a subject (hereinafter referred to as Egfl7 polypeptide), Examples include a method for detecting a polynucleotide that is all or part of the Egfl7 gene in a sample derived from a subject (referred to herein as an Egfl7 polynucleotide), particularly Egfl7 mRNA. Here, detection of Egfl7 mRNA includes detection of cDNA or cRNA converted from the RNA.

  That is, the method for detecting cancer in the present invention uses Egfl7 polynucleotide, Egfl7 polypeptide or a fragment thereof as a marker.

  In one embodiment, the marker consisting of an Egfl7 polynucleotide consists of a polynucleotide comprising all or part of the base sequence of SEQ ID NO: 1 or 2. The base length of the polynucleotide comprising the whole or part of the base sequence represented by SEQ ID NO: 1 or 2 is not particularly limited as long as it is the whole or part of the Egfl7 gene. The part of the base sequence is a base sequence of a part of each base sequence and has a length of the base sequence sufficient to hybridize under stringent conditions, for example, at least 15 bases, The sequence is preferably at least 100 bases, more preferably at least 200 bases. Preferably, it is a sequence of at least 15 bases, preferably at least 100 bases, more preferably at least 200 bases continuous in each base sequence. The stringent conditions are as described above.

  In one embodiment, the Egfl7 polynucleotide is under stringent conditions with a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 1 or 2, or a polynucleotide comprising a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 1 or 2 And a nucleotide sequence comprising a base sequence encoding a polypeptide having a biological function equivalent to that of Egfl7.

  In one embodiment, the marker consisting of an Egfl7 polypeptide or fragment thereof consists of a polypeptide comprising all or part of the amino acid sequence of SEQ ID NO: 3. The amino acid chain length of the polypeptide comprising all or part of the amino acid sequence of SEQ ID NO: 3 is not particularly limited as long as it has a function equivalent to that of the Egfl7 polypeptide. The part of the amino acid sequence is one having an amino acid chain length sufficient to have the activity of the Egfl7 polypeptide, for example, a sequence of at least 10 residues, preferably 100 residues, more preferably at least 200 residues. It is. Preferably, each amino acid sequence is a sequence of at least 10 residues, preferably 100 residues, more preferably at least 200 residues. The polypeptide comprising all or part of the amino acid sequence of SEQ ID NO: 3 includes a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 3 and at least 50% identity with the amino acid sequence represented by SEQ ID NO: 3. A polypeptide comprising an amino acid sequence having preferably 75% or more identity, more preferably 85% or more identity, more preferably 95% or more identity.

  In another embodiment, the marker consisting of Egfl7 polypeptide or a fragment thereof consists of an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 3, It consists of a polypeptide having an equivalent biological function. Here, the term “several” means usually 1 to 10, preferably 1 to 5, more preferably 1 to 3.

  In the present specification, the polynucleotide includes an oligonucleotide, and the polypeptide includes an oligopeptide and a protein.

1. Detection of Egfl7 polypeptide Methods for detecting Egfl7 polypeptide in a sample include methods well known to those skilled in the art, such as enzyme linked immunoassay (ELISA), double monoclonal antibody sandwich immunoassay (US Pat. No. 4,376). 110), monoclonal polyclonal antibody sandwich assay (Wide et al., Kirkham and Hunter, “Radioimmunoassay”, E. and S. Livingstone, Edinburgh, (1970)), immunofluorescence, Western blotting , Dot blotting method, immunoprecipitation method, protein chip analysis method (protein nucleic acid enzyme Vol. 47 No. 5 (2002), protein nucleic acid enzyme Vol. 47 No. 8 (20 02)) Examples include, but are not limited to, two-dimensional electrophoresis and SDS polyacrylamide electrophoresis.

  Hereinafter, a method for detecting the expression of Egfl7 polypeptide using an antibody that specifically binds to Egfl7 polypeptide or a fragment thereof will be described in detail. Since an antibody that specifically reacts with an Egfl7 polypeptide or a fragment thereof can bind to an Egfl7 polypeptide expressed in an organ such as the liver, the antibody is used to detect a reaction with the Egfl7 polypeptide in a sample. Thus, cancer can be detected.

The antibody that specifically reacts with the Egfl7 polypeptide or a fragment thereof is a polyclonal or monoclonal antibody, and can bind to an epitope of the Egfl7 polypeptide, respectively. The globulin type of the antibody is not particularly limited as long as it has the above characteristics, and may be any of IgG, IgM, IgA, IgE, and IgD, but IgG and IgM are preferred. The monoclonal antibodies in the present invention include, in particular, a portion of the heavy and / or light chain that is derived from a specific species, or a specific antibody class or subclass, and the remaining portion of the chain is another species, or another antibody Included are “chimeric” antibodies (immunoglobulins) that are of class or subclass, as well as immunoreactive antibody fragments such as Fab, F (ab ′) ₂ , Fv fragments so long as they have the desired biological activity ( U.S. Pat. No. 4,816,567).

  In producing the antibody of the present invention, a polypeptide to be used as an immunogen (antigen) is prepared. As the immunogenic polypeptide, an Egfl7 polypeptide or a fragment thereof is used. The amino acid sequence of an Egfl7 polypeptide that can be used as an immunogen in the present invention and the cDNA sequence encoding the polypeptide have been published as described above. Therefore, Egfl7 polypeptide or a fragment thereof for use as an immunogen can be synthesized by publicly known methods such as solid phase peptide synthesis using publicly available amino acid sequence information. . The fragment may be any fragment having the immunogenicity of Egfl7 polypeptide. Specifically, it is a portion consisting of at least 6 amino acids, preferably 6 to 200 amino acids, more preferably 8 to 50 amino acids in Egfl7 polypeptide. Peptides are mentioned. When an Egfl7 polypeptide fragment is used as an immunogen, it is preferably used by linking to a carrier protein such as KLH or BSA.

  It is also possible to produce Egfl7 polypeptide using information on cDNA encoding Egfl7 polypeptide using known gene recombination techniques. Hereinafter, production of Egfl7 polypeptide using a recombinant technique will be described.

  The recombinant vector for producing Egfl7 polypeptide can be obtained by ligating the above-described publicly available cDNA sequence to an appropriate vector, and the transformant can be obtained by combining the recombinant vector for producing Egfl7 polypeptide with Egfl7 polypeptide. It can be obtained by introducing it into a host so that it can be expressed.

  As the vector, a phage or a plasmid capable of autonomously growing in a host microorganism is used. Plasmid DNA includes plasmids derived from E. coli (eg, pET21a, pGEX4T, pUC118, pUC119, pUC18, pUC19, etc.), plasmids derived from Bacillus subtilis (eg, pUB110, pTP5, etc.), yeast-derived plasmids (eg, YEp13, YEp24, YCp50, etc.) ) And the like, and examples of the phage DNA include λ phage (λgt11, λZAP, etc.). Furthermore, animal viruses such as vaccinia virus and insect virus vectors such as baculovirus can be used.

  In order to insert Egfl7 cDNA into a vector, first, a method is used in which the purified DNA is cleaved with an appropriate restriction enzyme, inserted into a restriction enzyme site or a multicloning site of an appropriate vector DNA, and ligated to the vector. .

  In addition, recombinant vectors for producing Egfl7 polypeptide used in mammalian cells include promoters, Egfl7 cDNA, cis elements such as enhancers, splicing signals, poly A addition signals, selectable markers, ribosome binding sequences (SD sequences) as required. ) Etc. may be linked.

  A known DNA ligase is used to link the DNA fragment and the vector fragment. Then, the DNA fragment and the vector fragment are annealed and then ligated to produce a recombinant vector for producing Egfl7 polypeptide.

  The host used for transformation is not particularly limited as long as it can express Egfl7 polypeptide. Examples include bacteria (E. coli, Bacillus subtilis, etc.), yeast, animal cells (COS cells, CHO cells, etc.) and insect cells.

  As an example, when a bacterium is used as a host, the recombinant vector for producing Egfl7 polypeptide can autonomously replicate in the bacterium, and at the same time, it is composed of a promoter, a ribosome binding sequence, Egfl7 DNA, and a transcription termination sequence. preferable. Moreover, the gene which controls a promoter may be contained. Examples of E. coli include Escherichia coli BRL. Examples of Bacillus subtilis include Bacillus subtilis. Any promoter can be used as long as it can be expressed in a host such as Escherichia coli. The method for introducing a recombinant vector into bacteria is not particularly limited as long as it is a method for introducing DNA into bacteria. Examples thereof include a method using calcium ions and an electroporation method.

  Similarly, when yeast, animal cells, insect cells, and the like are used as hosts, Egfl7 polypeptide can be produced according to techniques known in the art.

  The Egfl7 polypeptide used as an immunogen in the present invention can be obtained by culturing the transformant prepared above and collecting it from the culture. “Culture” means any of culture supernatant, cultured cells, cultured cells, or disrupted cells or cells. The method of culturing the transformant in a medium is performed according to a usual method used for host culture.

  As a medium for culturing transformants obtained using microorganisms such as Escherichia coli and yeast as a host, the medium contains a carbon source, nitrogen source, inorganic salts, etc. that can be assimilated by the microorganisms. As long as the medium can be used, either a natural medium or a synthetic medium may be used.

  The culture is usually performed at 37 ° C. for 6 to 24 hours under aerobic conditions such as shaking culture or aeration and agitation culture. During the culture period, the pH is kept near neutral. The pH is adjusted using an inorganic or organic acid, an alkaline solution, or the like. During culture, an antibiotic such as ampicillin or tetracycline may be added to the medium as necessary.

  When Egfl7 polypeptide is produced in cells or cells after culturing, Egfl7 polypeptide is extracted by disrupting the cells or cells. When the Egfl7 polypeptide is produced outside the cells or cells, the culture solution is used as it is, or the cells or cells are removed by centrifugation or the like. Thereafter, by using general biochemical methods used for protein isolation and purification, such as ammonium sulfate precipitation, gel chromatography, ion exchange chromatography, affinity chromatography, etc. alone or in appropriate combination, The Egfl7 polypeptide can be isolated and purified from Whether or not the Egfl7 polypeptide has been obtained can be confirmed by SDS-polyacrylamide gel electrophoresis or the like.

  The recombinant Egfl7 polypeptide obtained by the above method includes a fusion protein with any other protein. Examples thereof include a fusion protein with glutathione-S-transferase (GST) and green fluorescent protein (GFP). Furthermore, the peptide expressed in the transformed cell may be subjected to various modifications in the cell after being translated. Therefore, modified peptides can also be used as Egfl7 polypeptides. Examples of such post-translational modifications include elimination of N-terminal methionine, N-terminal acetylation, sugar chain addition, limited degradation by intracellular protease, myristoylation, isoprenylation, phosphorylation and the like.

  Next, the obtained protein is dissolved in a buffer solution to prepare an immunogen. If necessary, an adjuvant may be added for effective immunization. Examples of the adjuvant include commercially available complete Freund's adjuvant and incomplete Freund's adjuvant, and any of these may be mixed.

  The monoclonal antibody may be produced, for example, by a hybridoma method (KOHLER AND MILSTEIN, NATURE (1975) 256: 495) or a recombinant method (US Pat. No. 4,816,567). It may also be isolated from a phage antibody library. For example, it can be produced as follows.

i) Immunization and collection of antibody-producing cells The immunogen obtained as described above is administered to mammals such as rats, mice (for example, BALB / c of inbred mice), rabbits, and the like. The single dose of the immunogen is appropriately determined according to the type of immunized animal, the route of administration, etc., and is about 50 to 200 μg per animal. Immunization is performed mainly by injecting an immunogen intravenously, subcutaneously or intraperitoneally. Further, the immunization interval is not particularly limited, and after the first immunization, booster immunization is performed 2 to 6 times, preferably 3 to 4 times at intervals of several days to several weeks, preferably at intervals of 1 to 4 weeks. After the initial immunization, the antibody titer in the serum of the immunized animal is repeatedly measured by ELISA (Enzyme-Linked Immuno Sorbent Assay) method, etc. When the antibody titer reaches a plateau, the immunogen is intravenously or intraperitoneally Inject and give final immunization. Then, antibody-producing cells are collected 2 to 5 days, preferably 3 days after the last immunization day. Examples of antibody-producing cells include spleen cells, lymph node cells, peripheral blood cells, etc., but spleen cells or local lymph node cells are preferred.

ii) Cell fusion In order to obtain a hybridoma, cell fusion between an antibody-producing cell obtained from an immunized animal and a myeloma cell is performed as described above.

  As myeloma cells to be fused with antibody-producing cells, generally available cell lines of animals such as mice can be used. The cell line used has drug selectivity and cannot survive in a HAT selection medium (including hypoxanthine, aminopterin, thymine) in an unfused state, but can survive only in a state fused with antibody-producing cells. Those having the following are preferred. The cell line is preferably derived from an animal of the same species as the immunized animal. Specific examples of myeloma cells include P3X63-Ag., Which is a hypoxanthine / guanine / phosphoribosyltransferase (HGPRT) deficient cell line derived from BALB / c mice. 8 strains, P3X63-Ag. 8). U1 strain, P3 / NSI / 1-Ag4-1 strain, P3x63Ag8.653 strain, Sp2 / 0-Ag14 strain and the like can be mentioned.

  Next, the myeloma cell and the antibody-producing cell are fused. For cell fusion, antibody-producing cells and myeloma cells are mixed at a ratio of about 1: 1 to 20: 1 in an animal cell culture medium such as serum-free DMEM, RPMI-1640 medium, etc. The fusion reaction is performed in the presence of. As the cell fusion promoter, polyethylene glycol having an average molecular weight of 1,500 to 4,000 daltons or the like can be used at a concentration of about 10 to 80%. In some cases, an auxiliary agent such as dimethyl sulfoxide may be used in combination in order to increase the fusion efficiency. Furthermore, antibody-producing cells and myeloma cells can be fused using a commercially available cell fusion device utilizing electrical stimulation (for example, electroporation).

iii) Selection and cloning of hybridoma The target hybridoma is selected from the cells after cell fusion treatment. As the method, the cell suspension is appropriately diluted with, for example, fetal bovine serum-containing RPMI-1640 medium, and then plated on a microtiter plate at about 2 × 10 ⁵ cells / well, and a selective medium is added to each well. Cultivation is performed by appropriately changing the selection medium. The culture temperature is 20 to 40 ° C, preferably about 37 ° C. When the myeloma cells are of HGPRT-deficient strain or thymidine kinase (TK) -deficient strain, cells having the ability to produce antibodies and myeloma can be obtained by using a selective medium (HAT medium) containing hypoxanthine / aminopterin / thymidine. Only cell hybridomas can be selectively cultured and propagated. As a result, cells that grow from about 10 days after the start of culture in the selective medium can be obtained as hybridomas.

  Next, it is screened whether the target antibody exists in the culture supernatant of the hybridoma which has proliferated. Hybridoma screening is not particularly limited, and may be carried out according to ordinary methods. For example, a part of the culture supernatant contained in a well grown as a hybridoma can be collected and used by enzyme immunoassay (EIA: Enzyme Immuno Assay and ELISA), radioimmunoassay (RIA: Radio Immuno Assay) or the like. it can.

  Cloning of the fused cells is performed by limiting dilution or the like, and finally a hybridoma that is a monoclonal antibody-producing cell is established. As will be described later, the hybridoma of the present invention is stable in culture in a basic medium such as RPMI 1640, DMEM, etc., and produces and secretes a monoclonal antibody that specifically reacts with Egfl7 polypeptide.

iv) Collection of monoclonal antibody As a method for collecting a monoclonal antibody from an established hybridoma, a normal cell culture method or ascites formation method can be employed.

In the cell culture method, the hybridoma is cultured in an animal cell culture medium such as RPMI-1640 medium containing 10% fetal bovine serum, DMEM medium, or serum-free medium under normal culture conditions (eg, 37 ° C., 5% CO ₂ concentration). Cultivate for 2 to 10 days or more, and obtain antibody from the culture supernatant.

In the case of the ascites formation method, about 1 × 10 ⁷ hybridomas are administered into the abdominal cavity of a mammal derived from a myeloma cell and the same strain, and the hybridomas are proliferated in large quantities. Then, ascites or serum is collected after 1 to 4 weeks.

  In the above antibody collection method, when purification of the antibody is required, the purified monoclonal of the present invention can be obtained by singly or appropriately combining ammonium sulfate precipitation, gel chromatography, ion exchange chromatography, affinity chromatography and the like. Antibodies can be obtained.

v) Collection of polyclonal antibody In order to prepare a polyclonal antibody, an animal is immunized in the same manner as described above, and 6 to 60 days after the final immunization, enzyme immunoassay (EIA and ELISA), radioimmunoassay (RIA) ) And the like, and blood is collected on the day when the maximum antibody titer is shown to obtain antiserum. Thereafter, the reactivity of the polyclonal antibody in the antiserum is measured by an ELISA method or the like.

  When detecting the expression of the Egfl7 gene in a sample derived from a subject using an antibody against the Egfl7 polypeptide and detecting cancer, the antibody binds to an antibody against the Egfl7 polypeptide in the sample of the subject or a labeled antibody thereof. The presence of cancer can be detected by measuring the antigen polypeptide. And it can determine with a cancer patient or its high-risk person. That is, since the antibody or labeled antibody used here is an antibody that specifically binds to the Egfl7 polypeptide expressed in cells produced by cancer, compared to a sample derived from a non-cancer patient, A sample with an increased or decreased amount of antigen polypeptide that binds to an antibody can be determined as a sample containing cancerous cells and / or a sample from a cancer patient or a high-risk patient thereof. In this case, preferably, two or more types, preferably 5 types or more, more preferably 10 types or more, and most preferably 15 to 39 types of antibodies are determined to bind to the Egfl7 polypeptide in the sample. The sample derived from the subject can be a sample derived from a living body such as blood, an organ / tissue, or a cell.

  Among cancers, kidney cancer, lung cancer, small intestine cancer, colon cancer and ovarian cancer, particularly kidney cancer, lung cancer and colon cancer, in the sample, compared to the sample derived from a non-cancer patient, If the amount is reduced, it can be determined that the patient is a cancer patient or a high-risk patient thereof. On the other hand, among cancers, liver cancer is a cancer patient or a high-risk patient when the amount of the antigen polypeptide is increased in the sample compared to a sample derived from a non-cancer patient. Can be determined.

  Another embodiment is a method for binding an antibody to an Egfl7 polypeptide in a liquid phase system. For example, the labeled antibody is brought into contact with the sample to bind the labeled antibody and the Egfl7 polypeptide, the conjugate is separated, and the labeled signal is detected.

  Another method for detection in a liquid phase system is to contact an antibody against Egfl7 polypeptide (primary antibody) with a sample to bind the primary antibody and antigen polypeptide, and to this conjugate a labeled antibody (secondary antibody) And detect the label signal in the tripartite conjugate. Alternatively, in order to further enhance the signal, an unlabeled secondary antibody may first be bound to an antibody + antigen polypeptide conjugate, and a labeling substance may be bound to this secondary antibody. Such binding of the labeling substance to the secondary antibody can be performed, for example, by biotinylating the secondary antibody and avidinizing the labeling substance. Alternatively, an antibody (tertiary antibody) that recognizes a partial region (for example, Fc region) of the secondary antibody may be labeled and this tertiary antibody may be bound to the secondary antibody. The primary antibody and the secondary antibody may both be monoclonal antibodies, or one of the primary antibody and the secondary antibody may be a polyclonal antibody. Separation of the conjugate from the liquid phase and detection of the signal can be the same as described above.

  Another aspect is a method for testing the binding of an antibody to an Egfl7 polypeptide in a solid phase system. This method in the solid phase system is a preferable method for detecting a very small amount of Egfl7 polypeptide and simplifying the operation. That is, in this solid phase system, an antibody (primary antibody) against Egfl7 polypeptide is immobilized on a solid phase (resin plate, membrane, beads, etc.), Egfl7 polypeptide is bound to this immobilized antibody, and unbound peptide is bound. In this method, after washing and removing, a labeled antibody (secondary antibody) is bound to the antibody + Egfl7 conjugate remaining on the plate, and the signal of this secondary antibody is detected. This method is a so-called “sandwich method”, and is a method widely used as an ELISA when an enzyme is used as a marker. Both the primary antibody and the secondary antibody can be monoclonal antibodies, or one of the primary antibody and the secondary antibody can be a polyclonal antibody. Signal detection can be as described above.

2. Detection of Egfl7 mRNA As a method for detecting Egfl7 mRNA in a sample derived from a subject, at least a 15-base long continuous primer for specifically amplifying Egfl7 polynucleotide or at least a hybrid that specifically hybridizes to Egfl7 polynucleotide A method for detecting the expression of the Egfl7 gene in a sample derived from a subject using a continuous probe having a length of 15 bases is mentioned.

  The primer or probe specifically binds to mRNA of Egfl7 gene expressed in the sample derived from the subject or cDNA or cRNA synthesized from mRNA, so that they can be used to express expression of Egfl7 gene in the sample. Can be detected.

  The primer of the present invention may be any primer that amplifies at least a part of the Egfl7 polynucleotide. Primers and probes can be designed according to techniques known to those skilled in the art. As points to keep in mind when designing the primer and probe, the following can be pointed out, for example.

  The length having a substantial function as a primer is usually 15 bases or more, preferably 16 to 50 bases, more preferably 18 to 30 bases. The length having a substantial function as a probe is usually 15 bases or more, preferably 16 to 1000 bases, more preferably 50 to 500 bases.

  In designing, it is preferable to confirm the melting temperature (Tm) of the primer or probe. Tm means the temperature at which 50% of any polynucleotide strand hybridizes with its complementary strand, and in order to anneal or hybridize the template polynucleotide and primer or probe to form a duplex It is necessary to optimize the temperature of annealing or hybridization. On the other hand, if this temperature is lowered too much, a non-specific reaction occurs, so it is desirable that the temperature be as high as possible. Therefore, the Tm of the primer or probe to be designed is an important factor for performing an amplification reaction or hybridization. For confirmation of Tm, known primer or probe design software can be used. Examples of software that can be used in the present invention include Oligo ™ (manufactured by National Bioscience Inc. (USA)), GENETYX (software development). (Manufactured by Japan Co., Ltd.). Also, Tm can be confirmed by calculating by itself without using software. In that case, a calculation formula based on the nearest neighbor method, the Wallance method, the GC% method, or the like can be used. In this invention, it is preferable that average Tm is about 45-65 degreeC.

  Other conditions that allow specific annealing or hybridization as a primer or probe include GC content, and such conditions are well known to those skilled in the art.

  Primers and probes designed as described above can be prepared according to methods known to those skilled in the art. Further, as is well known to those skilled in the art, the primer or probe may contain additional sequences other than the portion to be annealed or hybridized, such as a tag sequence, and so on. Those to which additional sequences are added are also included within the scope of the present invention.

  As a primer for specifically amplifying human Egfl7 polynucleotide, an oligonucleotide consisting of at least two kinds of 15 or more consecutive base sequences selected from the base sequence of SEQ ID NO: 1 or a complementary sequence thereof, and Primer set consisting of oligonucleotides having an interval of 1 base or more between selected oligonucleotides, oligonucleotide consisting of at least two consecutive 15 bases or more base sequences selected from the base sequence of SEQ ID NO: 2 or their complementary sequences And a primer set composed of oligonucleotides having an interval of 1 base or more between the selected oligonucleotides.

  Specific examples of primers for specifically amplifying human Egfl7 polynucleotide include a primer set consisting of an oligonucleotide consisting of the base sequence of SEQ ID NO: 4 and an oligonucleotide consisting of the base sequence of SEQ ID NO: 5. Specific examples of primers for specifically amplifying Xenopus Egfl7 polynucleotide include a primer set consisting of an oligonucleotide consisting of the base sequence of SEQ ID NO: 8 and an oligonucleotide consisting of the base sequence of SEQ ID NO: 9. .

  In order to detect the expression of the Egfl7 gene in a sample derived from a subject, the above-described primer and / or probe are used in an amplification reaction or a hybridization reaction, respectively, and the amplification product or hybrid product is detected.

  The sample derived from the subject can be a sample derived from a living body such as blood, an organ / tissue, or a cell.

  When performing an amplification reaction or a hybridization reaction, a test polynucleotide is usually prepared from a sample derived from a test sample. The test polynucleotide may be either DNA or RNA. DNA or RNA can be extracted appropriately using a method well known in the art. For example, when extracting DNA, a method of phenol extraction and ethanol precipitation, a method using glass beads, etc., and when extracting RNA, guanidine-cesium chloride ultracentrifugation, hot phenol method, or thiocyanic acid A guanidinium-phenol-chloroform (AGPC) method or the like can be used. The following amplification reaction and / or hybridization reaction is performed using the sample or the test polynucleotide prepared as described above.

  The extracted RNA is preferably further purified and used as mRNA. Although the purification method is not particularly limited, since many mRNAs present in the cytoplasm of eukaryotic cells have a poly (A) sequence at the 3 ′ end thereof, this feature is used, for example, as follows. be able to. First, a biotinylated oligo (dT) probe is added to the extracted total RNA to adsorb poly (A) + RNA. Next, a paramagnetic particle carrier on which streptavidin is immobilized is added, and poly (A) + RNA is captured using the binding between biotin / streptavidin. After the washing operation, finally, poly (A) + RNA is eluted from the oligo (dT) probe. Alternatively, a method may be employed in which poly (A) + RNA is adsorbed using an oligo (dT) cellulose column, and this is eluted and purified. The eluted poly (A) + RNA may be further fractionated by sucrose density gradient centrifugation or the like.

  By performing an amplification reaction using the test polynucleotide as a template using a primer and detecting the specific amplification reaction, the expression of the Egfl7 gene in the sample can be detected.

  The amplification method is not particularly limited, and examples thereof include known methods utilizing the principle of the polymerase chain reaction (PCR) method, such as PCR method, RT-PCR method, real-time PCR method and the like. Other amplification methods include the LAMP (Loop-Mediated Isometric Amplification) method, the ICAN (Isothermal and Chimeric Primer-Initiated Amplification of Nucleic Acids) method, and the RCA (Rolling Acid Acids) method. (Strand Displacement Amplification) method and the like. Amplification is performed until the amplification product is at a detectable level.

  For example, in the PCR method, a base sequence between a pair of primers is synthesized by DNA polymerase using DNA as a test polynucleotide as a template. According to the PCR method, the amplified fragment can be amplified exponentially by repeating a cycle consisting of denaturation, annealing and synthesis. Those skilled in the art can easily determine the optimum conditions for the PCR method.

  In the RT-PCR method, first, cDNA is prepared by reverse transcriptase reaction using RNA as a test polynucleotide as a template, and then PCR is performed using a pair of primers using the prepared cDNA as a template. is there.

  In addition, quantitative detection is possible by adopting a quantitative PCR method such as a competitive PCR method or a real-time PCR method as an amplification method. In the real-time PCR (TaqMan PCR) method, a probe that hybridizes to a specific region of a target gene, which is labeled with a fluorescent dye (reporter) at the 5 'end and a quencher (quencher) at the 3' end, is used. In the normal state of the probe, the fluorescence of the reporter is suppressed by the quencher. In a state where this fluorescent probe is completely hybridized to the target gene, PCR is performed from the outside using Taq DNA polymerase. When the extension reaction by Taq DNA polymerase proceeds, the exonuclease activity hydrolyzes the fluorescent probe from the 5 'end, releases the reporter dye, and emits fluorescence. In the real-time PCR method, the initial amount of template DNA is quantified by monitoring the fluorescence intensity in real time.

  In the LAMP method, primers consisting of four types of oligonucleotides that recognize base sequences in a total of 6 regions, a nucleic acid synthase having a strand displacement synthesis activity, and a substrate are used, and are kept isothermal throughout without requiring a heat denaturation step. A gene amplification reaction with high specificity proceeds promptly. The optimum conditions for the LAMP method can be easily determined by those skilled in the art.

  In order to detect whether or not a specific amplification reaction has occurred after the amplification reaction, a known means capable of specifically recognizing an amplification product obtained by the amplification reaction can be used. For example, a specific amplification reaction can be detected by confirming whether an amplified fragment of a specific size is amplified using agarose gel electrophoresis or the like.

Alternatively, a label such as a radioisotope, a fluorescent substance, or a luminescent substance is allowed to act on dNTP taken in during the amplification reaction, and this label can be detected. As a radioactive isotope, ³² P, ¹²⁵ I, ³⁵ S, or the like can be used. As the fluorescent material, for example, fluorescein (FITC), sulforhodamine (SR), tetramethylrhodamine (TRITC) and the like can be used. As the luminescent substance, luciferin or the like can be used.

  There are no particular restrictions on the type of the labeled body, the method for introducing the labeled body, and the like, and various conventionally known means can be used. For example, as a method for introducing a label, a random prime method using a radioisotope can be mentioned.

  As a method for observing the amplification product incorporating the labeled dNTP, any method known in the art for detecting the above-described labeled body may be used. For example, when a radioisotope is used as the label, the radioactivity can be measured by, for example, a liquid scintillation counter or a γ-counter. In addition, when fluorescence is used as the label, the fluorescence can be detected using a fluorescence microscope, a fluorescence plate reader, or the like.

  As described above, the increase or decrease in the expression of the Egfl7 gene in the sample can be detected by the specific increase or decrease in the amplification reaction. It can be determined that cancer is present when the expression of the Egfl7 gene in the sample is increased or decreased compared to a sample derived from a non-cancer patient.

  Among the cancers, kidney cancer, lung cancer, small intestine cancer, colon cancer and ovarian cancer, particularly kidney cancer, lung cancer and colon cancer, in the sample, expression of Egfl7 gene is higher than that in a sample derived from a non-cancer patient. When it is reduced, it can be determined that the patient is a cancer patient or a high-risk patient thereof. On the other hand, among cancers, liver cancer is determined to be a cancer patient or a high-risk patient when the expression of the Egfl7 gene is increased in the sample compared to a sample derived from a non-cancer patient. can do.

  In addition, the expression of the Egfl7 gene can be detected by performing a hybridization reaction with a sample or a test polynucleotide using a probe and detecting its specific binding (hybrid).

  The hybridization reaction must be performed under conditions such that the probe specifically binds only to the Egfl7 polynucleotide, that is, under stringent conditions. Such stringent conditions are well known in the art and are not particularly limited. As stringent conditions, for example, the sodium concentration is 10 to 300 mM, preferably 20 to 100 mM, and the temperature is 25 to 70 ° C., preferably 42 to 65 ° C.

When performing hybridization, fluorescent label probe (fluorescein, rhodamine, and the like), (such as ^{32 P)} radiolabeled, enzyme-labeled (alkaline phosphatase, horseradish peroxidase, etc.), adds the appropriate labels such biotin-labeled be able to. Therefore, the detection kit of the present invention described below includes a probe to which the above label is added.

  The detection using the labeled probe includes contacting the sample or a test polynucleotide prepared therefrom with the probe so that they can be hybridized. “To be able to hybridize” means in an environment (temperature, salt concentration) in which specific binding occurs under the above-described stringent conditions. Specifically, the sample or test polynucleotide is immobilized on an appropriate solid phase such as a slide glass, membrane, or microtiter plate, and a probe to which a label is added is added to the probe and the sample or test polynucleotide. The probe is brought into contact with each other and subjected to a hybridization reaction to remove unhybridized probes, and then the label of the probe hybridized with the sample or the test polynucleotide is detected. When the label is detected, the Egfl7 gene is expressed in the sample.

  In addition, quantitative detection is possible by using the concentration of the label as an index. Examples of the detection method using a labeled probe include Southern hybridization method, Northern hybridization method, FISH (fluorescence in situ hybridization) method and the like.

  As a determination criterion in the detection method of the present invention as described above, typically, an ROC curve (receiver operating characteristic curve) is created, a cutoff value (pathological condition identification value) is set, and a predetermined cut Examples include a method for determining the presence of cancer, as well as a patient with cancer or a high-risk patient, based on an off value.

  Other detection methods include, for example, the subtraction method (Sive, HL and John, T. St. (1988) Nucleic Acids Research 16, 10937, Wang, Z., and Brown, DD (1991) Proc. Natl.Acad.Sci.U.S.A.88, 11505-11509), differential display method (Liang, P., and Pardee, AB (1992) Science 257, 967-971, Liang, P , Averboukh, L., Keimarsi, K., Sager, R., and Pardee, AB (1992) Cancer Research 52, 6966-6968), differential hybridisation. (John, T. St., and Davis, RW Cell (1979) 16, 443-452), and a cross-hybridization method using an appropriate probe ("Molecular Cloning, A Laboratory Manual" Maniatis) , T., Fritsch, EF, Sambrook, J. (1982) Cold Spring Harbor Lab.

Kit for Detecting Cancer The present invention also relates to a kit for detecting cancer. The kit includes, as a means of detecting expression of the Egfl7 gene, an antibody that specifically binds to an Egfl7 polypeptide or a fragment thereof, a continuous primer of at least 15 bases in length for specifically amplifying an Egfl7 polynucleotide, and Egfl7 At least one selected from consecutive probes of at least 15 bases in length that specifically hybridize to the polynucleotide encoding. As such a kit, various types are commercially available depending on the type of the test component, and the kit of the present invention is also known as a publicly known kit except that the above means for detecting the expression of the Egfl7 gene is used. It can be constituted by each element used. In addition to the primer, probe or antibody for detecting the expression of the Egfl7 gene, for example, a labeled secondary antibody, a carrier, a washing buffer, a sample diluent, an enzyme substrate, a reaction stop solution, a standard substance and the like can be included.

Pharmaceutical Compositions According to the present invention, since the expression of Egfl7 gene is increased in tissues of specific cancers, particularly liver cancer, cancer is treated by administering an antibody that specifically binds to Egfl7 polypeptide or a fragment thereof. It is considered possible. Accordingly, one embodiment of the invention relates to a pharmaceutical composition for treating cancer comprising an antibody that specifically binds to an Egfl7 polypeptide or fragment thereof.

  Furthermore, the present inventors expressed Egfl7 gene in cancer cells because expression of Egfl7 gene is reduced in cancer tissues of specific cancers, particularly kidney cancer, lung cancer, small intestine cancer, colon cancer and ovarian cancer. Or by administering an Egfl7 polynucleotide and / or a derivative thereof, or an Egfl7 polypeptide and / or a derivative thereof.

  Accordingly, in one embodiment of the present invention, a pharmaceutical composition for treating cancer comprising an Egfl7 polynucleotide and / or a derivative thereof, and a pharmaceutical composition for treating cancer comprising an Egfl7 polypeptide and / or a derivative thereof. .

  Examples of the polynucleotide derivative include a polynucleotide derivative in which a phosphodiester bond in a polynucleotide is converted to a phosphorothioate bond or an N3′-P5 ′ phosphoramidite bond, and a ribose and phosphodiester bond is converted to a peptide bond. Polynucleotide derivatives, polynucleotide derivatives in which uracil in the polynucleotide is substituted with C-5 propionyl uracil or C-5 thiazole uracil, cytosine in the polynucleotide is substituted with C-5 propionyl cytosine or phenoxazine modified cytosine, etc. And polynucleotide derivatives in which ribose in DNA is substituted with 2′-O-propylribose or 2′-methoxyethoxyribose. Examples of polypeptide derivatives include fusion polypeptides fused with other polypeptides, chemically modified polypeptides bound to polymers such as polyethylene glycol, and the like.

  Antibodies, Egfl7 polynucleotides and Egfl7 polypeptides and derivatives thereof that specifically bind to an Egfl7 polypeptide or a fragment thereof are usually mixed together with one or more pharmaceutically acceptable carriers, and the technical field of pharmaceuticals Preferably, it is provided as a pharmaceutical composition prepared by any method well known in the art. It is desirable to use the most effective route for treatment, and examples include oral administration and parenteral administration such as buccal, intratracheal, rectal, subcutaneous, intramuscular and intravenous.

  When the Egfl7 polynucleotide or a derivative thereof is used for the treatment of cancer diseases, a method of administering a vector incorporating the polynucleotide in addition to a method of directly administering the Egfl7 polynucleotide by injection can be mentioned. Examples of the vector include an adenovirus vector, an adeno-associated virus vector, a herpes virus vector, a vaccinia virus vector, a retrovirus vector, and the like, and can be efficiently administered by using these virus vectors. Alternatively, a method of introducing the gene of the present invention into a phospholipid vesicle such as a liposome and administering the liposome may be employed. As an administration route of Egfl7 polynucleotide, in addition to systemic administration such as normal intravenous and intraarterial administration, local administration can be performed on immune system tissues (bone marrow, lymph nodes, etc.). Furthermore, administration routes combined with catheter techniques, surgical operations, and the like can also be employed.

The present invention also relates to a method of screening for a substance that inhibits cancer. The screening method of the present invention comprises contacting a test sample with a cell expressing the Egfl7 gene, and selecting a substance that inhibits the expression of the Egfl7 gene or a substance that promotes the expression of the Egfl7 gene from the test sample. It is characterized by. The method for measuring the expression of the Egfl7 gene is as described above.

  When the Egfl7 gene expression cell is brought into contact with a test sample and the expression of the Egfl7 gene is inhibited, it is determined that the sample contains a substance that inhibits the progression of liver cancer. Can do. On the other hand, when the expression of the Egfl7 gene is promoted when the Egfl7 gene-expressing cell is brought into contact with the test sample, kidney cancer, lung cancer, small intestine cancer, colon cancer and ovarian cancer, It can be determined that a substance that inhibits the development of kidney cancer, lung cancer, and colon cancer is contained.

  The test sample to be screened is not particularly limited, and examples thereof include a cell extract, a synthetic low molecular weight compound library, a gene library expression product, and a synthetic peptide library.

(Method)
1) Preparation of fertilized eggs Sixteen hours before collection of fertilized eggs, 600 IU of gonatotropin was injected subcutaneously into Xenopus males and females, and fertilized eggs were obtained by natural mating. Steinberg's solution (58 mM NaCl, 0.67 mM KCl, 0.34 mM Ca (NO ₃ ) ₂ .24H ₂ O, 0.83 mM containing 4.5% L-cysteine hydrochloride (pH 7.8). Development was continued until desalination using MgSO ₄ .7H ₂ O (3.0 mM HEPES) and then placing under 20 ° C. conditions until the blastocyst stage (stage 9 according to the stage table of Nieuwkop and Faber).

2) Re-assembly culture From the embryo of the blastocyst stage (stage 9), after detaching the egg membrane using tweezers, 5 fragments of the undifferentiated ectoderm region (animal cap) having a size of 0.6 mm × 0.6 mm were obtained. The cells were dissociated by pipetting using a Steinberg solution (CMF-Steinberg solution) containing no Ca and Mg in a petri dish filled with Steinberg solution on 3% agar.

  The dissociated cells were treated alone with 0.4 ng / mL activin A dissolved in 4 mM hydrochloric acid solution containing 0.1% fatty acid-free bovine serum albumin (BSA), or the 0.4 ng / mL activin Co-treatment with A and 100 ng / mL Angiopoietin 2 (TECHNE) was performed for 1 hour in CMF-Steinberg solution containing 0.1% BSA. Then, it wash | cleaned 5 times with Mr. Steinberg liquid, and the aggregate was formed in Mr. Steinberg liquid.

3) Preparation of probe A plasmid prepared by cloning a cDNA fragment was digested with restriction enzymes SpeI or SacII, and RNA was synthesized using T7 RNA polymerase or Sp6 RNA polymerase to obtain a probe. At this time, 10 × DIG RNA labeling mix (Roche) was used as a substrate.

4) Detection by microarray A DNA microarray to which 60-mer oligo DNA was affixed from 8,091 clones of Xenopus cDNA was prepared and used for detection. On the other hand, the aggregate treated with 0.4 ng / mL activin A alone or 0.4 ng / mL activin A and 100 ng / mL angiopoietin 2 was cultured for 2 days at 20 ° C., and then all RNA was extracted. . From the extracted RNA, using a Low RNA Input linear amplification and labeling kit (manufactured by Agilent), cy3 labeling (cyanine 3-CTP (manufactured by Perkin Elmer)) and cy5 labeling (cyanine 5-CTP (manufactured by Perkin Elmer) )) Labeled RNA was prepared. The DNA microarray and the labeled RNA were applied to a hybridization chamber, and hybridization was performed at 60 ° C. for 17 hours. After completion of hybridization, analysis was performed using an Agilent microarray scanner.

5) Preparation of RNA Total RNA was extracted using ISOGEN (Nippon Gene).

6) Reverse transcription reaction The reverse transcription reaction was performed using a superscript first strand system (manufactured by Invitrogen) and a random primer (manufactured by Takara).

7) PCR
For the PCR reaction, an Expand long template PCR amplification system (Roche) was used. The primer sequences and annealing temperatures used are as follows.
(Human)
H. Egfl7:
5′-AGCAATATGCCAGCCGCCAT-3 ′ (SEQ ID NO: 4)
5'-TCACGAGTCTTTCTTGCAGG-3 '(SEQ ID NO: 5)
Annealing temperature 60 ° C., 35 cycles G3PDH:
5′-TGAAGGTCGGAGTCAACGGATTTGGT-3 ′ (SEQ ID NO: 6)
5′-CATGTGGGCCATGAGGTCACCACCAC-3 ′ (SEQ ID NO: 7)
Annealing temperature 68 ° C, 27 cycles (Xenopus laevis)
X. Egfl7:
5′-CCGATTGCAATGTCAGAACG-3 ′ (SEQ ID NO: 8)
5′-TCAAAGCTCCGTCTTGCATG-3 ′ (SEQ ID NO: 9)
Annealing temperature 60 ° C., 38 cycle ODC:
5′-CAGCTAGCTGTGGGTGG-3 ′ (SEQ ID NO: 10)
5′-CAACATGGGAAACTCACACC-3 ′ (SEQ ID NO: 11)
Annealing temperature 55 ° C., 24 cycles For human tissue expression, human multiple tissue cDNA panel (manufactured by BD Biosciences) was used as a template. For expression in human cancer tissue, a human cancer tissue cDNA panel (manufactured by BioChain) was used as a template.

8) Whole-mount in situ hybridization After normal embryos were fixed in 10% neutral buffered formalin solution, they were dehydrated using 100% methanol. After dehydration, it was replaced with a PBS solution containing Tween 20, denatured at 95 ° C., and fixed again with 4% paraformaldehyde. After re-fixation, the sample was replaced with a PBS solution containing Tween 20 again, and applied to an automatic whole-mount in situ hybridization apparatus in situ pro (manufactured by ABIMED). BM purple (Roche) was used for color development.

(result)
1) Induction of vascular endothelial cells from undifferentiated cells of Xenopus embryos By treating dissociated aggregates with 0.4 ng / mL activin alone or 0.4 ng / mL activin and 100 ng / mL angiopoietin 2 vascular endothelium-like We examined whether cells could be induced. As a result, it was found that the expression of msr and tie2 genes, which are markers of vascular endothelial cells, was remarkably increased in co-treatment with activin and angiopoietin 2 as compared with activin alone treatment. From this, it was considered that vascular endothelial cells were induced from the dissociated aggregate by co-treatment with activin and angiopoietin 2 (FIG. 1). Furthermore, as a result of examining the expression of the globin gene which is a marker for erythrocytes, the expression of the globin gene was not observed in any case. That is, by co-treatment with activin and angiopoietin 2, it was found that vascular endothelial cells were induced, but erythrocytes were not induced, and a system that induced only vascular endothelial cells could be created. As a result, an experimental system useful for vascular cell differentiation and gene analysis was established.

2) Analysis by microarray The gene expression by 0.4 ng / mL activin alone treatment and 0.4 ng / mL activin and 100 ng / mL angiopoietin 2 co-treatment is compared using a DNA microarray to search for genes involved in angiogenesis. Tried. As a result of comparing the gene expression level after 48 hours after 0.4 ng / mL activin alone treatment or 0.4 ng / mL activin and 100 ng / mL angiopoietin 2 co-treatment on the dissociated aggregate, As a gene in which a difference of 2 times or more was observed, 57 genes were obtained. Of these, 34 genes increased and 23 genes decreased by co-treatment. Table 1 shows gene groups with increased gene expression, and Table 2 shows gene groups with decreased gene expression.

3) Identification of gene expression site by whole-mount in situ hybridization Of 57 genes that showed a difference of more than 2 times as a result of analysis by DNA microarray, expressed in muscle from those that increased more than 2 times Expression sites in Xenopus embryos were identified for 12 genes excluding those that were present. As a result, two genes (xl.9480 and xl.23270) were expressed in blood vessels (FIG. 2). xl. As for 23270, it was an Egfl7 gene reported to be a gene involved in angiogenesis (Leon H. Parker et al. (2004), Nature, 428, 754-758).

4) X. Examination of temporal expression of Egfl7 Gene X. which was newly expressed in Xenopus blood vessels From which stage of development, Egfl7 was examined using RT-PCR (FIG. 3) and Whole-mount in situ hybridization (FIG. 4). As a result, gene X. It was found that Egfl7 was expressed. This time is the time when blood vessels are formed in Xenopus, ie, the time of blood vessel formation and the gene X. It was found that the time when the expression of Egfl7 was observed coincided.

5) H.H. Examination of tissue-specific expression of Egfl7 Furthermore, a primer for human Egfl7 was prepared, and it was examined whether expression was increased or decreased in human cancer tissue using a human cancer tissue cDNA panel (manufactured by BioChain) (FIG. 5). As a result, Egfl7 was found to increase in the expression level in cancer tissues in the liver. On the other hand, in other tissues such as kidney, lung, large intestine, small intestine, and ovary, the expression level in cancer tissues was reduced. In particular, a marked reduction is observed in kidney cancer, lung cancer, and colon cancer, and it is expected to become a novel cancer marker. In addition, it is expected that abnormal growth of cancer cells can be suppressed by expressing this gene product in cancer cells whose expression is reduced, and application to gene therapy is also considered.

The result of the expression analysis of msr and tie2 in a dissociation aggregate is shown. It is a photograph which shows the result of having performed 0.4 ng / mL activin A single processing to the dissociation | aggregation | aggregation, or the co-processing of 0.4 ng / mL activin A and 100 ng / mL angiopoietin 2. FIG. Gene xl. 9480 and xl. It is a photograph which shows that 23270 is expressed in the blood vessel part in a Xenopus embryo. Xenopus laevis st34 was used. msr is a control, and blood vessels are stained black. Gene X. It is a photograph which shows the result of having examined expression stage from which the expression is seen about Egfl7 using RT-PCR. Gene X. It is a photograph showing the results of examining the stage of development of Egfl7 using whole-mount in situ hybridization. H. It is a photograph which shows the result of having examined comparatively a normal tissue and a cancer tissue about the Egfl7mRNA using the human cancer tissue cDNA panel (made by BioChain).

SEQ ID NOs: 4-11: Synthetic DNA

Claims (11)

  A method for detecting cancer, comprising measuring expression of an Egfl7 gene.   The method according to claim 1, wherein the Egfl7 gene is a gene identified by the nucleotide sequence of SEQ ID NO: 1 or 2.   A marker for detecting cancer, comprising an Egfl7 polynucleotide.   The marker according to claim 3, wherein the Egfl7 polynucleotide is a polynucleotide comprising all or part of the base sequence of SEQ ID NO: 1 or 2.   A marker for detecting cancer, comprising an Egfl7 polypeptide or a fragment thereof.   6. The marker according to claim 5, wherein the Egfl7 polypeptide is a polypeptide comprising all or part of the amino acid sequence of SEQ ID NO: 3.   A kit for detecting cancer, comprising an antibody that specifically binds to an Egfl7 polypeptide or a fragment thereof.   A kit for detecting cancer comprising a continuous primer of at least 15 bases length or a continuous probe of at least 15 bases specifically hybridizing to an Egfl7 polynucleotide for specifically amplifying an Egfl7 polynucleotide.   A pharmaceutical composition for treating cancer comprising an antibody that specifically binds to an Egfl7 polypeptide or a fragment thereof.   A pharmaceutical composition for treating cancer comprising an Egfl7 polynucleotide and / or a derivative thereof, or an Egfl7 polypeptide and / or a derivative thereof.   A method for screening a substance that inhibits cancer, comprising contacting a test sample with a cell expressing Egfl7 gene, and selecting a substance that inhibits or promotes expression of Egfl7 gene from the test sample.

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