JP2007029019A - Diabetes-associated gene and its use - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for assaying the primary cause of diabetes, with which a marker gene useful for assaying the primary cause of diabetes is identified and the identified gene is used, a method for screening a compound having effect on prevention or treatment of diabetes, with which expression of the gene is used as an index and to obtain a medicine for preventing or treating diabetes. <P>SOLUTION: A marker gene useful for judging diabetes is newly identified in order to develop a method for accurately and simply detecting crisis of diabetes in early stages. The method for assaying the primary cause of diabetes is developed by using the expression level of the marker gene as an index. In particular, amounts of gene transcription expressed in bloods of normal rat and GK (Goto-Kakizaki) rat of type II diabetes are compared and a great number of genes recognized to have expression variation specifically to diabetes are selected. These genes are useful as marker genes for assaying the primary cause of diabetes. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a marker gene that can be used to test for a predisposition to diabetes, a method for testing a predisposition for diabetes using the gene, and a screening for a compound having an effect of preventing or treating diabetes using the expression of the gene as an index. Regarding the method.

  In recent years, the number of diabetic patients in Japan is rapidly increasing due to westernization of eating habits, changes in the environment, changes in stress and lifestyle habits. According to a survey conducted by the Ministry of Health, Labor and Welfare in 2004, there are approximately 7.4 million people who are strongly suspected of having diabetes, and more than 16.2 million people including those who cannot deny the possibility of diabetes (diabetes reserve army). Over the past five years, the number of people who are suspected of having increased diabetes has increased by 500,000, and it is estimated that it will exceed 10 million in 2015, assuming that the current upward trend will continue.

  Diabetes is steadily increasing due to the lack of subjective symptoms such as pain at the early stages of the onset and lack of superficial changes, and it is difficult for the patient to be aware of it. However, there are cases where the blood glucose level temporarily approaches a normal value in the early stage of the onset, and the treatment is interrupted because the patient himself is completely cured. However, if the state of high blood sugar level is neglected even if the symptoms are poor, nephropathy, retinopathy, neuropathy, etc., which are three major complications specific to diabetes, are caused, and cardiovascular diseases such as ischemic heart disease Promote the onset and progression of stroke. Diabetes is never cured once it develops, and these complications significantly reduce the patient's quality of life. Among the complications, the neuropathy that develops at the earliest stage results in the progression of gangrene due to sensory abnormalities such as pain in the limbs, numbness, and coldness, often leading to amputation. Currently, there are more than 30,000 patients undergoing artificial dialysis due to diabetic nephropathy, accounting for the largest number of dialysis patients. In addition, more than 4,000 people are blinded annually due to diabetic retinopathy, which is the leading cause of blindness in adults.

  In order to prevent such complications caused by diabetes, it is most important to detect diabetes early. Currently, diabetes is diagnosed by comprehensively determining the measurement results of a plurality of marker substances by combining blood tests, urine tests, and the like. Since this diagnostic method has a problem that it is difficult to detect early-onset or mild diabetes, several tests are required. For example, urine sugar is generally not detected unless the blood glucose level is about 160 to 180 mg / dL or more. Regarding the blood glucose level, there are cases where fasting blood glucose falls to a normal range at the early stage of onset of diabetes, and there is a high possibility that the patient will miss diabetes.

  In addition, the following techniques have been disclosed so far with regard to a method for diagnosing diabetes or a diagnostic marker for diabetes (see Patent Documents 1 to 20).

JP-A-9-140398 Japanese National Patent Publication No. 9-511398 Japanese Patent Laid-Open No. 11-221077 JP 2001-204476 A JP-A-8-103280 JP 2003-259870 A JP 2004-49033 A JP 2004-283086 A JP 2004-121245 A JP 2004-105167 A JP 2004-81156 A JP 2004-81196 A JP 2004-57199 A JP 2004-57169 A JP 2003-235573 A Special Table 2002-537775 JP 2004-215647 A JP 2003-245087 A WO98 / 54361 JP 2004-154136 A

  This invention is made | formed in view of such a condition, and it aims at identifying the marker gene which can be utilized for the test | inspection of the predisposition of diabetes. Furthermore, the present invention relates to a method for examining a predisposition to diabetes using the identified marker gene, a method for screening a compound having an effect of preventing or treating diabetes using the expression of the gene as an index, and prevention or treatment of diabetes The issue is to provide drugs for the purpose.

  The present inventors have intensively studied to develop a method that can accurately and easily detect the onset of diabetes at an early stage, and succeeded in newly identifying a genetic marker that can be used for the determination of diabetes. Furthermore, the present inventors have developed a test method for diabetes predisposition using the expression level of the gene marker as an index.

  Specifically, the amount of gene transcription expressed in the blood of normal rats and type II diabetes model GK (Goto-Kakizaki) rats was compared, and genes that showed expression changes specifically in diabetes were observed. A number of were selected. These genes are useful as genetic markers for testing for predisposition to diabetes.

  Furthermore, from these selected gene groups, genes that selectively change in pancreatic Langerhans islet and are also observed in type I diabetes model rats administered with streptozotocin (STZ), a drug that causes type I diabetes, are selected. did. By quantifying the expression levels of these multiple genes, it was possible to detect early diabetes tests and pre-diabetes that were difficult to detect with diagnostic methods such as blood tests.

  A diabetes marker gene developed by the present inventors, or a gene having high homology with the gene (homologue gene) can be used for early examination and prediction of diabetes in humans and other mammals. It is expected. Furthermore, by using the expression level of these genes as an index, it is possible to appropriately screen for compounds that can become pharmaceuticals, functional foods, and the like targeting diabetes.

  It is also possible to produce a diabetes model animal by artificially modifying the expression of the marker gene of the present invention in an experimental animal such as a mouse. It is also possible to carry out the above screening method by using the animal.

The present invention relates to a marker gene that can be used to test for a predisposition to diabetes, a method for testing a predisposition for diabetes using the marker gene, and a screening for a compound having an effect of preventing or treating diabetes using the expression of the gene as an index. More specifically, a method and a drug for preventing or treating diabetes,
[1] A test method for a predisposition to type II diabetes using the expression level of the gene according to any one of SEQ ID NOS: 1 to 8 and 10 to 27, or a homologous gene of the gene as an index,
[2] The test method according to [1], wherein when the gene expression level is the following (a) or (b), the test animal is determined to have a predisposition to type II diabetes:
(A) The expression level of the gene according to any one of SEQ ID NOs: 1 to 3, 7, 8, or 11 or a homologous gene of the gene is decreased as compared with the control (b) SEQ ID NOs: 4 to The expression level of the gene according to any one of 6, 10, 12 to 27 or a homologous gene of the gene is increased compared to the control [3] For the test animals, SEQ ID NOs: 3, 6, 7 Or a method for testing a predisposition to type I diabetes, using the expression level of the gene according to any one of 17 or a homologous gene of the gene as an index,
[4] The test method according to [3], wherein when the gene expression level is (a) or (b) below, the test animal is determined to have a predisposition to type I diabetes,
(A) The expression level of the gene described in SEQ ID NO: 3 or 7 or a homologous gene of the gene is reduced compared to the control (b) the gene described in SEQ ID NO: 6 or 17, or the gene [5] The test method according to any one of [1] to [4], wherein the expression level of the homologous gene is increased as compared to the control [5] A biological sample derived from the test animal is used as the test sample ,
[6] A diagnostic agent for predisposition to diabetes, comprising any of the following substances (a) to (d) as an active ingredient:
(A) SEQ ID NO: 1 of SEQ ID NO: 1-8, oligonucleotide which hybridizes to the DNA of the gene according to any one of 10 to 27 and has a chain length of at least 15 nucleotides (b) SEQ ID NO: of [1] : A solid phase on which a nucleotide probe that hybridizes with DNA of the gene according to any one of 1 to 8 and 10 to 27 is immobilized (c) SEQ ID NO: 1 to 8 or 10 to 27 of [1] Primer oligonucleotide for amplifying DNA of the gene described (d) [1] SEQ ID NO: 1 to 8, an antibody against the protein encoded by the gene according to any one of 10 to 27 [7] a test kit for a predisposition to type II diabetes, comprising any of a) to (d) as a constituent;
(A) SEQ ID NO: 1 of SEQ ID NO: 1-8, oligonucleotide which hybridizes to the DNA of the gene according to any one of 10 to 27 and has a chain length of at least 15 nucleotides (b) SEQ ID NO: of [1] : A solid phase on which a nucleotide probe that hybridizes with DNA of the gene according to any one of 1 to 8 and 10 to 27 is immobilized (c) SEQ ID NO: 1 to 8 or 10 to 27 of [1] Primer oligonucleotide (d) for amplifying the DNA of the described gene (d) [1] SEQ ID NO: 1 to 8, an antibody against the protein encoded by the gene according to any one of 10 to 27 [8] SEQ ID NO: A substance that enhances the expression of the gene according to any one of 1 to 3, 7, 8, 11 or a homologous gene of the gene, or the function of a protein encoded by the gene; A drug for treating or preventing type II diabetes, which is contained as an active ingredient,
[9] SEQ ID NOS: 4 to 6, 10, or a homologous gene of the gene or a substance that suppresses the function of the protein encoded by the gene as an active ingredient A drug for treating or preventing type II diabetes,
[10] Treatment of type I diabetes comprising, as an active ingredient, a substance that enhances the expression of the gene of SEQ ID NO: 3 or 7 or a homologous gene of the gene, or the function of a protein encoded by the gene, or Drugs for prevention,
[11] An agent of type I diabetes comprising a substance that suppresses the expression of the gene of SEQ ID NO: 6 or 17 or the expression of a homologous gene of the gene or the function of the protein encoded by the gene as an active ingredient Drugs for treatment or prevention,
[12] The drug according to [9] or [11], wherein the substance that suppresses gene expression is a compound selected from the group consisting of the following (a) to (c):
(A) an antisense nucleic acid for the transcription product of the gene or a part thereof (b) a nucleic acid having a ribozyme activity that specifically cleaves the transcription product of the gene (c) an action of inhibiting the expression of the gene by the RNAi effect Nucleic acid [13] The agent according to [9] or [11], wherein the substance that suppresses the function of the protein is the following compound (a) or (b):
(A) an antibody that binds to the protein (b) a low molecular weight compound that binds to the protein [14] The gene-modified diabetes, wherein the expression level of the gene according to [1] is artificially modified Model non-human animals,
[15] A compound having a therapeutic or prophylactic effect for type II diabetes (candidate compound for treatment or prevention), which comprises selecting a compound that changes the expression level of any of the genes according to [1] Screening method,
[16] A compound having a therapeutic or prophylactic effect for type I diabetes (candidate compound for treatment or prevention), which comprises selecting a compound that changes the expression level of any of the genes described in [3] Screening method,
[17] The screening method according to [15] or [16], comprising the following steps (a) to (c):
(A) a step of contacting a test compound with a cell expressing any one of the genes according to [1] or [3] (b) a step of measuring the expression level of the gene (c) non-test compound [18] The step of selecting a compound that changes the expression level of the gene as compared with the case where it is measured in the presence [18] The method according to [15] or [16], comprising the following steps (a) to (c): Screening method,
(A) contacting a test compound with a cell or a cell extract containing DNA having a structure in which the transcriptional regulatory region of any of the genes according to [1] or [3] and a reporter gene are functionally linked Step (b) Step of measuring the expression level of the reporter gene (c) Step of selecting a compound that changes the expression level of the reporter gene as compared to the case of measuring in the absence of the test compound [19] A screening method using a non-human animal, comprising the following steps (a) to (c): [15] or [16]
(A) a step of administering a test compound to a non-human animal expressing any of the genes according to [1] or [3] (b) a step of measuring the expression level of the gene in the animal (c) A step of selecting a compound that changes the expression level of the gene as compared to the case where no test compound is administered [20] Treatment of type II diabetes comprising the following steps (a) to (c): A screening method for compounds having prophylactic effects (candidate compounds for treatment or prevention),
(A) contacting a test compound with a protein encoded by the gene according to any one of SEQ ID NOs: 2, 13, 14 or a homologous gene of the gene, or a cell expressing the protein (b) The step of measuring the activity of the protein (c) The step of selecting a compound that changes the activity of the protein as compared with the case of measuring in the absence of the test compound.

  Diagnosis of diabetes currently being performed in hospitals is mainly based on blood glucose levels, but in early stages of diabetes, fasting blood glucose often drops to normal levels, and oversight of diabetes is overlooked. Probability is high. As described above, it is difficult for the diagnostic method using the blood glucose level as an index to detect a subtle change actually occurring in the living body.

  However, by using an analysis method developed by the present invention using the diabetes marker gene as an index (a method for testing a predisposition to diabetes), it can be expected that early diagnosis and prediction of the onset of diabetes can be accurately and simply performed. It becomes possible to treat diabetes early. In addition, it is expected that diabetes and early onset of diabetes can be predicted in humans and other mammals by using the diabetes marker gene provided by the present invention or a gene highly homologous to the gene. . In addition, these diabetes marker genes may be widely applicable to the evaluation of the effectiveness of functional foods targeting diabetes, evaluation of drug efficacy, and the like.

  Further, the provision of these genes related to diabetes is highly expected to elucidate the mechanism of diabetes and to develop drugs for the prevention or treatment of diabetes.

  The present inventors have identified genes that are useful as markers for diabetes. Although these genes and the base sequences of these genes are already known, the present inventors have shown for the first time that they have a relationship with diabetes and can be used for testing of diabetes.

The present invention provides a test method for a predisposition to diabetes using, as an index, the expression level of a gene marker identified by the present inventors for a test animal.
In the present invention, diabetes generally refers to type I diabetes or type II diabetes.
The nucleotide sequences of 26 marker genes of type I diabetes or type II diabetes that can be used in the test method of the present invention are shown in SEQ ID NOs: 1 to 8, and 10 to 27, respectively.
In addition, Tables 1 to 3 show the name of the diabetes marker gene, Tigr Definition number and NCBI Definition number of the gene, sequence information in rats, and homologous sequence information in other animals.

Table 2 is a continuation table of Table 1.

Table 3 is a continuation table of Table 2.

  In addition, about the gene for which the name (official or common name) that is usually used is not particularly known, “unknown” is described in the column of “diabetes marker gene name” in the above table. In addition, the various marker genes described above may be referred to as “the marker gene of the present invention” in the present specification.

Primer sequences capable of isolating the marker gene of the present invention are shown below. Therefore, even if the name is “unknown”, those skilled in the art can appropriately obtain (isolate) these marker genes of the present invention using the following primer sets. Moreover, the name for specifying a gene was also described.
Diabetes marker gene 1: Rattus norvegicus 13 BAC CH230-151K3 (Children's Hospital Oakland Research Institute) (primer sequence number: 145, 146)
Diabetes marker gene 2: R. norvegicus mRNA for cathepsin B (primer SEQ ID NO: 147, 148)
Diabetes marker gene 3: Mus musculus cyclin I, mRN (primer sequence numbers: 149, 150)
Diabetes marker gene 4: Mus musculus RIKEN cDNA 1110032C13 gene, mRNA (primer sequence numbers: 151, 152)
Diabetes marker gene 5: Mus musculus mitochondrial ribosomal protein S7, mRNA (primer SEQ ID NOs: 153, 154)
Diabetes marker gene 6: Mouse DNA sequence from clone RP23-423P8 on chromosome 4 contains the 3 'end of the Astn2 gene for astrotactin 2 and the 3' end of the Pappa gene for pregnancy-associated plasma protein A (primer SEQ ID NO: 155 156)
Diabetes marker gene 7: Mus musculus nuclear receptor coactivator 4, mRNA (primer sequence number: 157, 158)
Diabetes marker gene 8: Rat mRNA for the cysteine proteinase inhibitor cystatin C (primer SEQ ID NOs: 159 and 160)
Diabetes marker gene 9: Mus musculus Sac domain-containing inositol phosphatase 3, mRNA (primer SEQ ID NO: 161, 162)
Diabetes marker gene 10: R. norvegicus mRNA for coupling factor 6 of mitochondrial ATP synthase complex. (Primer SEQ ID NO: 163, 164)
Diabetes marker gene 11: Rattus norvegicus Chymotrypsinogen B (Ctrb), mRNA (primer SEQ ID NO: 165, 166)
Diabetes marker gene 12: Rattus norvegicus clone RP31-249D7 strain Brown Norway (primer SEQ ID NO: 167, 168)
Diabetes marker gene 13: Rat carboxypeptidase B gene, exons 1 and 2. (Primer SEQ ID NO: 169, 170)
Diabetes marker gene 14: Rat C1-tetrahydrofolate synthase mRNA (primer sequence numbers: 171, 172)
Diabetes marker gene 15: Rattus norvegicus AIF mRNA for apoptosis-inducing factor (primer SEQ ID NOs: 173 and 174)
Diabetes marker gene 16: Rattus norvegicus LIS1-interacting protein NUDE2 mRNA (Primer SEQ ID NO: 175, 176)
Diabetes marker gene 17: Mus musculus chromosome 3 clone RP24-344F8 (primer SEQ ID NO: 177, 178)
Diabetes marker gene 18: Mus musculus ring-box 1, mRNA (primer sequence numbers: 179, 180)
Diabetes marker gene 19: Mus musculus Brca1 associated protein 1 (BAP1), mRNA (primer sequence numbers: 181 and 182)
Diabetes marker gene 20: Mus musculus chromosome 14 clone RP23-187G1 (primer sequence numbers: 183, 184)
Diabetes marker gene 21: Messenger RNA for rat preproalbumin. (Primer SEQ ID NO: 185, 186)
Diabetes marker gene 22: Mus musculus mRNA for resistin-like molecule gamma (retn1g gene). (Primer SEQ ID NO: 187, 188)
Diabetes marker gene 23: Rattus norvegicus 1 BAC CH230-362O18 (Children's Hospital Oakland Research Institute) (Primer SEQ ID NOs: 189, 190)
Diabetes marker gene 24: Mus musculus RAB24, member RAS oncogene family, mRNA (primer sequence numbers: 191 and 192)
Diabetes marker gene 25: Mus musculus chromosome 7 clone RP24-344N22 (primer SEQ ID NOs: 193 and 194)
Diabetes marker gene 26: Rat angiotensin receptor (AT1) gene, single exon. (Primer SEQ ID NO: 195, 196)
Diabetes marker gene 27: Rattus norvegicus Nphs2 mRNA for podocin (primer SEQ ID NO: 197, 198)

  Regarding the homologous genes in other organisms corresponding to the DNA consisting of the nucleotide sequence of SEQ ID NOs: 1-27, the accession numbers in the public gene database of these homologous genes, and the homology of these homologous genes to the rat sequence, the above table 1-3.

  Information on the base sequence of these homologous genes and the amino acid sequence of the protein encoded by the gene can be easily obtained from the accession numbers of the databases shown in Tables 1 to 3. Moreover, those skilled in the art relate to the nucleotide sequence of a gene from the public gene database or literature database, and the amino acid sequence of the protein encoded by the gene, based on the gene names (gene notations) shown in Tables 1-3. Information can be easily obtained.

  The DNA in the genome usually has a double-stranded DNA structure complementary to each other. Therefore, in the present specification, even when a DNA sequence in one strand is disclosed for convenience, it is understood that a sequence complementary to the sequence (base sequence) is also disclosed as a matter of course. For those skilled in the art, if one DNA sequence (base sequence) is known, a sequence complementary to the sequence (base sequence) is obvious.

  Each of the above-described marker genes of the present invention (the genes described in SEQ ID NOs: 1 to 8 and 10 to 27 or homologs of the genes) can be used for examination of type II diabetes. In addition, in the marker gene described above, the gene described in any of SEQ ID NOs: 3, 6, 7, or 17 or a homologue thereof can be used for testing for type I diabetes.

  In a preferred embodiment of the test method of the present invention, the expression level (expression) of the gene according to any one of SEQ ID NOs: 1 to 8 and 10 to 27, or a homologous gene of the gene, inherent in the test animal (target animal). This is a method for testing a predisposition to type II diabetes, characterized by using (quantity) as an index. That is, by detecting the expression level of the gene according to any one of SEQ ID NOs: 1 to 8 and 10 to 27 or a homologous gene of the gene contained in the test animal, the test animal can have type II diabetes. Can be tested for predisposition.

  In the present specification, “the gene described in any of SEQ ID NOs: 1 to 8 and 10 to 27 or homologous genes of the gene” may be collectively described as “the marker gene of the present invention”. .

  As another aspect of the present invention, the expression level of the gene according to any one of SEQ ID NO: 3, 6, 7, or 17 or a homologous gene of the gene, which is inherent in the test animal, is used as an index. This is a test method for predisposition to type I diabetes. That is, by detecting the expression level of the gene according to any one of SEQ ID NO: 3, 6, 7, or 17 or a homologous gene of the gene contained in the test animal, type I diabetes mellitus for the test animal. It is possible to check whether or not there is a predisposing factor.

  In general, “type I diabetes” refers to insulin-dependent diabetes, and “type II diabetes” refers to non-insulin-dependent diabetes. In the present invention, when “diabetes” is simply described, it is understood that it refers to both or both of type I diabetes and type II diabetes.

  In the present invention, "examination of diabetes predisposition" means that a test animal (subject) usually has an innate predisposition to diabetes (genetic predisposition) or is likely to suffer from diabetes. Refers to whether or not it is a constitution, but is not limited thereto, for example, whether it is currently predisposed to diabetes due to an acquired cause, or whether it is already suffering from diabetes In addition, the examination of whether or not there is a high possibility of suffering in the future is included in the examination of the present invention. Therefore, the test method of the present invention is, for example, “test method for presence / absence of diabetes predisposition”, “test method for degree of susceptibility to diabetes”, “determination method for high or low probability of suffering from diabetes”, etc. can do.

  Therefore, in the present invention, “having a predisposition to diabetes” means having an innate predisposition (genetic predisposition) of diabetes, being predisposed to suffering from diabetes, and currently being susceptible to suffering from diabetes. Or that the patient already has diabetes or has a high possibility of suffering in the future.

  Further, according to the present invention, when a test animal already has diabetes, it is possible to predict the type of diabetes. That is, by determining the type of genetic marker whose expression is changed in the test animal, it is possible to predict with high probability whether the affected diabetes is type I or type II. It is useful for etc.

  Diabetes is never cured once it develops, and the complications that diabetes causes can significantly reduce the patient's quality of life. Therefore, using the test method of the present invention, the test animal is tested for a predisposition to diabetes, for a test animal determined to have a predisposition to diabetes, select an appropriate treatment before the onset of diabetes, It is thought that the onset of diabetes and various complications caused by diabetes can be prevented in advance.

  In the test method of the present invention, the gene serving as an expression index is at least one gene selected from the genes described in SEQ ID NOs: 1 to 8 and 10 to 27, or a homologous gene of the gene. That is, the type of gene used in the method of the present invention is not necessarily limited to one, and a plurality of types of genes can be appropriately selected to carry out the method of the present invention. For example, it is possible to further increase the reliability of the test by evaluating the expression level of a plurality of types of the genes of the present invention.

  In the present invention, the “test animal” refers to the test subject animal of the present invention, and is usually a mammal. The test animal of the present invention is not particularly limited as long as it is an animal in which the marker gene of the present invention (the gene described in SEQ ID NOs: 1 to 8, 10 to 27 or a homolog of the gene) is endogenous. For example, mammals such as humans, chimpanzees, pigs, cows, dogs, cats, mice, rats and the like, and rodents such as mice and rats are more preferable.

  For example, when the test animal of the present invention is a rat, the method of the present invention can be carried out using the expression level of the gene described in any one of SEQ ID NOs: 1 to 8 and 10 to 27 as an index. It is. That is, the genes described in SEQ ID NOs: 1 to 8 and 10 to 27 are genes endogenous to the rat, as shown in Examples described later, and the present invention can be suitably implemented using these marker genes. it can.

  In addition, when the test animal of the present invention is an animal other than a rat, the marker gene of the present invention (for example, the gene described in SEQ ID NOs: 1 to 8, 10 to 27, or the gene) The method of the present invention can be carried out with the expression level of the

  The information regarding “homologue genes” in animals other than rats is exemplified in Tables 1 to 3 above. However, even if it is not exemplified in the table, those skilled in the art will know, for example, the presence or absence of an endogenous marker gene serving as an index for the test animal or the base sequence information related to the gene in this specification. Based on the information disclosed in (Sequence Listing), it is possible to know appropriately using known literatures or databases.

  In the present invention, the “homolog gene” is usually a DNA having a structure in which one or a plurality of bases are added, deleted or substituted in the gene described in any one of SEQ ID NOs: 1 to 8 and 10 to 27. The “homolog gene” of the present invention usually has high homology with the base sequence of the gene (DNA) described in any one of SEQ ID NOs: 1 to 8 and 10 to 27. High homology means 50% or more, preferably 70% or more, more preferably 80% or more, more preferably 90% or more (for example, 95% or more, or even 96%, 97%, 98% or 99% or more) Means homology. This homology is the mBLAST algorithm (Altschul et al. (1990) Proc. Natl. Acad. Sci. USA 87: 2264-8; Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90: 5873-7) Can be determined by. In addition, the “homolog gene” is considered to hybridize with the gene (DNA) described in any one of SEQ ID NOs: 1 to 8 and 10 to 27 under stringent conditions. Here, as "stringent conditions", for example, "2 x SSC, 0.1% SDS, 50 ° C", "2 x SSC, 0.1% SDS, 42 ° C", "1 x SSC, 0.1% SDS, 37 ° C" More stringent conditions include “2 x SSC, 0.1% SDS, 65 ° C.”, “0.5 x SSC, 0.1% SDS, 42 ° C.” and “0.2 x SSC, 0.1% SDS, 65 ° C.” be able to.

  In the person skilled in the art, an endogenous gene corresponding to the gene described in any one of SEQ ID NOs: 1 to 8 and 10 to 27 or a homologous gene of the gene in another organism is represented by SEQ ID NOs: 1 to 8, 10 It can be appropriately obtained based on the nucleotide sequence of the gene according to any one of 27 or a homologous gene of the gene.

In the present invention, “expression” of a gene includes both “transcription” from the gene and “translation” from the transcript into the polypeptide.
When measuring the expression level of a gene using the amount of translation product (protein) produced by the gene as an index, for example, a protein sample is prepared from a test sample and encoded by the marker gene of the present invention contained in the protein sample. Measure the amount of protein. Such methods include methods well known to those skilled in the art, such as enzyme-linked immunoassay (ELISA), double monoclonal antibody sandwich immunoassay, monoclonal polyclonal antibody sandwich assay, immunofluorescence, western blotting, dot blotting. Method, immunoprecipitation, protein chip analysis (protein nucleic acid enzyme Vol.47 No.5 (2002), protein nucleic acid enzyme Vol.47 No.8 (2002)), two-dimensional electrophoresis, SDS polyacrylamide electrophoresis Law, but not limited to.

  When measuring the expression level of a gene using the amount of transcription product (mRNA) of the gene as an index, for example, an RNA sample is prepared from a test sample, and the marker gene of the present invention contained in the RNA sample is encoded. Measure the amount of RNA. It is also possible to evaluate the expression level by preparing a cDNA sample from the test sample and measuring the amount of cDNA encoding the marker gene of the present invention contained in the cDNA sample. RNA samples and cDNA samples from test samples can be prepared from biological samples derived from test animals by methods well known to those skilled in the art. Examples of such methods include methods well known to those skilled in the art, such as Northern blotting, RT-PCR, and DNA array.

  The “control” usually refers to the expression level of the marker gene of the present invention in a biological sample derived from a healthy animal. In the present invention, “marker gene expression” means expression of mRNA transcribed from the marker gene (generation of a transcription product) or expression of a protein encoded by the marker gene (generation of a translation product). Means both. In the present invention, the “healthy animal” is usually an animal that does not have a predisposition to diabetes, for example, an animal that has not suffered from diabetes at present or in the past, and preferably has a current healthy state that has no history of diabetes. Is an animal.

In a preferred embodiment of the test method of the present invention, when the expression level of the marker gene of the present invention measured as described above is (a) or (b) below, the test animal is predisposed to type II diabetes. It is determined to have.
(A) The expression level of the gene according to any one of SEQ ID NOs: 1 to 3, 7, 8, or 11 or a homologous gene of the gene is decreased as compared with the control (b) SEQ ID NOs: 4 to The expression level of the gene according to any one of 6, 10, 12 to 27 or a homologous gene of the gene is increased compared to the control.

In a preferred embodiment of the test method of the present invention, when the expression level of the gene according to any one of SEQ ID NO: 3, 6, 7, or 17 or a homologous gene of the gene is (a) or (b) below: In addition, it is determined that the test animal has a predisposition to type I diabetes.
(A) The expression level of the gene described in SEQ ID NO: 3 or 7 or a homologous gene of the gene is reduced compared to the control (b) the gene described in SEQ ID NO: 6 or 17, or the gene The expression level of homologous gene is increased compared to the control

The test sample used for the test method of the present invention is usually a sample derived from the test animal, and is preferably a sample previously separated from the test animal. Examples of biological samples include blood, skin, oral mucosa, tissues or cells collected or excised by surgery, body fluids collected for the purpose of examination, or cDNA extracted from the biological sample, Examples include RNA and protein samples.
Preparation of cDNA, RNA, and protein samples from biological samples can be appropriately performed by those skilled in the art using known techniques.

  The present invention also relates to a reagent (test agent) for the test method of the present invention and a kit for the test method of the present invention, each of which comprises a substance used in the test method of the present invention.

In a preferred embodiment of the present invention, a reagent (test drug) for testing for a predisposition to diabetes, and a kit for testing a predisposition to diabetes, comprising any of the following substances (a) to (d) as an active ingredient: I will provide a.
(A) an oligonucleotide that hybridizes to the gene of any one of SEQ ID NOs: 1 to 8, 10 to 27, or a homologue of the gene, and has a chain length of at least 15 nucleotides (b) SEQ ID NO: 1 A solid phase on which a nucleotide probe that hybridizes with the gene according to any one of -8, 10-27, or a homologue of the gene is immobilized (c) any one of SEQ ID NOs: 1-8, 10-27 Primer oligonucleotide (d) for amplifying the DNA of the described gene or homologue of the gene (d) the gene according to any one of SEQ ID NOs: 1 to 8, 10 to 27, or a protein encoded by the homologue of the gene Antibodies against

The oligonucleotide, solid phase, or protein can be used for measuring the expression level of the marker gene of the present invention.
The oligonucleotide (a) above specifically hybridizes to the DNA of the marker gene of the present invention (the gene described in any of SEQ ID NOs: 1 to 8, 10 to 27, or a homolog of the gene). Preferably there is. Here, “specifically hybridize” means normal hybridization conditions, preferably stringent hybridization conditions (for example, Sambrook et al., Molecular Cloning, Cold Spring Harbor Laboratory Press, New York, USA, In the condition described in the second edition 1989), it means that cross-hybridization does not occur significantly with DNA encoding other proteins. If specific hybridization is possible, the oligonucleotide does not necessarily have to be completely complementary to the base sequence of the gene to be detected.

  As hybridization conditions in the present invention, for example, “2 × SSC, 0.1% SDS, 50 ° C.”, “2 × SSC, 0.1% SDS, 42 ° C.”, “1 × SSC, 0.1% SDS, 37 ° C.” More stringent conditions include “2 × SSC, 0.1% SDS, 65 ° C.”, “0.5 × SSC, 0.1% SDS, 42 ° C.” and “0.2 × SSC, 0.1% SDS, 65 ° C.” Can do. More specifically, as a method using Rapid-hyb buffer (Amersham Life Science), prehybridization is performed at 68 ° C for 30 minutes or more, and then a probe is added and kept at 68 ° C for 1 hour or more for hybridization. Then wash 3 times for 20 minutes at room temperature in 2 x SSC, 0.1% SDS, then 3 times for 20 minutes at 37 ° C in 1 x SSC, 0.1% SDS, and finally 1 x SSC Washing can be performed twice in 0.1% SDS at 50 ° C for 20 minutes. In addition, for example, after prehybridization in Expresshyb Hybridization Solution (CLONTECH) at 55 ° C for 30 minutes or more, add a labeled probe and incubate at 37-55 ° C for 1 hour or more in 2 × SSC, 0.1% SDS In addition, washing for 20 minutes at room temperature can be performed three times, and washing for 20 minutes at 37 ° C. in 1 × SSC and 0.1% SDS can be performed once. Here, for example, by setting the temperature at the time of pre-hybridization, hybridization, or the second washing higher, the conditions can be made more stringent. For example, the prehybridization and hybridization temperatures can be 60 ° C., and more stringent conditions can be 68 ° C. Those skilled in the art can set conditions by taking into account other conditions such as probe concentration, probe length, probe base sequence configuration, and reaction time in addition to conditions such as buffer salt concentration and temperature. can do.

  The oligonucleotide can be used as a probe or primer in the test method of the present invention. When the oligonucleotide is used as a primer, the length is usually 15 bp to 100 bp, preferably 17 bp to 30 bp. The primer is not particularly limited as long as it can amplify at least part of the DNA of the marker gene of the present invention.

  In addition, polynucleotides, antibodies, cell lines, or model animals that can be used in the various screening methods of the present invention can be combined in advance to form a kit. These kits can be packaged with reagents used to detect the label, media and containers for cell culture, positive and negative standard samples, and instructions describing how to use the kit. it can.

The present invention provides a primer for amplifying the DNA of the marker gene of the present invention, and a probe that hybridizes to the DNA.
A person skilled in the art can design a primer according to the analysis method based on the base sequence information about the DNA of the marker gene of the present invention. The base sequence constituting the primer of the present invention can be appropriately modified as well as a base sequence completely complementary to the genomic base sequence.

  In addition to the base sequence complementary to the genomic base sequence, an arbitrary base sequence can be added to the primer of the present invention. For example, a primer having a modified base sequence is included in the primer of the present invention. Furthermore, the primer of the present invention can be modified. For example, a fluorescent substance or a primer labeled with a binding affinity substance such as biotin or digoxigenin is used in various genotyping methods. Primers having these modifications are also included in the present invention.

  Preferable specific examples of the primer of the present invention include the primers (SEQ ID NOs: 145 to 198) described in Tables 4 and 5. The primer can be suitably used when the test animal of the present invention is a rat.

  The primer of the present invention is an oligonucleotide that can hybridize with the DNA of the marker gene of the present invention, wherein one or a few bases are substituted in the base sequence described in any of SEQ ID NOs: 145 to 198, Examples thereof include oligonucleotides composed of deleted and added nucleotide sequences. The “minority” represents, for example, a numerical value of about 2 to 5, preferably about 2 to 3.

  When an animal other than a rat is used as a test animal, the oligonucleotide used in the method of the present invention is prepared by appropriately modifying the oligonucleotide described in SEQ ID NOs: 145 to 198. Is also possible.

  On the other hand, in the present invention, the probe that hybridizes to the DNA of the marker gene of the present invention refers to a probe that can hybridize to a polynucleotide having the base sequence of the DNA of the marker gene. More specifically, a probe containing all or part of the DNA of the marker gene in the base sequence of the probe is preferred as the probe of the present invention. In the probe of the present invention, modification of the base sequence, addition of the base sequence, or modification is allowed in the same manner as the primer.

  The base sequence constituting the probe of the present invention can be designed according to the analysis method based on the base sequence of the DNA of the marker gene of the present invention in the genome.

  The primer or probe of the present invention can be synthesized by any method based on the base sequence constituting it. The length of the base sequence complementary to the genomic DNA of the primer or probe of the present invention is usually 15 to 100, generally 15 to 50, usually 15 to 30. A technique for synthesizing an oligonucleotide having the base sequence based on the given base sequence is known. Furthermore, in the synthesis of the oligonucleotide, any modification can be introduced into the oligonucleotide using a nucleotide derivative modified with a fluorescent dye or biotin. Alternatively, a method of binding a fluorescent dye or the like to a synthesized oligonucleotide is also known.

  When the oligonucleotide of the present invention is used as a probe, it is appropriately labeled with a radioisotope or a non-radioactive compound. When used as a primer, for example, the 3 ′ end region of the oligonucleotide is complementary to the target sequence, and a restriction enzyme recognition sequence, tag, etc. are added to the 5 ′ end side. Can be designed to Such a polynucleotide comprising a base sequence containing at least 15 consecutive bases can hybridize to the mRNA of the marker gene of the present invention.

  Oligonucleotides of the present invention are non-natural bases such as 4-acetylcytidine, 5- (carboxyhydroxymethyl) uridine, 2′-O-methylcytidine, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxyl. Methylaminomethyluridine, dihydrouridine, 2'-O-methyl pseudouridine, β-D-galactosyl cueosine, 2'-O-methylguanosine, inosine, N6-isopentenyladenosine, 1-methyladenosine, 1- Methyl pseudouridine, 1-methyl guanosine, 1-methyl inosine, 2,2-dimethyl guanosine, 2-methyl adenosine, 2-methyl guanosine, 3-methyl cytidine, 5-methyl cytidine, N6-methyl adenosine, 7-methyl Guanosine, 5-methylaminomethyluridine, 5-methoxyaminomethyl-2-thiouridine, β-D-mannosylqueosin, 5- Toxicarbonylmethyl-2-thiouridine, 5-methoxycarbonylmethyluridine, 5-methoxyuridine, 2-methylthio-N6-isopentenyladenosine, N-((9-β-D-ribofuranosyl-2-methylliopurine-6- Yl) carbamoyl) threonine, N-((9-β-D-ribofuranosylpurin-6-yl) N-methylcarbamoyl) threonine, uridine-5-oxyacetic acid-methyl ester, uridine-5-oxyacetic acid, wybutoxocin , Pseudouridine, cueosin, 2-thiocytidine, 5-methyl-2-thiouridine, 2-thiouridine, 4-thiouridine, 5-methyluridine, N-((9-β-D-ribofuranosylpurin-6-yl) carbamoyl ) Threonine, 2′-O-methyl-5-methyluridine, 2′-O-methyluridine, wybutosine, 3- (3-amino-3-carboxypropyl) uridine and the like may be contained as necessary.

  Furthermore, another embodiment of the test agent (reagent) and test kit in the present invention is a test for the predisposition to diabetes, comprising as an active ingredient a solid phase on which a nucleotide probe that hybridizes to the DNA of the marker gene of the present invention is immobilized. The present invention relates to a medicine (reagent) and a test kit.

  The solid phase on which the nucleotide probe is immobilized can be used for expression level analysis by a known microarray method, for example. Specifically, after preparing a solid phase on which the nucleotide probe is immobilized, the test sample is brought into contact with the solid phase, and nucleic acid hybridized to the nucleotide probe immobilized on the solid phase is detected. The inspection method of the invention can be carried out.

  In the present invention, “solid phase” means a material capable of fixing nucleotides. The solid phase of the present invention is not particularly limited as long as nucleotides can be immobilized. Specifically, solid phases including microplate wells, plastic beads, magnetic particles, substrates and the like may be exemplified. it can. As the “solid phase” of the present invention, a substrate generally used in DNA array technology can be preferably used. In the present invention, the “substrate” means a plate-like material capable of fixing nucleotides. In the present invention, the nucleotide includes oligonucleotides and polynucleotides.

  Furthermore, another embodiment of the test agent (reagent) and test kit in the present invention is an antibody against a protein encoded by the marker gene of the present invention (SEQ ID NO: 1-8, gene according to any one of 10-27, Alternatively, a test agent (reagent) and a test kit for predisposition to diabetes containing an antibody that recognizes a protein encoded by a homologue of the gene as an active ingredient.

  Antibodies against the protein encoded by the marker gene of the present invention can be prepared by methods known to those skilled in the art. If it is a polyclonal antibody, it can obtain as follows, for example. A small protein such as a rabbit is immunized with a natural protein encoded by a marker gene or a recombinant (recombinant) protein expressed in a microorganism such as Escherichia coli or a partial peptide thereof as a fusion protein with GST. This is prepared, for example, by purification using ammonium sulfate precipitation, protein A, protein G column, DEAE ion exchange chromatography, an affinity column coupled with a protein encoded by the marker gene of the present invention or a synthetic peptide, or the like. In the case of a monoclonal antibody, for example, a protein encoded by the marker gene of the present invention or a partial peptide thereof is immunized to a small animal such as a mouse, the spleen is removed from the mouse, and this is ground to isolate cells. The cells and mouse myeloma cells are fused using a reagent such as polyethylene glycol, and an antibody that binds to the protein encoded by the marker gene of the present invention is produced from the resulting fused cells (hybridoma). Select a clone. Subsequently, the obtained hybridoma is transplanted into the abdominal cavity of the mouse, and ascites is collected from the mouse, and the obtained monoclonal antibody is obtained by, for example, ammonium sulfate precipitation, protein A, protein G column, DEAE ion exchange chromatography, It can be prepared by purification with an affinity column coupled with a protein encoded by a marker gene or a synthetic peptide.

  The form of the antibody of the present invention is not particularly limited, as long as it binds to or recognizes the protein encoded by the marker gene of the present invention, in addition to the polyclonal antibody and the monoclonal antibody, a human antibody, a human by gene recombination A typed antibody, an antibody fragment thereof, and an antibody modification product are also included.

  The protein encoded by the marker gene of the present invention used as a sensitizing antigen for antibody acquisition is not limited with respect to the animal species from which it is derived, but is preferably derived from the animal species to be tested.

  In the present invention, the protein used as the sensitizing antigen may be a complete protein or a partial peptide of the protein. Examples of the partial peptide of the protein include an amino group (N) terminal fragment and a carboxy (C) terminal fragment of the protein, or a kinase active site at the center. As used herein, “antibody” means an antibody that reacts with the full length or fragment of a protein.

  Polyclonal antibodies, monoclonal antibodies, chimeric antibodies, single chain antibodies (scFv) (Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85: 5879-83; The Pharmacology of Monoclonal Antibody, Vol. 113, Rosenburg and Moore ed., Springer Verlag (1994) pp. 269-315), humanized antibody, multispecific antibody (LeDoussal et al. (1992) Int. J. Cancer Suppl. 7: 58 -62; Paulus (1985) Behring Inst. Mitt. 78: 118-32; Millstein and Cuello (1983) Nature 305: 537-9; Zimmermann (1986) Rev. Physiol. Biochem. Pharmacol. 105: 176-260; VanDijk et al. (1989) Int. J. Cancer 43: 944-9), and antibody fragments such as Fab, Fab ′, F (ab ′) 2, Fc, Fv and the like. Such an antibody may be modified with PEG or the like as necessary. It can also be produced as a fusion protein with β-galactosidase, maltose binding protein, GST, green fluorescent protein (GFP), etc. so that it can be detected without using a secondary antibody. Further, by labeling the antibody with biotin or the like, it can be modified so that the antibody can be detected and recovered using avidin, streptavidin or the like.

  In addition to immunizing non-human animals with antigens to obtain the above hybridomas, human lymphocytes such as human lymphocytes infected with EB virus are sensitized with proteins, protein-expressing cells or lysates thereof in vitro. Lymphocytes can be fused with human-derived myeloma cells having permanent mitotic activity, such as U266, to obtain hybridomas that produce desired human antibodies having binding activity to proteins.

  As described above, the antibody against the protein encoded by the marker gene of the present invention can be suitably used for the test of the present invention, and is useful as a constituent of the test drug or test kit of the present invention.

  In addition, as shown in Examples described later, in the model animal of type II diabetes, the expression level of the genes described in SEQ ID NOs: 1-3, 7, 8, 11 is lower than that of the control. I understood that. Therefore, a substance that enhances the expression (expression level) of the gene according to any one of SEQ ID NOs: 1 to 3, 7, 8, and 11 or a homologous gene of the gene is a drug for treating or preventing type II diabetes It is expected to be

  The present invention relates to a substance that enhances the function (activity) of the gene according to any one of SEQ ID NOs: 1 to 3, 7, 8, and 11, or the homologous gene of the gene, or the protein encoded by the gene. Provided is a drug for treating or preventing type II diabetes, which is contained as an active ingredient.

  More specifically, when the present invention is intended for rats, the gene expression enhancer according to any one of SEQ ID NOs: 1-3, 7, 8, and 11 inherent in rats is used as an active ingredient, Provided a drug / pharmaceutical composition for treating or preventing type II diabetes containing a substance that enhances the expression of a homologous gene of the gene endogenous to the animal as an active ingredient for animals other than rats To do.

  Moreover, in the experimental animals exhibiting the pathological condition of type I diabetes, the expression level of the gene described in SEQ ID NO: 3 or 7 was found to be lower than that of the control. Therefore, a substance that enhances the expression (expression level) of the gene described in SEQ ID NO: 3 or 7 or a homologous gene of the gene is expected to be a drug for the treatment or prevention of type I diabetes.

  The present invention relates to type I diabetes comprising, as an active ingredient, a substance that enhances the expression (SEQ ID NO: 3 or 7) or a homologous gene of the gene, or a function (activity) of a protein encoded by the gene. Provides a drug for the treatment or prevention of

  More specifically, in the present invention, when a rat is a subject, the gene expression enhancer of the gene described in SEQ ID NO: 3 or 7 that is present in the rat is used as an active ingredient, and an animal other than a rat is a subject. In this case, a pharmaceutical / pharmaceutical composition for treating or preventing type I diabetes is provided, which contains, as an active ingredient, a substance that enhances the expression of a homologous gene of the gene endogenous to the animal.

  Moreover, in the model animal of type II diabetes, it turned out that the expression level of the gene as described in any one of sequence number: 4-6, 10, 12-27 is a high level compared with a control | contrast. Therefore, the substance that suppresses the expression (expression level) of the gene described in any of SEQ ID NOs: 4 to 6, 10, 12 to 27 or a homologous gene of the gene is a drug for the treatment or prevention of type II diabetes It is expected to be

  The present invention relates to a substance that suppresses the expression (SEQ ID NO: 4-6, 10, 12-27) of a gene or a homologous gene of the gene, or a function (activity) of a protein encoded by the gene. Provided is a drug for treating or preventing type II diabetes, which is contained as an active ingredient.

  More specifically, the present invention, when intended for a rat, comprises as an active ingredient a substance that suppresses the expression of a gene according to any one of SEQ ID NOs: 4-6, 10, 12-27, When targeting animals other than rats, an agent (pharmaceutical composition) for treating or preventing type II diabetes containing a substance that suppresses the expression of a homologous gene of the gene endogenous to the animal as an active ingredient provide.

  Moreover, in the experimental animals exhibiting the pathological condition of type I diabetes, the expression level of the gene described in SEQ ID NO: 6 or 17 was found to be higher than that of the control. Therefore, a substance that suppresses the expression (expression level) of the gene described in SEQ ID NO: 6 or 17 or a homologous gene of the gene is expected to be a drug for the treatment or prevention of type I diabetes.

  The present invention relates to type I diabetes comprising, as an active ingredient, a substance that suppresses the expression of the gene shown in SEQ ID NO: 6 or 17 or a homologous gene of the gene, or the function (activity) of the protein encoded by the gene. Provides a drug for the treatment or prevention of

  More specifically, in the present invention, when the subject is a rat, the gene described in SEQ ID NO: 6 or 17 or the substance that suppresses the expression of the gene is used as an active ingredient, and the subject is an animal other than a rat. In such a case, a drug (pharmaceutical composition) for treating or preventing type I diabetes, comprising as an active ingredient a substance that suppresses the expression of a homologous gene of the gene endogenous to the animal is provided.

  “Treatment” as used herein generally means obtaining a pharmacological and / or physiological effect. The effect may be preventive in that the disease or symptom is completely or partially suppressed (improved), or may be therapeutic in that the symptom of the disease is completely or partially treated. . “Treatment” as used herein includes all treatment of disease in animals, preferably mammals, more preferably rodents, and even more preferably rats. In addition, the prevention of the onset of test animals that have a predisposition to the disease but have not yet been diagnosed, suppress the progression of the disease, reduce the disease, delay the onset, etc. Included in “treatment”.

  The expression-suppressing substance of the present invention includes transcription of the marker gene of the present invention (for example, the gene described in any of SEQ ID NOs: 4-6, 10, 12-27, or a homologous gene of the gene) or the transcription product. Substances capable of suppressing the translation of are included.

Preferred embodiments of the expression-suppressing substance of the present invention include, for example, compounds (nucleic acids) selected from the group consisting of the following (a) to (c).
(A) SEQ ID NOs: 4-6, 10, and antisense nucleic acids for the transcript of the homologous gene of the gene or part thereof, or a part thereof (b) SEQ ID NOs: 4-6, 10 , A nucleic acid having a ribozyme activity that specifically cleaves a transcription product of the gene according to any one of 12 to 27 or a homologous gene of the gene (c) SEQ ID NO: 4-6, 10, 12-27 Nucleic acid having an action of inhibiting the expression of the gene or homologous gene of the gene by RNAi effect

  The “nucleic acid” in the present invention means RNA or DNA. Further, chemically synthesized nucleic acid analogs such as so-called PNA (peptide nucleic acid) are also included in the nucleic acid of the present invention. PNA is obtained by replacing the pentose / phosphate skeleton, which is the basic skeleton structure of nucleic acids, with a polyamide skeleton having glycine as a unit, and has a three-dimensional structure very similar to nucleic acids.

  As a method for suppressing the expression of a specific endogenous gene, a method using an antisense technique is well known to those skilled in the art. There are several factors as described below for the action of an antisense nucleic acid to suppress the expression of a target gene. Inhibition of transcription initiation by triplex formation, inhibition of transcription by hybridization with a site where an open loop structure was locally created by RNA polymerase, inhibition of transcription by hybridization with RNA undergoing synthesis, intron and exon Splicing inhibition by hybridization at splice point, splicing inhibition by hybridization with spliceosome formation site, inhibition of translocation from nucleus to cytoplasm by hybridization with mRNA, hybridization with capping site and poly (A) addition site Inhibition of splicing by RNA, inhibition of translation initiation by hybridization with a translation initiation factor binding site, translation inhibition by hybridization with a ribosome binding site near the initiation codon, peptide by hybridization with an mRNA translation region or polysome binding site Elongation inhibition, and the like inhibiting gene expression by hybrid formation with the site of interaction between nucleic acids and proteins. In this way, antisense nucleic acids inhibit the expression of target genes by inhibiting various processes such as transcription, splicing or translation (Hirashima and Inoue, Shinsei Kagaku Kenkyu 2 Lecture and Expression of Nucleic Acid IV Gene, Japan Biochemical) (Academic Society, Tokyo Chemical Doujin, 1993, 319-347).

  The antisense nucleic acid used in the present invention may suppress the expression of the marker gene of the present invention by any of the actions described above. In one embodiment, if an antisense sequence complementary to the untranslated region near the 5 ′ end of the mRNA of the marker gene of the present invention is designed, it is considered effective for inhibiting gene translation. In addition, a sequence complementary to the coding region or the 3 ′ untranslated region can also be used. Thus, the nucleic acid containing the antisense sequence of the sequence of the untranslated region as well as the translated region of the marker gene of the present invention is also included in the antisense nucleic acid used in the present invention. The antisense nucleic acid used is linked downstream of a suitable promoter, and preferably a sequence containing a transcription termination signal is linked on the 3 ′ side. The nucleic acid thus prepared can be transformed into a desired animal by using a known method. The sequence of the antisense nucleic acid is preferably a sequence complementary to the endogenous marker gene of the animal to be transformed, or a part thereof, but is completely complementary as long as the gene expression can be effectively suppressed. It does n’t have to be. The transcribed RNA preferably has a complementarity of 90% or more, most preferably 95% or more, to the transcript of the target gene. In order to effectively suppress the expression of a target gene (for example, the gene described in any of SEQ ID NOs: 4-6, 10, 12-27 or a homologous gene of the gene) using an antisense nucleic acid, antisense The length of the nucleic acid is preferably at least 15 bases and less than 25 bases, but the antisense nucleic acid of the present invention is not necessarily limited to this length.

  Although the antisense of the present invention is not particularly limited, for example, it can be prepared based on the nucleotide sequence of the gene described in SEQ ID NOs: 4-6, 10, 12-27 or a homologous gene of the gene.

  In addition, the suppression of the expression of the marker gene of the present invention can be carried out using a ribozyme or a DNA encoding a ribozyme. A ribozyme refers to an RNA molecule having catalytic activity. Some ribozymes have a variety of activities, but research focusing on ribozymes as enzymes that cleave RNA has made it possible to design ribozymes that cleave RNA in a site-specific manner. Some ribozymes have a size of 400 nucleotides or more, such as group I intron type and M1 RNA contained in RNase P, but some have an active domain of about 40 nucleotides called hammerhead type or hairpin type. (Makoto Koizumi and Eiko Otsuka, Protein Nucleic Acid Enzymes, 1990, 35, 2191.)

  For example, the self-cleaving domain of the hammerhead ribozyme cleaves 3 ′ of C15 in the sequence G13U14C15, but base pairing between U14 and A9 is important for its activity, and instead of C15, A15 or U15 However, it has been shown that it can be cleaved (Koizumi, M. et al., FEBS Lett, 1988, 228, 228.). By designing a ribozyme whose substrate binding site is complementary to the RNA sequence in the vicinity of the target site, it is possible to create a restriction RNA-cleaving ribozyme that recognizes the sequence UC, UU or UA in the target RNA (Koizumi, M. et al., FEBS Lett, 1988, 239, 285., Makoto Koizumi and Eiko Otsuka, Protein Nucleic Acid Enzymes, 1990, 35, 2191., Koizumi, M. et al., Nucl Acids Res, 1989, 17, 7059 .).

  Hairpin ribozymes are also useful for the purposes of the present invention. This ribozyme is found, for example, in the minus strand of tobacco ring spot virus satellite RNA (Buzayan, JM., Nature, 1986, 323, 349.). It has been shown that hairpin ribozymes can also produce target-specific RNA-cleaving ribozymes (Kikuchi, Y. & Sasaki, N., Nucl Acids Res, 1991, 19, 6751., Hiroshi Kikuchi, Chemistry and Biology, 1992, 30, 112.). Thus, the expression of the gene can be suppressed by specifically cleaving the transcription product of the marker gene of the present invention using a ribozyme.

  Inhibition of endogenous gene expression can also be carried out by RNA interference (RNAi) using double-stranded RNA having the same or similar sequence as the target gene sequence. The nucleic acid having an inhibitory action by the RNAi effect of the present invention is generally also referred to as siRNA. RNAi induces destruction of target gene mRNA by introducing double-stranded RNA consisting of a sense RNA consisting of a sequence homologous to the mRNA of the target gene and an antisense RNA consisting of a complementary sequence into the cell. This is a phenomenon that can suppress the expression of the target gene. As described above, RNAi can suppress the expression of a target gene, so that it can be used as a simple gene knockout method instead of the conventional complicated and low-efficiency gene disruption method by homologous recombination, or as a method applicable to gene therapy. It attracts attention. The RNA used for RNAi is not necessarily completely identical to the marker gene of the present invention or a partial region of the gene, but preferably has complete homology.

  As a preferred embodiment of the nucleic acid of (c) of the present invention, there can be mentioned double-stranded RNA (siRNA) having an RNAi (RNA interference) effect on the marker gene of the present invention. More specifically, a double-stranded RNA (siRNA) comprising a sense RNA and an antisense RNA for a partial sequence of the base sequence in the marker gene of the present invention can be mentioned.

  The details of the RNAi mechanism are still unknown, but an enzyme called DICER (a member of the RNase III nuclease family) contacts double-stranded RNA, and the double-stranded RNA is called a small interfering RNA or siRNA. It is thought to be broken down into pieces. The double-stranded RNA having the RNAi effect in the present invention includes a double-stranded RNA before being degraded by DICER as described above. That is, it is expected that even a long-chain RNA that does not have an RNAi effect with the same length is decomposed into siRNA having an RNAi effect in a cell. The length is not particularly limited.

  For example, a long double-stranded RNA corresponding to the full-length or almost full-length region of the mRNA of the marker gene of the present invention can be decomposed in advance with, for example, DICER, and the degradation product can be used as the drug of the present invention. is there. This degradation product is expected to contain a double-stranded RNA molecule (siRNA) having an RNAi effect. According to this method, a region on mRNA that is expected to have an RNAi effect need not be selected. That is, the region on the mRNA of the marker gene of the present invention having the RNAi effect is not necessarily defined accurately.

  In addition, a molecule having one end closed in the above RNA molecule, for example, a siRNA (shRNA) having a hairpin structure is also included in the present invention. That is, a single-stranded RNA molecule capable of forming a double-stranded RNA structure within the molecule is also included in the present invention.

  The above-described “double-stranded RNA that can be suppressed by the RNAi effect” of the present invention can be appropriately prepared by those skilled in the art based on the base sequence of the marker gene of the present invention that is the target of the double-stranded RNA. . For example, the double-stranded RNA of the present invention can be prepared based on the base sequence of the gene described in any of SEQ ID NOs: 4-6, 10, 12-27 or a homologous gene of the gene. it can. That is, based on the base sequence of the gene according to any one of SEQ ID NOs: 4-6, 10, 12-27 or a homologous gene of the gene, any continuous RNA region of mRNA that is a transcription product of the sequence It is possible for those skilled in the art to appropriately perform the selection and preparation of double-stranded RNA corresponding to this region within the range of ordinary trials. In addition, selection of siRNA sequences having a stronger RNAi effect from mRNA sequences that are transcripts of the sequences can also be appropriately performed by those skilled in the art by known methods. Moreover, if one strand (for example, the nucleotide sequence of the gene described in any one of SEQ ID NOs: 4-6, 10, 12-27 or a homologous gene of the gene) is known, those skilled in the art can easily perform it. The base sequence of the other strand (complementary strand) can be known. The siRNA can be appropriately prepared by those skilled in the art using a commercially available nucleic acid synthesizer. In addition, for synthesis of a desired RNA, a general synthesis contract service can be used.

  Furthermore, a DNA (vector) capable of expressing the RNA of the present invention is also included in a preferred embodiment of the compound capable of suppressing the expression of the marker gene of the present invention. For example, the DNA (vector) capable of expressing the double-stranded RNA of the present invention is a DNA encoding one strand of the double-stranded RNA and a DNA encoding the other strand of the double-stranded RNA, Each DNA has a structure linked to a promoter so that it can be expressed. Those skilled in the art can appropriately prepare the above DNA of the present invention by general genetic engineering techniques. More specifically, the expression vector of the present invention can be prepared by appropriately inserting DNA encoding the RNA of the present invention into various known expression vectors.

  In addition, the expression-suppressing substance of the present invention binds to, for example, the expression regulatory region (eg, promoter region) of the gene described in any of SEQ ID NOs: 4-6, 10, 12-27 or a homologous gene of the gene. Thus, a compound that suppresses the expression of the gene is included. The compound uses, for example, a promoter DNA fragment of the gene described in any of SEQ ID NOs: 4 to 6, 10, 12 to 27 or a homologous gene of the gene, and the binding activity with the DNA fragment is used as an index. It can be obtained by a screening method. Moreover, those skilled in the art can appropriately determine whether or not to suppress the expression of the marker gene of the present invention for a desired compound by a known method such as a reporter assay method.

  The present invention also provides a diabetic agent comprising, as an active ingredient, a substance that suppresses the function of a protein encoded by the gene set forth in any of SEQ ID NOs: 4-6, 10, 12-27 or a homologous gene of the gene. Provide drugs for treatment or prevention.

Examples of the function-suppressing substance include the following compounds (a) or (b).
(A) an antibody that binds to a protein encoded by the gene according to any one of SEQ ID NOs: 4 to 6, 10, 12 to 27 or a homologous gene of the gene (b) SEQ ID NOs: 4 to 6, 10, 12 A low molecular weight compound that binds to a protein encoded by the gene according to any one of -27 or a homologous gene of the gene

  The above antibody of the present invention binds to a protein encoded by the marker gene of the present invention, thereby suppressing the function of the protein, and is expected to have an effect of treating, preventing or improving diabetes, for example. When the obtained antibody is used for the purpose of administering it to the human body (antibody treatment), a human antibody or a human-type antibody is preferable in order to reduce immunogenicity.

  In addition, as the above-described function inhibitory substance, as a substance capable of suppressing the function of the protein encoded by the gene according to any of SEQ ID NOs: 4-6, 10, 12-27 or a homologous gene of the gene, The low molecular compound (low molecular weight substance) to couple | bond can be mentioned. The low molecular weight compound that binds to a protein encoded by the gene according to any one of SEQ ID NOs: 4 to 6, 10, 12 to 27 of the present invention or a homologous gene of the gene may be a natural or artificial compound. Good. Usually, it is a compound that can be produced or obtained by using methods known to those skilled in the art. The compound of the present invention can also be obtained by the screening method described later.

  Examples of the low molecular weight compound (b) include compounds having high affinity for the protein.

  Furthermore, as the function-suppressing substance of the present invention, for example, a property of dominant negative with respect to a protein encoded by the gene described in any one of SEQ ID NOs: 4-6, 10, 12-27 or a homologous gene of the gene And a mutant of the protein (dominant negative mutant) having The dominant negative mutant refers to a protein having a function of eliminating or reducing the activity of an endogenous wild-type protein when expressed.

  It is also known that the function of the protein encoded by the marker gene of the present invention (for example, the gene described in any of SEQ ID NOs: 4-6, 10, 12-27 or a homologous gene of the gene) is inhibited. The substance (compound) used can be suitably used as the function-suppressing substance of the present invention.

  For example, as a substance capable of inhibiting the function of a protein encoded by the marker gene (C1-tetrahydrofolate synthase) described in SEQ ID NO: 14 of the present invention, Pemetrexed (LY231514) (references; Shih C, Habeck LL, Mendelsohn LG , Chen VJ, Schultz RM., Multiple folate enzyme inhibition: mechanism of a novel pyrrolopyrimidine-based antifolate LY231514 (MTA)., Adv Enzyme Regul. 1998; 38: 135-52.).

  Moreover, as a substance capable of inhibiting the function of the protein encoded by the marker gene (angiotensin II receptor, type 1) described in SEQ ID NO: 26 of the present invention, Losartan (EXP 3174), Valsartan, Irbesartan, Candesartan cilexetil, Telmisaetan, (Reference: M. Burnier, Angiotensin II Type 1 Receptor Blockers, Circulation, February 13, 2001; 103 (6): 904-912.) Is known. These compounds are preferred specific examples of the above-described function-suppressing substances of the present invention. These compounds have not been known to be related to diabetes so far, and for the first time, the present inventors have found the possibility of new uses as drugs for the prevention or treatment of diabetes.

  In addition, the present invention is characterized in that the expression level of the marker gene of the present invention (the gene described in any of SEQ ID NOs: 1 to 8, 10 to 27 or a homologous gene of the gene) is artificially modified. A genetically modified diabetes model non-human animal (in the present specification, sometimes referred to as “gene modified non-human animal” or simply “animal of the present invention”) is provided.

  The genetically modified non-human animal of the present invention can be used for screening for drugs for the treatment or prevention of diabetes, for example. It is also very useful as a disease model animal for elucidating the mechanism of diabetes.

  For example, a model animal in which the expression of the marker gene of the present invention is changed can be used for screening a substance having an action of returning the expression level of the gene to normal. The substance is expected to be a medicament for the prevention or treatment of diabetes.

Preferred embodiments of the genetically modified diabetes model animal of the present invention include, for example, the following animals.
(A) Type II diabetes model animal in which expression of the gene according to any one of SEQ ID NOs: 1 to 3, 7, 8, or 11 or a homologous gene of the gene is suppressed (b) SEQ ID NOs: 4 to 6, A type II diabetes model animal (c) having increased expression of the gene according to any one of 10, 12 to 27 or a homologous gene of the gene (c) The gene according to SEQ ID NO: 3 or 7, or the homologous gene of the gene Type I diabetes model animal with suppressed expression (d) Type I diabetes model animal with increased expression of the gene according to SEQ ID NO: 6 or 17 or homologous gene of the gene

  Therefore, the genetically modified non-human animal in the present invention includes a gene knockout non-human animal in which the expression of the marker gene of the present invention is artificially suppressed. This knockout non-human animal includes so-called “knockdown animals” in which the expression of the marker gene of the present invention is suppressed by the action of antisense RNA or siRNA.

In addition, specifically, the expression of the marker gene of the present invention is artificially suppressed, specifically,
(1) Insertion, deletion, substitution, etc. of nucleotides into one or both of the gene sequence of SEQ ID NOS: 1-8, 10-27 or a homologous gene of the gene existing in the model animal A state in which expression of the gene is suppressed by having a gene mutation,
(2) Examples include a state in which the expression of the marker gene of the present invention is suppressed by the action of the nucleic acid (for example, antisense RNA or siRNA) in the present invention.

  In the “suppression” of the present invention, when the expression of the marker gene of the present invention is completely suppressed, the expression level of the gene is significantly reduced compared to the expression level of the gene in a wild-type animal. Is included.

  The above (1) includes the case where only the expression of one gene of the marker gene gene pair of the present invention is suppressed. The site where the gene mutation is present in the present invention is not particularly limited as long as the expression of the gene is suppressed, and examples thereof include an exon site and a promoter site.

  The genetically modified non-human animal of the present invention can be produced by those skilled in the art by generally known genetic engineering techniques. For example, a gene knockout mouse can be prepared as follows. First, DNA containing the exon portion of the marker gene of the present invention is isolated from a mouse, and an appropriate marker gene is inserted into this DNA fragment to construct a targeting vector. This targeting vector is introduced into a mouse ES cell line by electroporation or the like, and a cell line in which homologous recombination has occurred is selected. The marker gene to be inserted is preferably an antibiotic resistance gene such as a neomycin resistance gene. When an antibiotic resistance gene is inserted, a cell line that has undergone homologous recombination can be selected simply by culturing in an antibiotic-containing medium. In addition, in order to perform more efficient selection, it is possible to bind a thymidine kinase gene or the like to a targeting vector. Thereby, the cell line which caused non-homologous recombination can be excluded. In addition, homologous recombinants can be assayed by PCR and Southern blotting to efficiently obtain a cell line in which one of the gene pairs of the marker gene of the present invention is inactivated.

  When selecting a cell line in which homologous recombination has occurred, it is preferable to produce a chimera using a plurality of clones because there is a risk of unknown gene disruption due to gene insertion in addition to the homologous recombination site. The obtained ES cell line can be injected into mouse blastoderm to obtain a chimeric mouse. By crossing this chimeric mouse, a mouse in which one of the gene pairs of the marker gene of the present invention is inactivated can be obtained. Furthermore, by crossing this mouse, it is possible to obtain a mouse in which both gene pairs are inactivated. In animals where ES cells other than mice are established, genetic modification can be performed by the same technique.

  Furthermore, the genetically modified diabetes model animal of the present invention includes an animal in which the expression of the marker gene of the present invention has been increased, or an animal into which an exogenous marker gene of the present invention has been introduced.

  As a method for producing the animal, for example, a method of microinjecting DNA (eg, DNA having a structure in which the marker gene of the present invention is functionally linked downstream of a promoter) into a fertilized egg pronucleus under a microscope is common. is there. In this case, a fertilized egg into which DNA has been injected is transplanted into the oviduct of a female mouse in a pseudopregnant state to obtain a transgenic mouse. At that time, in order to express the target gene in a specific tissue in the living body, it is possible to use a construct that expresses the target gene under the control of a tissue-specific promoter.

  The kind of the genetically modified non-human animal of the present invention is not particularly limited as long as it is a non-human animal, but is usually a mammal, preferably a rodent. More specifically, the genetically modified non-human animal of the present invention is preferably a mammal such as a rat, mouse, chimpanzee, pig, cow, or dog, and more preferably a rodent such as a rat or mouse. Most preferably, it is a rat.

  In addition, the present invention selects an expression level indicator of the marker gene of the present invention (the gene described in any of SEQ ID NOs: 1 to 8, 10 to 27, or a homologous gene of the gene), and a compound that changes the expression level. The present invention provides a method for screening a compound having an effect of treating or preventing diabetes. The “compound having a therapeutic or prophylactic effect for diabetes” includes not only a drug (medicine) having the effect but also a (functional) food or drink having the effect or function.

  The present invention also provides a method for screening a compound having an effect of treating or preventing diabetes, using the expression level of the marker gene of the present invention as an index. The compound is expected to be a prophylactic or therapeutic agent for diabetes. Moreover, this compound is useful also as a functional food-drinks which has a diabetes prevention effect.

  A preferred embodiment of the screening method of the present invention includes a method for screening a compound having the effect of treating or preventing type II diabetes, which comprises selecting a compound that changes the expression level of the marker gene of the present invention.

  More preferably, in the above method, the expression level of the gene according to any one of SEQ ID NOs: 1 to 3, 7, 8, and 11 or a homologous gene of the gene is measured, and the expression level is increased compared to the control. This is a method of selecting a substance to be made to be.

  In another embodiment, the method comprises measuring the expression level of the gene according to any one of SEQ ID NOs: 4-6, 10, 12-27 or a homologous gene of the gene, and comparing the expression level with a control. This is a method for selecting a substance to be reduced.

  In another aspect of the screening method of the present invention, the present invention relates to a method for screening a compound having a therapeutic or prophylactic effect for type I diabetes, comprising selecting a compound that changes the expression level of the marker gene of the present invention. .

  The method is preferably a method of measuring the expression level of the gene set forth in SEQ ID NO: 3 or 7 or a homologous gene of the gene, and selecting a substance that increases the expression level compared to a control.

  Further, the method is a method of measuring the expression level of the gene shown in SEQ ID NO: 6 or 17 or the expression level of a homologous gene of the gene and selecting a substance that reduces the expression level as compared with a control.

The screening method of the present invention is, for example, a screening method for a compound having a therapeutic or prophylactic effect for diabetes including the following steps (a) to (c).
(A) contacting a test compound with a cell expressing the marker gene of the present invention (the gene according to any one of SEQ ID NOs: 1 to 8 and 10 to 27 or a homologous gene of the gene) (b) A step of measuring the expression level of the gene (c) a step of selecting a compound that changes the expression level as compared with the case of measuring in the absence of the test compound

  In this screening method, first, a test compound is brought into contact with a cell expressing the marker gene of the present invention (the gene described in any of SEQ ID NOs: 1 to 8, 10 to 27, or a homologous gene of the gene). Examples of the origin of the “cell” used include, but are not particularly limited to, cells derived from mice, rats, humans, cats, dogs, cows, sheep, birds, pets, livestock, and the like. As the “cell expressing the marker gene of the present invention”, a cell expressing the endogenous gene or a cell into which the foreign gene is introduced and expressing the gene can be used. . A cell in which the exogenous gene is expressed can be usually prepared by introducing an expression vector into which the gene has been inserted into a host cell. The expression vector can be prepared by general genetic engineering techniques.

  There is no restriction | limiting in particular as a test compound used for this screening method. For example, natural compounds, organic compounds, inorganic compounds, proteins, peptides and other single compounds, as well as compound libraries, gene library expression products, cell extracts, cell culture supernatants, published microbial products, marine organism extracts , Plant extracts and the like, but are not limited thereto.

  The “contact” of the test compound to the cell expressing the marker gene of the present invention is usually performed by adding the test compound to the culture medium of the cell expressing the gene, but is not limited to this method. When the test compound is a protein or the like, “contact” can be performed by introducing a DNA vector expressing the protein into the cell.

  In this screening method, the expression level of the marker gene of the present invention is then measured. Here, “gene expression” includes both transcription and translation. The gene expression level can be measured by methods known to those skilled in the art. For example, mRNA can be extracted from cells expressing the gene according to a standard method, and the transcription level of the gene can be measured by performing Northern hybridization or RT-PCR using this mRNA as a template. It is also possible to measure the translation level of the gene by recovering the protein fraction from cells expressing the gene and detecting the expression of the protein encoded by the gene by electrophoresis such as SDS-PAGE. .

  Furthermore, it is also possible to measure the translation level of a gene by detecting the expression of the protein by carrying out Western blotting using an antibody against the protein encoded by the marker gene of the present invention. The antibody used for detection of the protein encoded by the gene is not particularly limited as long as it is a detectable antibody. For example, both a monoclonal antibody and a polyclonal antibody can be used.

  In this screening method, a compound that changes the expression level of the gene is then selected as compared with the case where the test compound is not contacted (control). The compound to be changed becomes a drug for the treatment or prevention of diabetes.

Another preferred embodiment of the screening method of the present invention is a method for identifying a compound that changes the expression level of the marker gene of the present invention using the expression of the reporter gene as an index. This method is, for example, a screening method for a compound having an effect of treating or preventing diabetes, including the following steps (a) to (c).
(A) a step of bringing a test compound into contact with a cell containing DNA having a structure in which the transcriptional regulatory region of the marker gene of the present invention and the reporter gene are functionally linked, and (b) measuring the expression level of the reporter gene Step (c) A step of selecting a compound that changes the expression level as compared with the case where it is measured in the absence of the test compound.

  In this screening method, first, a test compound is contacted with a cell or cell extract containing DNA having a structure in which a transcriptional regulatory region of a marker gene of the present invention and a reporter gene are functionally linked. Here, “functionally linked” means that the transcriptional regulatory region of the gene is bound to the reporter gene so that expression of the reporter gene is induced by binding of a transcription factor to the transcriptional regulatory region of the gene. Indicates what you are doing. Therefore, even when the reporter gene is bound to another gene and forms a fusion protein with another gene product, a transcription factor binds to the transcriptional regulatory region of the gene, so that the fusion protein Any expression that is induced is included in the meaning of “functionally linked”. Based on the cDNA base sequence of the gene, those skilled in the art can obtain the transcriptional regulatory region of the gene present in the genome by a well-known method.

  The reporter gene used in this method is not particularly limited as long as its expression can be detected, and examples thereof include a CAT gene, a LacZ gene, a luciferase gene, and a GFP gene. Examples of the “cell containing a DNA having a structure in which the transcriptional regulatory region of the marker gene of the present invention and a reporter gene are functionally linked” include a cell into which a vector having such a structure inserted is introduced. Such vectors can be prepared by methods well known to those skilled in the art. Introduction into a cell can be performed by a general method such as a calcium phosphate precipitation method, an electric pulse perforation method, a lipofectamine method, a microinjection method and the like. “Cells containing DNA having a structure in which the transcriptional regulatory region of the marker gene of the present invention and a reporter gene are functionally linked” include cells in which the structure is inserted into the chromosome. The DNA structure can be inserted into the chromosome by a method generally used by those skilled in the art, for example, a gene introduction method utilizing homologous recombination.

  `` A cell extract containing DNA having a structure in which the transcriptional regulatory region of the marker gene of the present invention and a reporter gene are functionally linked '' refers to, for example, a cell extract contained in a commercially available in vitro transcription translation kit, Examples include those to which DNA having a structure in which a transcriptional regulatory region of the gene and a reporter gene are functionally linked is added.

  “Contact” in this method means that a test compound is added to a culture solution of “a cell containing a DNA having a structure in which a transcriptional regulatory region of a marker gene of the present invention and a reporter gene are functionally linked”, or the DNA It can be performed by adding a test compound to the above-described commercially available cell extract. When the test compound is a protein, it can also be carried out, for example, by introducing a DNA vector that expresses the protein into the cell.

  In this method, the expression level of the reporter gene is then measured. The expression level of the reporter gene can be measured by methods known to those skilled in the art depending on the type of the reporter gene. For example, when the reporter gene is a CAT gene, the expression level of the reporter gene can be measured by detecting acetylation of chloramphenicol by the gene product. When the reporter gene is a lacZ gene, by detecting the color development of the dye compound catalyzed by the gene expression product, and when the reporter gene is a luciferase gene, the fluorescent compound catalyzed by the gene expression product By detecting fluorescence, and in the case of a GFP gene, the expression level of the reporter gene can be measured by detecting fluorescence due to the GFP protein.

  In this method, a compound that changes (including an increase or decrease) is then selected as compared to the case where the measured expression level of the reporter gene is measured in the absence of the test compound. The compound to be changed is expected to be a drug for the treatment or prevention of diabetes.

  Another aspect of the screening method of the present invention is a method using the activity of the protein encoded by the marker gene of the present invention as an index.

A preferred embodiment of the above method is, for example, a screening method for a compound having an effect of treating or preventing type II diabetes, comprising the following steps (a) to (c).
(A) a protein encoded by the marker gene of the present invention (preferably, the gene according to SEQ ID NO: 2, 13, or 14 or a homologous gene of the gene), or a cell expressing the protein, A step of contacting a test compound (b) a step of measuring the activity of the protein (c) a step of selecting a compound that changes the activity of the protein as compared to the case of measuring in the absence of the test compound

  In this method, first, a protein encoded by the marker gene of the present invention (preferably, the gene described in SEQ ID NO: 2, 13, or 14 or a homologous gene of the gene), or a cell expressing the protein Alternatively, the cell extract is brought into contact with the test compound. The protein activity is then measured. Information on the activity of the protein and a method for measuring the activity can be appropriately obtained by those skilled in the art from publicly known literatures and public databases based on the contents disclosed in the present specification.

  In the above method, for example, a protein whose activity has already been found (for example, a protein encoded by a gene described in any of SEQ ID NOs: 2, 13, 14 or a homologous gene of the gene) is preferably used. be able to.

  For example, when the protein to be used as an index is a protein (cathepsin B) encoded by the gene described in SEQ ID NO: 2 or a homologous gene thereof, for example, 100 mM sodium phosphate (pH 6.0), 1 Z-Arg-Arg- by the action of catthepsin B in a reaction solution containing mM EDTA, 10 mM dithiothleitol, 0.8 mM N-bezyloxycarbonyl-L-arginyl-L-arginine-p-nitroanilide (Z-Arg-Arg-pNA) By detecting p-nitroaniline generated from pNA as a change in absorbance at a light wavelength of 410 nm, the activity of the protein can be evaluated (measured) (S Hasnain, T Hirama, A Tam, and JS Mort, Characterization of recombinant rat cathepsin B and nonglycosylated mutants expressed in yeast., New insights into the pH dependence of cathepsin B-catalyzed hydrolyses, J. Biol. Chem., Mar 1992; 267: 4713-4721).

  When the protein to be used as an index is a protein (carboxypeptidase B) encoded by the gene shown in SEQ ID NO: 13 or a homologous gene thereof, for example, 25 mM Tris-HCl (pH 7.65), 100 mM NaCl, 1 By detecting hippuric acid produced by the action of carboxylpeptidase B in a reaction solution containing mM hippuryl-L-arginine at a light wavelength of 254 nm, the activity of the protein can be evaluated (measured) (JE Folk, Karl A Piez, William R. Carroll, and Jules A. Gladner, Carboxypeptidase B. IV. PURIFICATION AND CHARACTERIZATION OF THE PORCINE ENZYME, J. Biol. Chem., Aug 1960; 235: 2272-2277).

  When the protein used as an index is a protein (C1-tetrahydrofolate synthase) encoded by the gene of SEQ ID NO: 14 or a homologous gene thereof, for example, 50 mM sodium 2- (N-morpholino) sthanesulfonate (pH 6.0), 0.1 mM tetrahydrofolate, 2.5 mM Mg-ATP, 50 mM sodium formate, 25 mM ammonium sulfate, 5 mM 2-mercaptoethanol, 10 μg cyclohydrolase produces 10-formyltetrahydrofolate by cyclohydrolase. By converting to 5, 10-methyltetrahydrofolate and detecting the change as an increase in absorbance at an optical wavelength of 355 nm, the activity of the protein can be evaluated (measured) (E Villar, B Schuster, D Peterson, and V Schirch, C1-Tetrahydrofolate synthase from rabbit liver. Structural and kinetic properties of the enzyme and its two domains, J. Biol. Chem., Feb 1985; 260: 2245-2252.).

  In the above method of the present invention, a compound that changes the activity is then selected as compared to the case where it is measured in the absence of the test compound. Compounds that alter activity become candidate agents for the treatment or prevention of diabetes.

Furthermore, the screening method of the present invention can be performed using the genetically modified animal of the present invention.
That is, it is possible to screen for a substance that changes the expression of the marker gene to a normal level using a modified animal in which the expression of the marker gene of the present invention is reduced or increased from the normal level.

In the preferable aspect of the said method of this invention, it is a method including the process of the following (a)-(c).
(A) The step of administering a test compound to the genetically modified non-human animal of the present invention (b) The step of measuring the expression level of the gene in the animal (c) Compared with the case where the test compound is not administered And selecting a compound that changes the expression level of the gene.

  In this method, first, a test compound is administered to the non-human animal. The test compound can be administered either orally or parenterally, but parenteral administration is preferred when the test compound is a peptide. Specific examples include vascular administration, nasal administration, pulmonary administration, and transdermal administration. Examples of vascular administration include intravenous injection, intramuscular injection, intraperitoneal injection, subcutaneous injection, and the like, and can be administered systemically or locally.

  When the test compound is DNA, a viral vector such as a retrovirus, adenovirus, or Sendai virus, or a non-viral vector such as a liposome can be used when administered in vivo. Examples of administration methods include in vivo methods and ex vivo methods.

  Next, the expression level or activity of the marker gene of the present invention in the non-human animal is measured. Next, a compound that changes the expression level or activity of the marker gene of the present invention (for example, restores the gene expression level in a wild-type animal) is selected as compared with the case where the test compound is not administered (control).

More specifically, when the following animal (a) is used as the genetically modified non-human animal in the above method, a compound that increases the expression level is selected. The compound is expected to be a candidate compound for the treatment or prevention of type II diabetes.
(A) Type II diabetes model animal in which expression of the gene according to any one of SEQ ID NOs: 1-3, 7, 8, 11 or a homologous gene of the gene is suppressed

When the following animal (b) is used as the genetically modified non-human animal, a compound that reduces the expression level is selected. The compound is expected to be a candidate compound for the treatment or prevention of type II diabetes.
(B) Type II diabetes model animal in which expression of SEQ ID NO: 4-6, 10, 12-27 or a homologous gene of the gene is increased

When the following animal (c) is used as the genetically modified non-human animal, a compound that increases the expression level is selected. The compound is expected to be a candidate compound for the treatment or prevention of type I diabetes.
(C) Type I diabetes model animal in which expression of the gene of SEQ ID NO: 3 or 7 or a homologous gene of the gene is suppressed

When the following animal (d) is used as the genetically modified non-human animal, a compound that reduces the expression level is selected. The compound is expected to be a candidate compound for the treatment or prevention of type I diabetes.
(D) Type I diabetes model animal in which the expression of the gene of SEQ ID NO: 6 or 17 or the expression of the homologous gene of the gene is increased

  The agent of the present invention can be mixed with a physiologically acceptable carrier, excipient, diluent or the like and administered orally or parenterally as a pharmaceutical composition. As oral preparations, dosage forms such as granules, powders, tablets, capsules, solvents, emulsions or suspensions can be used. As the parenteral preparation, a dosage form such as an injection, an infusion, an external medicine, or a suppository can be selected. Examples of injections include subcutaneous injections, intramuscular injections, intraperitoneal injections, and the like. Examples of the medicine for external use include a nasal administration agent or an ointment. The preparation technology for making the above-mentioned dosage form so as to include the drug of the present invention as the main component is known.

  For example, a tablet for oral administration can be produced by adding an excipient, a disintegrant, a binder, a lubricant, and the like to the drug of the present invention, mixing, and compressing and shaping. As the excipient, lactose, starch, mannitol or the like is generally used. As the disintegrant, calcium carbonate, carboxymethyl cellulose calcium and the like are generally used. As the binder, gum arabic, carboxymethylcellulose, or polyvinylpyrrolidone is used. As the lubricant, talc, magnesium stearate and the like are known.

  The tablet containing the drug of the present invention can be coated with a known coating for masking or enteric preparation. As the coating agent, ethyl cellulose, polyoxyethylene glycol, or the like can be used.

  An injection can be obtained by dissolving the drug of the present invention as a main component together with an appropriate dispersant, or dissolving or dispersing in a dispersion medium. Depending on the selection of the dispersion medium, either a water-based solvent or oil-based solvent can be used. In order to use an aqueous solvent, distilled water, physiological saline, Ringer's solution, or the like is used as a dispersion medium. In the oil solvent, various vegetable oils and propylene glycol are used as a dispersion medium. At this time, a preservative such as paraben may be added as necessary. In addition, known isotonic agents such as sodium chloride and glucose can be added to the injection. Further, a soothing agent such as benzalkonium chloride or procaine hydrochloride can be added.

  Moreover, it can be set as an external preparation by making the chemical | medical agent of this invention into a solid, liquid, or semi-solid composition. About a solid or liquid composition, it can be set as an external preparation by setting it as the composition similar to what was described previously. A semi-solid composition can be prepared by adding a thickener to an appropriate solvent as required. As the solvent, water, ethyl alcohol, polyethylene glycol, or the like can be used. As the thickener, bentonite, polyvinyl alcohol, acrylic acid, methacrylic acid, or polyvinylpyrrolidone is generally used. A preservative such as benzalkonium chloride can be added to the composition. Also, a suppository can be obtained by combining an oily base material such as cacao butter or an aqueous gel base material such as a cellulose derivative as a carrier.

  The necessary amount of the drug of the present invention is administered to mammals including humans within the safe dose range. The dosage of the drug of the present invention can be appropriately determined finally based on the judgment of a doctor or veterinarian in consideration of the type of dosage form, administration method, patient age and weight, patient symptoms, and the like.

EXAMPLES Hereinafter, although an Example demonstrates this invention in detail, this invention is not restrict | limited by these Examples.
[Example 1] Search for genes whose expression varies in type II diabetes
Blood was collected from male 8-week-old rats of spontaneously diabetic rats (GK rats) (CLEA Japan) and normal Wister rats (CLEA Japan) as type II diabetes model rats and used for the following experiments .

PAXgene Blood RNA Kit (QIAGEN) was used for total RNA extraction from blood, and total RNA was obtained according to the attached protocol. Purification of mRNA was performed using the obtained total RNA and Oligotex-MAG mRNA Purification Kit (TaKaRa) according to the attached protocol. Select approximately 3000 clones from the rat 3 ′ end-specific cDNA library prepared by the present inventors, 3 μl of E. coli culture solution as a template, 100 μl of the total reaction solution, and PCR in a 96-well plate (ABgene) went. The reaction solution final concentration of 10 mM Tris-HCl (pH8.3) , 50 mM KCl, 0.2 mM dNTPs, 1.5 mM MgCl 2, 0.2μM Primer Mix, 0.025 U / μl AmpliTaq Gold so that the DNA polymerase (Applied Biosystems) Prepared. PCR conditions were as follows: heat denaturation at 95 ° C. for 10 minutes, followed by 40 reactions of 95 ° C. for 30 seconds, 55 ° C. for 1 minute, 72 ° C. for 1 minute, and finally 72 ° C. for 7 minutes. After the reaction, an equal amount of isopropanol was added and mixed, and then allowed to stand overnight at -20 ° C, followed by centrifugation at 3000 rpm x 1 hour at 4 ° C. The supernatant was discarded and dried, and then dissolved in 50% DMSO. Each of the clones was spotted twice using a SpotArray24 (PerkinElmer) on a coated glass DMSO-compatible TYPE1 densified amino group introduction type (Matsunami Glass) for DNA microarray. After spotting, the plate was allowed to stand at room temperature for 30 minutes or more and dried completely, and then UV-crosslinked with an energy of 250 mJoules using a UV crosslinker (STRATAGENE) to fix the cDNA to the slide. Slides were transferred to a slide rack and washed with gentle shaking for 1 minute in 3 × SSC containing 1% SDS. The slide was transferred into sterile distilled water and allowed to stand at room temperature for 5 minutes for washing. After performing this operation twice, the slide was immersed in boiling distilled water for 2 minutes. The slide was quickly removed and allowed to stand until it was completely dry while being shielded from light at room temperature. This was made into rat cDNA Chip, shielded from light and dried until used, and stored at room temperature.

  Using 0.5 μg of mRNA extracted from GK rat blood and Wister rat blood as a template, these were fluorescently labeled with Cy3 (PerkinElmer) and Cy5, or vice versa, according to the Label Star Array Kit (QIAGEN) protocol. After labeling, the probe was purified using a purification column in the kit to obtain 24 μl of a fluorescently labeled cDNA probe. To this, 20 × SSC (300 mM sodium chloride, 300 mM sodium citrate) was added to a final concentration of 5 × SSC, and heat denaturation was performed at 95 ° C. for 3 minutes. Immediately after heat denaturation, it was rapidly cooled at 4 ° C., and 10% (w / v) SDS was added to a final concentration of 0.5% and mixed well. A gap cover glass (24 × 50 mm) (Matsunami Glass) washed with ethanol was placed so as to cover the entire spot cDNA chip spotted surface. The purified probe solution was poured so as not to allow air to enter through the gap cover glass. After incubation for 18 hours at 65 ° C with SYNTHETECH OVEN HA-1 (BM instrument), gently peel off the gap cover glass in 2x SSC solution containing 0.1% SDS, and then 2x SSC containing 0.1% SDS for 20 minutes. Washing was performed in the order of 0.2 × SSC containing 0.1% SDS for 20 minutes, 0.2 × SSC for several tens of seconds, and 0.05 × SSC for several tens of seconds. All washing was performed in a dark room. After washing, the mixture was lightly centrifuged at 300 rpm and dried at room temperature. Thereafter, the fluorescence pattern was read with ScanArray Express (PerkinElmer), and the signal intensity of each was digitized. As a result, 27 genes were selected that showed a difference in gene expression more than double between normal rats and type II diabetes model rats. The names of these 27 genes and the sequence numbers of the primer sequences of each gene are shown below. The primer sequences are shown in Tables 4 and 5 together with the primer names.

<Gene selected by microarray> (homology search results)
Diabetes marker gene 1: Rattus norvegicus 13 BAC CH230-151K3 (Children's Hospital Oakland Research Institute) (primer sequence number: 145, 146)
Diabetes marker gene 2: R. norvegicus mRNA for cathepsin B (primer SEQ ID NO: 147, 148)
Diabetes marker gene 3: Mus musculus cyclin I, mRN (primer sequence numbers: 149, 150)
Diabetes marker gene 4: Mus musculus RIKEN cDNA 1110032C13 gene, mRNA (primer sequence numbers: 151, 152)
Diabetes marker gene 5: Mus musculus mitochondrial ribosomal protein S7, mRNA (primer SEQ ID NOs: 153, 154)
Diabetes marker gene 6: Mouse DNA sequence from clone RP23-423P8 on chromosome 4 contains the 3 'end of the Astn2 gene for astrotactin 2 and the 3' end of the Pappa gene for pregnancy-associated plasma protein A (primer SEQ ID NO: 155 156)
Diabetes marker gene 7: Mus musculus nuclear receptor coactivator 4, mRNA (primer sequence number: 157, 158)
Diabetes marker gene 8: Rat mRNA for the cysteine proteinase inhibitor cystatin C (primer SEQ ID NOs: 159 and 160)
Diabetes marker gene 9: Mus musculus Sac domain-containing inositol phosphatase 3, mRNA (primer SEQ ID NO: 161, 162)
Diabetes marker gene 10: R. norvegicus mRNA for coupling factor 6 of mitochondrial ATP synthase complex. (Primer SEQ ID NO: 163, 164)
Diabetes marker gene 11: Rattus norvegicus Chymotrypsinogen B (Ctrb), mRNA (primer SEQ ID NO: 165, 166)
Diabetes marker gene 12: Rattus norvegicus clone RP31-249D7 strain Brown Norway (primer SEQ ID NO: 167, 168)
Diabetes marker gene 13: Rat carboxypeptidase B gene, exons 1 and 2. (Primer SEQ ID NO: 169, 170)
Diabetes marker gene 14: Rat C1-tetrahydrofolate synthase mRNA (primer sequence numbers: 171, 172)
Diabetes marker gene 15: Rattus norvegicus AIF mRNA for apoptosis-inducing factor (primer SEQ ID NOs: 173 and 174)
Diabetes marker gene 16: Rattus norvegicus LIS1-interacting protein NUDE2 mRNA (Primer SEQ ID NO: 175, 176)
Diabetes marker gene 17: Mus musculus chromosome 3 clone RP24-344F8 (primer SEQ ID NO: 177, 178)
Diabetes marker gene 18: Mus musculus ring-box 1, mRNA (primer sequence numbers: 179, 180)
Diabetes marker gene 19: Mus musculus Brca1 associated protein 1 (BAP1), mRNA (primer sequence numbers: 181 and 182)
Diabetes marker gene 20: Mus musculus chromosome 14 clone RP23-187G1 (primer sequence numbers: 183, 184)
Diabetes marker gene 21: Messenger RNA for rat preproalbumin. (Primer SEQ ID NO: 185, 186)
Diabetes marker gene 22: Mus musculus mRNA for resistin-like molecule gamma (retn1g gene). (Primer SEQ ID NO: 187, 188)
Diabetes marker gene 23: Rattus norvegicus 1 BAC CH230-362O18 (Children's Hospital Oakland Research Institute) (Primer SEQ ID NOs: 189, 190)
Diabetes marker gene 24: Mus musculus RAB24, member RAS oncogene family, mRNA (primer sequence numbers: 191 and 192)
Diabetes marker gene 25: Mus musculus chromosome 7 clone RP24-344N22 (primer SEQ ID NOs: 193 and 194)
Diabetes marker gene 26: Rat angiotensin receptor (AT1) gene, single exon. (Primer SEQ ID NO: 195, 196)
Diabetes marker gene 27: Rattus norvegicus Nphs2 mRNA for podocin (primer SEQ ID NO: 197, 198)

Table 5 is a continuation table of Table 4.

Reverse transcription was performed using 100 ng of mRNA according to the protocol attached to ReverTra Dash (TOYOBO) (using Oligo (dT) primer) to synthesize cDNA. Specific primers were designed for each of the 27 genes selected from the microarray experiments, and real-time PCR was performed using ABI PRISM 7900 Sequence Detection System (Applied Biosystems) using 5 times diluted cDNA as a template. Primer design support site Primer3 (http://frodo.wi.mit.edu/cgi-bin/primer3_www.cgi) was used for primer design. The composition of the reaction solution is 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 0.2 mM dNTPs, 2.5 mM MgCl ₂ , 0.3 μM Primer mix, 0.2 × SYBR Green I (SIGMA), 0.16 pmol / μl 5 -ROX, 0.0225 U / μl AmpliTaq Gold DNA Polymerase (Applied Biosystems) was prepared. PCR conditions were as follows: heat denaturation at 95 ° C. for 10 minutes, and then a reaction of 94 ° C .: 30 seconds, 60 ° C .: 40 seconds, 72 ° C .: 1 minute was repeated 40 times. After the reaction, the gene expression was analyzed with the analysis software ABI PRISM 7000 SDS Software. The expression level of the target gene was normalized by the expression level of the internal standard gene β-globin, and this value was compared between normal rats and type II diabetic rats.
As a result, it was confirmed that the expression level of 6 genes was decreased in the blood of type II diabetic rats and the expression level of 20 genes was increased (see FIG. 1).

《Gene with low expression level in type II diabetic rats》
Diabetes marker gene 1: Rattus norvegicus 13 BAC CH230-151K3 (Children's Hospital Oakland Research Institute)
Diabetes marker gene 2: R. norvegicus mRNA for cathepsin B
Diabetes marker gene 3: Mus musculus cyclin I, mRNA
Diabetes marker gene 7: Mus musculus nuclear receptor coactivator 4, mRNA
Diabetes marker gene 8: Rat mRNA for the cysteine proteinase inhibitor cystatin C
Diabetes marker gene 11: Rattus norvegicus Chymotrypsinogen B (Ctrb), mRNA

《Gene with high expression level in type II diabetic rats》
Diabetes marker gene 4: Mus musculus RIKEN cDNA 1110032C13 gene, mRNA
Diabetes marker gene 5: Mus musculus mitochondrial ribosomal protein S7, mRNA
Diabetes marker gene 6: Mouse DNA sequence from clone RP23-423P8 on chromosome 4 contains the 3 'end of the Astn2 gene for astrotactin 2 and the 3' end of the Pappa gene for pregnancy-associated plasma protein A
Diabetes marker gene 10: R. norvegicus mRNA for coupling factor 6 of mitochondrial ATP synthase complex.
Diabetes marker gene 12: Rattus norvegicus clone RP31-249D7 strain Brown Norway
Diabetes marker gene 13: Rat carboxypeptidase B gene, exons 1 and 2.
Diabetes marker gene 14: Rat C1-tetrahydrofolate synthase mRNA
Diabetes marker gene 15: Rattus norvegicus AIF mRNA for apoptosis-inducing factor
Diabetes marker gene 16: Rattus norvegicus LIS1-interacting protein NUDE2 mRNA
Diabetes marker gene 17: Mus musculus chromosome 3 clone RP24-344F8
Diabetes marker gene 18: Mus musculus ring-box 1, mRNA
Diabetes marker gene 19: Mus musculus Brca1 associated protein 1 (BAP1), mRNA
Diabetes marker gene 20: Mus musculus chromosome 14 clone RP23-187G1
Diabetes marker gene 21: Messenger RNA for rat preproalbumin.
Diabetes marker gene 22: Mus musculus mRNA for resistin-like molecule gamma (retn1g gene).
Diabetes marker gene 23: Rattus norvegicus 1 BAC CH230-362O18 (Children's Hospital Oakland Research Institute)
Diabetes marker gene 24: Mus musculus RAB24, member RAS oncogene family, mRNA
Diabetes marker gene 25: Mus musculus chromosome 7 clone RP24-344N22
Diabetes marker gene 26: Rat angiotensin receptor (AT1) gene, single exon.
Diabetes marker gene 27: Rattus norvegicus Nphs2 mRNA for podocin

[Example 2] Diagnosis of onset of type I diabetes model rat (STZ-administered rat) using diabetes marker gene Examination of whether the gene selected in the above experiment can be used not only for type II diabetes but also for type I diabetes did.
Unlike non-insulin-dependent type II diabetes caused by lifestyle and genetic factors, type I diabetes is caused by a decrease in insulin secretion caused by destruction of β cells in the pancreas. Therefore, the present inventors administered streptozotocin (STZ) having a pancreatic β-cell destruction action to male 8-week-old Wister rats in stages of 25 mg, 45 mg, and 65 mg per kg body weight, and the onset level of diabetes is increased. Different rats were made. Diabetes onset level was confirmed by collecting blood from the tail vein on an empty stomach and measuring the blood glucose level. As a result, finally, type I diabetes model rats with different onset levels at 4 stages could be prepared (see Table 6 “Average blood glucose level before and after STZ administration (mg / dl)”).

  The water was given as free drinking water, and the food was 12 g (CE-2 Nippon Claire Co., Ltd.) per day. After 14 days of rearing, the animals were dissected and blood samples were taken. Extraction of mRNA from blood and analysis by real-time PCR followed the method described above.

As a result, four diabetes marker genes whose diabetes onset level and gene expression level are linked were identified (see FIG. 2).
Diabetes marker gene 3: Mus musculus cyclin I, mRNA
Diabetes marker gene 7: Mus musculus nuclear receptor coactivator 4, mRNA
Diabetes marker gene 6: Mouse DNA sequence from clone RP23-423P8 on chromosome 4 contains the 3 'end of the Astn 2 gene for astrotactin 2 and the 3' end of the Pappa gene for pregnancy-associated plasma protein A
Diabetes marker gene 17: Mus musculus chromosome 3 clone RP24-344F8

  As diabetes became more severe, there was a tendency for diabetes marker genes 3 and 7 to decrease in gene expression in stages, while diabetes marker genes 6 and 17 increased (see FIG. 2). In this experiment, there was no difference in blood glucose level between normal rats and extremely mild diabetic rats, but this method using gene expression level as an index was able to detect a clear difference between the two. . This confirms that the above four diabetes marker genes can diagnose not only the onset of type II diabetes but also early stage diabetes that is not reflected in blood glucose levels in type I diabetes. It was.

FIG. 1 is a graph showing the expression level of a diabetes marker gene in a type II diabetes model rat. The expression level of the target gene was normalized with the expression level of the internal standard gene β-globin, and this value was compared between normal rats and type II diabetic rats. The genes shown in graphs A and B are genes whose expression level is low in type II diabetes model rats, and the genes shown in C to G are genes whose expression level is high in type II diabetes model rats. FIG. 2 is a graph showing changes in the expression level of diabetes marker genes (3, 6, 7, and 17) in rats administered with type I diabetes model STZ.

Claims (20)

  A test method for a predisposition to type II diabetes, using as an index the expression level of the gene according to any one of SEQ ID NOs: 1 to 8 and 10 to 27 or a homologous gene of the gene. The test method according to claim 1, wherein when the gene expression level is (a) or (b) below, the test animal is determined to have a predisposition to type II diabetes.
(A) The expression level of the gene according to any one of SEQ ID NOs: 1 to 3, 7, 8, or 11 or a homologous gene of the gene is decreased as compared with the control (b) SEQ ID NOs: 4 to The expression level of the gene according to any one of 6, 10, 12 to 27 or a homologous gene of the gene is increased compared to the control.   A test method for a predisposition to type I diabetes, using as an index the expression level of the gene according to SEQ ID NO: 3, 6, 7, or 17 or a homologous gene of the gene. The test method according to claim 3, wherein the test animal is determined to have a predisposition to type I diabetes when the expression level of the gene is the following (a) or (b).
(A) The expression level of the gene described in SEQ ID NO: 3 or 7 or a homologous gene of the gene is reduced compared to the control (b) the gene described in SEQ ID NO: 6 or 17, or the gene The expression level of homologous gene is increased compared to the control   The test | inspection method in any one of Claims 1-4 which uses for the test | inspection the biological sample derived from a test animal as a test sample. A test agent for predisposition to diabetes comprising any of the following substances (a) to (d) as an active ingredient.
(A) an oligonucleotide that hybridizes to the DNA of the gene of any one of claims 1 to 8 and 10 to 27 and has a chain length of at least 15 nucleotides (b) A solid phase (c) on which a nucleotide probe that hybridizes with DNA of the gene according to any one of 1 to 8 and 10 to 27 is immobilized (SEQ ID NO: 1 to 8 or 10 to 27) Primer oligonucleotide (d) for amplifying the DNA of the gene according to claim 1. Antibody against the protein encoded by the gene according to any one of SEQ ID NOs: 1 to 8 and 10 to 27. A test kit for a predisposition to type II diabetes comprising any of the following (a) to (d) as a constituent:
(A) an oligonucleotide that hybridizes to the DNA of the gene of any one of claims 1 to 8 and 10 to 27 and has a chain length of at least 15 nucleotides (b) A solid phase (c) on which a nucleotide probe that hybridizes with DNA of the gene according to any one of 1 to 8 and 10 to 27 is immobilized (SEQ ID NO: 1 to 8 or 10 to 27) Primer oligonucleotide (d) for amplifying the DNA of the gene according to claim 1. Antibody against the protein encoded by the gene according to any one of SEQ ID NOs: 1 to 8 and 10 to 27.   Containing as an active ingredient a substance that enhances the expression of the gene according to SEQ ID NO: 1-3, 7, 8, 11, or a homologous gene of the gene, or the function of the protein encoded by the gene, Drugs for the treatment or prevention of type II diabetes.   Containing as an active ingredient a substance that suppresses the expression of the gene according to any one of SEQ ID NOs: 4-6, 10, 12-27 or the homologous gene of the gene, or the function of the protein encoded by the gene, Drugs for the treatment or prevention of type II diabetes.   For the treatment or prevention of type I diabetes, comprising as an active ingredient a substance that enhances the expression of the gene of SEQ ID NO: 3 or 7 or a homologous gene of the gene, or the function of the protein encoded by the gene Drugs.   Treatment or prevention of type I diabetes containing, as an active ingredient, a substance that suppresses the expression of the gene shown in SEQ ID NO: 6 or 17 or the expression of a homologous gene of the gene, or the function of the protein encoded by the gene Drug for. The drug according to claim 9 or 11, wherein the substance that suppresses gene expression is a compound selected from the group consisting of the following (a) to (c).
(A) an antisense nucleic acid for the transcription product of the gene or a part thereof (b) a nucleic acid having a ribozyme activity that specifically cleaves the transcription product of the gene (c) an action of inhibiting the expression of the gene by the RNAi effect Nucleic acid The drug according to claim 9 or 11, wherein the substance that suppresses the function of the protein is the following compound (a) or (b).
(A) an antibody that binds to the protein (b) a low molecular weight compound that binds to the protein   A gene-modified diabetes model non-human animal, wherein the expression level of the gene according to claim 1 is artificially modified.   A method for screening a compound having an effect of treating or preventing type II diabetes, comprising selecting a compound that changes the expression level of any of the genes according to claim 1.   A method for screening a compound having a therapeutic or prophylactic effect for type I diabetes, comprising selecting a compound that changes the expression level of any one of the genes according to claim 3. The screening method according to claim 15 or 16, comprising the following steps (a) to (c).
(A) a step of bringing a test compound into contact with a cell expressing any of the genes according to claim 1 or 3; (b) a step of measuring the expression level of the gene; and (c) in the absence of the test compound. The step of selecting a compound that changes the expression level of the gene as compared to the case of measuring in The screening method according to claim 15 or 16, comprising the following steps (a) to (c).
(A) a step of contacting a test compound with a cell or a cell extract containing DNA having a structure in which the transcriptional regulatory region of the gene according to claim 1 or 3 and a reporter gene are functionally linked ( b) a step of measuring the expression level of the reporter gene (c) a step of selecting a compound that changes the expression level of the reporter gene as compared to the measurement in the absence of the test compound The screening method according to claim 15 or 16, which is a screening method using a non-human animal, comprising the following steps (a) to (c).
(A) a step of administering a test compound to a non-human animal expressing any of the genes according to claim 1 or 3 (b) a step of measuring the expression level of the gene in the animal (c) a test A step of selecting a compound that changes the expression level of the gene as compared to the case where the compound is not administered. A screening method for a compound having an effect of treating or preventing type II diabetes, comprising the following steps (a) to (c).
(A) contacting a test compound with a protein encoded by the gene according to any one of SEQ ID NOs: 2, 13, 14 or a homologous gene of the gene, or a cell expressing the protein (b) A step of measuring the activity of the protein (c) a step of selecting a compound that changes the activity of the protein as compared to the case of measuring in the absence of the test compound