JP2004159653A - Oncogene and use of the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for producing a cancer cell by using an oncogene, to provide a method for detecting cancer by using the oncogene, to provide a method for screening a compound having effects of treating and/or preventing the cancer, and to provide a pharmaceutical composition for treating and/or preventing the cancer. <P>SOLUTION: This method for producing the cancer cell by excessively expressing MGC2742 genes in cells and selecting a cell strain in which proliferation potency of the cell is changed, the method for detecting the cancer by using expression of the MGC2742 as an indicator, the method for screening the new compound having the effects of treating and/or preventing the cancer by using the MGC2742, the pharmaceutical composition for treating and/or preventing the cancer, and a method for treating the cancer by using the pharmaceutical composition are provided, respectively. <P>COPYRIGHT: (C)2004,JPO

Description

  The present invention relates to a method for producing cancer cells using an oncogene, a method for detecting cancer using the oncogene, a method for screening for a compound having a therapeutic and / or prophylactic effect, and a pharmaceutical composition for treating and / or preventing cancer. About things, etc.

  Cancer remains one of the leading causes of death in developed countries such as Europe, the United States and Japan. Clinically, various medical approaches have been taken, including surgical resection of cancerous tissue, radiation therapy and chemotherapy, but no one based on a sufficiently accurate understanding of malignant transformation and cancer cell growth. Unfortunately, in many cases, the effect is partial and not satisfactory for many patients. With regard to such malignant transformation and cancer cell growth mechanism, intensive research has been carried out in many research facilities over the past several decades. At present, it is thought that the growth of normal cells is controlled by the balance between an oncogene (Oncogene) (proliferation promoting gene) and a tumor suppressor gene (Tumor suppressor gene) (growth inhibitory gene) (for example, Non-patent). Reference 1).

  Cellular endogenous oncogenes generally do not necessarily have a strong effect of causing normal cells to become cancerous, but if their regulatory functions are lost due to structural mutations, they become oncogenes, which lead to abnormal growth and direct canceration. Cause. For example, Ras, which causes cancer by point mutation, and Abl gene, which causes abnormal enhancement of enzyme activity by chromosomal translocation, are examples. In addition, even without mutation, increased expression of an oncogene often triggers canceration. Increased expression of the growth factor receptor HER2 / neu by gene amplification or the like is induced in metastatic breast cancer, and its inhibitor is used for the treatment of metastatic breast cancer. The above facts strongly suggest the working hypothesis that these oncogenes provide extremely valid target molecules as targets for cancer therapy.

  Among cancer diseases, prostate cancer is the most frequently diagnosed cancer, especially in the United States, and is the second leading cause of cancer death in men. Approximately 300,000 men are newly diagnosed with prostate cancer each year, and more than 40,000 men die from the disease. Although death from prostate cancer is primarily due to metastatic disease, nearly 60% of newly diagnosed patients have primary tumors confined to the prostate. Surgery and radiation therapy are often effective for patients with such localized cancers, but most are incurable for patients with metastatic spread of cancer. Despite intensive research to date, very little is known about the biological mechanisms that cause the development and progression of prostate cancer.

  As described above, the discovery of oncogenes involved in the development and progression of these cancers (including prostate cancer) is extremely useful in elucidating the mechanism and developing therapeutic drugs.

  As an alternative method of knowing the effect of inhibiting the function of a target molecule, a method of reducing the expression of the gene by some method is used. Examples thereof include a method using an antisense oligonucleotide which is a single-stranded DNA having a sequence complementary to the target gene and a ribozyme which is a single-stranded RNA having RNA cleavage activity. In addition to these, RNA interference (RNAi) using double-stranded RNA (dsRNA) has recently become available (see, for example, Non-Patent Document 2).

  RNA interference (RNAi) is a phenomenon in which homologous mRNAs present in cells are specifically degraded by dsRNA, and was reported as a phenomenon occurring in nematodes for the first time in 1998 (for example, see Non-Patent Document 3). . In mammals, long dsRNA causes an interferon response (see, for example, Non-Patent Documents 4 and 5), and it is initially considered difficult to use the RNAi effect as a specific gene suppression technique. However, in 2001, Elbashir et al. Reported that the use of a short dsRNA (siRNA) of 21-23mer, which is an intermediate product of RNAi, could induce the RNAi effect while avoiding the interferon response (for example, see Non-Patent Documents). 6)), RNAi using this siRNA has attracted attention as an unprecedented simple and powerful method for suppressing gene expression.

It is considered that some genes whose functions are unknown may be related to cancer, and it is important to clarify the relationship between these genes whose functions are unknown and cancer. Hypothetical protein MGC2742 (registered in GenBank under accession number: BC000765) was estimated as a gene encoding a protein as a result of genome analysis, but its function has not been clarified at all.
Yojiro Niitsu and Jun Yokota, “Oncogenes / Cancer Suppressor Genes for Clinicians”, Nankodo, October 15, 1999, p. 1-46 Kazumasa Tahira, ed., “Experimental method for inhibiting gene function”, Youtosha, September 20, 2001, p. 190-203 Nature, February 19, 1998, Vol. 391, No. 6669, p. 806-811 "Microbiology and Molecular Biology Reviews", December 1998, Vol. 62, No. 4, p. 1415-1434 "The International Journal of Biochemistry and Cell Biology", 1997, July, Vol. 29, No. 7, p. 945-949 "Jeans and Developments", January 15, 2001, Volume 15, Issue 2, p. 188-200

  Cancer cells and genes related to the development and / or proliferation of cancer cells are found, a method for producing cancer cells using the cancer genes is developed, a method for detecting cancer using the cancer genes, cancer treatment and / or It is an object of the present invention to provide a method for screening a compound having a prophylactic effect, and a pharmaceutical composition for treating and / or preventing cancer.

The present inventors have found that a cancer cell can be produced by overexpressing the MGC2742 gene, and also, using MGC2742, to detect a cancer, and to screen for a compound having a novel cancer treatment and / or cancer prevention effect. The present invention was completed by developing a method and providing a substance that suppresses the expression of MGC2742.
That is, the present invention
(1) A method for producing a cancer cell, comprising the following steps 1) and 2):
1) a step of transforming a cell with the polynucleotide of the following (i) or (ii);
(I) a polynucleotide having a nucleotide sequence represented by nucleotide numbers 165 to 1970 of SEQ ID NO: 1 in the sequence listing,
(Ii) a polynucleotide that hybridizes under stringent conditions to a polynucleotide consisting of a nucleotide sequence complementary to the polynucleotide of (i) above, and encodes a protein substantially identical to MGC2742;
2) a step of selecting a cell line in which the cell proliferation ability has changed in step 1);
(2) The method for producing a cancer cell according to (1), wherein the cell is an animal cell;
(3) The method for producing a cancer cell according to (2), wherein the animal cell is derived from a mammal.
(4) The method for producing a cancer cell according to (3), wherein the mammal is a human, monkey, mouse, or rat;
(5) The method for producing a cancer cell according to (3), wherein the mammal is a human.
(6) The method for producing a cancer cell according to (1), wherein the cell is a mouse fibroblast cell line NIH3T3 cell,
(7) The method for producing a cancer cell according to any one of (1) to (6), wherein the cell is transformed with a recombinant vector.
(8) A cancer cell obtained by the method for producing a cancer cell according to any one of (1) to (7),
(9) a non-human mammal into which the cancer cell according to (8) has been introduced,
(10) A method for detecting cancer, comprising a step of analyzing the expression level of the polynucleotide according to 1) or 2) in a sample collected from a subject or a test animal:
1) a polynucleotide having a nucleotide sequence represented by nucleotide numbers 165 to 1970 of SEQ ID NO: 1 in the sequence listing,
2) a polynucleotide that hybridizes under stringent conditions to a polynucleotide consisting of a nucleotide sequence complementary to the polynucleotide of 1) above, and that encodes a protein substantially identical to MGC2742;
(11) A method for detecting cancer, comprising a step of analyzing the expression level of the protein described in 1) or 2) in a sample collected from a subject or a test animal:
1) a protein having an amino acid sequence represented by amino acid numbers 1 to 601 of SEQ ID NO: 2 in the sequence listing;
2) a protein substantially identical to MGC2742, comprising an amino acid sequence in which one or several amino acids have been deleted, substituted or added in the amino acid sequence of the protein described in 2) above;
(12) A method for detecting cancer, comprising the following steps 1) to 4):
1) a step of extracting a total RNA fraction from a specimen collected from a subject or a test animal;
2) a step of extracting a total RNA fraction from a specimen collected from a normal human or a normal animal;
3) a step of measuring the expression level of the polynucleotide according to the following (i) or (ii) in the total RNA fraction derived from the above step 1) and the total RNA fraction derived from the above step 2);
(I) a polynucleotide having a nucleotide sequence represented by nucleotide numbers 165 to 1970 of SEQ ID NO: 1 in the sequence listing,
(Ii) a polynucleotide consisting of a nucleotide sequence that hybridizes with a polynucleotide consisting of a nucleotide sequence complementary to the polynucleotide described in (i) above under stringent conditions and encodes a protein substantially identical to MGC2742 ;
4) The difference in the expression level of the polynucleotide measured in the above step 3) between the total RNA fraction derived from the above step 1) and the total RNA fraction derived from the above step 2) is analyzed, and the above step 1) is analyzed. A step of detecting cancer in a subject or a test animal according to,
(13) A method for detecting cancer, comprising the following steps 1) to 3):
1) a step of measuring the expression level of the protein described in (i) or (ii) below in a sample collected from a subject or a test animal;
(I) a protein comprising an amino acid sequence represented by amino acid numbers 1 to 601 of SEQ ID NO: 2 in the sequence listing,
(Ii) a protein consisting of an amino acid sequence in which one or several amino acids have been deleted, substituted or added in the amino acid sequence of the protein described in (i) above, and which is substantially the same as MGC2742;
2) a step of measuring the expression level of the protein according to (i) or (ii) in the above step 1) in a sample collected from a normal human or a normal animal;
3) analyzing the difference between the expression level of the protein measured in the step 1) and the expression level of the protein measured in the step 2), and detecting cancer in the subject or the test animal;
(14) The method for detecting cancer according to any one of (10) to (13), wherein the cancer is prostate cancer.
(15) A method for screening a substance having a therapeutic and / or prophylactic effect against cancer, comprising the following steps 1) to 4):
1) a step of extracting a total RNA fraction from cultured cells derived from mammals cultured in a medium to which a test substance has been added;
2) a step of extracting a total RNA fraction from mammalian-derived cultured cells cultured without adding a test substance;
3) a step of measuring the expression level of the polynucleotide according to the following (i) or (ii) in the total RNA fraction derived from the above step 1) and the total RNA fraction derived from the above step 2);
(I) a polynucleotide having a nucleotide sequence represented by nucleotide numbers 165 to 1970 of SEQ ID NO: 1 in the sequence listing,
(Ii) a polynucleotide consisting of a nucleotide sequence that hybridizes with a polynucleotide consisting of a nucleotide sequence complementary to the polynucleotide described in (i) above under stringent conditions and encodes a protein substantially identical to MGC2742 ;
4) Analyzing the difference in the expression level of the polynucleotide measured in the above step 3) between the total RNA fraction derived from the above step 1) and the total RNA fraction derived from the above step 2), A step of determining a therapeutic effect and / or a preventive effect on cancer;
(16) A method for screening a substance having a therapeutic and / or prophylactic effect for cancer, comprising the following steps 1) to 4):
1) a step of extracting a total RNA fraction from a specimen collected from a mammalian individual to which the test substance has been administered;
2) a step of extracting a total RNA fraction from a specimen collected from an animal to which the test substance has not been administered;
3) a step of measuring the expression level of the polynucleotide according to the following (i) or (ii) in the total RNA fraction derived from the above step 1) and the total RNA fraction derived from the above step 2);
(I) a polynucleotide having a nucleotide sequence represented by nucleotide numbers 165 to 1970 of SEQ ID NO: 1 in the sequence listing,
(Ii) a DNA consisting of a nucleotide sequence that hybridizes under stringent conditions to a polynucleotide consisting of a nucleotide sequence complementary to the polynucleotide described in (i) above and encodes a protein substantially identical to MGC2742;
4) Analyze the difference in the expression level of the polynucleotide measured in the above step 3) between the total RNA fraction derived from the above step 1) and the total RNA fraction derived from the above step 2), and A step of determining a therapeutic effect and / or a preventive effect on cancer;
(17) A method for screening a substance having a therapeutic and / or prophylactic effect for cancer, comprising the following steps 1) to 3):
1) a step of measuring the expression level of the protein described in the following (i) or (ii) in cultured cells derived from a mammal cultured in a medium to which a test substance is added;
(I) a protein comprising an amino acid sequence represented by amino acid numbers 1 to 601 of SEQ ID NO: 2 in the sequence listing,
(Ii) a protein consisting of the amino acid sequence of the protein according to (i), wherein one or several amino acids are deleted, substituted or added, and which is substantially the same as MGC2742;
2) a step of measuring the expression level of the protein according to (i) or (ii) in the above step 1) in cultured cells derived from a mammal cultured in a medium to which a test substance is not added;
3) Analyze the difference between the expression level of the protein measured in step 1) and the expression level of the protein measured in step 2) to determine the therapeutic and / or prophylactic effect of the test substance on cancer. Process,
(18) A method for screening a substance having a therapeutic and / or prophylactic effect for cancer, comprising the following steps 1) to 3):
1) a step of measuring the expression level of the protein described in the following (i) or (ii) in a specimen collected from a mammal individual to which the test substance has been administered;
(I) a protein comprising an amino acid sequence represented by amino acid numbers 1 to 601 of SEQ ID NO: 2 in the sequence listing,
(Ii) a protein consisting of an amino acid sequence in which one or several amino acids have been deleted, substituted or added in the amino acid sequence of the protein described in (i) above, and which is substantially the same as MGC2742;
2) a step of measuring the expression level of the protein according to (i) or (ii) in the above step 1) in a sample collected from a mammal individual to which the test substance has not been administered;
3) Analyze the difference between the expression level of the protein measured in step 1) and the expression level of the protein measured in step 2) to determine the therapeutic and / or prophylactic effect of the test substance on cancer. Process,
(19) The screening method according to (15) or (17), wherein the cultured mammalian cell is the cancer cell according to (8).
(20) The screening method according to (16) or (18), wherein the mammal individual is the non-human mammal according to (9).
(21) the screening method according to any one of (15) to (19), wherein the cancer is prostate cancer;
(22) A kit for determining the therapeutic and / or prophylactic effect of a test substance on cancer and / or for detecting cancer, comprising at least one or more selected from the group consisting of the following 1) to 3):
1) a continuous oligonucleotide primer having a length of 15 to 30 bases for specifically amplifying a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 1 in the sequence listing;
2) a continuous polynucleotide probe of 15 nucleotides or more for hybridizing to a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 1 in the sequence listing under stringent conditions and detecting the polynucleotide;
3) a solid-phased sample on which a polynucleotide consisting of the nucleotide sequence of nucleotide numbers 165 to 1970 of SEQ ID NO: 1 in the sequence listing is fixed,
(23) A kit for determining the therapeutic and / or prophylactic effect of a test substance on cancer and / or detecting cancer, comprising at least one of the following 1) and 2):
1) an antibody that specifically binds to a protein consisting of the amino acid sequence of SEQ ID NO: 2 in the sequence listing and detects the protein;
2) a secondary antibody capable of binding to the antibody of 1) above;
(24) The method for measuring the expression level of a polynucleotide is Northern blot, dot blot, slot blot, RT-PCR, ribonuclease protection assay or run-on assay, (10), ( (12) The method according to any one of (14), (15), (16), (19) and (20).
(25) The method for measuring the expression level of a polynucleotide uses a gene chip or an array made of a DNA consisting of a complementary DNA group derived from animal tissues or animal cells or a partial sequence of each DNA of the DNA group. The method according to any one of (10), (12), (14), (15), (16), (19) and (20),
(26) The method for measuring the expression level of a protein uses an antibody or ligand that specifically binds to the protein, (11), (13), (14), (17), (18), (19) The method according to any one selected from (20) and (21),
(27) (11), (13), wherein the method for measuring the expression level of the protein is Western blotting, dot blotting, slot blotting or enzyme-linked immunosorbent assay (ELISA). (14) The method according to any one selected from (17), (18), (19), (20) and (21),
(28) A pharmaceutical composition for treating and / or preventing cancer comprising an oligonucleotide having a nucleotide sequence represented by nucleotide numbers 165 to 1970 of SEQ ID NO: 1 in the sequence listing or a nucleotide sequence complementary to a partial sequence of the sequence. ,
(29) a pharmaceutical composition for treating and / or preventing cancer, comprising an antibody that specifically recognizes MGC2742;
(30) a pharmaceutical composition for treating and / or preventing cancer, comprising an siRNA against the nucleotide sequence shown in SEQ ID NO: 1 of the sequence listing or a partial sequence thereof;
(31) the siRNA is an siRNA consisting of a combination of an oligonucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 7 in the sequence listing and an oligonucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 8 in the sequence listing, (30) The pharmaceutical composition for treating and / or preventing cancer according to (30),
(32) The pharmaceutical composition according to any one of (28) to (31), wherein the cancer is prostate cancer.
(33) A method for treating cancer, using the pharmaceutical composition according to at least one selected from (28) to (32).
(34) The method for treating cancer according to (33), wherein the cancer is prostate cancer.
About.

  According to the present invention, cancer cells can be produced using human MGC2742, and specific detection of cancer has also become possible. In addition, by developing an inhibitor targeting the MGC2742 gene, it has become possible to screen for a novel anticancer agent having extremely high therapeutic and / or preventive effects against cancer. Furthermore, an antibody that specifically binds to MGC2742 can be a therapeutic and / or prophylactic agent for cancer.

  As used herein, the compound having a cancer therapeutic and / or cancer preventive effect refers to a compound having an activity of inhibiting the growth of cancer, an activity of reducing cancer, and / or an activity of preventing the occurrence of cancer. . In this specification, “cancer” and “tumor” are used interchangeably. In this specification, the term "gene" includes not only DNA but also its mRNA, cDNA and its cRNA. Therefore, the “MGC2742 gene” in the present invention includes MGC2742 DNA, mRNA, cDNA and cRNA. In the present specification, the term "polynucleotide" is used synonymously with nucleic acid, and also includes DNA, RNA, probes, oligonucleotides, and primers. In the present specification, "polypeptide" and "protein" are used without distinction. Further, in the present specification, the “RNA fraction” refers to a fraction containing RNA. In addition, in the present specification, “cells” include cells in animal individuals and cultured cells. As used herein, "cancer of a cell" refers to a cell exhibiting abnormal proliferation, such as a cell losing sensitivity to a contact inhibition phenomenon, or exhibiting anchorage-independent proliferation, Cells showing such abnormal growth are called "cancer cells". As used herein, the term “substantially the same protein” as MGC2742 refers to a protein having the same function as that of MGC2742, such as a canceration activity of cells. The term oncogene in the present invention includes proto-oncogene and proto-oncogene in addition to oncogene.

1. Acquisition of MGC2742 gene The nucleotide sequence of the human MGC2742 cDNA used in the present invention is shown in, for example, nucleotide numbers 1 to 2279 of SEQ ID NO: 1 in the sequence listing. Human MGC2742 is, for example, a protein encoded by a nucleotide sequence represented by nucleotide numbers 165 to 1970 of SEQ ID NO: 1 in the sequence listing. The amino acid sequence of human MGC2742 is represented by, for example, amino acid numbers 1 to 601 of SEQ ID NO: 2 in the sequence listing. The nucleotide sequence of human MGC2742 cDNA is registered in GenBank under the accession number: BC000765 (version: BC000765.1.). That is, in the present specification, the “MGC2742 gene” refers to a gene consisting of the nucleotide sequence represented by nucleotide numbers 165 to 1970 of SEQ ID NO: 1 in the sequence listing, or a nucleotide sequence complementary to the gene consisting of the nucleotide sequence And a gene consisting of a nucleotide sequence encoding a protein substantially identical to MGC2742, which hybridizes under stringent conditions with a polynucleotide consisting of In the present specification, “MGC2742” refers to a protein consisting of the amino acid sequence represented by amino acid numbers 1 to 601 in SEQ ID NO: 2 in the sequence listing, or one or several amino acids in the amino acid sequence of the protein. Is a protein consisting of a deleted, substituted or added amino acid sequence and substantially the same as MGC2742. The “total RNA fraction” in the present specification refers to a fraction containing total RNA, and is extracted from blood, various organs, various tissues, cultured cells and the like by a solvent for extracting RNA by a usual method. A fraction containing total RNA.

  The MGC2742 gene can be obtained by the following method.

(1) When using a human cDNA library A full-length cDNA is obtained from a cDNA library expressing the MGC2742 gene according to a known method such as a colony hybridization method. Human MGC2742 cDNA can be obtained by PCR using this full-length cDNA as a template. As the cDNA library, for example, a cDNA library derived from human prostate cancer, human lung cancer, human breast cancer, human stomach cancer, human colorectal cancer, human malignant melanoma, human pancreatic cancer, and each non-cancerous normal tissue can be used. Can be. As commercially available human cDNA libraries, Prostate-Leiomyosarcoma cDNA (Invitrogen: 11598-018), Fetal Brain cDNA (Invitrogen: 10662-013), Marathon-Ready cDNA, Normal Prostate, -Coloed: -74 1-1) can be used or can be prepared by itself. In addition, MGC2742 cDNA can also be obtained by directly performing PCR using the cDNA library as a template. Any PCR primer may be used as long as it can amplify MGC2742 cDNA, and an appropriate primer can be selected by a known method. As a PCR primer for amplifying the MGC2742 cDNA, for example, an oligonucleotide having the following nucleotide sequence can be selected.
5′-caccatgcccgagagggagctgt-3 ′ (primer 1: SEQ ID NO: 3 in the sequence listing) and
5'-cagaacaatattaggtggcag-3 '(primer 2: SEQ ID NO: 4 in the sequence listing).
The following method can be mentioned as a method for preparing cDNA.

  When extracting the total RNA fraction from blood, various tissues, and organs taken from humans, a solvent for extracting RNA from blood, various tissues, and organs (including components having an action to inactivate ribonuclease, such as phenol) It is preferred to dissolve directly in Alternatively, the cells in the tissue are carefully scraped with a scraper or gently extracted from the tissue using a protease such as trypsin so as not to destroy the cells. Then, the process proceeds to the RNA fraction extraction step.

  Methods for extracting the RNA fraction include guanidine thiocyanate / cesium chloride ultracentrifugation, guanidine thiocyanate / hot phenol method, guanidine hydrochloride method, and guanidine acid thiocyanate / phenol / chloroform method (Chomczynski, P. and Sacchi, N. , (1987) Anal. Biochem., 162, 156-159), but the guanidine acid thiocyanate-phenol-chloroform method is preferred. In addition, commercially available reagents for RNA extraction (for example, ISOGEN (manufactured by Nippon Gene), TRIzol reagent (manufactured by Invitrogen)) and the like can be used according to the protocol attached to the reagent. For example, a total RNA fraction can be extracted from a human prostate cancer cell line LNCaP (American Tissue Culture Collection ATCC No .: CRL-1740) using a TRIzol reagent.

  It is preferable that the obtained total RNA fraction is further purified and used only for mRNA if necessary. Although the purification method is not particularly limited, it is known that many mRNAs present in the cytoplasm of eukaryotic cells have a poly (A) sequence at the 3′-terminal. The mRNA is adsorbed on the oligo (dT) probe thus obtained, and the mRNA is captured on the paramagnetic particles on which streptavidin is immobilized using the binding between biotin / streptavidin, and after washing, the mRNA is eluted. , MRNA can be purified. Alternatively, a method in which mRNA is adsorbed on an oligo (dT) cellulose column and then eluted and purified may be employed. Further, mRNA can be further fractionated by sucrose density gradient centrifugation or the like.

  cDNA can be synthesized by a known method using reverse transcriptase (Reverse transcriptase) using total RNA or mRNA as a template. For example, Omniscript Reverse Transcriptase (QIAGEN) ) Can be used in accordance with the attached protocol to synthesize cDNA. MGC2742 cDNA can be obtained by performing PCR on the obtained cDNA using PCR primers specific to the amplification of MGC2742 gene (for example, a combination of primers of SEQ ID NO: 9 and SEQ ID NO: 10 in the sequence listing). . PCR can be performed under ordinary reaction conditions.

  Anyone having ordinary knowledge in the technical field to which the present invention pertains may substitute a part of the natural nucleotide sequence of the human MGC2742 gene with another nucleotide or delete or add a nucleotide to the nucleotide sequence by modifying the nucleotide sequence. It is possible to prepare a polynucleotide having the same biological activity as that of the natural MGC2742 gene to promote canceration of cells. Thus, a polynucleotide having a nucleotide sequence in which a nucleotide is substituted, deleted, or added in a natural nucleotide sequence and showing expression fluctuation equivalent to that of the natural MGC2742 gene can also be used in the present invention. Modification of the nucleotide sequence includes, for example, introduction of a deletion by a restriction enzyme or DNA exonuclease, mutagenesis by a site-directed mutagenesis method, modification of the nucleotide sequence by a PCR method using a mutation primer, direct introduction of a synthetic mutant DNA, and the like. It can be done by a method. Alternatively, a polynucleotide that hybridizes with a polynucleotide having a nucleotide sequence complementary to the MGC2742 gene under stringent conditions and has the same cancer cell growth activity as MGC2742 can be used.

  In the present invention, “hybridize under stringent conditions” refers to hybridization at 68 ° C. in a commercially available hybridization solution ExpressHyb Hybridization Solution (manufactured by Clontech), or using a filter on which DNA is immobilized. After hybridization at 68 ° C. in the presence of 0.7-1.0 M NaCl, 0.1-fold concentration SSC solution (1x concentration SSC is composed of 150 mM NaCl and 15 mM sodium citrate). As used herein, refers to hybridization under conditions that can be identified by washing at 68 ° C. or under conditions equivalent thereto.

2. Expression of MGC2742 gene In order to express the MGC2742 gene in an animal individual, a method of incorporating the obtained full-length cDNA into a viral vector and administering it to an animal can be mentioned. Examples of the method of gene transfer using a viral vector include a method of incorporating cDNA into a retrovirus, an adenovirus, an adeno-associated virus, a herpes virus, a vaccinia virus, a pox virus, a polio virus, or another DNA virus, or an RNA virus to introduce cDNA. . Among them, a method using a retrovirus, an adenovirus, an adeno-associated virus, and a vaccinia virus is preferable.

  Examples of non-viral gene transfer methods include a method of directly administering an expression plasmid intramuscularly (DNA vaccine method), a liposome method, a lipofection method, a microinjection method, a calcium phosphate method, an electroporation method, and the like. The DNA vaccine method and the liposome method are preferred.

  In addition, cells that differentiate full-length cDNA into muscle cells, hepatocytes, adipocytes, prostate cells or muscle cells, hepatocytes, adipocytes derived from humans, mice, rats, etc., for cultured cells (eg, fibroblasts) Can be introduced into cells and highly expressed to examine the functions of each target cell, specifically, what effects appear on cell morphology or cell differentiation such as canceration of cells. . Conversely, an antisense nucleic acid against the gene to be tested can be introduced into cells, and the effect on the function, morphology or differentiation of each target cell can be examined.

  In introducing the full-length cDNA into an animal or a cell, the cDNA is incorporated into a vector containing an appropriate promoter and a sequence involved in expression of a gene, and a host cell is transformed with the vector. As a vertebrate cell expression promoter, those having a promoter, an RNA splice site, a polyadenylation site, and a transcription termination sequence, which are usually located upstream of the gene to be expressed, can be used. May be provided. Examples of the expression vector include pSV2dhfr having the early promoter of SV40 (Subramani, S. et al. (1981) Mol. Cell. Biol 1, 854-864), retrovirus vectors pLNCX, pLNSX, pLXIN, pSIR (Clontech ( Clontech), but are not limited thereto. The expression vector is prepared by a diethylaminoethyl (DEAE) -dextran method (Luthman, H. and Magnusson, G. (1983) Nucleic Acids Res, 11, 1295-1308), a calcium phosphate-DNA coprecipitation method (Graham, FL and van der). Eb, AJ (1973) Virology 52, 456-457), electric pulse drilling (Neumann, E. et al. (1982) EMBO J. 1, 841-845), or Lipofectamine 2000 (Invitrogen), Lipofectamine PLUS (Invitrogen) ), DMRIE-C Reagent (manufactured by Invitrogen), FuGENE6 (manufactured by Roche Diagnostics) or the like can be incorporated into COS cells or mouse fibroblast cell line NIH3T3 (ATCC No .: CRL-1658). Or, in the case of a retroviral vector, a packaging cell line, For example, 293-10A1 (manufactured by IMGENEX) or PT67 (manufactured by Clontech) may be incorporated or 293 cells (manufactured by Takara Shuzo) or NIH3T3 cells may be packaged with a packaging plasmid such as pCL-10A1 or pCL-Eco (manufactured by IMGENEX) By incorporating (co-transfecting) together to produce a virus and infecting it with COS cells or mouse fibroblast NIH3T3, desired transformed cells can be obtained.

  It is also possible to prepare knockout animals in which the target gene is disrupted in normal animals by genetic manipulation, and to examine what effects appear on tumor development and / or tumor growth mechanisms. Conversely, it is also possible to examine the function of MGC2742 by preparing a transgenic animal so that the target gene is highly expressed in an animal having a tumor and observing the morphological change of the tumor. The transgenic animal can be obtained by obtaining a fertilized egg from the animal, transferring the gene, transplanting the animal into a pseudopregnant animal, and developing the animal. The procedure is known in the art (Developmental Engineering Experiment Manual (Tatsuji Nomura Supervision, edited by Motoya Katsuki, published in 1987), and JP-A-5-48093. Specifically, for example, in the case of a mouse, first, an ovulation-inducing agent is administered to a female mouse, then mated with a male of the same strain, and the pronuclear fertilized egg is collected from the oviduct of the female mouse the next day. Next, the DNA fragment solution to be introduced is injected into the pronucleus of a fertilized egg using a micro glass tube. Regulatory genes such as promoters and enhancers for expressing the gene to be introduced in animal cells are not particularly limited as long as they function in the cell of the introduced animal. The fertilized egg into which the DNA has been injected is transplanted into the oviduct of a pseudopregnant foster female mouse (Slc: ICR, etc.), and is born about 20 days later by spontaneous delivery or cesarean section.

  As a method for confirming that the thus obtained animal carries the transgene, for example, DNA is extracted from the tail of the animal, and the DNA is extracted using specific sense and antisense primers. After digesting the DNA with a restriction enzyme, gel electrophoresis, blotting the DNA in the gel onto a nylon membrane or the like, and performing Southern blot analysis using all or a part of the labeled transgene as a probe Methods and the like can be mentioned.

3. Method for Producing Cancer Cell Using MGC2742 Gene Whether or not a target gene can function as an oncogene, that is, whether or not it has a malignant trait, is determined by a contact inhibition phenomenon in a cell line in which they are expressed (Contact Inhibition). Sensitivity, or the presence or absence of anchorage-independent growth (Anchorage independent growth), etc. (Atsushi Yokota, Masaru Yamamoto, Bio Manual UP Series Cancer Research Protocol Yodosha p.168-174, 1995 Published October 15). That is, genes that cause cells to lose sensitivity to the contact inhibition phenomenon or exhibit anchorage-independent growth can be said to be oncogenes.

  For example, when the MGC2742 gene is overexpressed in the mouse fibroblast cell line NIH3T3 cell line, the above-mentioned loss of sensitivity to contact inhibition ability and anchorage-independent growth are confirmed, and the MGC2742 gene may function as an oncogene. It became clear. In many cases, injection of NIH3T3 cells overexpressing an oncogene into a nude mouse forms a tumor at the injection site, so that a cell line overexpressing the MGC2742 gene can also be used for tumorigenicity experiments.

That is, cancer cells can be produced by using the MGC2742 gene. Examples of the method for producing a cancer cell include a method for producing a cancer cell including the following steps 1) and 2).
1) a step of transforming a cell with the polynucleotide of the following (i) or (ii);
(I) a polynucleotide having a nucleotide sequence represented by nucleotide numbers 165 to 1970 of SEQ ID NO: 1 in the sequence listing,
(Ii) a polynucleotide that hybridizes under stringent conditions to a polynucleotide consisting of a nucleotide sequence complementary to the polynucleotide described in (i) above, and has a nucleotide sequence encoding a protein substantially identical to MGC2742 ,
2) a step of selecting a cell line in which the cell growth ability has changed in step 1);

  In the above-mentioned cell transformation in step 1), cells in an animal individual can be transformed to generate cancer cells in the animal individual, or cultured cells can be transformed into cancer cells. Cell transformation can be performed using animals or cultured cells, for example, by the method described in the above section “2. Expression of MGC2742 gene”.

  The change in the proliferation ability of the cell in the above step 2) means that the transformed cell loses the sensitivity to the cell contact inhibition phenomenon or exhibits anchorage-independent growth. Changes in the cell growth ability also indicate that the number of foci was significantly increased in the transformed cells compared to the untransformed cells in the foci formation test described below, and the number of colonies in the colony formation test was It can also be confirmed by a remarkable increase and / or a remarkable increase in the diameter of the spheroid in the spheroid proliferation test, but as long as the change in cell proliferation ability can be examined. Are not limited to these methods. A cell line in which the proliferation ability of the cell has changed can be selected as a cancer cell.

  A cell line that overexpresses MGC2742 has one or more of the following properties (1) to (3).

(1) Focus formation Normal fibroblasts such as NIH3T3 usually do not overlap even if they proliferate and become dense, and proliferation stops when a monolayer is formed. On the other hand, transformed cells and cancer cells have lost their susceptibility to the contact inhibition phenomenon and can continue to grow while overlapping, forming a layered cell population and forming a monolayer of cells. Has the property of creating a high-density cell mass, Focus, in the spread of the human body (Jun Yokota, Masaru Yamamoto, Bio Manual UP Series Cancer Research Protocol Yodosha 168-174 (Oct. 15, 1995) Since this phenomenon is observed, for example, when a cultured cell is infected with a cancer virus, it is not only used for the quantification of the cancer virus but also used (Fundamental Techniques in Virology, Academic Press (1969) , 198-211), which can also be observed by transfecting cells with DNA fragments containing viruses or cell-derived oncogenes. Used for cleaning.

  A cell line in which the MGC2742 gene is overexpressed and a cell line in which the MGC2742 gene is not overexpressed are cultured in a usual manner, and replaced with a fresh culture solution, and then placed in a multiwell plate (for example, a 96-well plate: 3598 manufactured by Corning-Costar). After dispensing and culturing for a certain time, the number of focuses per well is measured. Statistical processing is performed after totaling the number of focuses per well, and the average number of focuses and standard deviation per well are calculated.

  It is observed that the number of foci is significantly increased in the NIH3T3 cells in which the MGC2742 gene is overexpressed as compared with the cells in which the MGC2742 gene is not overexpressed.

(2) Colony formation test It is known that normal cells cannot proliferate unless they have a scaffold to adhere, except for blood cells. In contrast, transformed cells and cancer cells have the property of being able to proliferate without a scaffold. This characteristic change can be easily examined by the formation of a proliferating colony in a soft agar medium (Jun Yokota, Masaru Yamamoto, Bio Manual UP Series Cancer Research Protocol Yodosha 168-174 (Oct. 1995) Published on March 15).

A cell line in which the MGC2742 gene is overexpressed and a cell line in which the MGC2742 gene is not overexpressed are cultured in a usual manner, and replaced with a fresh culture solution. % RPMS containing RPMI 1640) and overlay on an agar medium (eg, RPMI 1640 containing 0.66% Bactogar and 20% FCS). The cells are cultured under normal conditions (for example, 37 ° C., 5% CO ₂ ), and the number of colonies growing in the soft agar formed is measured. When culturing is performed using a multiwell plate (for example, 12-well plate: 3512 manufactured by Corning-Costar), the number of colonies per well is measured after culturing for a certain period of time. After totaling the number of colonies per well, statistical processing is performed, and the average number of colonies per standard and the standard deviation are calculated.

  It is observed that the number of colonies is remarkably increased in the NIH3T3 cell line overexpressing the MGC2742 gene, as compared with the cells not overexpressing the MGC2742 gene.

(3) Spheroid proliferation test The characteristic of anchorage-independent growth of transformed cells and cancer cells can be easily evaluated by culturing in a spheroid state. Spheroids are aggregates of a large number of cells and can be easily formed by culturing on a culture plate in which cell adhesion is extremely low. Culture in a spheroid state is different from ordinary monolayer culture, and is similar to culture in an anchorage-independent state, as shown in (2) of 3 above, and functions under conditions close to in vivo. It can be observed (Sumitomo Bakelite Co., Ltd., SUMILON physical and chemical instrument general catalog).

A cell line in which the MGC2742 gene is overexpressed and a cell line in which the MGC2742 gene is not overexpressed are cultured by a usual method, replaced with a fresh culture solution, and a cell non-adherent multiwell plate (for example, spheroid 96U: MS manufactured by Sumitomo Bakelite Co., Ltd.) -0096S). The cells are cultured under normal conditions (for example, 37 ° C., 5% CO ₂ ), and the size of spheroids formed on the plate is measured after a certain period of time from the start of the culture. The size of the spheroid can be measured using, for example, an ocular micrometer (S-6, manufactured by Sankeisha) under a microscope, and the diameter of the spheroid can be used as an index of the size of the spheroid.

  In the NIH3T3 cell line in which the MGC2742 gene was overexpressed, a remarkable increase in the diameter of the spheroid was observed as compared with the cells in which the MGC2742 gene was not overexpressed.

4. The tumor marker gene MGC2742 is considered to be involved in canceration of cells and / or proliferation of cancer cells. Therefore, by measuring the expression level of MGC2742 in each cell and / or each tissue, it is possible to determine the state of canceration and / or proliferation of cancer cells caused by overexpression of MGC2742. Such cancers include, for example, prostate cancer, testicular cancer, penis cancer, bladder cancer, kidney cancer, oral cancer, pharyngeal cancer, lip cancer, tongue cancer, gingival cancer, nasopharyngeal cancer, esophageal cancer, gastric cancer, small intestine cancer , Colorectal cancer, colon cancer, liver cancer, gallbladder cancer, pancreatic cancer, nasal cavity cancer, lung cancer, osteosarcoma, soft tissue cancer, skin cancer, melanoma, breast cancer, uterine cancer, ovarian cancer, brain tumor, thyroid cancer, lymphoma, leukemia And the like.

  That is, the MGC2742 gene can be used as a marker gene for tumor formation. As a method for measuring the expression level of a gene, cRNA or cDNA is prepared from total RNA extracted from a sample, and labeled with an appropriate label, whereby the signal intensity can be detected.

  Hereinafter, as a method of analyzing the expression level of a gene, an analysis method using a solid-phased sample and some other analysis methods will be described.

(1) Analysis method using immobilized sample i) Immobilized sample Examples of the immobilized sample include the following.
a) Gene chip:
A gene chip on which an antisense oligonucleotide synthesized based on an EST (expressed sequence tag) sequence or an mRNA sequence on a database is immobilized can be used. As such a gene chip, a gene chip manufactured by Affymetrix (Lipshutz, RJ et al. (1999) Nature genet. 21, suppliment, 20-24) can be used. It may be produced based on the method. It is most preferable that the EST sequence or mRNA sequence serving as the base of the antisense oligonucleotide immobilized on the gene chip be derived from an animal of the same species as the animal or animal cell to be analyzed. When a mouse or a mouse-derived cultured cell is used, a mouse-derived cell is desirable, and when a human specimen or a human-derived cultured cell is used, a human-derived cell is desirable. When analyzing mRNA of a mouse tumor, a mouse-derived mRNA is most preferable. For example, a mouse 19K set (Murine 19K set: 19KA, 19KB, 19KC) or a mouse 11K set (Murine 11K set: 11KA, 11kB) manufactured by Affymetrix Co., Ltd. Can be used. When analyzing mRNA of a human tumor (for example, prostate cancer cell line LNCaP: ATCC No. CRL-1740, PC-3: ATCC No. CRL-1435, DU-145: ATCC No. HTB-81, etc.), a human-derived mRNA is used. Preferably, for example, a human U95 set or U133 set manufactured by Affymetrix can be used. However, the present invention is not limited thereto, and for example, those derived from animals of closely related species can also be used. Examples of closely related species include, but are not limited to, mammals, such as humans and mice, and humans and monkeys.

b) Array or membrane filter on which cDNA or RT-PCR product prepared from mouse or human, total RNA or total RNA obtained from a specific tissue is immobilized:
The cDNA or RT-PCR product is cloned by performing a reverse transcriptase reaction or PCR using primers prepared based on sequence information such as a mouse or human EST database. This cDNA or RT-PCR product may hybridize with only the MGC2742 gene. In addition, a commercially available array (eg, Intelligene: manufactured by Takara Shuzo Co., Ltd.) may be used for the array or the filter, or the above cDNA or RT-PCR product may be used with a commercially available spotter (eg, GMS417 Alayer: manufactured by Takara Shuzo Co., Ltd.). , Etc.) to produce a solid phase.

ii) Preparation and analysis of probe Not a specific mRNA clone but a label of all expressed mRNAs is used as a labeled probe. Unpurified mRNA may be used as a starting material for preparing the probe, but it is preferable to use poly (A) + RNA purified by the above-described method. Hereinafter, a method for preparing a labeled probe and a method for detecting and analyzing a labeled probe when various types of immobilized samples are used will be described.

a) Gene chip manufactured by Affymetrix:
A biotin-labeled cRNA probe is prepared according to the protocol (Affymetrix Expression Analysis Technical Manual) attached to the gene chip manufactured by Affymetrix. Next, hybridization is performed using an analysis device (GeneChip Fluidics Station 400) manufactured by Affymetrix Co., Ltd. according to the protocol (expression analysis technical manual) attached to the gene chip manufactured by Affymetrix Co., and the light emission by avidin is detected and analyzed.

b) Array:
When cDNA is prepared from poly (A) ⁺ RNA by the reverse transcriptase reaction, it is necessary to label the cDNA so that the cDNA can be detected. For example, cDNA is fluorescently labeled by adding d-UTP or the like labeled with Cy3, Cy5, or the like. In this case, be used as a mixture of both the if labeled test substance added cell derived poly (A) + and ^RNA to control cell-derived poly (A) + ^RNA with different dyes, after hybridization when it can. For example, when a commercially available array of Takara Shuzo Co., Ltd. is used as an array, hybridization and washing are performed according to the protocol of the company, and after detecting a fluorescent signal with a fluorescent signal detector (for example, GMS418 array scanner (Takara Shuzo) or the like). Perform the analysis. However, the array to be used is not limited to a commercially available array, and may be a homemade array or a specially manufactured array.

c) Membrane filter:
In making cDNA from poly ^(A) + RNA by reverse transcriptase, radioisotopes ^{(e.g., d-CTP [α- 33 P} ]) labeled probes were prepared by adding the like, hybridization in a conventional manner After performing hybridization and washing using, for example, an atlas system (manufactured by Clontech), which is a commercially available filter microarray, detection is performed using an analyzer (for example, an atlas image: manufactured by Clontech). Perform analysis.

  In the case of using fluorescently labeled probes, if each probe is labeled with a different fluorescent dye, a mixture of both probes can be hybridized to one immobilized sample at a time, and the fluorescence intensity can be read (Brown, PO et al. (1999) Nature genet, 21, supplement, 33-37).

(2) Other analysis methods a) RT-PCR
First, a reverse transcriptase reaction using the mRNA of MGC2742 as a template is performed, and then PCR is performed to specifically amplify the DNA fragment. In this method, in order to specifically amplify the nucleotide sequence of interest, an antisense primer complementary to a specific partial sequence of the mRNA of interest and a sequence of cDNA generated by reverse transcriptase from the antisense primer are used. A sense primer complementary to a specific partial sequence therein is used. RT-PCR can be performed using a commercially available kit (for example, RNA PCR kit AMV ver2.1: manufactured by Takara Shuzo Co., Ltd.) according to the manual attached to the kit.

  The antisense primer used in both the reverse transcriptase reaction and the PCR is substantially 15 to 40 contiguous nucleotides, preferably at least 18 to 30 nucleotides, more preferably, in the antisense sequence of the nucleotide sequence of the MGC2742 gene. Consists of a nucleotide sequence of 23 to 30 nucleotides.

  On the other hand, in the nucleotide sequence of the MGC2742 gene, the sequence of the sense primer used in the PCR is the sequence located in the 5 'terminal region further than the most 5' terminal position in the sequence corresponding to the complementary strand of the antisense primer. Consists of any partial sequence of 15 to 40 nucleotides, preferably 18 to 35 nucleotides, more preferably 21 to 30 nucleotides. However, if the sense primer and the antisense primer have mutually complementary sequences, non-specific sequences may be amplified by annealing the primers, which may hinder detection of a specific polynucleotide. It is preferable to design primers that avoid such combinations.

  In each of the antisense primer and the sense primer, a nucleotide sequence irrelevant to the nucleotide sequence of the MGC2742 gene may be added as a linker at the 5 'end of the nucleotide sequence defined above. However, it is preferable that the linker does not cause non-specific annealing with the nucleic acid in the reaction solution during the reaction so as not to hinder the detection of the specific polynucleotide.

In the embodiment in which the nucleic acid is detected by RT-PCR, the conditions of each reaction are as described below. Note that the sample for detection by RT-PCR does not usually need to be purified to poly (A) ⁺ RNA.

i) Reverse transcriptase reaction Example of reaction solution composition (total volume 20 μl):
Total RNA as appropriate;
Magnesium chloride 2.5 to 5 mM (preferably 5 mM);
1 × RT-PCR buffer (10 mM Tris-HCl (pH 8.3 to 9.0 (preferably 8.3) at 25 ° C.), 50 mM potassium chloride);
dNTPs 0.5 to 1 mM (preferably 1 mM);
Antisense primer 1 μM (2.5 μM of a commercially available random primer or oligo (dT) primer (12-20 nucleotides) can be added as a substitute for the antisense primer);
Reverse transcriptase 0.25 to 1 unit / μl (preferably 0.25 unit / μl);
Adjust to 20 μl with sterile water.

Reaction temperature conditions:
After incubating at 30 ° C for 10 minutes (only when using a random primer), incubating at 42 to 60 ° C (preferably 42 ° C) for 15 to 30 minutes (preferably 30 minutes), and further heating at 99 ° C for 5 minutes, the enzyme And then cooled at 4-5 ° C (preferably 5 ° C) for 5 minutes.

ii) PCR
Example of reaction solution composition:
Magnesium chloride 2 to 2.5 mM (preferably 2.5 mM);
1 × PCR buffer (10 mM Tris-HCl (pH 8.3 to 9.0 (preferably 8.3) at 25 ° C.)), 50 mM potassium chloride;
dNTPs 0.2 to 0.25 mM (preferably 0.25 mM);
Antisense primer and sense primer 0.2 to 0.5 μM (preferably 0.2 μM);
1 to 2.5 units (preferably 2.5 units) of Taq polymerase;
The total volume is adjusted to 80 μl by adding sterile water, and the total volume is added to the total volume of the reaction solution after the completion of the reverse transcription reaction before PCR is started.

  Reaction temperature conditions: First, heating at 94 ° C for 2 minutes, then at 90 to 95 ° C (preferably 94 ° C) for 30 seconds, 40 to 60 ° C (preferably from the dissociation temperature (Tm) calculated from the characteristics of the primer). A temperature cycle of 70 to 75 ° C. (preferably 72 ° C.) for 1.5 seconds at 28 to 50 cycles (preferably 28 cycles) at a temperature lower than 20 ° C. for 30 seconds, then cooling to 4 ° C. I do.

  After completion of the PCR, the reaction solution is subjected to electrophoresis to detect whether or not a band of a desired size has been amplified. In order to perform quantitative detection, PCR is performed under the same conditions using a serially diluted cDNA clone as a standard template DNA, and the number of temperature cycles at which quantitative detection can be performed is determined. A part of the reaction solution is sampled and subjected to electrophoresis. Also, for example, by using radiolabeled dCTP during the PCR reaction, quantification can be performed using the amount of radioactivity incorporated in the band as an index. As methods for improving the reliability of gene quantification, competitive RT-PCR (Souaze et al., BioTechniques 21,280-285 (1996)), which is an improvement of the above RT-PCR, and TaqMan PCR (Heid et al., Genom) Res. 6, 986-994 (1996)).

  b) Ribonuclease protection assay: a labeled probe only in the RNA sample having the nucleotide sequence according to any one of the above 1) to 5) (where t in the sequence is replaced by u). After hybridizing to form a double-stranded polynucleotide and then incubating the sample with ribonuclease, the probe-hybridized mRNA is not digested by ribonuclease due to its double-stranded form. Since the other RNAs are digested, only the double-stranded polynucleotide remains (if the probe is shorter than the mRNA to be detected, the double-stranded polynucleotide corresponding to the length of the probe remains). By quantifying the double-stranded polynucleotide, the expression level of the target polynucleotide is measured. Specifically, for example, the method described below is used.

  To ensure that the extra labeled probe that does not form a double strand is separated from the double-stranded polynucleotide to facilitate quantification, the extra labeled probe is preferably digested with ribonuclease. The labeling probe may be DNA or RNA as long as it is capable of digesting the main-strand DNA. The method for preparing a labeled probe can be performed according to the above-mentioned conventional method, but the length of the probe used in the ribonuclease assay is preferably about 50 to 500 nucleotides. Further, a probe in which complementary strands are mixed, such as a probe obtained by directly labeling a double-stranded DNA and thermally denaturing it, is not suitable for this method.

  The RNA probe is prepared, for example, according to the following method. First, the template DNA is inserted into a plasmid vector (for example, pGEM-T (promega)) having a bacteriophage promoter (T7, SP6, T3 promoter, etc.). Next, this recombinant plasmid vector is digested with a restriction enzyme so that it is cleaved only one point immediately downstream of the inserted fragment. Using the obtained linear DNA as a template, an in vitro transcription reaction is carried out in the presence of radiolabeled ribonucleotides. In this reaction, an enzyme such as T7, SP6 or T3 polymerase is used in accordance with the promoter in the vector. The above operation can be performed using, for example, Riboprobe System-T7, -SP6 or -T3 (all manufactured by Promega).

The steps up to the preparation of the RNA sample can be carried out according to the method described in the section “1. A ribonuclease protection assay is performed using 10 to 20 μg of the prepared total RNA sample and an excess amount of the labeled probe corresponding to 5 × 10 ⁵ cpm. This operation can be performed using a commercially available kit (HybSpeed RPA Kit, manufactured by Ambion). The obtained ribonuclease digested sample is electrophoresed on a 4 to 12% polyacrylamide gel containing 8 M urea, the gel is dried, and autoradiography is performed with an X-ray film. By the above operation, the band of the double-stranded polynucleotide which escaped the ribonuclease digestion can be detected, and the quantification can be carried out according to a conventional method. Furthermore, if the expression level of the β-actin gene is simultaneously measured for the purpose of correcting the variation caused by the difference in the RNA amount between the samples, a more precise evaluation can be performed.

  c) Run-on assay (See Greenberg, ME and Ziff, EBB (1984) Nature 311, 433-438, Groudine, M. et al. (1981) Mol. Cell Biol. 1, 281-288) : This method is a method for measuring the transcriptional activity of a target gene by isolating a nucleus from a cell, and is not a method for detecting mRNA in a cell. However, in the present invention, the "method for measuring the expression level of a gene" ]. When a transcription reaction is performed in a test tube using the isolated cell nucleus, only the reaction in which transcription is already started before the nucleus is isolated and the one where the mRNA chain is being generated elongates proceeds. I do. At the time of isolating the nucleus by adding a radiolabeled ribonucleotide during this reaction to label the elongating mRNA and detecting the mRNA contained therein that hybridizes to the unlabeled probe The transcription activity of the target polynucleotide in can be measured. The specific operation method is the same as that described in the above-mentioned reference except that an unlabeled probe is prepared. A measurement result is compared between a sample derived from a sample collected from a subject or a test animal and a sample derived from a normal person or a normal animal. If the transcriptional activity of the MGC2742 gene is higher than that of a normal person or a normal animal, the patient has a tumor. Can be determined to be.

  d) Under the control of the promoter of the MGC2742 gene, the expression of the MGC2742 gene or MGC2742 can also be detected indirectly by using a gene capable of detecting the promoter activity (hereinafter referred to as “reporter gene”). Hereinafter, a detection method using a reporter gene will be described.

d-1) Reporter gene The reporter gene may be any that encodes a reporter protein that can be specifically distinguished from any other protein that the host cell can produce in a series of steps of this test method. Preferably, cells before transformation do not have a gene encoding the same or similar protein as the reporter protein. For example, even if the reporter protein is toxic to the cell or if the cell confers resistance to sensitive antibiotics, the presence or absence of reporter gene expression will determine the viability of the cell. Can be determined. However, a more preferable reporter gene used in the present invention can specifically and quantitatively detect the expression level (for example, a specific antibody against the protein encoded by the reporter gene has been obtained). Na) structural gene. More preferably, it is a gene encoding an enzyme or the like which specifically reacts with a foreign substrate to generate a metabolite which can be easily measured quantitatively. Examples of such reporter genes include, for example, genes encoding the following proteins, but the present invention is not limited thereto.

d-1-1) Chloramphenicol acetyltransferase:
An enzyme that adds an acetyl group to chloramphenicol, and can be detected by a so-called CAT assay or the like. As a vector for preparing a reporter assay vector simply by incorporating a promoter, a pCAT3-Basic vector (promega) is commercially available.

d-1-2) Firefly luciferase:
It can be quantified by measuring bioluminescence generated when luciferin is metabolized. As a vector for the reporter assay, a pGL3-Basic vector (promega) is commercially available.

d-1-3) β-galactosidase:
There are substrates that can be measured by color reaction, fluorescence or chemiluminescence, respectively. As a vector for a reporter assay, pβgal-Basic (promega) is commercially available.

d-1-4) Secreted alkaline phosphatase:
There are substrates that can be measured by color reaction, bioluminescence or chemiluminescence, respectively. As a vector for a reporter assay, pSEAP2-Basic (manufactured by Clontech) is commercially available.

d-1-5) Green-fluorescent protein:
Although it is not an enzyme, it can quantify directly because it emits fluorescence. Similarly, pEGFP-1 (manufactured by Clontech) is commercially available as a vector for a reporter assay.

d-2) Introduction of Reporter Gene According to a known method, a cell is transfected with a reporter expression plasmid capable of expressing the reporter gene under the control of the MGC2742 gene promoter.

  As a method for introducing an expression plasmid into cells, a diethylaminoethyl (DEAE) -dextran method (Luthman, H. and Magnusson, G. (1983) Nucleic Acids Res. 11, 1295-1308), a calcium phosphate-DNA coprecipitation method ( Graham, FL and van der Eb, AJ (1973) Virology 52, 456-457), electric pulse drilling (Neumann, E. et al. (1982) EMBO J. 1, 841-845), lipofection (Lopata et al. al. (1984) Nucl. Acids Res. 12, 5707-5717, Sussman and Milman (1984) Mol. Cell. Biol. 4, 1641-1643), and the like, but are not limited thereto. Any method widely used in the technical field to which the present invention belongs can be adopted. However, when the cells are so-called floating cells, it is preferable to use a method other than the calcium phosphate-DNA coprecipitation method. In any method, it is necessary to use optimized transfection conditions according to the cells used.

d-3) Evaluation Thus, when cells transfected with the MGC2742 gene reporter expression plasmid are cultured, the transcription of the reporter gene is promoted in correlation with the expression of the MGC2742 gene. Therefore, the expression level of the MGC2742 gene can be evaluated by observing the change in the expression level of the reporter gene between the case where the test substance is added to the medium and the case where the test substance is not added under the conditions where the reporter gene can be expressed. Here, the "conditions under which the reporter gene can be expressed" may be any conditions under which cells transfected with the reporter expression vector can survive and produce a transcript (reporter protein) of the reporter gene. Preferably, in a medium suitable for the cell line to be used (a serum component such as fetal bovine serum may be added) in the presence of air containing 4 to 6% (most preferably 5%) carbon dioxide, Incubate at 36-38 ° C (most preferably 37 ° C) for 2-3 days (most preferably 2 days).

5. Tumor marker protein MGC2742 is considered to cause canceration of cells and to increase the expression level in tumor cells. Therefore, it can be used as a tumor marker protein for detecting the presence of a tumor.

  As a preferred embodiment of the method for detecting MGC2742, a method using an antibody will be described. First, the protein in the sample is immobilized on the inner bottom of a well of a multi-well plate such as a 96-well plate or a 384-well plate, or on a membrane, and then detection using an antibody that specifically recognizes MGC2742 is performed. . Among them, a multi-well plate such as a 96-well plate or a 384-well plate is generally used as a method called an enzyme-linked immunosorbent assay (ELISA) or a radioisotope immunoassay (RIA). On the other hand, as a method for immobilizing a sample on a membrane, a method of transferring a protein onto a membrane via polyacrylamide electrophoresis of a sample (Western blotting), or a so-called dot blot in which a sample or a dilution thereof is directly permeated into the membrane. And a slot blot method.

(1) Sample preparation Samples include whole blood, serum, liver, prostate, testis, penis, bladder, kidney, oral cavity, pharynx, lips, tongue, gingiva, nasopharynx of non-human mammals such as humans and mice. , Esophagus, stomach, small intestine, large intestine, colon, liver, gallbladder, pancreas, nasal cavity, lung, soft tissue, skin, breast, uterus, ovary, brain, thyroid, lymph, etc. Can be arbitrarily selected. In addition, it is necessary to perform sampling in accordance with the experimental ethical guidelines of each laboratory. The sample is subjected to high-speed centrifugation as necessary to remove insoluble substances, and then prepared as an ELISA / RIA sample or a Western blot sample as described below.

  Examples of samples for ELISA / RIA include, for example, collected serum, liver, prostate, testis, penis, bladder, kidney, oral cavity, pharynx, lips, tongue, gingiva, nasopharynx, esophagus, stomach, small intestine, large intestine, colon, liver , Gall bladder, pancreas, nasal cavity, lung, soft tissue, skin, breast, uterus, ovary, brain, thyroid, lymph, etc. may be used as they are or appropriately diluted with a buffer. Samples for Western blot (for electrophoresis) include, for example, serum, liver, prostate, testis, penis, bladder, kidney, oral cavity, pharynx, lips, tongue, gingiva, nasopharynx, esophagus, stomach, small intestine, large intestine, colon, SDS-polyacrylamide gel electrophoresis using the extract of liver, gallbladder, pancreas, nasal cavity, lung, soft tissue, skin, breast, uterus, ovary, brain, thyroid, lymph, etc. as it is or appropriately diluting it with a buffer solution And a sample buffer containing 2-mercatorethanol for use (eg, manufactured by Sigma). In the case of dot / slot blot, for example, collected serum, liver, prostate, testis, penis, bladder, kidney, oral cavity, pharynx, lips, tongue, gingiva, nasopharynx, esophagus, stomach, small intestine, large intestine, colon, liver, Extracts of the gallbladder, pancreas, nasal cavity, lungs, soft tissue, skin, breast, uterus, ovary, brain, thyroid, lymph, etc., or those appropriately diluted with a buffer solution, directly using a blotting device, etc. Adsorb to the membrane.

(2) Immobilization of the sample In order to specifically detect the protein in the sample obtained as described above, the sample is precipitated by immunoprecipitation, a method utilizing the binding of ligand, and the like. The detection can be performed without phase conversion, or the sample to be detected can be solid-phased as it is. When a protein is immobilized, a nitrocellulose membrane (for example, manufactured by Bio-Rad) or a nylon membrane (for example, Hybond-ECL (Amersham) is used as a membrane for Western blotting, dot blotting, or slot blotting. Pharmacia), a cotton membrane (for example, a blot absorbent filter (manufactured by Bio-Rad), etc.) or a polyvinylidene difluoride (PVDF) membrane (for example, manufactured by Bio-Rad, etc.).

  The so-called blotting method for transferring proteins from a gel after electrophoresis to a membrane includes a wet blotting method (CURRENT PROTOCOLS IN IMMUNOLOGY volume 2 ed by JE Coligan, AM Kruisbeek, DH Margulies, EM Shevach, W. Strober), and a semi-dry method. Blotting method (see CURRENT PROTOCOLS IN IMMUNOLOGY volume 2 above) and the like can be mentioned. Equipment for dot blotting and slot blotting is also commercially available (for example, Biodot (Biorad)).

  On the other hand, in order to perform detection and quantification by the ELISA method / RIA method, a sample or a diluent thereof (eg, 0.05% adjuvant) is placed in a dedicated 96-well plate (eg, Immunoplate Maxisorp (manufactured by Nunc)). A phosphate buffered saline solution containing sodium chloride (diluted with “PBS”) is added and left at 4 ° C. to room temperature overnight, or at 37 ° C. for 1 to 3 hours to form a bottom surface of the well. Is immobilized by adsorbing protein.

(3) Antibody The antibody used specifically recognizes MGC2742 or a part thereof. Such antibodies include, for example, antibodies that bind to MGC2742 but do not bind to any other mouse or human protein.

  The antibody can be obtained by using a conventional method (for example, Shinsei Kagaku Kenkyusho 1, Protein 1, p. 389-397, 1992) to obtain a polypeptide serving as an antigen or any polypeptide selected from its amino acid sequence. , And collecting and purifying antibodies produced in the living body. In addition, an antibody against MGC2742 is produced according to a known method (eg, Kohler and Milstein, Nature 256, 495-497, 1975, Kennet, R. ed., Monoclonal Antibody p. 365-367, 1980, Prenum Press, NY). Hybridomas can be established by fusing the antibody-producing cells with the myeloma cells to obtain monoclonal antibodies.

  Examples of an antigen for preparing an antibody include MGC2742 or a polypeptide comprising at least six consecutive partial amino acid sequences thereof, or a derivative obtained by adding an arbitrary amino acid sequence or a carrier thereto.

  The antigen polypeptide can be obtained by synthesizing MGC2742 of the present invention in vitro, or by producing it in host cells by genetic engineering. Specifically, after MGC2742 is incorporated into a vector capable of expressing, it is synthesized in a solution containing enzymes, substrates and energetic substances necessary for transcription and translation, or other prokaryotic or eukaryotic host cells are prepared. The protein can be obtained by expressing MGC2742 by transformation.

  In vitro synthesis of polypeptides includes, but is not limited to, for example, the Rapid Translation System (RTS) manufactured by Roche Diagnostics. For example, when RTS is used, a target gene is cloned into an expression vector controlled by a T7 promoter, and this is added to an in vitro reaction system. First, mRNA is transcribed from a template DNA by T7 RNA polymerase, Thereafter, translation is performed by ribosomes or the like in the E. coli lysate, and the target polypeptide is synthesized in the reaction solution (Biochemica, 1, 20-23 (2001), Biochemica, 2, 28-29 (2001)).

  Examples of prokaryotic host cells include Escherichia coli and Bacillus subtilis. To transform a gene of interest into these host cells, the host cells are transformed with a plasmid vector that contains replicons or origins of replication from species compatible with the host and regulatory sequences. Further, the vector is preferably a vector having a sequence capable of imparting phenotypic (phenotypic) selectivity to transformed cells.

  For example, K12 strain or the like is often used as Escherichia coli, and pBR322 or a pUC-type plasmid is generally used as a vector. However, the present invention is not limited thereto, and any known various strains and vectors can be used.

  Examples of the promoter include, in Escherichia coli, tryptophan (trp) promoter, lactose (lac) promoter, tryptophan lactose (tac) promoter, lipoprotein (lpp) promoter, polypeptide chain elongation factor Tu (tufB) promoter, and the like. Any promoter can be used to produce the polypeptide of interest.

  As Bacillus subtilis, for example, strain 207-25 is preferable, and as a vector, pTUB228 (Ohmura, K. et al. (1984) J. Biochem. 95, 87-93) is used, but it is not limited thereto. is not. By ligating a DNA sequence encoding the signal peptide sequence of Bacillus subtilis α-amylase, extracellular secretory expression becomes possible.

  Eukaryotic host cells include cells such as vertebrates, insects, and yeast. Examples of vertebrate cells include COS cells, which are monkey cells (Gluzman, Y. (1981) Cell 23, 175- 182, ATCC CRL-1650), mouse fibroblast NIH3T3 (ATCC No. CRL-1658) and Chinese hamster ovary cells (CHO cells, ATCC CCL-61) deficient strains (Urlaub, G. and Chasin). Natl. Acad. Sci. USA 77, 4126-4220) and the like, but are not limited thereto.

  As a vertebrate cell expression promoter, those having a promoter, an RNA splice site, a polyadenylation site, and a transcription termination sequence, which are usually located upstream of the gene to be expressed, can be used. May be provided. Examples of the expression vector include pCR3.1 (Invitrogen) having a cytomegalovirus early promoter and pSV2dhfr having an SV40 early promoter (Subramani, S. et al. (1981) Mol. Cell. Biol. 1, 854-864), but are not limited thereto.

  When a COS cell or a NIH3T3 cell is used as a host cell, for example, the expression vector has an SV40 origin of replication, is capable of autonomous growth in a COS cell or an NIH3T3 cell, and further has a transcription promoter and a transcription termination. Those provided with a signal and an RNA splice site can be used. The expression vector is prepared by a DEAE-dextran method (Luthman, H. and Magnusson, G. (1983) Nucleic Acids Res., 11, 1295-1308), a calcium phosphate-DNA coprecipitation method (Graham, FL and van der Eb, AJ). (1973) Virology 52, 456-457) and electric pulse perforation (Neumann, E. et al. (1982) EMBO J. 1, 841-845) can be incorporated into COS cells or NIH3T3 cells. Thus, a desired transformed cell can be obtained. When a CHO cell is used as a host cell, a vector capable of expressing a neo gene functioning as an antibiotic G418 resistance marker together with an expression vector, for example, pRSVneo (Sambrook, J. et al. (1989): “Molecular Cloning A Laboratory Manual “Cold Spring Harbor Laboratory, NY” and pSV2neo (Southern, PJ and Berg, P. (1982) J. Mol. Appl. Genet. 1, 327-341) are co-transfected and G418 resistant. By selecting a colony, a transformed cell stably producing the desired polypeptide can be obtained.

  When insect cells are used as host cells, ovarian cell-derived cell lines (Sf-9 or Sf-21) of Spodoptera frugiperda of the Lepidoptera Noctuidae and High Five cells derived from Trichoplusia ni egg cells (Wickham, TJ et al. 1992) Biotechnol. Prog. I: 391-396) is often used as a host cell, and as a baculovirus transfer vector, pVL1392 / 1393 using a polyhedrin protein promoter of autographa nucleopolyhedrovirus (AcNPV) is often used. (Kidd, IM and VC Emery (1993) The use of baculoviruses as expression vectors. Applied Biochemistry and Biotechnology 42, 137-159). In addition, vectors using baculovirus P10 or a promoter of the same basic protein can also be used. Furthermore, the recombinant protein can be expressed as a secreted protein by linking the secretory signal sequence of the envelope surface protein GP67 of AcNPV to the N-terminal side of the target protein (Zhe-mei Wang, et al. (1998)). Biol. Chem., 379, 167-174).

  As an expression system using a eukaryotic microorganism as a host cell, yeast is generally well known. Among them, yeast of the genus Saccharomyces, for example, baker's yeast Saccharomyces cerevisiae and petroleum yeast Pichia pastoris are preferable. Examples of expression vectors for eukaryotic microorganisms such as the yeast include, for example, an alcohol dehydrogenase gene promoter (Bennetzen, JL and Hall, BD (1982) J. Biol. Chem. 257, 3018-3025) and an acid phosphatase gene. A promoter (Miyanohara, A. et al. (1983) Proc. Natl. Acad. Sci. USA 80, 1-5) can be preferably used. When expressed as a secretory protein, it can be expressed as a recombinant having a secretory signal sequence and a cleavage site of an endogenous protease or a known protease possessed by the host cell at the N-terminal side. For example, in a system in which human mast cell tryptase of a trypsin-type serine protease is expressed in petroleum yeast, the activity is achieved by connecting the secretory signal sequence of α-factor of yeast and the cleavage site of KEX2 protease possessed by petroleum yeast to the N-terminal side and expressing it. It is known that type tryptase is secreted into the medium (Andrew, L. Niles, et al. (1998) Biotechnol. Appl. Biochem. 28, 125-131).

  The transformant obtained as described above can be cultured according to a conventional method, and the culture produces the target polypeptide inside or outside the cell. The medium used for the culture can be appropriately selected from various ones commonly used depending on the host cell used. For example, in the case of the above-mentioned COS cells, RPMI1640 medium or Dulbecco's modified Eagle medium (hereinafter referred to as “DMEM”) ), To which a serum component such as fetal bovine serum may be added as necessary.

Recombinant proteins produced in the cells of the transformants or outside the cells by the above culture can be separated and purified by various known separation procedures utilizing the physical properties and chemical properties of the proteins. it can. As the method, specifically, for example, treatment with a normal protein precipitant, ultrafiltration, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, affinity chromatography, high-performance liquid chromatography ( Examples thereof include various liquid chromatography such as HPLC, dialysis, and combinations thereof. In addition, by connecting histidine consisting of 6 residues to the recombinant protein to be expressed, it can be efficiently purified with a nickel affinity column. By combining the above methods, the desired polypeptide can be easily produced in large quantities with high yield and high purity.
As an example of the antibody that specifically binds to MGC2742, a monoclonal antibody that specifically binds to MGC2742 can be mentioned, and the method for obtaining the antibody is as described below.

In the production of monoclonal antibodies, the following working steps are generally required. That is,
(A) purification of a biopolymer used as an antigen,
(B) after immunizing the animal by injecting the antigen, collecting blood and assaying the antibody titer to determine the timing of splenectomy, and then preparing antibody-producing cells;
(C) preparation of myeloma cells (hereinafter referred to as “myeloma”);
(D) cell fusion between antibody-producing cells and myeloma,
(E) selecting a hybridoma group producing the desired antibody,
(F) division into single cell clones (cloning),
(G) In some cases, culturing a hybridoma to produce a large amount of a monoclonal antibody, or breeding an animal into which the hybridoma has been transplanted,
(H) Examination of the physiological activity of the monoclonal antibody thus produced and its recognition specificity, or assay of properties as a labeling reagent.

  Hereinafter, a method for preparing a monoclonal antibody will be described in detail along the above steps, but the method for preparing the antibody is not limited thereto. For example, antibody-producing cells other than splenocytes and myeloma can also be used.

(A) Purification of antigen As the antigen, MGC2742 prepared by the method described above or a part thereof can be used. Furthermore, a partial peptide of the protein of the present invention can be chemically synthesized using a method well known to those skilled in the art and used as an antigen.

(B) Preparation of antibody-producing cells The antigen obtained in the step (a) is mixed with an auxiliary such as Freund's complete or incomplete adjuvant or alum, and the animal is immunized as an immunogen. A mouse is most preferably used as an experimental animal, but is not limited thereto.

  The method of immunogen administration for mouse immunization may be any of subcutaneous injection, intraperitoneal injection, intravenous injection, intradermal injection, and intramuscular injection, but subcutaneous injection or intraperitoneal injection is preferred.

  Immunization can be performed once or multiple times at appropriate intervals (preferably at one to five week intervals). Thereafter, the antibody titer to the antigen in the serum of the immunized animal is measured, and the effect of the subsequent operations can be enhanced by using the animal whose antibody titer is sufficiently high as a source of antibody-producing cells. Generally, it is preferable to use antibody-producing cells derived from an animal 3 to 5 days after the final immunization for subsequent cell fusion.

  Examples of the method for measuring the antibody titer used herein include various known techniques such as RIA, ELISA, fluorescent antibody, and passive hemagglutination, and the detection sensitivity, speed, accuracy, and automation of the operation are known. In view of the possibility of the above, the RIA method or the ELISA method is more preferable.

  The measurement of the antibody titer in the present invention can be performed, for example, by the following procedure according to the ELISA method. First, a purified or partially purified antigen is adsorbed on a solid surface such as a 96-well plate for ELISA, and the solid surface on which the antigen is not adsorbed is a protein unrelated to the antigen, for example, bovine serum albumin (hereinafter referred to as “BSA”). ), And after washing the surface, it is brought into contact with a serially diluted sample (for example, mouse serum) as the first antibody to bind the monoclonal antibody in the sample to the antigen. Further, an antibody against a mouse antibody that is enzyme-labeled as a second antibody is added to bind to the mouse antibody, a substrate of the enzyme is added after washing, and a change in absorbance due to color development based on the decomposition of the substrate is measured. calculate.

(C) Myeloma preparation step As a myeloma, a cell line generally obtained from a mouse, for example, an 8-azaguanine-resistant mouse (derived from BALB / c) myeloma strain P3X63Ag8U.1 (P3-U1) [Yelton, DE et al. Current Topics in Microbiology and Immunology, 81, 1-7 (1978)], P3 / NSI / 1-Ag4-1 (NS-1) [Kohler, G. et al. European J. Immunology, 6, 511 -519 (1976)], Sp2 / O-Ag14 (SP-2) [Shulman, M. et al. Nature, 276, 269-270 (1978)], P3X63Ag8.653 (653) [Kearney, JF et al. J. Immunology, 123, 1548-1550 (1979)], and P3X63Ag8 (X63) [Horibata, K. and Harris, AW Nature, 256, 495-497 (1975)]. These cell lines are prepared by adding 8-azaguanine to an appropriate medium, for example, 8-azaguanine medium [RPMI-1640 medium supplemented with glutamine, 2-mercaptoethanol, gentamicin, and fetal calf serum (hereinafter referred to as “FCS”). Medium), Iscove's Modified Dulbecco's Medium (hereinafter referred to as "IMDM"), or Dulbecco's Modified Eagle Medium (hereinafter referred to as "DMEM"). Four to four days before, the cells are subcultured in a normal medium [for example, ASF104 medium containing 10% FCS (manufactured by Ajinomoto Co., Inc.)], and a cell count of 2 × 10 ⁷ or more is secured on the day of fusion.

(D) Cell fusion Antibody-producing cells are plasma cells and lymphocytes which are precursor cells thereof, which may be obtained from any site of an individual, and are generally spleen, lymph nodes, peripheral blood, or these cells. Although splenocytes can be obtained from those appropriately combined, splenocytes are most commonly used.

  After the final immunization, a site where antibody-producing cells are present, for example, a spleen is removed from a mouse having a predetermined antibody titer, and spleen cells as antibody-producing cells are prepared. Currently, the most commonly used means for fusing the spleen cells with the myeloma obtained in step (c) is a method using polyethylene glycol, which has relatively low cytotoxicity and simple fusion operation. This method includes, for example, the following procedure.

  The spleen cells and myeloma are thoroughly washed with a serum-free medium (for example, RPMI 1640) or a phosphate buffered saline (hereinafter, referred to as “PBS”), and the ratio of spleen cells to myeloma is 5: 1 to 10: 1. Mix to the extent necessary and centrifuge. After removing the supernatant and thoroughly loosening the precipitated cell group, 1 ml of a serum-free medium containing 50% (w / v) polyethylene glycol (molecular weight: 1,000 to 4,000) is added dropwise with stirring. Thereafter, 10 ml of serum-free medium is slowly added, followed by centrifugation. The supernatant was discarded again, and the precipitated cells were placed in a normal medium (hereinafter referred to as "HAT") containing an appropriate amount of hypoxanthine / aminopterin / thymidine (hereinafter referred to as "HAT") solution and mouse interleukin-2 (hereinafter referred to as "IL-2"). Medium)), dispensed into each well of a culture plate (hereinafter, referred to as “plate”), and cultured at 37 ° C. for about 2 weeks in the presence of 5% carbon dioxide gas. HAT medium is supplemented as needed on the way.

(E) Selection of hybridoma group When the myeloma cells are 8-azaguanine resistant strains, that is, when they are hypoxanthine guanine phosphoribosyltransferase (HGPRT) deficient strains, the unfused myeloma cells and myeloma A fused cell cannot survive in a HAT-containing medium. On the other hand, a fusion cell of antibody-producing cells or a hybridoma of antibody-producing cells and myeloma cells can survive, but a fusion cell of antibody-producing cells has a long life. Therefore, by continuing the culture in the HAT-containing medium, only the hybridoma of the antibody-producing cell and the myeloma cell survives, and as a result, the hybridoma can be selected.

  With respect to the hybridomas that have grown in a colony, the medium is replaced with a medium obtained by removing aminopterin from the HAT medium (hereinafter referred to as “HT medium”). Thereafter, a part of the culture supernatant is collected, and the antibody titer is measured by, for example, an ELISA method.

  The method using an 8-azaguanine-resistant cell line has been described above, but other cell lines can also be used according to the selection method of the hybridoma, and in that case, the composition of the medium used also changes.

(F) Cloning A hybridoma that has been found to produce a specific antibody by measuring the antibody titer in the same manner as described in step (b) is transferred to another plate and cloned. The cloning method includes a limiting dilution method in which one hybridoma is diluted and cultured so that one hybridoma is contained in one well of the plate, a soft agar method in which the colony is collected by culturing in a soft agar medium, and a micromanipulator. And a cell sorter for separating one cell using a cell sorter. The limiting dilution method is simple and often used.

  For the wells in which the antibody titer has been recognized, for example, cloning by the limiting dilution method is repeated 2 to 4 times, and those having a stable antibody titer are selected as the monoclonal antibody-producing hybridoma strain of the present invention.

(G) Preparation of Monoclonal Antibody by Hybridoma Culture The cloned hybridoma is cultured by changing the medium from HT medium to normal medium. Large-scale culture is performed by rotary culture using a large culture bottle or spinner culture. The monoclonal antibody that specifically binds to the protein of the present invention can be obtained by purifying the supernatant from this large-scale culture using a method known to those skilled in the art such as gel filtration. In addition, ascites containing a large amount of the monoclonal antibody of the present invention can be obtained by growing the hybridoma in the abdominal cavity of a mouse of the same strain (for example, BALB / c described above) or a Nu / Nu mouse. . As a simple purification method, a commercially available monoclonal antibody purification kit (for example, MAbTrap GII kit; manufactured by Pharmacia) or the like can also be used.

  The monoclonal antibody thus obtained has a high antigen specificity for MGC2742.

(H) Assay of monoclonal antibody The isotype and subclass of the monoclonal antibody thus obtained can be determined as follows. First, examples of the identification method include the Ouchterlony method, the ELISA method, and the RIA method. The Otterlony method is simple but requires a concentration operation when the concentration of the monoclonal antibody is low. On the other hand, when the ELISA method or the RIA method is used, the culture supernatant is allowed to react with the antigen-adsorbed solid phase as it is, and further, antibodies corresponding to various immunoglobulin isotypes and subclasses are used as secondary antibodies. Isotypes and subclasses can be identified. As a simpler method, a commercially available kit for identification (for example, Mouse Typer Kit; manufactured by Bio-Rad) can be used.

  Further, the protein can be quantified by the Foreign Lowry method and a method of calculating from the absorbance at 280 nm [1.4 (OD280) = 1 mg / ml of immunoglobulin].

  The thus obtained monoclonal antibody of the present invention can be used for detection, separation and purification of MGC2742 using its specificity.

When the obtained antibody is used as a drug for humans, it is desirable to prepare a humanized anti-human MGC2742 antibody in view of antigenicity. The production of a humanized anti-human MGC2742 antibody can be obtained by the method described below. That is, (i) human lymphocytes collected from human peripheral blood or spleen are sensitized in vitro with human MGC2742 as an antigen in the presence of IL-4, and the sensitized human lymphocytes are heterohybridomas of mice and humans. A target antibody-producing hybridoma is screened by cell fusion with K ₆ H ₆ / B ₅ (ATCC CRL-1823). The antibody produced by the obtained antibody-producing hybridoma is a humanized anti-human MGC2742 monoclonal antibody. An antibody that neutralizes the activity of human MGC2742 is selected from these antibodies. However, in the method of sensitizing human lymphocytes in vitro as described above, it is generally difficult to obtain an antibody having a high affinity for an antigen. Therefore, in order to obtain a monoclonal antibody having a high affinity for human MGC2742, it is necessary to increase the affinity of the low-affinity human anti-human MGC2742 monoclonal antibody obtained as described above. To do so, a random mutation is introduced into the CDR region (particularly CDR-3) of a humanized anti-human MGC2742 monoclonal antibody which is obtained as described above and is a low-affinity neutralizing antibody, and expressed in phage. A phage that strongly binds to human MGC2742 as an antigen is selected by a phage display method using a plate on which human MGC2742 is immobilized, and the phage is expanded in E. coli. May be determined. A human-type anti-human MGC2742 monoclonal antibody can be obtained by inserting the gene encoding the human-type anti-human MGC2742 monoclonal antibody thus obtained into a generally used expression vector for mammalian cells and expressing it. . From these, the desired human anti-MGC2742 monoclonal antibody that neutralizes the biological activity of human MGC2742 and has high affinity can be selected. Further, (ii) a mouse-type anti-human MGC2742 monoclonal antibody was prepared using a Balb / c mouse by a conventional method (Kohler et al .: Nature 256, p. 495-497, 1975) in the same manner as in the present invention. A monoclonal antibody that neutralizes the biological activity of human MGC2742 and has high affinity is selected. CDR-grafting (Winter and Milstein: Nature 349, p293-299, 1991) grafting the CDR-regions (CDR-1, 2 and 3) of this high-affinity mouse anti-human MGC2742 monoclonal antibody into the CDR regions of human IgG. Humanization is possible by making full use of the method described above. In addition to the above, (iii) human peripheral blood lymphocytes are transplanted into Severe combined immunodeficiency (SCID) mice, and the transplanted SCID mice produce humanized antibodies (Mosier DE et al .: Nature 335, p256). -259, 1988; Duchosal MA et al .: Nature 355, p258-262, 1992), producing human monoclonal antibodies specific to human MGC2742 by sensitizing and screening with human MGC2742 as an antigen. Lymphocytes can be collected from the mouse. Lymphocytes obtained in the same manner as the preparation method of the aforementioned human antibody _{(i), K 6 H 6} / B 5 (ATCC CRL-1823) is a hetero hybridoma of mouse and human as to cell fusion, obtained By screening the hybridomas, a hybridoma producing the desired humanized monoclonal antibody can be obtained. By culturing the hybridoma thus obtained, the desired humanized monoclonal antibody can be produced in large quantities, and a purified product can be obtained by purification in the same manner as described above. Also, by cloning the gene (cDNA) encoding the desired humanized monoclonal antibody, incorporating this gene into an appropriate vector by genetic engineering techniques, and expressing it in various animal cells, co-injected cells, or Escherichia coli as a host. Thus, a large amount of recombinant humanized monoclonal antibodies can be produced. By purifying from the obtained culture solution in the same manner as described above, a large amount of purified human monoclonal antibody can be obtained. Furthermore, from the anti-MGC2742 monoclonal antibodies obtained by the above-described method, an antibody that neutralizes the biological activity of MGC2742 can be obtained. Since the antibody that neutralizes the biological activity of MGC2742 inhibits the biological activity of MGC2742 in vivo, that is, the canceration of cells, the antibody is expected as a medicine, particularly as a therapeutic and / or prophylactic agent for cancer. The activity of neutralizing the biological activity of MGC2742 by an anti-MGC2742 antibody in vitro can be measured, for example, by the activity of suppressing the canceration of cells in cells overexpressing MGC2742. For example, mouse fibroblast cell line NIH3T3 overexpressing MGC2742 is cultured, anti-MGC2742 antibodies are added to the culture system at various concentrations, and the inhibitory activity on focus formation, colony formation and spheroid proliferation can be measured. . The therapeutic and / or prophylactic effect of anti-MGC2742 antibody on cancer using experimental animals in vivo can be measured by, for example, administering the MGC2742 antibody to a transgenic animal overexpressing MGC2742 and measuring the change in cancer cells. can do.

  The thus obtained antibody that neutralizes the biological activity of MGC2742 can be used as a pharmaceutical, particularly as a pharmaceutical composition for the treatment and / or prevention of cancer, or for the immunological diagnosis of such diseases. Useful as antibodies. The antibody of the present invention can be formulated and administered orally or parenterally. The preparation containing the antibody of the present invention can be safely administered to humans or animals as a pharmaceutical composition containing an antibody that recognizes MGC2742 as an active ingredient. Examples of the form of the pharmaceutical composition include injection preparations including infusion, suppositories, nasal preparations, sublingual preparations, transdermal absorption agents and the like. Since monoclonal antibodies are high-molecular proteins, they are remarkably adsorbed on glass containers such as vials and syringes and are unstable, and are easily absorbed by various physicochemical factors such as heat, pH and humidity. To be deactivated. Therefore, a stabilizer, a pH adjuster, a buffer, a solubilizing agent, a surfactant and the like are added in order to formulate the composition in a stable form. Examples of the stabilizer include amino acids such as glycine and alanine, sugars such as dextran 40 and mannose, and sugar alcohols such as sorbitol, mannitol, and xylitol, and two or more of these may be used in combination. These stabilizers are preferably added in an amount of 0.01 to 100 times, especially 0.1 to 10 times the weight of the antibody. By adding these stabilizers, the storage stability of the liquid preparation or freeze-dried preparation can be improved. Examples of the buffer include a phosphate buffer and a citrate buffer. The buffer adjusts the pH of the aqueous solution after re-dissolving the liquid preparation or the lyophilized preparation, and contributes to the stability and solubility of the antibody. The amount of the buffer added is preferably, for example, 1 to 10 mM based on the amount of water after adopting a liquid preparation or a lyophilized preparation. Examples of the surfactant include preferably polysorbate 20, Pluronic F-68, polyethylene glycol, and the like, particularly preferably polysorbate 80, and two or more of these may be used in combination. High-molecular proteins such as antibodies are easily adsorbed to glass or resin, which is a material of the container. Therefore, by adding a surfactant, adsorption of the antibody after re-dissolving the liquid preparation or the freeze-dried preparation to the container can be prevented. The surfactant is preferably added in an amount of 0.001 to 1.0% based on the weight of water after reconstitution of the liquid preparation or freeze-dried preparation. The preparation of the antibody of the present invention can be prepared by adding the above-mentioned stabilizing agent, buffering agent, or anti-adsorption agent. The osmotic pressure ratio is preferably from 1 to 2. The osmotic pressure ratio can be adjusted by increasing or decreasing sodium chloride during formulation. The antibody content in the preparation can be appropriately adjusted according to the disease to be applied, the administration route, and the like. The dose of the human antibody to humans depends on the affinity of the antibody for human MGC2742, that is, the dissociation constant for human MGC2742 ( The higher the affinity (lower the Kd value) with respect to the (Kd value), the smaller the dose to humans, and the more effective the drug. When the human anti-MGC2742 antibody is administered to a human, about 0.1 to 100 mg / kg may be administered once every 1 to 30 days.

(4) Detection The antibody obtained by the method described in (3) above can be directly labeled, or can be used as a primary antibody to specifically recognize the antibody (antibody derived from the animal in which the antibody is produced is used). It is used for detection in cooperation with a labeled secondary antibody.

Preferred examples of the type of label include, but are not limited to, an enzyme (alkaline phosphatase or horseradish peroxidase) or biotin (however, an operation of further binding enzyme-labeled streptavidin to biotin of the secondary antibody is added). Various pre-labeled antibodies (or streptavidin) are commercially available for methods that use labeled secondary antibodies (or labeled streptavidin). In the case of RIA, an antibody labeled with a radioisotope such as I ¹²⁵ is used, and the measurement is performed using a liquid scintillation counter or the like.

  By detecting the activity of these labeled enzymes, the amount of the polypeptide that is the antigen is measured. In the case of alkaline phosphatase or horseradish peroxidase, substrates that emit color or emit light by the catalyst of those enzymes are commercially available.

  When a color-developing substrate is used, it can be visually detected by Western blotting or dot / slot blotting. In the ELISA method, quantification is preferably performed by measuring the absorbance (measurement wavelength varies depending on the substrate) of each well using a commercially available microplate reader. Also preferably, a dilution series of the antigen used for the production of the antibody in the above (3) is prepared, and this is used as a standard antigen sample, and the detection operation is performed simultaneously with other samples, and the standard antigen concentration and the measured value are plotted. By creating a curve, it is possible to quantify the antigen concentration in another sample.

  On the other hand, when a substrate that emits light is used, it can be detected by Western radiography or dot / slot blotting by autoradiography using an X-ray film or an imaging plate, or photography using an instant camera, Quantitation using densitometry, Molecular Imager Fx system (manufactured by Bio-Rad), or the like is also possible. When a luminescent substrate is used in the ELISA method, the enzyme activity is measured using a luminescent microplate reader (for example, manufactured by Bio-Rad).

(5) Measurement operation i) In the case of Western blot, dot blot or slot blot First, in order to prevent nonspecific adsorption of the antibody, the membrane must be previously treated with a substance that inhibits such nonspecific adsorption (skim milk, casein, bovine). An operation (blocking) of dipping in a buffer solution containing serum albumin, gelatin, polyvinylpyrrolidone, etc. for a certain period of time is performed. The composition of the blocking solution is, for example, phosphate buffered saline (PBS) or Tris buffered saline (TBS) containing 5% skim milk, 0.05 to 0.1% Tween 20. Instead of skim milk, Block Ace (Dainippon Pharmaceutical), 1 to 10% bovine serum albumin, 0.5 to 3% gelatin, 1% polyvinylpyrrolidone, or the like may be used. The blocking time is 16 to 24 hours at 4 ° C. or 1 to 3 hours at room temperature.

  Next, the membrane is washed with PBS or TBS containing 0.05 to 0.1% Tween 20 (hereinafter referred to as “washing solution”) to remove an excess blocking solution, and then manufactured by the method described in (3) above. The antibody thus obtained is immersed in a solution appropriately diluted with a blocking solution for a certain period of time to bind the antibody to the antigen on the membrane. The dilution ratio of the antibody at this time can be determined, for example, by performing a preliminary Western blotting experiment using a sample obtained by serially diluting the recombinant antigen described in 3) above. This antibody reaction operation is preferably performed at room temperature for 2 hours. After completion of the antibody reaction operation, the membrane is washed with a washing solution. Here, when the used antibody is labeled, the detection operation can be performed immediately. When an unlabeled antibody is used, a secondary antibody reaction is subsequently performed. When using a labeled secondary antibody, for example, when a commercially available product is used, it is used after diluting 2,000 to 20,000-fold with a blocking solution (if a suitable dilution ratio is described in the attached instruction, follow the description). After washing and removing the primary antibody, the membrane is immersed in a secondary antibody solution at room temperature for 45 minutes to 1 hour, washed with a washing solution, and then subjected to a detection operation according to the labeling method. The washing operation is performed, for example, by first shaking the membrane in the washing solution for 15 minutes, exchanging the washing solution for a new one, shaking for 5 minutes, exchanging the washing solution again, and shaking for 5 minutes. If necessary, the washing solution may be further exchanged for washing.

ii) ELISA / RIA
First, as in the case of Western blotting, blocking is performed in advance to prevent nonspecific adsorption of the antibody to the bottom surface in the well of the plate on which the sample is immobilized by the method (2). The blocking conditions are as described in the section of Western blot.

  Next, after the inside of the well is washed with PBS or TBS containing 0.05 to 0.1% Tween 20 (hereinafter referred to as “washing solution”) to remove an excess blocking solution, the well is prepared by the method described in (3) above. A solution obtained by appropriately diluting the washed antibody with a washing solution is dispensed and incubated for a certain period of time to allow the antibody to bind to the antigen. The dilution ratio of the antibody at this time can be determined, for example, by performing a preliminary ELISA experiment using a serially diluted recombinant antigen described in (3) above as a sample. This antibody reaction operation is preferably performed at room temperature for about 1 hour. After completion of the antibody reaction operation, the inside of the well is washed with a washing solution. Here, when the used antibody is labeled, the detection operation can be performed immediately. When an unlabeled antibody is used, a secondary antibody reaction is subsequently performed. For example, when a commercially available product is used, the labeled secondary antibody is diluted 2,000 to 20,000 times with a washing solution and used (if a suitable dilution ratio is described in the attached instruction, follow the description). After washing and removing the primary antibody, a secondary antibody solution is dispensed into the wells, incubated at room temperature for 1 to 3 hours, washed with a washing solution, and then subjected to a detection operation according to the labeling method. The washing operation is performed by, for example, dispensing a washing solution into a well and shaking for 5 minutes, replacing the washing solution with a new one, shaking for 5 minutes, exchanging the washing solution again and shaking for 5 minutes. If necessary, the washing solution may be further exchanged for washing.

  In the present invention, the so-called sandwich ELISA can be carried out, for example, by the method described below. First, in any one of the amino acid sequences of MGC2742, two regions having high hydrophilicity were selected, and a partial peptide consisting of 6 or more amino acids in each region was synthesized. Get different types of antibodies. One of the antibodies is labeled as described in (4) above. The unlabeled antibody is immobilized on the bottom surface in the well of a 96-well ELISA plate according to the method described in (2) above. After blocking, the sample solution is placed in a well and incubated at room temperature for 1 hour. After washing the wells, the labeled antibody diluent is dispensed into each well and incubated. After washing the inside of the well again, a detection operation according to the labeling method is performed.

(6) MGC2742 can also be quantified by using a ligand such as a substrate, a coenzyme, or a regulator that specifically binds to MGC2742, in addition to the ligand antibody.

(7) Judgment According to the method described above, the detection result is compared between a sample collected from a subject or a test animal and a sample collected from a normal person or a normal animal. The amount of the polypeptide to which the produced antibody or ligand specifically binds is defined as the expression level of each MGC2742. When the expression level of MGC2742 is increased in a normal individual, it is determined that the possibility of having a tumor is high. can do.

6. Function of MGC2742 The physiological function of MGC2742 obtained by genetic engineering using the MGC2742 gene in this way can be examined by expression experiments using the following viruses.

  The MGC2742 gene is amplified, for example, by the PCR method, and incorporated into an expression vector for adenovirus or a retrovirus vector. At that time, a commercially available kit can also be used (for example, adenovirus expression vector kit (manufactured by Takara Shuzo) or Retro-X System (manufactured by Clontech)). The thus obtained virus is inoculated into, for example, a diabetic mouse (for example, Balb / c mouse (CLEA Japan)) via the tail vein, and the control is carried out between the mouse and a mouse that has received the adenovirus containing no MGC2742 gene as a control. The ALT value (alanine aminotransferase value) and the expression level of the tumor marker gene are measured. In addition, various tissues are taken out from the mouse to examine the state of the tissues. At this time, a tissue-specific promoter (for example, rat probasin promoter (Greenberg, et al., Mol Endocrinol (1994) p. 230-239), mouse mammary tumor virus When a promoter (Wynshaw-Boris, Cancer Handbook 2 (2002), Nature Publishing Group, London, p. 891-902, etc.) is used, a tissue in which the gene is specifically expressed is taken out and the state of the tissue is examined. If there is a difference between the measured values, the physiological function of MGC2742 can be grasped.

7. How to detect cancer
MGC2742 is involved in canceration of cells and / or proliferation of cancer cells, and is considered to be highly expressed in cancer cells. Therefore, it is considered that a substance that suppresses the expression level of MGC2742 in a cell in which MGC2742 is overexpressed suppresses cell carcinogenesis and also suppresses cancer cell proliferation. In addition, "sample" refers to blood, body fluid, prostate, testis, penis, bladder, kidney, oral cavity, pharynx, lips, tongue, gingiva, nasopharynx, esophagus, Stomach, small intestine, large intestine, colon, liver, gallbladder, pancreas, nose, lung, bone, soft tissue, skin, breast, uterus, ovary, brain, thyroid, lymph node, muscle, adipose tissue, etc. Although it means a sample, in the present invention, the prostate is more preferable.

  In the present invention, normal humans and normal animals refer to humans and animals without cancer. Whether or not the animal is a normal person or a normal animal can be determined by measuring the concentration of MGC2742 and determining whether or not the value falls within a numerical range determined in advance as a normal person value. By previously examining the correlation between the expression level of MGC2742 and the degree of cancer formation in a normal human or animal, the subject or test animal is determined by measuring the expression level of MGC2742 in a sample collected from the test subject or test animal. It can be determined whether the animal is a normal person or a normal animal.

7.1 Method for detecting cancer using MGC2742 gene A method for detecting cancer using MGC2742 is specifically as described in 7.1.1 or 7.1.2.
7.1.1 Including the following steps 1) to 4):
1) a step of extracting a total RNA fraction from a specimen collected from a subject or a test animal;
2) a step of measuring the difference in the expression level of the MGC2742 gene between the total RNA fraction derived from the sample described in the above step 1) and the total RNA fraction extracted from a sample collected from a normal human or a normal animal;
3) A step of analyzing the difference in the expression level of the gene described in the above step 2) and detecting cancer in the subject or the test animal described in the above step 1).
7.1.2 Including the following steps 1) to 4):
1) a step of extracting a total RNA fraction from a specimen collected from a subject or a test animal;
2) a step of extracting a total RNA fraction from a specimen collected from a normal human or a normal animal;
3) a step of measuring the expression level of the MGC2742 gene in the total RNA fraction described in the above step 1) and the total RNA fraction described in the above step 2);
4) Analyzing the difference in the expression level of the gene measured in the above step 3) between the total RNA fraction derived from the above step 1) and the total RNA fraction derived from the above step 2), 2. The step of detecting cancer in a subject or a test animal according to the above.
7.1.3 Here, the extraction of the total RNA fraction in step 1) of 7.1.1 and step 1) and step 2) of 7.1.2 is the same as described in “1. Acquisition of MGC2742 gene” above. It can be carried out according to the method described in the section, and it is preferable that the extracted total RNA fraction is further purified and used as mRNA. In addition, the measurement of the expression level of the MGC2742 gene in the step 2) of 7.1.1 and the step 3) of 7.1.2 is measured using the method described in the section “4. Tumor marker gene” above. It can be measured using nucleic acid hybridization using solid-phased samples such as Northern blot, dot blot, slot blot, RT-PCR, ribonuclease protection assay, run-on assay, gene chip, and cDNA array. It is desirable.

7.2 Method for detecting cancer using MGC2742 The method for detecting cancer using MGC2742 of the present invention is specifically described in the following 7.2.1, 7.2.2, 7.2.3 or 7.2.3. 2.4.
7.2.1 Including the following steps 1) and 2):
1) a step of measuring the expression level of MGC2742 in a sample collected from a subject or a test animal;
2) Analyze the difference between the expression level of the protein detected in the above step 1) and the expression level of the protein in a sample collected from a normal human or normal animal, and detect cancer in the subject or the test animal in the above step 1). Process.

7.2.2 Including the following steps 1) to 3):
1) A step of measuring the expression level of MGC2742 in a sample collected from a subject or a test animal:
2) a step of measuring the expression level of the protein according to 1) in a sample collected from a normal human or a normal animal:
3) a step of analyzing the difference between the expression level of the protein measured in the above step 1) and the expression level of the protein measured in the above step 2), and detecting cancer in the subject or test animal.

7.2.3 Including the following steps 1) to 3):
1) immobilizing all proteins obtained from a specimen collected from a subject or a test animal;
2) a step of measuring the expression level of MGC2742 in the immobilized protein according to the above step 1);
3) Analyze the difference between the expression level of the protein detected in the above step 2) and the expression level of the protein in the total protein obtained from a sample collected from a normal human or a normal animal, and the subject or test animal in the above step 1) is analyzed. Detecting the cancer of the subject.

7.2.4 A method for detecting cancer, comprising the following steps 1) to 5):
1) immobilizing all proteins obtained from a specimen collected from a subject or a test animal;
2) immobilizing all proteins obtained from a specimen collected from a normal human or a normal animal;
3) The step of measuring the expression level of MGC2742 in the immobilized protein according to the above step 1):
4) a step of measuring the expression level of the protein whose expression level has been measured in step 3) in the immobilized protein according to step 2);
5) analyzing the difference between the expression level of the protein detected in step 3) and the expression level of the protein detected in step 4), and detecting the cancer of the subject or test animal according to step 1);

  7.2.5 Here, 7.2.1 step 1), step 2), 7.2.2 step 1), step 2), 7.2.3 step 2), step 3), For the measurement of the expression level of the protein in step 3) and step 4) of 7.2.4, the method described in the above section “5. Tumor marker protein” can be used, and preferably, it specifically binds to MGC2742. Methods using antibodies or ligands, more preferably Western blotting, dot blotting, slot blotting or enzyme-linked immunosorbent assay (ELISA) can be used.

7.3 Judgment 7.1.1 Step 3), 7.1.2 Step 4), 7.2.1 Step 1), 7.2.2 Step 3), 7.2.3 Step It is desirable that the detection of cancer in the step 3) and the step 5) of 7.2.4 is performed by comprehensively evaluating the difference in expression level. The “difference in expression level” refers to the expression ratio even if the expression level is different, as long as the expression level of MGC2742 in a sample derived from a subject or a test animal and a sample derived from a normal person or normal animal can be quantitatively compared. It may be.

  As a result of the above analysis, the measurement results are compared between a sample derived from a sample collected from a subject or a test animal and a sample derived from a normal person or a normal animal, and when the expression level of MGC2742 is higher than that of a normal person or a normal animal, It can be determined that the subject has cancer.

8. MGC2742 gene and / or kit for detecting MGC2742 The MGC2742 gene and / or MGC2742 can be detected using a kit containing at least one of the following 1) or 2).
1) a continuous oligonucleotide primer having a length of 15 to 30 bases for specifically amplifying a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 1 in the sequence listing;
2) a continuous polynucleotide probe of 15 nucleotides or more for hybridizing to a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 1 in the sequence listing under stringent conditions and detecting the polynucleotide;
3) a solid-phased sample on which a polynucleotide consisting of the nucleotide sequence of nucleotide numbers 165 to 1970 of SEQ ID NO: 1 in the sequence listing has been immobilized;
4) an antibody that specifically binds to a protein consisting of the amino acid sequence of SEQ ID NO: 2 and detects the protein;
5) A secondary antibody capable of binding to the antibody according to 4).

  The primer described in 1) above can be easily prepared by a conventional method such as using commercially available primer design software (for example, Wisconsin GCG package Version 10.2) based on the nucleotide sequence of the MGC2742 gene (the nucleotide sequence shown in SEQ ID NO: 1 in the sequence listing). Can be designed and amplified. As such a primer, for example, in order to amplify a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 1 in the Sequence Listing, an oligonucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 3 in the Sequence Listing and SEQ ID NO: A combination of oligonucleotides consisting of the nucleotide sequence shown in No. 4 can be used. The probe described in 2) above is a polynucleotide that specifically hybridizes to MGC2742, and has a length of 100 to 1500 bases, preferably 300 to 600 bases. These primers and probes may be labeled with an appropriate label (for example, an enzyme label, a radioactive label, a fluorescent label, and the like), and may be added with a linker.

The solid-phased sample described in 3) above is prepared by fixing the probe described in 2) above to a solid phase such as a glass plate or a nylon membrane. The method for preparing such an immobilized sample is as described in the section “(1) Analysis method using immobilized sample” in the section “4. Tumor marker gene”. Gene chips, cDNA arrays, oligo arrays, membrane filters and the like can be mentioned.
The kit of the present invention can further include a thermostable DNA polymerase, dNTP (a mixture of dATP, dCTP, dGTP, and dTTP) and a buffer. Examples of the heat-resistant DNA polymerase include Taq DNA polymerase, LA Taq DAN polymerase (manufactured by Takara Shuzo), Tth DNA polymerase, and Pfu DNA polymerase. The buffer is selected depending on the DNA polymerase to be used, and Mg ²⁺ or the like is added as necessary.

  The antibodies described in 4) and 5) above can be prepared by the method described in 3.2. The antibody may be labeled with a suitable label (for example, an enzyme label, a radioactive label, a fluorescent label, etc.).

  The kit of the present invention can be used for detecting the MGC2742 gene and / or MGC2742, and can also be used for determining the presence or absence of cancer and for screening for a substance that suppresses cancer growth.

9. Screening method for substance having therapeutic and / or preventive effect on cancer By measuring the expression level of at least one of MGC2742 gene or MGC2742, a substance having a therapeutic and / or preventive effect on cancer can be screened. .

  The “test substance” refers to a substance whose cancer growth inhibitory activity is to be examined by the screening method of the present invention. The test substance includes compounds, metabolites of microorganisms, extracts of plant and animal tissues, derivatives thereof, and mixtures thereof. In addition, a nucleic acid designed to reduce the expression level of MGC2742 or a derivative thereof (including an antisense oligonucleotide, a ribozyme, a dsRNA, an siRNA, and the like) can be used as a test substance. The dose and concentration of the test substance may be set as appropriate, or a plurality of doses may be set, for example, by creating a dilution series, and the test substance may be administered in an appropriate state such as a solid or liquid. Or a stabilizer or the like may be added. In the case of a screening method using cultured cells, the cells can be added to a medium and cultured. When it is added to the medium, it may be added from the start of the culture or during the culture, and the number of additions is not limited to one. The period for culturing in the presence of the test substance may be appropriately set, but is preferably 30 minutes to 2 weeks, and more preferably 30 minutes to 48 hours. When a test substance is administered to a mammal individual, administration forms such as oral administration, intravenous injection, intraperitoneal injection, transdermal administration, and subcutaneous injection are used depending on the properties of the test substance. It can also be applied or injected directly to the cancer. The time from the administration of the test substance until the specimen is obtained can be appropriately selected.

  The cultured cells used in the screening method of the present invention may be mammalian cells expressing MGC2742, and may be normal cells or cells exhibiting abnormal cell proliferation such as cancer cells. Cell line NIH3T3 cells, human prostate cancer cell line LNCaP, mouse, rat and human free adipocytes, mouse, rat and human primary hepatocytes, but are not limited thereto. The cultured cell mammalian species is preferably human, mouse or hamster, more preferably human or mouse, but is not limited thereto. It is more preferable to use mammalian cells overexpressing MGC2742 as cultured cells, and examples include NIH3T3 cells that overexpress MGC2742 by introducing the MGC2742 gene together with its promoter region.

  The screening method of the present invention includes a method of administering a test substance to a mammalian individual without using cultured cells, and then detecting the expression of the MGC2742 gene of the present invention in the organ or tissue cell extracted from the animal individual. Is also included. The organ or tissue to be detected for gene expression may be any organ or tissue that expresses MGC2742, but is preferably a tissue in which cancer has occurred. As the mammalian species, non-human mammals can be used, and mice, rats or hamsters are preferred, and mice or rats are more preferred. In addition, tumor-bearing animals (nude mice transplanted with human cancer strains, allografted mice, etc.), which are tumor model animals in which tumors derived from humans, monkeys, mice or rats are transplanted subcutaneously, intradermally, or intraperitoneally, or Animals of a line that naturally causes cancer (including naturally identified animals and genetically modified animals. For example, p53 knockout mice (Donehower et al., Nature (1992), 356, pp. 215-221), Myc transgenic mice (Adams et al., Nature (1985), vol. 318, p. 533-538), TRAMP mouse (Foster et al., Cancer Res. (1997), 57, p. 3325-3330), and the like. An animal in which MGC2742 is overexpressed can be used, and an animal that has been subjected to a cancer induction treatment can also be used.

  The cultured cells used in the method of the present invention may be cultured under any conditions that can express MGC2742 when no test substance is added. For example, known culture conditions are known for the cultured cells, and when the cells express MGC2742 under the conditions, the cells may be cultured under the conditions. In addition, when detecting the expression of MGC2742 in an organ or tissue extracted from a mammal individual, the breeding condition of the animal may be any condition that allows expression of MGC2742 when no test substance is added.

  In order to examine the effect of the test substance on the expression of the MGC2742 gene, there are a method of measuring the expression amount of the MGC2742 gene and a method of measuring the expression amount of MGC2742, which is a translation product of the MGC2742 gene.

  Regarding the extraction of the total RNA fraction from the cultured cells, the measurement of the expression level of the MGC2742 gene, and the measurement of the expression level of the MGC2742, the above-mentioned “1. Acquisition of the MGC2742 gene”, “4. Tumor marker gene” and “5. Tumor marker protein ”.

9.1 Method Using MGC2742 Gene As the screening method of the present invention, for example, a method using cultured cells derived from a mammal and a method using a mammalian individual are as follows.

A) Method using cultured cells derived from mammals A-1) The following steps 1) to 3) are included.
1) a step of extracting a total RNA fraction from cultured cells derived from mammals cultured in a medium to which a test substance has been added;
2) a step of detecting a difference in the expression level of the MGC2742 gene between the total RNA fraction derived from the above step 1) and the total RNA fraction derived from cultured mammalian cells cultured without adding a test substance;
3) a step of analyzing the difference in the expression level of the MGC2742 gene and determining the therapeutic and / or prophylactic effect of the test substance on cancer.

A-2) It includes the following steps 1) to 4).
1) a step of extracting a total RNA fraction from cultured cells derived from mammals cultured in a medium to which a test substance has been added;
2) a step of extracting a total RNA fraction from mammalian-derived cultured cells cultured in a medium to which a test substance is not added;
3) a step of measuring the expression level of the MGC2742 gene in the total RNA fraction derived from step 1) and the total RNA derived from step 2);
4) The difference in the expression level of the MGC2742 gene between the total RNA fraction derived from the above step 1) and the total RNA fraction derived from the above step 2) was analyzed, and the cancer of the test substance according to the above step 1) was analyzed. Determining the therapeutic and / or prophylactic effect of the drug.

B) Method using a mammalian individual B-1) The following steps 1) to 3) are included.
1) extracting a total RNA fraction from a specimen collected from a mammal to which the test substance has been administered;
2) a step of detecting a difference in the expression level of the MGC2742 gene between the total RNA fraction derived from the above step 1) and the total RNA fraction obtained from a sample collected from an animal to which the test substance has not been administered;
3) a step of analyzing the difference in the expression level of the MGC2742 gene described in the step 2) and determining the therapeutic and / or preventive effect of the test substance on cancer.

B-2) The following steps 1) to 4) are included.
1) extracting a total RNA fraction from a specimen collected from a mammal to which the test substance has been administered;
2) extracting a total RNA fraction from a sample collected from a mammal to which the test substance has not been administered;
3) measuring the expression level of the MGC2742 gene in the total RNA fraction derived from the above step 1) and the total RNA fraction derived from the above step 2);
4) The difference between the expression level of the MGC2742 gene measured in the above step 3) between the total RNA fraction derived from the above step 1) and the total RNA fraction derived from the above step 2) is analyzed, and A step of determining a therapeutic and / or preventive effect on cancer.

9.2 Method Using MGC2742 A screening method using measurement of the expression level of MGC2742 includes the following steps for a method using mammalian cultured cells and a method using animal individuals. The measurement of the expression level of the MGC2742 gene and the measurement of the MGC2742 in each step are described in the above section “7. Method for detecting cancer”.

A) Method using cultured cells derived from mammals A-1) Including the following steps 1) and 2):
1) a step of measuring the expression level of MGC2742 in cultured cells derived from a mammal cultured in a medium to which a test substance is added;
2) Analyze the difference between the expression level of MGC2742 measured in 1) and the expression level of the protein in cultured cells derived from mammals cultured in a medium to which the test substance was not added, and analyzed the therapeutic effect of the test substance on cancer and / or A step of determining a preventive effect;

A-2) including the following steps 1) to 3):
1) a step of measuring the expression level of MGC2742 in cultured cells derived from a mammal cultured in a medium to which a test substance is added;
2) a step of measuring the expression level of MGC2742 in cultured cells derived from mammals cultured in a medium to which no test substance is added;
3) analyzing the difference between the expression level of MGC2742 measured in the above step 1) and the expression level of MGC2742 measured in the above step 2) to determine the therapeutic effect and / or preventive effect of the test substance on cancer .

A-3) Including the following steps 1) to 3):
1) immobilizing all proteins obtained from cultured cells derived from mammals cultured in a medium to which a test substance is added;
2) detecting the expression level of MGC2742 of the present invention in the immobilized protein using an antibody or ligand that specifically binds to the protein;
3) The difference between the expression level of MGC2742 detected in the above 2) and the expression level of the protein in the total protein obtained from cultured cells derived from mammals cultured in a medium without the test substance was analyzed, and the cancer of the test substance was analyzed. Determining the therapeutic and / or prophylactic effect of the drug.

A-4) Including the following steps 1) to 5):
1) immobilizing all proteins obtained from cultured cells derived from mammals cultured in a medium to which a test substance is added;
2) immobilizing all proteins obtained from cultured cells derived from mammals cultured in a medium to which no test substance is added;
3) a step of measuring the expression level of MGC2742 in the solid-phased protein according to 1) above using an antibody or a ligand that specifically binds to the protein;
4) a step of measuring the expression level of MGC2742 in the immobilized protein according to 2) above, using an antibody or a ligand that specifically binds to the protein;
5) analyzing the difference between the expression level of the protein measured in 3) and the expression level of the protein measured in 4) to determine the therapeutic and / or prophylactic effect of the test substance on cancer.

B) Method using a mammalian individual B-1) comprising the following steps 1) and 2):
1) a step of detecting the expression level of MGC2742 in a specimen collected from a mammalian individual to which the test substance has been administered, using an antibody or a ligand that specifically binds to the protein;
2) Analyze the difference between the expression level of MGC2742 detected in 1) and the expression level of the protein in a sample collected from a mammal individual to which the test substance was not administered, and analyzed the therapeutic effect of the test substance on cancer and / or A step of determining a preventive effect;

B-2) Including the following steps 1) to 3):
1) a step of measuring the expression level of MGC2742 in a sample collected from a mammalian individual to which the test substance has been administered;
2) a step of measuring the expression level of the protein in a sample collected from a mammal individual to which the test substance has not been administered;
3) analyzing the difference between the expression level of MGC2742 measured in the above step 1) and the expression level of MGC2742 measured in the above step 2) to determine the therapeutic effect and / or preventive effect of the test substance on cancer .

B-3) Including the following steps 1) to 3):
1) immobilizing all proteins in a sample collected from a mammal individual to which the test substance has been administered;
2) a step of detecting the expression level of MGC2742 in the immobilized protein using an antibody or ligand that specifically binds to the protein;
3) Analyze the difference between the expression level of MGC2742 detected in 2) and the expression level of the protein in a sample collected from a mammal individual to which the test substance has not been administered, and treat the test substance for cancer and / or A step of determining a preventive effect;

B-4) Including the following steps 1) to 5):
1) immobilizing all proteins in a sample collected from a mammal individual to which the test substance has been administered;
2) immobilizing all proteins in a sample collected from a mammal individual to which the test substance has not been administered;
3) a step of detecting the expression level of MGC2742 in the solid-phased protein according to 1), using an antibody or a ligand that specifically binds to the protein;
4) a step of detecting the expression level of MGC2742 in the solid-phased protein according to 2) above, using an antibody or a ligand that specifically binds to the protein;
5) analyzing the difference between the expression level of MGC2742 detected in 3) and the expression level of MGC2742 detected in 4), and determining the therapeutic and / or prophylactic effect of the test substance on cancer.

9.3 Other methods A) The incidence of cancer, the size of cancer, and / or the survival rate over time when a test substance is administered to a mammal individual overexpressing MGC2742 and when no test substance is administered. Measure etc. The incidence of cancer is significantly reduced in the mammal to which the test substance is administered, the size of the cancer is significantly smaller, and / or the survival rate is about 10% or more, preferably about 30% or more, more preferably When the test substance increases by about 50% or more, the test substance can be selected as a compound having a therapeutic and / or prophylactic effect on cancer against cancer.

10. Search for Directly Interacting Substances Another embodiment of the present invention includes a drug design technique based on the three-dimensional structure of the protein for the purpose of obtaining a substance that suppresses the activity of MGC2742. . Such a technique is known as a rational drug design method, and is used for searching for a compound that efficiently inhibits or activates functions such as enzyme activity and binding to a ligand, cofactor, or DNA. ing. As an example of this, inhibitors of proteases, which are already marketed anti-HIV agents, are well known. In the three-dimensional structure analysis of the MGC2742 of the present invention, it is considered that generally well-known techniques such as X-ray crystallography and nuclear magnetic resonance can be used. Furthermore, in order to search for a substance that suppresses the function of MGC2742, a design utilizing computer drug design (CADD) is also possible. As this example, a low molecular weight compound (International Patent Application Publication No. WO 99/58515) which inhibits the function of AP-1 which is expected as a new genomic drug for the treatment of rheumatoid arthritis is known. By such a method, it is possible to obtain a substance which directly binds to MGC2742 or inhibits the interaction between MGC2742 and other factors, thereby suppressing the function of MGC2742.

  Further, another embodiment relates to a polypeptide to which the MGC2742 of the present invention associates, that is, a partner protein of the MGC2742. That is, the present invention relates to a method for screening a partner protein that regulates the activity of MGC2742.

  One embodiment of this screening method includes a step of bringing a test protein sample into contact with MGC2742 and selecting a protein that binds to MGC2742. As such a method, for example, a method of performing affinity purification of a protein that binds to MGC2742 is used. An example of a specific method is as follows. A sequence obtained by fusing a sequence consisting of six histidines with MGC2742 as an affinity tag is prepared, and this is extracted from a cell (a nickel-agarose column is charged in advance, and this column is charged). (Passed fraction) at 4 ° C. for 12 hours, and then separately add a nickel-agarose carrier to the mixture and incubate at 4 ° C. for 1 hour. After sufficiently washing the nickel-agarose carrier with a washing buffer, 100 mM imidazole is added to elute and purify the protein in the cell extract specifically binding to MGC2742, and the structure is determined. In this way, a protein that directly binds to MGC2742 and a protein that has no binding activity to MGC2742, but forms a complex with a protein that directly binds to MGC2742 as a subunit to thereby indirectly bind to MGC2742 can be purified. [Experimental Medicine Separate Volume, Bio Manual Series 5, “Transcription Factor Research Methods”, pp. 215-219 (published by Yodosha)].

  As another method, a far western blot method [Experimental Medicine Separate Volume, “New Genetic Engineering Handbook”, pp76-81 (published by Yodosha)] and a two-hybrid system method using yeast or mammalian cells [Experimental Medicine Separate Volume, Cloning by "New Genetic Engineering Handbook" pp66-75 (published by Yodosha), "Checkmate Manmarian Two Hybrid System" (promega)] is also possible, but is not limited to these methods.

  If a cDNA of a partner protein that interacts directly or indirectly with MGC2742 is obtained in this way, it can be used for functional screening for a substance that inhibits the interaction between MGC2742 and the partner protein. Specifically, for example, a fusion protein of MGC2742 and glutathione S-transferase is prepared, bound to a microplate covered with an anti-glutathione S-transferase antibody, and then the biotinylated partner protein is combined with this fusion protein. After contact, the binding to the fusion protein is detected with streptavidinated alkaline phosphatase. When the biotinylated partner protein is added, a test substance is also added, and a substance that promotes or inhibits the binding between the fusion protein and the partner protein is selected. According to this method, a substance that directly acts on the fusion protein or a substance that directly acts on the partner protein is obtained.

  If the binding between the fusion protein and the partner protein is indirect and mediated by some other factor, the above assay is performed in the same manner, for example, in the presence of a cell extract containing the factor. In this case, a substance that acts on the factor may be selected.

  In addition, when the obtained partner protein has an activity of suppressing the function of MGC2742, an anticancer agent, for example, treatment or prevention of prostate cancer, according to the test method using the MGC2742 gene expression vector described above. A candidate substance useful as an agent can be screened. In addition, when the obtained partner protein has an activity of suppressing the function of MGC2742, the polynucleotide having a nucleotide sequence encoding such an inhibitor can be used for cancer gene therapy. .

  Such a polynucleotide can be obtained by, for example, analyzing the amino acid sequence of the identified inhibitor, synthesizing an oligonucleotide probe consisting of a nucleotide sequence encoding the amino acid sequence, and screening a cDNA library or genomic library. Can be obtained. When the peptide having the activity of inhibiting the function of MGC2742 is derived from a randomly synthesized artificial peptide library, a DNA consisting of a nucleotide sequence encoding the amino acid sequence of the peptide is chemically synthesized.

  In gene therapy, a gene encoding the inhibitor thus obtained is incorporated into, for example, a viral vector, and a virus (detoxified) having the recombinant viral vector is transmitted to a patient. Since an anticancer factor is produced in the patient and has a function of suppressing the growth of cancer cells, cancer can be treated.

  Methods for introducing a gene therapy agent into cells include a gene transfer method using a virus vector and a non-viral gene transfer method (Nikkei Science, April 1994, pp. 20-45, Experimental Medicine Special Edition, 12 (15) (1994), Experimental Medicine Supplement “Basic Technology for Gene Therapy”, Youtosha (1996)).

  As a method for gene transfer using a virus vector, for example, TR4 or mutant TR4 is encoded in a DNA virus or RNA virus such as retrovirus, adenovirus, adeno-associated virus, herpes virus, vaccinia virus, pox virus, polio virus, and simbis virus. And a method of incorporating and introducing DNA to be incorporated. Among them, a method using a retrovirus, an adenovirus, an adeno-associated virus, and a vaccinia virus is particularly preferable. Examples of non-viral gene transfer methods include a method of directly administering an expression plasmid intramuscularly (DNA vaccine method), a liposome method, a lipofectin method, a microinjection method, a calcium phosphate method, and an electroporation method. The vaccine method and the liposome method are preferred.

  Further, in order for a gene therapy agent to actually act as a medicine, an in vivo method of directly introducing DNA into the body, a method of removing certain cells from a human, introducing the DNA outside the body, and then introducing the cells into the body. There is an ex vivo method (Nikkei Science, April 1994, pp. 20-45, Monthly Pharmaceutical Affairs, 36 (1), 23-48 (1994), extra edition of experimental medicine, 12 (15) (1994) )).

  For example, when the gene therapy agent is administered by an in vivo method, it is administered by an appropriate administration route, such as vein, artery, subcutaneous, intradermal, intramuscular, etc., depending on the disease, condition and the like. When administered by an in vivo method, the gene therapy agent is generally used as an injection or the like, but if necessary, a conventional carrier may be added. In the case of a liposome or a membrane-fused liposome (Sendai virus-liposome or the like), a liposome preparation such as a suspension, a freezing agent, a centrifugal concentrated frozen agent and the like can be obtained.

  A nucleotide sequence complementary to the nucleotide sequence shown in nucleotide numbers 165 to 1970 of SEQ ID NO: 1 in the sequence listing or a nucleotide sequence complementary to a partial sequence of the sequence can be used for so-called antisense therapy. The antisense molecule is a DNA consisting of a nucleotide sequence of usually 15 to 30 mer, which is complementary to a part of the nucleotide sequence of nucleotide numbers 1 to 2279 of SEQ ID NO: 1 in the sequence listing, or a phosphorothioate, methylphosphonate or morpholino derivative thereof. And stable RNA derivatives such as 2′-O-alkyl RNA. Such antisense molecules can be introduced into cells by methods well known in the art of the present invention, such as by microinjection, liposome encapsulation, or expression using a vector having an antisense sequence. Such antisense therapy is useful for treating or preventing a disease caused by excessively increasing the activity of the protein encoded by the nucleotide sequence represented by nucleotide numbers 165 to 1970 of SEQ ID NO: 1 in the sequence listing.

  A composition useful as a medicine containing the antisense oligonucleotide can be produced by a known method such as mixing a pharmaceutically acceptable carrier. Examples of such carriers and manufacturing methods are described in Applied Antisense Oligonucleotide Technology (1998 Wiley-Liss, Inc.). The preparation containing the antisense oligonucleotide is itself or a mixture with an appropriate pharmacologically acceptable excipient, diluent, or the like, and is orally administered as a tablet, capsule, granule, powder, or syrup. Alternatively, it can be administered parenterally by injection, suppository, patch, or external preparation. These formulations include excipients (e.g., sugar derivatives such as lactose, sucrose, glucose, mannitol, sorbitol; starch derivatives such as corn starch, potato starch, alpha starch, dextrin; cellulose derivatives such as crystalline cellulose; Gum arabic; dextran; organic excipients such as pullulan; and silicate derivatives such as light anhydrous silicic acid, synthetic aluminum silicate, calcium silicate, magnesium metasilicate aluminate; phosphates such as calcium hydrogen phosphate; Inorganic excipients such as carbonates such as calcium; sulfates such as calcium sulfate; and lubricants (eg, metal stearate such as stearic acid, calcium stearate, magnesium stearate). Salt; talc; colloidal silica; beeswax; Boric acid; adipic acid; sulfates such as sodium sulfate; glycol; fumaric acid; sodium benzoate; DL leucine; lauryl sulfates such as sodium lauryl sulfate and magnesium lauryl sulfate; Silicic acids such as silicic acid hydrate; and the above-mentioned starch derivatives.), Binders (for example, hydroxypropylcellulose, hydroxypropylmethylcellulose, polyvinylpyrrolidone, macrogol, and the same as the above-mentioned excipients) And disintegrants (for example, cellulose derivatives such as low-substituted hydroxypropylcellulose, carboxymethylcellulose, carboxymethylcellulose calcium, and internally cross-linked sodium carboxymethylcellulose); Starch, celluloses such as sodium carboxymethyl starch, crosslinked polyvinylpyrrolidone, and emulsifiers (eg, colloidal clays such as bentonite and veegum); magnesium hydroxide, hydroxide Metal hydroxides such as aluminum; anionic surfactants such as sodium lauryl sulfate and calcium stearate; cationic surfactants such as benzalkonium chloride; and polyoxyethylene alkyl ethers and polyoxyethylene sorbitan fatty acids Esters, nonionic surfactants such as sucrose fatty acid esters, and stabilizers (paraoxybenzoic acid esters such as methylparaben and propylparaben; chlorobutanol, benzyl alcohol, phenylethyl) Alcohols such as alcohol; benzalkonium chloride; phenols, phenols such as cresol; thimerosal; dehydroacetic acid; and sorbic acid and the. ), Flavoring agents (for example, commonly used sweeteners, sour agents, flavors, etc.) and diluents can be used in a well-known method.

  As for the method of introducing the compound of the present invention into a patient, a colloid dispersion system can be used in addition to the above. The colloidal dispersion system is expected to have the effect of increasing the stability of the compound in vivo and the effect of efficiently transporting the compound to a specific organ, tissue or cell. Colloidal dispersions are not limited as long as they are commonly used, but are based on polymer complexes, nanocapsules, microspheres, beads, and lipids including oil-in-water emulsifiers, micelles, mixed micelles, and liposomes. Dispersed systems can be mentioned, and are preferably a plurality of liposomes and artificial membrane vesicles having an effect of efficiently transporting a compound to a specific organ, tissue or cell (Mannino et al., Biotechniques, 1988). , 6, 682; Blume and Cevc, Biochem. Et Biophys. Acta, 1990, 1029, 91; Lappalainen et al., Antiviral Res., 1994, 23, 119; Chonn and Cullis, Current Op. Biotech., 1995, 6, 698. ).

  Unilamellar liposomes, ranging in size from 0.2-0.4 μm, can encapsulate a significant proportion of the aqueous buffer containing macromolecules, and the compound is encapsulated in this aqueous inner membrane, Is transported to brain cells in a more active form (Fraley et al., Trends Biochem. Sci., 1981, 6, 77). The composition of the liposome is usually a complex of a lipid, especially a phospholipid, especially a phospholipid having a high phase transition temperature, with one or more steroids, especially cholesterol. Examples of lipids useful for liposome production include phosphatidyl compounds such as phosphatidylglycerol, phosphatidylcholine, phosphatidylserine, sphingolipids, phosphatidylethanolamine, cerebroside and ganglioside. Particularly useful are diacylphosphatidylglycerols, where the lipid moiety contains 14-18 carbon atoms, especially 16-18 carbon atoms, and is saturated (with a double bond within the 14-18 carbon atom chain). Lack). Representative phospholipids include phosphatidylcholine, dipalmitoyl phosphatidylcholine, and distearoyl phosphatidylcholine.

  Targeting colloidal dispersion systems, including liposomes, may be either passive or active. Passive targeting is achieved by taking advantage of the liposome's natural tendency to distribute to cells of the reticuloendothelial system of organs containing sinusoidal capillaries. Active targeting, on the other hand, includes, for example, protein coats of the virus (Morishita et al., Proc. Natl. Acad. Sci. (USA), 1993, 90, 8744), monoclonal antibodies (or appropriate binding portions thereof). Binding specific ligands, such as sugars, glycolipids or proteins (or appropriate oligopeptide fragments thereof) to liposomes, or to achieve distribution to organs and cell types other than the naturally occurring localized sites Examples of the method include modifying the liposome by changing the composition of the liposome. The surface of the targeted colloidal dispersion can be modified in various ways. In liposome-targeted delivery systems, lipid groups can be incorporated into the lipid bilayer of the liposome to maintain the target ligand in intimate association with the lipid bilayer. Various linking groups can be used to link the lipid chain to the targeting ligand. Target ligands that bind to specific cell surface molecules that are predominantly found on cells where delivery of the oligonucleotides of the invention are desired are, for example, (1) predominantly expressed by cells where delivery is desired Polyclonal or monoclonal antibodies that specifically bind to hormones, growth factors or suitable oligopeptide fragments thereof, or (2) antigenic epitopes predominantly found on target cells, which bind to specific cell receptors Or an appropriate fragment thereof (eg, Fab; F (ab ') 2). The two or more bioactive agents can also be complexed and administered within a single liposome. Agents that increase the intracellular stability and / or targeting of the contents can also be added to the colloidal dispersion.

  The dosage varies depending on symptoms, age, etc., but in the case of oral administration, the lower limit is 1 mg (preferably 30 mg) and the upper limit is 2000 mg (preferably 1500 mg) per dose. A lower limit of 0.1 mg (preferably 5 mg) and an upper limit of 1000 mg (preferably 500 mg) can be administered by subcutaneous injection, intramuscular injection or intravenous injection.

  Hereinafter, the present invention will be described with reference to examples, but the present invention is not limited to these examples. In the following examples, unless otherwise specified, each operation relating to gene manipulation is referred to as "Molecular Cloning" [Sambrook, J., Fritsch, EF and Maniatis, T., Cold Spring Harbor Laboratory Press, 1989 Published in the year], or when a commercially available reagent or kit is used, it was used according to the instructions of a commercially available product.

Example 1. Acquisition of human MGC2742 cDNA clone a) Isolation of total RNA A human prostate cancer cell line LNCaP (American Tissue Culture Collection ATCC No .: CRL-1740) was used in 10% fetal bovine serum (FCS: Hyclone). Were cultured in a 75 cm ² tissue culture flask (manufactured by Sumitomo Bakelite: MS-23250) using an RPMI1640 medium (manufactured by Asahi Techno Glass Co., Ltd.). The cells were cultured under conditions of 5% CO ₂ and 37 ° C. while taking care not to become confluent. During the logarithmic growth phase, the cells were peeled off from the tissue culture flask using a trypsin-EDTA solution (manufactured by Sigma) and collected. The part was transferred to a new tissue culture flask and subcultured. Isolation of total RNA was performed by collecting human prostate cancer cell line LNCaP in logarithmic growth phase and using TRIzol (Invitrogen) according to the attached protocol.

b) First-strand cDNA synthesis First-strand cDNA was synthesized from the total RNA obtained in Example 1a) using Omniscript Reverse Transcriptase (manufactured by QIAGEN) according to the attached protocol. The reaction was performed in a volume of 20 μl.

c) PCR reaction As primer for amplifying MGC2742 cDNA by PCR
5'-caccatgcccgagagggagctgt-3 '(Primer 1: SEQ ID NO: 3 in Sequence Listing)
and,
5'-ctactccttcaacagtccca-3 '(Primer 2: SEQ ID NO: 4 in Sequence Listing)
An oligonucleotide having the sequence was synthesized according to a conventional method. The primer 1 is an oligonucleotide having 4 bases and a CACC added as a Kozak sequence upstream of the start codon of the MGC2742 gene, and 4 bases on the 5 ′ side of the nucleotide sequence consisting of nucleotide numbers 165 to 183 of SEQ ID NO: 1 in the sequence listing. An oligonucleotide consisting of a base sequence to which a sequence (CACC) is added. This CACC sequence forms a complementary chain with the 3 'end of the vector upon integration into the cloning vector pENTR / SD / D-TOPO, thereby enabling integration into the vector maintaining the orientation of the gene. Primer 2 is an oligonucleotide comprising a complementary strand of a nucleotide sequence consisting of nucleotide numbers 1951 to 1970 of SEQ ID NO: 1 in the sequence listing.

  The PCR reaction was performed using ProofStart DNA Polymerase (Qiagen) according to the attached protocol. Specifically, 1 μl of the obtained first-strand cDNA was added with 0.5 μl each of synthetic primer 1 and synthetic primer 2 at a concentration of 100 pmol / μl, 5 μl of 10X ProofStart PCR Buffer, 1.5 μl of 10 mM dNTP Mix, and 1.5 μl of ProofStart DNA Polymerase. Then, 10 μl of 5 × Q-solution and 29.5 μl of sterilized purified water were added to prepare a 50 μl PCR reaction solution. The PCR reaction was performed by GeneAmp PCR System 9700 (manufactured by Applied Biosystems). After heating at 95 ° C for 5 minutes, a temperature cycle of 94 ° C for 30 seconds, 57 ° C for 30 seconds and 72 ° C for 4 minutes was repeated 35 times, and finally, the temperature was kept at 72 ° C for 20 minutes, and then 4 ° C. Saved. The target cDNA was electrophoresed on a 1% agarose gel to confirm the amplification of MGC2742 cDNA (1806 bp), and then the DNA was purified from the agarose gel by using QIAquick Gel Extraction Kit (manufactured by Qiagen) according to the protocol attached thereto. Purified. The concentration of the purified MGC2742 cDNA was measured with an absorptiometer (Gene Spec I: manufactured by Hitachi Keiki Service Co., Ltd.).

d) Cloning of MGC2742 cDNA into pENTR / SD / D-TOPO vector MGC2742 cDNA obtained by Example 1c) was obtained using pENTR Directional TOPO Cloning Kits (manufactured by Invitrogen) according to the attached protocol, and pENTR / SD / D-TOPO. Cloned into vector. MGC2742 cDNA was mixed with pENTR / SD / D-TOPO vector conjugated with Topoisomerase in a reaction buffer provided with the kit, and incubated at room temperature for 5 minutes. Escherichia coli OneShot TOP10 Chemically Competent E.C. E. coli (Invitrogen) was transformed and cultured on an LB agar medium containing 50 μg / ml kanamycin. As a result, Escherichia coli colonies that had grown showing kanamycin resistance were selected and cultured overnight at 37 ° C. in 0.3 ml of liquid LB medium containing 50 μg / ml of kanamycin, and BIO ROBOT 9600 (manufactured by Qiagen) was obtained. Plasmid DNA was isolated and purified by utilizing it. The obtained plasmid DNA was subjected to nucleotide sequence analysis by ABI PRISM 3700 DNA Analyzer (manufactured by Applied Biosystems), and cDNA having Open Reading Frame of the nucleotide sequence shown in GenBank Accession No. BC000765 (SEQ ID NO: in the sequence listing) It was confirmed that 1) was incorporated into the pENTR / SD / D-TOPO vector.

e) Cloning into retrovirus vector pLNCX DNA fragment, Reading Frame Cassette A, contained in GATEWAY Vector Conversion System (manufactured by Invitrogen), and Retrovirus vector pLNCX, contained in RetroXpress System (manufactured by Clontech), pLHCI (Fig. 1) The site was inserted in the forward direction with respect to the CMV promoter on the plasmid to prepare a modified vector pLNCX-GW. Using LR CLONASE Enzyme Mix (manufactured by Invitrogen) according to the attached protocol, a recombinant vector was prepared by transferring MGC2742 cDNA cloned in the pENTR / SD / D-TOPO vector to pLNCX-GW. Escherichia coli OneShot TOP10 Chemically Competent E.C. E. coli was transformed and cultured on LB agar medium containing 50 μg / ml ampicillin. As a result, Escherichia coli colonies that showed resistance to ampicillin were selected and cultured overnight at 37 ° C. in 0.3 ml of liquid TBG medium containing 50 μg / ml of ampicillin. The cells were cultured overnight in an LB medium containing 50 μg / ml of ampicillin, and the plasmid was purified from the culture using Endo Free Plasmid Maxi Kits (Qiagen) according to the attached protocol. This plasmid was designated as pLNCX-GW-MGC2742. It was confirmed that the inserted gene of this plasmid was MGC2742 cDNA by assaying the length of the DNA fragment after digestion with a restriction enzyme by agarose electrophoresis.

Example 2 Preparation of MGC2742 Gene-Expressing Retrovirus and Establishment of MGC2742 Stable Expression Cell Line a) Cell line and its subculture packaging cell line 293-10A1 (manufactured by IMGENEX) and mouse fibroblast cell line NIH3T3 (American Tissue Culture) Collection was cultured using RPMI1640 medium (manufactured by Asahi Techno Glass) containing 10% fetal calf serum (FCS: manufactured by Hyclone) using a 25, 75 to 225 cm ² tissue culture flask (manufactured by Corning Costar or (Sumitomo Bakelite). The cells were cultured under conditions of 5% CO ₂ and 37 ° C. while taking care not to become confluent. During the logarithmic growth phase, the cells were peeled off from the tissue culture flask using a trypsin-EDTA solution (manufactured by Sigma) and collected. The part was transferred to a new tissue culture flask and subcultured.

b) Preparation of Gene-Expressing Retrovirus and Establishment of Stable Expression Strain 2 × 10 ⁶ 293-10A1 cells were seeded on a 10 cm-diameter tissue culture dish (manufactured by Asahi Techno Glass Co.) coated with type I collagen and 10 ml of 10 The cells were cultured overnight in RPMI1640 medium containing% FCS. After replacing the culture supernatant with 2 ml of fresh RPMI1640 medium, about 10 μg of the pLNCX-GW-MGC2742 vector was transfected using Lipofectamine 2000 (manufactured by Invitrogen) according to the attached protocol. After culturing for 6 hours, 6 ml of RPMI1640 medium containing 20% FCS was added, and the cells were further cultured overnight. The culture solution was replaced with a fresh 20% FCS-containing RPMI 1640 medium, and the cells were further cultured for 24 hours to produce a virus. The culture supernatant containing the virus was collected, filtered through a 0.45 μm pore size filter (MILLEX-HV: manufactured by Millipore), and the filtrate was doubled in volume with fresh 10% FCS-containing RPMI1640 medium and a final concentration of 8 μg / ml. Was added and mixed to prepare a virus infectious solution. This virus infection solution was added to a tissue culture dish (430293: manufactured by Corning Costar) in which 1.2 × 10 ⁶ NIH3T3 cells were sowed and cultured overnight, and the cells were infected with the virus. This infection procedure was repeated four times every 12 hours. Further, after culturing for 3 days, in order to remove non-infected cells, that is, cells having no expression of the target gene, the culture solution was replaced with RPMI1640 medium containing 10% FCS containing Geneticin (manufactured by Invitrogen) at 500 μg / ml. Subsequently, once every 2 to 3 days, the culture solution was replaced with a fresh Geneticin-containing medium, and the culture was continued for 7 days, thereby establishing a cell line stably expressing the target gene. Expression of the target gene in the present cell line is determined by a primer set capable of amplifying a fragment inserted into pLNCX-GW,
5'-ccaaaatgtcgtaacaactc-3 '(Primer 3: SEQ ID NO: 5 in Sequence Listing)
5'-gaccttgatctgaacttctc-3 '(Primer 4: SEQ ID NO: 6 in Sequence Listing)
Was confirmed by RT-PCR. Primers 3 and 4 are oligonucleotides designed based on the pLNCX nucleotide sequence, and can amplify a DNA fragment inserted into pLNCX.

Example 3 Focus formation test The MGC2742 gene stable expression strain established in Example 2 and the NIH3T3 cell line not transfected were recovered by trypsin-EDTA treatment, washed twice with a fresh culture solution, and then washed with a fresh culture solution. The cell suspension was prepared and dispensed into a 96-well plate (Corning-Costar 3598) in a volume of about 1,000 cells per well in a volume of 100 μl. Six similar wells were prepared. After culturing for 14 days at 37 ° C. and 5% CO _{2, the} number of focuses per well was measured.

  As a result, in the NIH3T3 cell line into which the control gene was not introduced, only 0.5 ± 0.5 (mean ± standard deviation) focuses could be detected per well, whereas MGC2742 was expressed. 34.0 ± 3.1 (mean ± standard deviation) foci were detected in the recovered cells (Table 1), strongly suggesting that MGC2742 has an activity to significantly induce the focus trait.

Example 4 Colony formation test The established MGC2742 gene stably expressing strain and the non-transgenic parent strain as a control were recovered by trypsin-EDTA treatment, washed twice with a fresh medium, and 50,000 cells per well. And warmed to about 38-39 ° C., suspended in 1 ml of liquefied RPMI 1640 containing 0.33% Bactogar and 20% FCS, and quickly suspended in advance with RPMI 1640 containing 0.66% Bactogar (manufactured by Difco) and 20% FCS. The whole amount was dispensed into a dispensed and solidified 12-well plate (3512: manufactured by Corning Coster). Three similar wells were prepared. After standing at room temperature for 30 minutes to completely solidify the Bactogar, the cells were cultured at 37 ° C. and 5% CO ₂ for 17 days, and the number of colonies growing in the soft agar was measured. Microscopic observation after culture The image (DIAPHOTO300 manufactured by Nikon Corporation) is shown in FIG. In the control NIH3T3 parent strain, only 2.0 ± 2.0 (mean ± standard deviation) colonies could be detected per well, whereas in the cells expressing MGC2742, 225.0 ± 53.1 (mean). ± standard deviation) colonies were detected (Table 2), strongly suggesting that MGC2742 has an activity to induce colony formation.

Example 5 Spheroid Proliferation Test The established MGC2742 gene stable expression strain and the NIH3T3 parent strain into which no gene was introduced were recovered by trypsin-EDTA treatment, washed twice with fresh culture solution, and then washed with fresh culture solution. The suspension was prepared and dispensed into a non-cell-adherent 96-well plate (Spheroid 96U: MS-0096S manufactured by Sumitomo Bakelite Co., Ltd.) in a volume of about 1,000 cells per well in a volume of 200 μl. Three similar wells were prepared. The cells were cultured under the conditions of 37 ° C. and 5% CO ₂ , and the spherical cell mass (spheroid) formed on the plate 1, 4, 5, 6, 7, 8, and 11 days after the start of the culture was collected using a microscope (manufactured by Nikon Corporation). Under DIAPHOTO300), the diameter was measured using an eyepiece micrometer (manufactured by Sankeisha: S-6).
The results are shown in Table 3 and FIG. In the control parent strain, the spheroid diameter remained constant, and no growth ability was observed in the spheroid state on the non-adherent plate, whereas the cells expressing MGC2742 showed a remarkable spheroid diameter. Increase over time was observed. This strongly suggested that MGC2742 induces proliferation of NIH3T3 cells in a spheroid state.

Example 6 Growth Inhibition of Human Prostate Cancer Cell Line LNCaP by siRNA (1) Cell Line and Subculture thereof The human prostate cancer cell line LNCaP (American Tissue Culture Collection ATCC No .: CRL-1740) was cultured in 10% fetal bovine serum ( Using an RPMI 1640 medium (manufactured by Asahi Techno Glass) containing FCS (manufactured by Hyclone), pay attention not to become confluent in a 75 cm ² tissue culture flask (manufactured by Sumitomo Bakelite; MS-23250) so as not to become confluent. The cells were cultured under the conditions of% CO ₂ and 37 ° C., transferred to a new tissue culture flask using a trypsin-EDTA solution (manufactured by Sigma) during the logarithmic growth phase, and subcultured.

(2) Growth inhibition test and gene expression suppression test of human prostate cancer cell line LNCaP by siRNA Poly-D-lysine coated tissue culture 24-well Petri dish (Poly-D-Lysine Cellware 24-Well Plate: manufactured by Becton Dickinson) 40,000 human prostate cancer cell lines LNCaP were seeded and cultured overnight in 0.4 ml of RPMI1640 medium containing 10% FCS. After the culture supernatant was replaced with 0.4 ml of RPMI1640 medium, the culture supernatant was further replaced with 0.2 ml of RPMI1640 medium containing siRNA homologous to any of the genes, 200 nM and 1.2% DMRIE-C Reagent (Invitrogen). By culturing for 4 hours, the siRNA was introduced into the cells.

Here, the nucleotide sequence of the siRNA for MGC2742 is homologous to nucleotide numbers 1081 to 1099 of SEQ ID NO: 1 in the sequence listing, and the following sequence in the sequence listing:
5'-GAUGAGCCUGGACUCCACUTT-3 '(SEQ ID NO: 7 in the sequence listing) and
5'-AGUGGAGUCCAGGCUCAUCTT-3 '(SEQ ID NO: 8 in the sequence listing).

The culture solution was replaced with 0.4 ml of fresh RPMI1640 medium containing 10% FCS and cultured overnight. The cells were collected using a trypsin-EDTA solution (manufactured by Sigma), suspended in 2 ml of RPMI1640 medium containing 10% FCS, and then dispersed in a 96-well plate (Catalog No. 3598 or 3917 manufactured by Corning Coaster Co., Ltd.) in 0.1 ml portions. The cells were poured and cultured under conditions of 5% CO ₂ and 37 ° C. Twenty-four hours and six days after the introduction of the siRNA into the cells, the amount of intracellular ATP was measured using CellTiter-Glo ^™ Luminescent Cell Viability Assay (promega) according to the attached protocol. The rate of change, ie, the rate of cell proliferation, was calculated. As a negative control that does not suppress cell growth, siRNA against a luciferase gene that does not exist in humans was used, and as a positive control that suppresses cell growth, siRNA against an Eg5 gene, which is a human essential gene, was used.

The nucleotide sequence of the siRNA for luciferase is as follows:
5'-CGUACGCGGAAUACUUCGATT-3 '(SEQ ID NO: 9 in the sequence listing),
5'-UCGAAGUAUUCCGCGUACGTT-3 '(SEQ ID NO: 10 in the sequence listing).
The nucleotide sequence of the siRNA against human Eg5 is as follows:
5'-CUGGAUCGUAAGAAGGCAGTT-3 '(SEQ ID NO: 11 in the sequence listing),
5'-CUGCCUUCUUACGAUCCAGTT-3 '(SEQ ID NO: 12 in Sequence Listing).
Two days after the introduction of the siRNA into the cells, total intracellular RNA was extracted using the RNeasy Mini Kit (Qiagen) according to the attached protocol. The expression level of the target gene was measured for the obtained total RNA using QuantiTect SYBR Green RT-PCR Kit (manufactured by Qiagen) according to the attached protocol. Specifically, an oligonucleotide DNA capable of specifically amplifying the internal sequence of the gene of interest was used for the measurement of the expression level. The expression level of the human Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene in the total RNA obtained by the same method was measured. The ratio of the expression level of the target gene to the human GAPDH gene level was calculated, and the rate of change in the gene expression level was determined based on this numerical value.

RT-PCR primers for amplification of MGC2742 have the following sequence:
5′-ACTGACGAGTCTGAGCCAGC-3 ′ (Primer 5: SEQ ID NO: 13 in Sequence Listing),
5'-TGTCCTTGGCGTTCGTCCTT-3 '(primer 6: SEQ ID NO: 14 in the sequence listing).
RT-PCR primers for human GAPDH amplification have the following sequence:
5'-GAAGGTGAAGGTCGGAGTC-3 '(Primer 7: SEQ ID NO: 15 in Sequence Listing)
5'-GAAGATGGTGATGGGATTTC-3 '(Primer 8: SEQ ID NO: 16 in Sequence Listing).
Table 1 shows the results.

Eg5, a human essential gene used as a positive control, assuming that the cell growth rate in the 5 days from 1 to 6 days after the introduction of the siRNA corresponding to the luciferase gene sequence into the human prostate cancer cell line LNCaP is 100%. When siRNAs corresponding to the gene sequence and siRNAs corresponding to MGC2742 gene sequences 1, 2, and 3 were used, 25.9% ± 1.1% and 45.6% ± 1.6% (mean ± standard deviation (SD)), respectively. The cell proliferation rate decreased. In the measurement of the gene expression amount performed two days after the introduction of the siRNA, the expression amount using the siRNA corresponding to the sequence of the target gene MGC2742 was 100% when the gene expression amount when the siRNA corresponding to the luciferase gene sequence was introduced was 100%. , Respectively, was suppressed to 12%. It was suggested that suppressing the expression of the target gene MGC2742 would suppress the growth of the human prostate cancer cell line LNCaP.

Example 7 Growth Inhibition of Human Cancer Cell Line LNCaP by siRNA (1) Cell Line and Subcultured Human Prostate Cancer Cell Line LNCaP (American Tissue Culture Collection ATCC No .: CRL-1740), PC-3 (American Tissue Culture) Collection ATCC No .: CRL-1435), WI-38 VA-13 subline 2RA (VA13: American Tissue Culture Collection ATCC No .: CCL-75.1), A549 (American Tissue Culture Collection ATCC No .: CCL-185), HCT116 (American Tissue Culture Collection ATCC No .: CCL-247), MCF7 (American Tissue Culture Collection ATCC No .: HTB-22), ACHN (American Tissue Culture Collection ATCC No .: CRL-1611) with 10% fetal bovine serum ( Using an RPMI 1640 medium (manufactured by Asahi Techno Glass) containing FCS (manufactured by Hyclone), pay attention not to become confluent in a 75 cm ² tissue culture flask (manufactured by Sumitomo Bakelite; MS-23250) so as not to become confluent. Cultured at 37 ° C,% CO ₂ , logarithmic growth During the period, the cells were transferred to a new tissue culture flask using a trypsin-EDTA solution (manufactured by Sigma) and subcultured.

(2) Growth inhibition test and gene expression suppression test using siRNA for human cancer cell lines ⁴ × 10 ⁴ cancer cell lines LNCaP, PC-3, VA13, A549, HCT116, MCF7, and ACHN were seeded at a time and cultured overnight in 0.4 ml of RPMI1640 medium containing 10% FCS. After the culture supernatant was replaced with 0.4 ml of RPMI1640 medium, the culture supernatant was further replaced with 0.2 ml of RPMI1640 medium containing siRNA homologous to any of the genes, 200 nM and 1.2% DMRIE-C Reagent (Invitrogen). By culturing for 4 hours, the siRNA was introduced into the cells.

  Here, the same siRNA as in Example 6 was used for MGC2742.

The culture solution was replaced with 0.4 ml of fresh RPMI1640 medium containing 10% FCS and cultured overnight. The cells were collected using a trypsin-EDTA solution (manufactured by Sigma), suspended in 2 ml of RPMI1640 medium containing 10% FCS, and then dispersed in a 96-well plate (Catalog No. 3598 or 3917 manufactured by Corning Coaster Co., Ltd.) in 0.1 ml portions. The cells were poured and cultured under conditions of 5% CO ₂ and 37 ° C. Twenty-four hours and six days after the introduction of the siRNA into the cells, the amount of intracellular ATP was measured using CellTiter-Glo ^™ Luminescent Cell Viability Assay (promega) according to the attached protocol. The rate of change, ie, the rate of cell proliferation, was calculated. As a negative control that does not suppress cell growth, siRNA against a luciferase gene that does not exist in humans was used, and as a positive control that suppresses cell growth, siRNA against an Eg5 gene, which is a human essential gene, was used.
The same siRNA as described in Example 6 was used for siRNA for luciferase and siRNA for human Eg5.
Table 5 shows the results.

Assuming that the cell growth rate over 5 days from the introduction of the siRNA corresponding to the luciferase gene sequence to LNCaP in the first to sixth days is 100%, the siRNA for the MGC2742 gene sequence is the same as the siRNA for the Eg5 gene sequence, which is an essential gene. While cell growth was suppressed to approximately the same level of 38% (growth inhibition rate: 62%), when introduced into PC-3, VA-13, A549, MCF7, and ACHN, the inhibitory effect was at most 11 to 11. 34%, suggesting that it is selective for prostate cancer LNCaP cells. These results indicate that when the expression or function of MGC2742 gene is inhibited in prostate cancer in which MGC2742 gene is highly expressed, its growth can be selectively suppressed.

Expression vector pLNCX. The colony formation figure by the MGC2742 expression strain. Spheroid proliferation test.

SEQ ID NO: 3: PCR primer for MGC2742 gene SEQ ID NO: 4: PCR antisense primer for MGC2742 gene SEQ ID NO: 5: Sense primer for RT-PCR for detecting MGC2742 SEQ ID NO: 6: Antisense for RT-PCR for detecting MGC2742 gene Primer SEQ ID NO: 7: siRNA sense strand for MGC2742 SEQ ID NO: 8: siRNA antisense strand for MGC2742 SEQ ID NO: 9: siRNA sense strand for luciferase SEQ ID NO: 10: siRNA antisense strand for luciferase SEQ ID NO: 11: siRNA for Eg5 Sense strand SEQ ID NO: 12: siRNA antisense strand for Eg5 SEQ ID NO: 13: RT-PCR sense primer 2 for MGC2742
SEQ ID NO: 14: RT-PCR antisense primer 2 for MGC2742
SEQ ID NO: 15: RT-PCR sense primer for GAPDH SEQ ID NO: 16: RT-PCR antisense primer for GAPDH

Claims (34)

A method for producing a cancer cell, comprising the following steps 1) and 2):
1) a step of transforming a cell with the polynucleotide of the following (i) or (ii);
(I) a polynucleotide having a nucleotide sequence represented by nucleotide numbers 165 to 1970 of SEQ ID NO: 1 in the sequence listing,
(Ii) a polynucleotide that hybridizes under stringent conditions to a polynucleotide consisting of a nucleotide sequence complementary to the polynucleotide of (i) above, and encodes a protein substantially identical to MGC2742;
2) a step of selecting a cell line in which the cell growth ability has changed in step 1); The method for producing a cancer cell according to claim 1, wherein the cell is an animal cell. The method for producing a cancer cell according to claim 2, wherein the animal cell is derived from a mammal. The method for producing cancer cells according to claim 3, wherein the mammal is a human, monkey, mouse, or rat. The method for producing cancer cells according to claim 3, wherein the mammal is a human. The method for producing a cancer cell according to claim 1, wherein the cell is a mouse fibroblast cell line NIH3T3 cell. The method for producing a cancer cell according to any one of claims 1 to 6, wherein the cell is transformed with a recombinant vector. A cancer cell obtained by the method for producing a cancer cell according to any one of claims 1 to 7. A non-human mammal into which the cancer cell according to claim 8 has been introduced. A method for detecting cancer, comprising the step of analyzing the expression level of the polynucleotide according to 1) or 2) in a sample collected from a subject or a test animal:
1) a polynucleotide having a nucleotide sequence represented by nucleotide numbers 165 to 1970 of SEQ ID NO: 1 in the sequence listing,
2) A polynucleotide that hybridizes under stringent conditions to a polynucleotide consisting of a nucleotide sequence complementary to the polynucleotide described in 1) above, and encodes a protein substantially identical to MGC2742. A method for detecting cancer, comprising a step of analyzing the expression level of the protein described in 1) or 2) below in a sample collected from a subject or a test animal:
1) a protein having an amino acid sequence represented by amino acid numbers 1 to 601 of SEQ ID NO: 2 in the sequence listing;
2) A protein substantially identical to MGC2742, comprising an amino acid sequence in which one or several amino acids have been deleted, substituted or added in the amino acid sequence of the protein described in 2) above. A method for detecting cancer, comprising the following steps 1) to 4):
1) a step of extracting a total RNA fraction from a specimen collected from a subject or a test animal;
2) a step of extracting a total RNA fraction from a specimen collected from a normal human or a normal animal;
3) a step of measuring the expression level of the polynucleotide according to the following (i) or (ii) in the total RNA fraction derived from the above step 1) and the total RNA fraction derived from the above step 2);
(I) a polynucleotide having a nucleotide sequence represented by nucleotide numbers 165 to 1970 of SEQ ID NO: 1 in the sequence listing,
(Ii) a polynucleotide consisting of a nucleotide sequence that hybridizes with a polynucleotide consisting of a nucleotide sequence complementary to the polynucleotide described in (i) above under stringent conditions and encodes a protein substantially identical to MGC2742 ;
4) The difference in the expression level of the polynucleotide measured in the above step 3) between the total RNA fraction derived from the above step 1) and the total RNA fraction derived from the above step 2) is analyzed, and the above step 1) is analyzed. 2. The step of detecting cancer in a subject or a test animal according to the above. A method for detecting cancer, comprising the following steps 1) to 3):
1) a step of measuring the expression level of the protein described in (i) or (ii) below in a sample collected from a subject or a test animal;
(I) a protein comprising an amino acid sequence represented by amino acid numbers 1 to 601 of SEQ ID NO: 2 in the sequence listing,
(Ii) a protein consisting of an amino acid sequence in which one or several amino acids have been deleted, substituted or added in the amino acid sequence of the protein described in (i) above, and which is substantially the same as MGC2742;
2) a step of measuring the expression level of the protein according to (i) or (ii) in the above step 1) in a sample collected from a normal human or a normal animal;
3) a step of analyzing the difference between the expression level of the protein measured in the above step 1) and the expression level of the protein measured in the above step 2) to detect cancer in a subject or a test animal. 14. The method for detecting cancer according to claim 10, wherein the cancer is prostate cancer. A method for screening a substance having a therapeutic and / or prophylactic effect on cancer, comprising the following steps 1) to 4):
1) a step of extracting a total RNA fraction from cultured cells derived from mammals cultured in a medium to which a test substance has been added;
2) a step of extracting a total RNA fraction from mammalian-derived cultured cells cultured without adding a test substance;
3) a step of measuring the expression level of the polynucleotide according to the following (i) or (ii) in the total RNA fraction derived from the above step 1) and the total RNA fraction derived from the above step 2);
(I) a polynucleotide having a nucleotide sequence represented by nucleotide numbers 165 to 1970 of SEQ ID NO: 1 in the sequence listing,
(Ii) a polynucleotide consisting of a nucleotide sequence that hybridizes with a polynucleotide consisting of a nucleotide sequence complementary to the polynucleotide described in (i) above under stringent conditions and encodes a protein substantially identical to MGC2742 ;
4) The difference in the expression level of the polynucleotide measured in the above step 3) between the total RNA fraction derived from the above step 1) and the total RNA fraction derived from the above step 2) is analyzed. A step of determining a therapeutic effect and / or a preventive effect on cancer. A method for screening a substance having a therapeutic and / or prophylactic effect on cancer, comprising the following steps 1) to 4):
1) a step of extracting a total RNA fraction from a specimen collected from a mammalian individual to which the test substance has been administered;
2) a step of extracting a total RNA fraction from a specimen collected from an animal to which the test substance has not been administered;
3) a step of measuring the expression level of the polynucleotide according to the following (i) or (ii) in the total RNA fraction derived from the above step 1) and the total RNA fraction derived from the above step 2);
(I) a polynucleotide having a nucleotide sequence represented by nucleotide numbers 165 to 1970 of SEQ ID NO: 1 in the sequence listing,
(Ii) a DNA consisting of a nucleotide sequence that hybridizes under stringent conditions to a polynucleotide consisting of a nucleotide sequence complementary to the polynucleotide described in (i) above and encodes a protein substantially identical to MGC2742;
4) Analyze the difference in the expression level of the polynucleotide measured in the above step 3) between the total RNA fraction derived from the above step 1) and the total RNA fraction derived from the above step 2), and analyze the test substance. A step of determining a therapeutic effect and / or a preventive effect on cancer. A method for screening a substance having a therapeutic and / or prophylactic effect on cancer, comprising the following steps 1) to 3):
1) a step of measuring the expression level of the protein described in the following (i) or (ii) in cultured cells derived from a mammal cultured in a medium to which a test substance is added;
(I) a protein comprising an amino acid sequence represented by amino acid numbers 1 to 601 of SEQ ID NO: 2 in the sequence listing,
(Ii) a protein consisting of the amino acid sequence of the protein according to (i), wherein one or several amino acids are deleted, substituted or added, and which is substantially the same as MGC2742;
2) a step of measuring the expression level of the protein according to (i) or (ii) in the above step 1) in cultured cells derived from a mammal cultured in a medium to which a test substance is not added;
3) Analyze the difference between the expression level of the protein measured in step 1) and the expression level of the protein measured in step 2) to determine the therapeutic and / or prophylactic effect of the test substance on cancer. Process. A method for screening a substance having a therapeutic and / or prophylactic effect on cancer, comprising the following steps 1) to 3):
1) a step of measuring the expression level of the protein described in the following (i) or (ii) in a specimen collected from a mammal individual to which the test substance has been administered;
(I) a protein comprising an amino acid sequence represented by amino acid numbers 1 to 601 of SEQ ID NO: 2 in the sequence listing,
(Ii) a protein consisting of an amino acid sequence in which one or several amino acids have been deleted, substituted or added in the amino acid sequence of the protein described in (i) above, and which is substantially the same as MGC2742;
2) a step of measuring the expression level of the protein according to (i) or (ii) in the above step 1) in a sample collected from a mammal individual to which the test substance has not been administered;
3) Analyze the difference between the expression level of the protein measured in step 1) and the expression level of the protein measured in step 2) to determine the therapeutic and / or prophylactic effect of the test substance on cancer. Process. The screening method according to claim 15 or 17, wherein the cultured mammalian cell is the cancer cell according to claim 8. 19. The screening method according to claim 16, wherein the mammal individual is the non-human mammal according to claim 9. The screening method according to any one of claims 15 to 19, wherein the cancer is prostate cancer. A kit for determining the therapeutic and / or prophylactic effect of a test substance on cancer and / or detecting cancer, comprising at least one or more selected from the group consisting of 1) to 3) below:
1) a continuous oligonucleotide primer having a length of 15 to 30 bases for specifically amplifying a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 1 in the sequence listing;
2) a continuous polynucleotide probe of 15 nucleotides or more for hybridizing to a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 1 in the sequence listing under stringent conditions and detecting the polynucleotide;
3) An immobilized sample on which a polynucleotide consisting of the nucleotide sequence shown by nucleotide numbers 165 to 1970 of SEQ ID NO: 1 in the sequence listing is immobilized. A kit for determining the therapeutic and / or prophylactic effect of a test substance on cancer and / or detecting cancer, comprising at least one of the following 1) and 2):
1) an antibody that specifically binds to a protein consisting of the amino acid sequence of SEQ ID NO: 2 in the sequence listing and detects the protein;
2) A secondary antibody capable of binding to the antibody according to 1). The method for measuring the expression level of a polynucleotide is a Northern blot method, a dot blot method, a slot blot method, an RT-PCR, a ribonuclease protection assay or a run-on assay, wherein: The method according to any one of 15, 16, 19 and 20. The method for measuring the expression level of a polynucleotide uses a gene chip or an array made of a DNA group consisting of a complementary DNA group derived from animal tissue or animal cell or a partial sequence of each DNA of the DNA group. 21. The method according to any one of items 10, 12, 14, 15, 16, 19 and 20. 22. The method according to any one of claims 11, 13, 14, 17, 18, 19, 20, and 21, wherein the method for measuring the expression level of the protein uses an antibody or a ligand that specifically binds to the protein. Item 2. The method according to item 1. The method for measuring the expression level of a protein is Western blotting, dot blotting, slot blotting, or enzyme-linked immunosorbent assay (ELISA), characterized in that it is characterized in that: , 19, 20, and 21. A pharmaceutical composition for treating and / or preventing cancer, comprising an oligonucleotide having a nucleotide sequence represented by nucleotide numbers 165 to 1970 of SEQ ID NO: 1 in the sequence listing or a nucleotide sequence complementary to a partial sequence of the sequence. A pharmaceutical composition for treating and / or preventing cancer, comprising an antibody that specifically recognizes MGC2742. A pharmaceutical composition for treating and / or preventing cancer, comprising an siRNA against the nucleotide sequence shown in SEQ ID NO: 1 of the Sequence Listing or a partial sequence thereof. 31. The siRNA, which is an siRNA comprising a combination of an oligonucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 7 in the sequence listing and an oligonucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 8 in the sequence listing. 8. The pharmaceutical composition for treating and / or preventing cancer according to the above. The pharmaceutical composition according to any one of claims 28 to 31, wherein the cancer is prostate cancer. A method for treating cancer, comprising using the pharmaceutical composition according to at least one selected from claims 28 to 32. The method for treating cancer according to claim 33, wherein the cancer is prostate cancer.