WO2004035777A1 - Adipocyte differentiation-associated genes and proteins - Google Patents

Abstract

Genes expressed within 6 hours after the initiation of differentiation from adipocyte precursors into adipocytes with showing a peak 3 hours thereafter are found out. A polynucleotide containing a base sequence which is the same as a base sequence represented by SEQ ID NO:1 or SEQ ID NO:3 or has a homology of at least 80% therewith, or a polynucleotide complementary to the above polynucleotide; and a protein encoded by a polynucleotide of SEQ ID NO:1 or SEQ ID NO:3 and containing an amino acid sequence which is the same as a protein represented by SEQ ID NO:2 or SEQ ID NO:4 over the full length or has a homology of at least 80% therewith.

Description

 Specification

 Adipocyte differentiation-related genes and proteins

 The present invention relates to a clone 104 polynucleotide and a clone 104 protein that are activated in the early stage of adipocyte differentiation, and further relates to an antibody, an angelite and an agonist of the clone 104 protein. , And their production, and pharmaceuticals and diagnostics containing these substances. Background art

 Patent Document 1: Japanese Patent Application Laid-Open No. H8-33333

 Non-Patent Document 1: Biochemical and Biophysical Research

 Communications 254, 299-305 (1999)

 Obesity due to overnutrition is the leading cause of serious lifestyle-related diseases such as diabetes, hypertension, and atherosclerosis. Elucidation of the molecular mechanism of obesity is an essential issue in considering future life sciences and health sciences. Adipocytes are directly involved in obesity, and if the process of producing adipocytes is elucidated, it will directly lead to the treatment of obesity. With the recent development of molecular biology, the mechanism of adipocyte differentiation is being elucidated. It is known that there are key genes that form a network for exchanging information skillfully.

The adipocyte differentiation process consists of complex steps, which differentiate into lipoblasts (Adipoblasts) → preadipocytes (Predipocytes) → adipocytes (Adipocytes). In recent years, transcription factors that are closely involved in adipocyte differentiation using cultured cells or systems using genetically modified individuals have been elucidated in recent years. PPAR (Peroxisome Pro I iferator-Activated Receptor) Binding Protein 1 or Adipocyte Determination and Differentiation-dependent

Factor 1) is said to be the most important transcription factor for adipocyte differentiation

PPARs have been shown to form families, among which PPARa has been shown to be particularly important for adipocyte differentiation. In other words, it is clear that forcible expression of PPARτ in lipoblasts and preadipocytes differentiates into adipocytes. This result proves that PPARA plays an important role in adipocyte differentiation.

 C / EBP also forms a family similar to PPAR, and recently, C / EBPα functions as a master regulator of adipocyte differentiation like PPARa. It is clear that there is. In addition, C / EBP; 8 and CEBP <5 are expressed in the early stage of differentiation, and S / RBP1 / ADD1, which is thought to regulate the expression of C / Ε Β Ρα and PPARa, While it is known to promote adipocyte differentiation, it has also been shown to be involved in the formation of PPARa ligands during adipocyte differentiation.

 It has been clarified that the above three types of transcription factor groups (PPAR, C / EBP, SREBP1 / ADD1) cross-talk to promote the differentiation process.

It has been shown that expression of the three types of transcription factors increases from relatively early stages of adipocyte differentiation, and expression of multiple target genes It is believed that the control of the trout is one of those. From the viewpoint of the timing of expression of these transcription factors, it is known that the expression of C / EBP iS and C / Ε Β Ρδ increases relatively early. However, what kind of genes are activated at the earliest stage of differentiation from preadipocytes to adipocytes, that is, within 6 hours from the start of induction of adipocyte differentiation to within 3 hours. Little is disclosed. Disclosure of the Invention-Problems to be Solved by the Invention-The earliest stage of differentiation from preadipocytes to adipocytes, that is, the presence or absence of the expression of the gene activated within 6 hours after the initiation of adipocyte differentiation induction, the degree of the gene Identifying the presence, amount, and location of expression of the protein as a product, and analyzing gene mutations can elucidate the mechanism of obesity and appropriately monitor the progress of obesity. It can be a critical factor in understanding and helping to prevent or treat obesity. For this purpose, it is desirable to identify the gene and the protein that is the product of the gene, and to detect or measure such a gene or protein.

 Thus, the present invention has found a gene that is activated within 6 hours from the initiation of differentiation induction from a preadipocyte to an adipocyte, a vector containing the gene, a transformant carrying the vector, It is intended to provide a protein produced from a transformant, an antibody specific to the protein, a method for producing the protein, an agonist or an agonist against the protein, a medicament containing these compounds, and a diagnostic agent. Aim.

 Means for solving the problem

As a result of intensive research to solve the above problems, Mouse 3T3-L1 cells (ATCC

In order to analyze changes in gene expression during the earliest stages of differentiation, the subtraction method was used before and 3 hours after adipocyte differentiation. Cloning was performed. As a result, a polynucleotide having the nucleotide sequence represented by SEQ ID NO: 1 was obtained as a gene activated within 6 hours with the peak at 3 hours after the initiation of adipocyte differentiation induction. The polynucleotide is referred to as mouse clone 104 nucleotide.

 That is, the polynucleotide of the present invention is a polynucleotide encoding a protein that is related to adipocyte differentiation and that is expressed within 6 hours after peaking at 3 hours after induction of adipocyte differentiation induction. is there.

 In the present specification, the term “activation” means expression or enhanced expression.

 A polynucleotide containing a nucleotide sequence having homology to the nucleotide sequence of the mouse represented by SEQ ID NO: 1 or a polynucleotide complementary to the polynucleotide is http: //Vww.ncbi. A search on nlra.nih.gov/BLAST/ revealed that the base sequence with homology was not registered at the time of filing the present application.

 The polynucleotide of the present invention is preferably identical to the nucleotide sequence represented by SEQ ID NO: 1 or at least 80%, 85%, 90% or 95% homologous. Is a polynucleotide containing a nucleotide sequence having an avidity or a polynucleotide complementary to the polynucleotide, within 6 hours with a peak at 3 hours after the initiation of adipocyte differentiation induction It is a polynucleotide that encodes the expressed protein and is activated within 12 hours after the initiation of adipocyte differentiation induction.

In addition, the polynucleotide of the present invention may be identical or at least 80 to nucleotides 61 to 368 of the nucleotide sequence represented by SEQ ID NO: 1. %, 85%, 90%, or 95%, a polynucleotide comprising a nucleotide sequence having homology or a polynucleotide complementary to the polynucleotide, which is a fat Peak with the third hour after the initiation of cell differentiation induction, and use a polynucleotide encoding a protein expressed within 6 hours.

 Based on the polynucleotide of the mouse nucleotide sequence represented by SEQ ID NO: 1, the human polynucleotide of the novel nucleotide sequence was determined by estimating the homology using a homology search. The nucleotide sequence is shown in SEQ ID NO: 3. That is, the human polynucleotide of the present invention is a polynucleotide comprising the base sequence represented by SEQ ID NO: 3 or a polynucleotide complementary to the polynucleotide. A polynucleotide encoding a protein associated with adipocyte differentiation, such as cDNA. The polynucleotide is referred to as human clone 104 nucleotide. The polynucleotide of the present invention has the identity or at least 80%, 85%, 90% or 95% homology to the base sequence represented by SEQ ID NO: 3. It is a polynucleotide or a polynucleotide that is complementary to the polynucleotide.

 In addition, the polynucleotide of the human of the present invention is the same as at least 80%, at least 80%, of the polynucleotide containing nucleotide numbers 58 to 3669 of the nucleotide sequence represented by SEQ ID NO: 3. It is a polynucleotide having a homology of 5%, 90% or 95%, or a polynucleotide complementary to the polynucleotide. Alternatively, it is a polynucleotide complementary to the polynucleotide, which encodes a protein associated with adipocyte differentiation.

The agreement between the mouse clone 104 represented by SEQ ID NO: 1 and the human clone 104 represented by SEQ ID NO: 3 was 82.4% ( 5 6 7 8/6 8 9 4). The polynucleotide of the mouse of the present invention can be extracted from mouse cells and other animal cells within 6 hours with a peak at 3 hours after the initiation of adipocyte differentiation induction. Similarly to the mouse polynucleotide, the human polynucleotide of the present invention peaks at 3 hours after the initiation of induction of adipocyte differentiation, and, within 6 hours after the initiation of induction of adipocyte differentiation, as well as the mouse polynucleotide. It is presumed that it can be extracted from animal cells. In addition, the polynucleotide of the present invention can be obtained from any of genomic DNA, cDNA derived from cells and tissues, cDNA library derived from cells and tissues, and synthetic DNA. The "polynucleotide" of the present invention includes DNA or RNA.

 When simply referred to herein as "clone", this may mean clone 104 nucleotide or clone 104 polynucleotide.

 The protein of the mouse of the present invention is a protein that is expressed within 6 hours from the peak at 3 hours after initiation of adipocyte differentiation induction.

 The protein of the mouse of the present invention is preferably at least 80%, 85%, 9% or more of the entire amino acid sequence of the protein represented by SEQ ID NO: 2 below. It is a protein containing an amino acid sequence having 0% or 95% homology. More preferably, the protein of the present invention is at least 80% identical to at least nucleotides 61 to 3681 of the nucleotide sequence of the polynucleotide represented by SEQ ID NO: 1. It is a gene translation product of a polynucleotide containing a nucleotide sequence having homology of%, 85%, 90% or 95%.

A polynucleotide containing a nucleotide sequence having 100% homology with the amino acid sequence of the protein represented by SEQ ID NO: 2 is activated in the early stages of mouse adipocyte differentiation. Protein. The protein is referred to as mouse clone 104 protein. The human protein of the present invention is a protein containing the amino acid sequence represented by SEQ ID NO: 4. Further, the protein of the present invention has an amino acid sequence having at least 80%, 85%, 90% or 95% homology with the amino acid sequence represented by SEQ ID NO: 4. Includes proteins containing acid sequences. The human protein of the present invention is a gene translation product of a polynucleotide containing the nucleotide sequence Nos. 58 to 3669 of the polynucleotide represented by SEQ ID NO: 3 below. is there. According to the present invention, from the viewpoint of SEQ ID NO: 3, at least 80%, 85%, 90%, 95% or 10% of the total length of the protein of the amino acid sequence represented by SEQ ID NO: 4 The protein or its salt containing an amino acid sequence having 0% homology is included.

 The protein represented by SEQ ID NO: 4 is a protein related to human adipocyte differentiation. The protein is referred to as human clone 104 protein.

 The polynucleotide of the present invention or a protein or peptide produced as a gene translation product of the polynucleotide may be used for obesity, hypertension, hyperlipidemia, diabetes, heart disease due to arteriosclerosis, or the like. It is expected to be used for the treatment or diagnosis of lifestyle-related diseases selected from strokes.

 Sequence homology

In another invention of the present invention, the reason that “at least 80% with respect to the base sequence” or “at least 80% with respect to the amino acid sequence” is that amino acids exist between mice and humans. It has already been reported that there is a homology of 84% at the acid level [Y. Zhang et al., Nature, Vol. 372, Vol. 4, pp. 25 (1994); 8—3 3 3 3 9 4 publication]. It is known that the homology of genes related to lifestyle-related diseases such as hypertension, diabetes mellitus, and obesity is conserved by 80% or more between animals (Japanese Patent Application Laid-Open No. 11-185865). From the polynucleotide of the present invention. Also, it is appropriate to consider that the homology of the primates such as humans and rodents such as rabbits based on the present sequence in the protein of the present invention is more than 80% or more. The ORF of nucleotide sequence Nos. 61-3681 of the mouse clone 104 represented by SEQ ID NO: 1 and the human clone represented by SEQ ID NO: 3 Among 104 polynucleotides, the matching rate with ΔRF of nucleotide sequence numbers 58 to 3669 is 82.4% (5678/6894).

 Cloning of Nucleotide

• As a means for cloning a polynucleotide encoding the protein of the present invention, for example, a PCR method using a synthetic DNA primer having a partial nucleotide sequence of the protein of the present invention may be used. Hybridization with a DNA fragment encoding a part or the whole region of the protein of the present invention or a DNA fragment that has been amplified using a DNA fragment or a DNA fragment encoding a partial or entire region of the protein of the present invention. Can be sorted by Hybridization methods are described, for example, in Molecular Cloning.

 J. Sarabrook et al., Cold Spring Harbor Lab. Press, 1989). When a commercially available library is used, it can be performed according to the method described in the attached instruction manual. The DNA encoding the cloned protein can be used as it is depending on the purpose, or can be digested with a restriction enzyme or added with a linker as desired. The DNA has an ATG as a translation initiation codon at its 5 'end and a TAA, TGA or TAG as a translation termination codon at its 3' end. You may. These translation initiation codon and translation termination codon can also be added using an appropriate synthetic DNA adapter.

Vectors that express polynucleotides, etc. The vector of the present invention has a nucleotide sequence having the same or at least 80%, 85%, 90% or 95% homology to the polynucleotide represented by SEQ ID NO: 1. Or a recombinant vector containing a polynucleotide complementary to the polynucleotide, and preferably a nucleotide sequence represented by SEQ ID NO: 1. Or a polynucleotide comprising a nucleotide sequence having at least 80%, 85%, 90% or 95% homology to 61 to 3681, It is a recombinant vector containing a polynucleotide that is complementary to the nucleotide. The vector of the present invention, which contains a polynucleotide relating to human adipocyte differentiation, is at least 80% identical to the nucleotide sequence represented by SEQ ID NO: 3. %, 85%. A recombinant vector containing a polynucleotide containing a nucleotide sequence having 90% or 95% homology, or a polynucleotide complementary to the polynucleotide. Preferably, at least 80%, 85%, 90%, or at least 80%, 85%, 90% or the same as the nucleotide sequence Nos. 58 to 3656 of the nucleotide sequence represented by SEQ ID NO: 3. It is a recombinant vector containing a polynucleotide containing a nucleotide sequence having a homology of 95% or a polynucleotide complementary to the polynucleotide.

The vector having the polynucleotide sequence of the present invention can be constructed by a known method. A vector suitable for such a purpose has a promoter region upstream of the insertion site of the polynucleotide of the present invention. Known promoters can be used and can be selected depending on the host cell. For example, when the host is a bacterium such as Escherichia coli, a lac promoter, a trp promoter, a T7 promoter, a tac promoter, an APL promoter, or the like can be used. When yeast is used as the host, GAP DH Promoter, ADH promoter, PGK promoter, PH05 promoter and the like. When animal-derived cells are used as a host, a human cytomegalovirus promoter, a SV40 virus-derived promoter, an EF-1 promoter, a ^ -actin promoter, a metathion thionine promoter, and the like can be mentioned. . It is desirable that a transcription termination signal be present downstream of the insertion site of the polynucleotide of the present invention in the vector that expresses the polynucleotide of the present invention. Further, it is desirable that the vector has an identification marker such as a drug resistance marker. -As the host usable in the present invention, for example, Escherichia bacteria, Bacillus bacteria, yeast, insect cells, insects, animal cells, and the like can be used.

Specific examples of Escherichia coli include Escherichia coli JM109 (ATCC53332, manufactured by Toyobo Co., Ltd.) and JM103 (Nucleic Acids Research, ( Nucleic Acids Research), vol. 9, 309 (19981)), Κ12 · D Η1 [Procedures • Ob 'The' National 'Academy' Ob ''Sciences' Ob. Natl. Acad. Sci. USA), Volume 60, ₁₆₀ ( ₁₉₆₆₈ )], JA2221 [Journal of Ob-Moleki Jura-Noology] (Journal of Molecular Biology)], Volume 120, 5 17 (19778)], HB 101 (Journal 'ob' 乇 Rekylura. Biology, Volume 41, Volume 4 59 (1 969)], C600 [Genetics, 39, 44 (1954)].

Examples of Bacillus species include, for example, Bacillus subtilis (Bacillus subtilis) MI 1 1 4 [Gene, 24, 25 5 (19 lo) 8 3)], 207-21 [Journal of Biochemistry, 95, Vol. 87 (1989)].

 Examples of yeast include Saccharomyces cerevisiae (

Saccharomyces cerevisiae) AH22, AH22R-, NA87-11A, DKD-5D, 20B-12, Schizosaccharomyces pombe NCYC1913, NCYC2 036, Saccharomyces piccia pastor is, and the like.

 As insect cells, for example, when the virus is Ac NPV, a cell line derived from a larva of a moth moth (Spodoptera frugiperda cell; Sf cell), MG1 cell derived from the midgut of Trichoplusia rn, Trichoplus Examples include High Five ™ cells derived from ia n_ eggs, cells derived from Mamestra brassicae, and cells derived from Estigmena acrea. When the virus is BmNPV, a silkworm-derived cell line (Bombyx mori cell; BmN cell) and the like can be mentioned. Examples of the Sf cells include Sf9 cells (ATCC CRL1711), Sf21 cells (Vaughn, JL et al., In Vivo, 13, 213-217, (1977) )). Insects include, for example, silkworm larvae [Maeda et al., Nature, Vol. 3115, 592 (19985)].

Examples of animal cells include monkey cells C 0 S-7, Vero, Chinese hamster cells CH 0 (hereinafter abbreviated as CHO cells), dhfr gene-deficient Chinese hamster cells CHO [abbreviation: CHO (dhfr) -) Cells], mouse L cells, mouse AtT-20, mouse myeloma cells, rat GH3, human FL cells, etc. The transformant of the present invention has the above-described recombinant vector retained in the above-mentioned host.

 To transform Escherichia sp., For example, Proc. Natl. Acad. Sci. USA, Proc. Natl. Acad. Sci. USA , 69, 2110 (1972) and Gene, 17th, 107 (i982).

 For transformation of Bacillus sp., For example, Molecular & General-Genetics, 1668, 111 (1977) The method can be performed according to the method described in the above.

 To transform yeast, for example, the methods described in Methods in Enzymology, Vol. 194, 1982, 182 — 187 (1991), Processings · The National 'Academi'-'Ob' Sciences' Ob ■ Described in Proc. Natl. Acad. Sci. USA, Vol. 75, 1929 (1992) Can be performed according to the method described in

 Insect cells or insects can be transformed, for example, according to the method described in Bio / Technology, 6, 47-55 (1988).

 To transform animal cells, for example, Cell Engineering Separate Volume 8 New Cell Science Experimental Protocol. 263-26 (1995) (published by Shujunsha), Virology, 5 It can be carried out according to the method described in Vol. 2, 456 (1973).

 Culture of transformants

The culturing of the transformant of the present invention can be carried out under conditions sufficient for protein production in consideration of the following conditions. As a result, a protein can be recovered from the culture medium or the transformant.When culturing a transformant whose host is Escherichia or Bacillus, a liquid medium is suitable as a culture medium. It contains a carbon source, a nitrogen source, an inorganic substance, and the like necessary for the growth of the transformant. Examples of carbon sources include glucose, dextrin, soluble starch, and sucrose.Examples of nitrogen sources include ammonium salts, nitrates, corn chips, peptone, casein, and meat. Inorganic or organic substances such as extracts, soybean meal and potato extract, and inorganic substances include, for example, calcium chloride, sodium dihydrogen phosphate, magnesium chloride and the like. In addition, yeast, vitamins, growth promoting factors and the like may be added. The pH of the medium is preferably about 5-8.

 As a culture medium for culturing Escherichia sp., For example, M9 medium containing glucose and casamino acid [Miller, Journal “Ob. Esperimen in” Molecular ”Genetics <4, Journal of Experiments in Molecular Genetics ;, 4d1-43, Cold Spring Harbor Laboratory, New York 1972]. If necessary, an agent such as 3-indolylacrylic acid can be added in order to make the promoter work efficiently. When the host is a bacterium belonging to the genus Escherichia, cultivation is usually performed at about 15 to 43 ° C for about 3 to 24 hours, and if necessary, aeration and stirring may be added.

When the host is a bacterium belonging to the genus Bacillus, the cultivation is usually carried out at about 30 to 40 ° C for about 6 to 24 hours, and if necessary, aeration and stirring can be applied. When the host is culturing a transformant that is a yeast, the culture medium may be, for example, Burkholder's minimum medium [Bostian, K. Shira, Proc. Natl. Acad. Sci. Provisions. The 'National' Academy 'Ob'Sciences' Ob the USA

USA), 775, 450 (1980)] or an SD medium containing 0.5% casamino acid [Bitter, GA et al., "Processing's of the National Academy." _Nats . Acad. Sci. USA, ₈₁ , 5

3 3 0 (1 98 4)]. The pH of the medium is preferably adjusted to about 5-8. Cultivation is usually performed at about 20 ° (up to 35 ° C for about 24 to 72 hours, with aeration and agitation as needed.) +--Cultivating insect cells or transformants whose host is insects As a culture medium, Grace's Insect Medium (Grace, T., Nature, 195, 788 (1962)) was supplemented with an additive such as 10% serum immobilized as appropriate. It is preferable to adjust the ρ 6 of the culture medium to about 6.2 to 6.4. Culture is usually performed at about 17 ° C for about 3 to 5 days, and aeration and agitation as necessary. Add.

 When culturing a transformant in which the host is an animal cell, the culture medium may be, for example, a MEM medium containing about 5 to 20% fetal bovine serum [Seience, 122 vol. (1952)], DMEM medium (Virology, 8 volumes, 396 (1955)), RPM 1 1640 medium [Journal 'ob' the 'American' Medical • Association ihe Jounal of the American Medical

 Association, Vol. 199, 1991 (19667)), 199 Medium [Procedure 'ob' the 'Society for' the 'biology' of the Society for the

 Biological Medicine), 73, 1 (1950)]. Preferably, the pH is about 6-8. Culture is usually about 30 ° C ~

Approximately 15 to 60 hours at 40 ° C, with ventilation and agitation as needed You. As described above, the protein of the present invention can be produced in the cells of the transformant.

 Protein production by cell-free system

 In addition to producing proteins by culturing the above-mentioned transformants, Invitrotech (http://www.inbitrotech.co.jp)

Generating the protein by using a cell-free culture system using a high-efficiency cell-free protein synthesis system (/) or Roche's invitro translation / transcription system, etc. Is also possible. · · '' —.-Separation and purification of evening protein

The protein of the present invention can be separated and purified from the culture by, for example, the following method. When the protein of the present invention is extracted from cultured cells or cells, after culturing, cells or cells are collected by a known method, suspended in an appropriate buffer, sonicated, and lysed. After the cells or cells are destroyed by zozyme and / or freeze-thawing, a method of obtaining a crude extract of protein by centrifugation or filtration is used as appropriate. The buffer may contain a protein denaturant such as urea or guanidine hydrochloride, or a surfactant such as Triton X-100 (registered trademark, manufactured by Union Carbide). These known separation and purification methods include methods using solubility such as salting out and solvent precipitation, dialysis, ultrafiltration, gel filtration, and SDS-polyacrylamide gel electrophoresis. Methods that mainly use differences in molecular weight, such as methods, methods that use differences in charges such as ion-exchange chromatography, methods that use specific affinities such as affinity chromatography, and vice versa. Methods utilizing the difference in hydrophobicity, such as phase high performance liquid chromatography, and methods utilizing the difference in isoelectric points, such as isoelectric focusing, are used. When the protein obtained by each of the above methods is obtained in a free form, it can be converted to a salt by a known method or a method analogous thereto. It can be converted to a free form or another salt by a method analogous thereto. The protein produced by the recombinant can be arbitrarily modified or the polypeptide can be partially removed by allowing a suitable protein modifying enzyme to act on the protein before or after purification. As the protein-modifying enzyme, for example, trypsin, chymotrypsin, arginylendopeptidase, protein kinase, glycosidase and the like are used. The presence of the thus-produced protein of the present invention or a salt thereof can be measured by a binding experiment with a labeled ligand, an enzyme immunoassay using a specific antibody, or the like.

 Nucleotide sequence and amino acid sequence of human homolog

NCB I Genome Sequenquence, a polynucleotide that is activated within 6 hours with a peak at 3 hours after the initiation of adipocyte differentiation induction, and is the full-length mouse DNA sequence represented by SEQ ID NO: 1 determined above. (Human Genome Database), it is possible to find a sequence with high homology by performing homology search and to predict the full length of the human homologue. The total length of the human homologue predicted and sequenced in this way is the nucleotide sequence shown in SEQ ID NO: 3. In the full-length nucleotide sequence of the human homologue represented by SEQ ID NO: 3, the ORFs of nucleotide sequence numbers 58 to 369 are genes encoding a protein consisting of 124 amino acids. The amino acid sequence of the protein is expected to be SEQ ID NO: 4. The human clone 104 polynucleotide of the present invention, like the mouse clone 104 polynucleotide, was activated within 6 hours, peaking at 3 hours after the initiation of adipocyte differentiation induction. Presumed to be Human homolog homology

 A polynucleotide comprising a nucleotide sequence having homology to the nucleotide sequence of clone 104 human homologue represented by SEQ ID NO: 3 (sometimes simply referred to as human homologue), or a polynucleotide complementary to said polynucleotide. Polynucleotide is not registered at the above-mentioned http: biwww.ncbi.rUm.nih.gov/BLAST/.

 Method for identifying gonist and agonist.

 The method for determining a substance includes the following steps (a) and (b);-(a) a cell or a cell expressing the protein of the present invention. Contacting a candidate compound with a cell responsive to the protein of the invention;

 (b) observing binding or stimulation or inhibition of a functional response; or comparing the ability of the cells contacted with the candidate compound with respect to the activity of the protein of the present invention to the unaffected cells of the same kind.

 According to the above identification method, the antagonist or agonist of the present invention can be identified.

 Polynucleotide detection method; Diagnosis using polynucleotide Diagnosis of a disease or a susceptibility to such a disease in a diagnostic subject related to activation of the protein of the present invention in the diagnostic subject can be performed as follows. . That is, to determine the presence or absence of a mutation in the nucleotide sequence encoding the protein of the present invention in the genome of the subject to be diagnosed; and / or to determine the presence or absence of the present invention in a sample derived from the subject. This is done by analyzing the presence or amount of protein.

In order to detect or measure the polynucleotide of the present invention, a primer having a continuous sequence of 8 to 100 bases is synthesized from the nucleotide sequence of the polynucleotide of the present invention, and the primer is synthesized from cells or blood. Extract mRNA and convert mRNA to DNA by RT-PCR Amplification with T7-based mRNA amplification (Van GelderR. N. et al. Proc. Natl. Acad. Sci. USA, 87: 1663-1667, 1990). It is possible to amplify the polynucleotide and detect the amplified polynucleotide by various electrophoresis. In addition, Cy 3 — d UTP or Cy 5 — d UTP is added at the time of polynucleotide amplification, and the amplified polynucleotide is fluorescently labeled, and the DNA chip or microarray (Brown , P.0. Et al .: Nature Genet., 21 sup .: 33-37, 1999).

 • As a method for quantifying the amount of extracted mRNA-, quantitative PCR using a fluorescent dye is performed (Livak, K. et al .: PCR Methods Applic, 4: 357-362). , 1995). By this method, it is possible to determine the level of mRNA in the cell. The degree of this occurrence can be used to determine the status of obesity.

 It is also possible to simultaneously detect and measure a plurality of polynucleotides using a DNA chip or a microarray. For example, a plurality of polynucleotides including the polynucleotide of the present invention, which are related to lifestyle-related diseases such as diabetes, hypertension, and obesity, are bound on a substrate, and the method described above is used. By reacting the labeled polynucleotide prepared from the sample, it is possible to determine which polynucleotide is activated or present. With this determination, it is possible to grasp the status of lifestyle-related diseases and use them for treatment. In addition, a plurality of obesity-related polynucleotides including the polynucleotide of the present invention are bound on a base, and it is possible to grasp the status and degree of obesity by the same procedure. It can be used for treatment.

In order to detect the polynucleotide of the present invention, ABIPRISM 310 or PRISM 310 manufactured by Applied Biosystems is used. Detection by capillary electrophoresis using 0000 or PRISM 370 is also possible.

 The substitution of a specific nucleobase in a polynucleotide with another base alters the activity of the protein as a gene product, thereby increasing the ability of the protein of the present invention to bind to its receptor. Gene polymorphism (e.g., altering, or altering the reactivity of the protein to a drug, altering the stability of the protein, or terminating the synthesis of the protein) Nucleotide polymorphism: single nucleotide polymorphisms (hereinafter SNPs) has been found to be very important, but the significance of detecting this SNPs in the polynucleotide of the present invention is very high. is important.

 If a high-density SNP genetic map of the adipocyte differentiation-related polynucleotide of the present invention is created, it is easy to compare between healthy subjects and patients using SNPs that can identify the causative gene of diabetes. The use of high-density SNP maps will enable the Whole Genome Association Study using large samples that are not related to families. In SNPs, in particular, the substitution of one base changes the corresponding amino acid, and this change causes a change in the physical properties of the original protein. For example, in the case of an enzyme, a change in amino acid near the active center causes a decrease or enhancement of the enzyme activity. Rise. Adjusting the dose of a drug or selecting a different drug according to the genetic polymorphism of each person is called tailored medicine.

As a method for detecting SNPs in the polynucleotide of the present invention, for example, a method using nanochip of America Nanogen (http://www.nanogen.com) (Gilles et al., Nature Biotechnology 365- 370 17 1999), a method for determining a polynucleotide sequence (http: 〃www. Pyrosequencing.com), a method using a DNA chip or a DNA array, and a method using a mass spectrometer (Legler et al., Transfusion, 36 : 426-431, 1996), the method using primer extension, and the Luminex method (Iannone et al., Cytometry, 39:13, 140, 2000).

 The diagnostic agent configured to detect each of the above polynucleotides is identical or at least 80%, 85% ヽ 90% to the nucleotide sequence represented by SEQ ID NO: 1. Or a diagnostic agent comprising a polynucleotide comprising a nucleotide sequence having a homology of 95% or a polynucleotide complementary to the polynucleotide. Preferably, it has at least 80%, 85%, 90% or 95% homology to the nucleotide sequence represented by SEQ ID NO: 1 from No. 61 to 36581. It is a diagnostic agent containing a polynucleotide having a nucleotide sequence having the same or a polynucleotide complementary to the polynucleotide.

 In addition, a polynucleotide containing a nucleotide sequence having at least 80%, 85%, 90% or 95% homology to the nucleotide sequence represented by SEQ ID NO: The diagnostic agent may include a polynucleotide having a nucleotide sequence of at least 10 consecutive nucleotides among the nucleotides complementary to the polynucleotide or the polynucleotide complementary to the polynucleotide. Preferably, at least 80%, 85%, 90% or 95% homology to the nucleotide sequence Nos. 61 to 3681 of SEQ ID NO: 1 Includes a polynucleotide having a nucleotide sequence having the property or a polynucleotide complementary to the polynucleotide, having a continuous nucleotide sequence of at least 10 nucleotides It can be used as a diagnostic.

In addition, diagnostic agents from the viewpoint of detecting human polynucleotides are: A polynucleotide comprising a nucleotide sequence having at least 80%, 85%, 90% or 95% homology to the nucleotide sequence shown in SEQ ID NO: 3, or It is a diagnostic agent that contains a polynucleotide that is complementary to the polynucleotide. Preferably, at least 80%, 85%, 90% or 95% homology to nucleotide sequence Nos. 58 to 3669 of SEQ ID NO: 3 A diagnostic comprising a polynucleotide comprising a nucleotide sequence having the formula: or a polynucleotide complementary to the polynucleotide.

 In addition, a diagnostic agent from the viewpoint of detection of human polynucleotides is at least 80%, 85%, or 90% identical or at least to the nucleotide sequence represented by SEQ ID NO: 3. Or at least 10 consecutive nucleotide sequences of a polynucleotide containing a nucleotide sequence having a homology of 95% or a polynucleotide complementary to the polynucleotide. It is a diagnostic agent containing a polynucleotide having Preferably, at least 80%, 85%, 90% or 95% homology to nucleotides Nos. 58 to 3656 of the nucleotide sequence represented by SEQ ID NO: 3 Of a polynucleotide comprising a nucleotide sequence having the following sequence or a polynucleotide complementary to the polynucleotide, the polynucleotide having at least 10 consecutive nucleotide sequences: Including diagnostic agents.

 Detection of proteins or peptides, and detection of antibodies to proteins or peptides, and diagnostic applications

In order to detect the protein of the present invention, an immunoassay is generally used. For example, a protein represented by SEQ ID NO: 2 or SEQ ID NO: 4, or a peptide consisting of three or more consecutive amino acid sequences contained in each of those sequences can be used for mouse, rat, egret, and goat. The antibody can be produced by immunizing pigs, pigs, horses, sheep, nits, and the like. When performing this immunization, substances usually called adjuvants are used. It is common to add As the adjuvant, various known adjuvants such as complete adjuvant of Freund, incomplete adjuvant, and alum can be used. If the mouse is immunized, after immunization for a certain period, the spleen of the immunized mouse is removed, and the spleen cell, which is an antibody-producing cell, is fused with myeloma cells, and cloned to obtain SEQ ID NO: 2 or SEQ ID NO: 2. Prepare a hybridoma that produces an antibody that specifically binds to the No. 4 protein.

 -By culturing the V-predomas, the -antibody produced is purified by various methods, and the obtained antibody is bound to a solid phase. Separately, the antibody is labeled with an enzyme, fluorescent dye, metal colloid, latex, DNA, RNA or the like to obtain a labeled antibody. The immobilized antibody and the labeled antibody are reacted with cells, blood, or fractionated fractions thereof as a sample, and the presence or amount of the label bound to the solid phase is measured. The amount of the protein of the present invention can be measured.Because the protein of the present invention is specifically found in fat cells, it is possible to grasp the degree and condition of obesity by measuring the amount. Become.

In order to measure the amount of the protein of the present invention contained in a sample, the protein or a peptide which is a part of the protein is used as an antigen for rabbits, rats, sheep, goats and the like. Immunize to prepare an antibody (this involves preparing a monoclonal antibody that can bind to a specific recognition site). The antibody obtained here is physically adsorbed on a material such as polystyrene, latex, or nitrocellulose, or biotin is introduced into the antibody in advance, and the antibody is previously bound to the solid phase. Streptavidin or avidin, etc. To prepare a solid phase, or by covalently binding the antibody via a carboxyl group, an amino group, a sulfhydryl group, etc., which are present on the solid phase. Prepare the phases. The antibody-bound solid phase obtained here is reacted with cells, blood or fractions thereof as a sample, and the antigen contained in the sample is allowed to bind to the solid phase. The antibody prepared by the above-described steps is reacted with a labeled antibody to which a label such as a radioisotope, an enzyme, a fluorescent dye, a nucleic acid, or biotin is bound. The step of reacting the antibody-bound solid phase with the sample and the labeled antibody can be performed simultaneously. By measuring the labeled substance bound to the solid phase in this way, the protein contained in the sample can be measured.

 Alternatively, the following method can be used for detecting the antibody. That is, the protein of the present invention or a peptide that is a part of the protein is immobilized as an antigen to form an antigen-immobilized solid phase, and then the antigen-immobilized solid phase is labeled with an antigen. Then, cells, blood or fractions thereof are reacted as a sample, and the amount of the bound label is quantified, whereby the amount of antibody contained in the sample can be measured. Therefore, a diagnostic agent that detects an immunologically specific antibody to the protein of the present invention can be constructed.

In the above, all of the reaction systems in which the antigen or antibody is directly immobilized have been described. However, as described in US Pat. No. 5,789,165, oligonucleotides are bound to the solid phase. However, a reaction system in which an antigen or an antibody in which an oligonucleotide complementary to the oligonucleotide is bound or an antibody is bound can also be applied. Furthermore, as described in Japanese Patent Application Laid-Open No. H10-2553032, EP 0 905 5 17 A1, different items can be detected or measured simultaneously. For example, it is possible to simultaneously detect markers related to lifestyle-related diseases such as diabetes, high blood pressure, and obesity. Thus, it will be possible to judge the level of lifestyle-related diseases based on which protein is detected and to what extent. Also, by simultaneously detecting and measuring markers such as obesity-related proteins or peptides, it will be possible to understand the status of obesity and use it for treatment.

 In order to immunologically detect the protein of the present invention or an antibody against the protein, in addition to the above-mentioned method, a method using an immunoagglutination method (Japanese Patent Application Laid-Open No. 6-33558), B i acore I nternat iona l AB (Sweden) d: (http: // www. bi aeore. co. jp-) ø BI Ac ore Surface plasmon resonance (Surface P l Asmon Resonance = SPR), a method using a crystal oscillator (Japanese Patent Application Laid-Open No. 9-122939), and the like.

 Examples of the diagnostic agent for detecting the protein of the present invention described above include the diagnostic agent containing the protein of the present invention or a salt thereof. In a preferred embodiment, the diagnostic agent of the present invention includes a diagnostic agent comprising a protein or a peptide consisting of at least 5 consecutive amino acids of the protein of the present invention or a salt thereof. . Another diagnostic agent for detecting the protein of the present invention includes a diagnostic agent containing an antibody against the protein of the present invention.

 Use for therapeutic purposes

The present invention relates to obesity, hypertension, hyperlipidemia, diabetes, kidney disease, which is related to the activity of an excessive or insufficient amount of the protein of the present invention or a peptide constituting a part of the protein. The present invention provides a method for treating abnormal conditions such as insulin resistance, lipodystrophy, CNS disease, and lifestyle-related diseases such as heart disease or stroke due to arteriosclerosis. When the activity of the protein of the present invention or the peptide constituting a part of the protein is excessive, several methods can be used. One means is to administer to a subject a medicament prepared by combining an effective amount of the above inhibitor compound (annogogonist) with a pharmaceutically acceptable carrier, to obtain a protein or a peptide of the present invention. Other means of blocking activation of ligand binding or inhibiting activation by inhibiting a second signal, thereby ameliorating abnormal conditions, are described in Administering a therapeutically effective amount of a compound (ie, agonist) which activates the polynucleotide of the invention, in combination with a pharmaceutically acceptable carrier, thereby producing an -abnormal- It is characterized by improving the condition.

 In another approach, administration of a soluble form of a protein or peptide of the present invention or a salt thereof capable of binding to a ligand in competition with the protein or peptide of the present invention can be performed. Good. Typical examples of such competitors include the proteins of the present invention or the peptides that form part of the proteins. That is, the medicament of the present invention can be a medicament containing the protein or a salt thereof, or a peptide comprising at least five consecutive amino acids of the protein or a salt thereof.

 Such a medicament of the present invention has homology of at least 80%, 85%, 90% or 95% to the full length of the protein represented by SEQ ID NO: 2. A medicament containing a protein or a salt thereof, or a medicament containing a peptide consisting of at least five consecutive amino acids of the protein.

Further, the medicament of the present invention has homology of at least 80%, 85%, 90% or 95% to the full length of the protein represented by SEQ ID NO: 4. A drug containing a protein or a salt thereof, or a peptide consisting of at least five consecutive amino acids of the protein Medicines.

 In yet another approach, the polynucleotide of the present invention or a polynucleotide complementary thereto is administered using an expression block method to activate the gene. May be inhibited.

5 Such a medicament of the present invention is at least 80%, 85%, 90% identical or at least to the nucleotide sequence of the clone 104 polynucleotide represented by SEQ ID NO: 1. Or a pharmaceutical comprising a polynucleotide comprising a nucleotide sequence having a homology of 95%, or a polynucleotide complementary to the polynucleotide. More preferably, at least 80%, 85%, 90%, or 95% of the nucleotide sequence represented by SEQ ID NO: 1 from nucleotides 61 to 36581. It is a drug containing a polynucleotide containing a nucleotide sequence having a homology of 5% or a polynucleotide complementary to the polynucleotide.

 In addition, a polynucleotide containing a nucleotide sequence having homology of at least 50%, 85%, 90% or 95% to the nucleotide sequence represented by SEQ ID NO: 1 A drug comprising a polynucleotide or a polynucleotide complementary to the polynucleotide and comprising a polynucleotide having at least 10 consecutive nucleotide sequences. Well, more preferably

Has 0 or at least 80%, 85%, 90% or 95% homology to nucleotide numbers 61 to 368 of the nucleotide sequence represented by SEQ ID NO: 1. A polynucleotide having at least 10 nucleotide sequences that are complementary and continuous to a polynucleotide containing a nucleotide sequence or a polynucleotide complementary to the polynucleotide. It may be a drug containing a nucleotide.

5 In particular, the human clone 104 polynucleotide is identical or at least 80%, 85%, 90% or 95% to the nucleotide sequence represented by SEQ ID NO: 3. Polynucleotides containing nucleotide sequences with homology of It is a drug containing a nucleotide or a polynucleotide complementary to the polynucleotide. Preferably, at least 80%, 85%, 90% or 95% homology to nucleotide sequence Nos. 58 to 3669 of SEQ ID NO: 3 And a drug containing a polynucleotide complementary to the polynucleotide or a polynucleotide comprising a nucleotide sequence having the formula:

 In addition, a polynucleotide containing a nucleotide sequence having homology of at least 80%, 85%, 90% or 95% to the nucleotide sequence represented by SEQ ID NO: 3 A drug which is complementary to the polynucleotide or the polynucleotide, and which comprises a polynucleotide having at least 10 contiguous base sequences among the polynucleotides; Or even more preferably

A nucleotide sequence having at least 80%, 85%, 90% or 95% homology to 58 to 3669 of the nucleotide sequence represented by SEQ ID NO: 3 And a polynucleotide complementary to and complementary to the polynucleotide comprising at least 10 nucleotides of at least 10 base sequences. It may be a medicine containing.

 These drugs utilize antisense of a sequence around ATG which is a translation initiation codon as a therapeutic drug. These medicines can use antisense sequences produced intracellularly or administered separately by known methods. For example,

01 igodeoxynucleotides as Ant i sense Inhibitors of Gene

Expression, CRC Press, Boca Raton, FL (1988), 0 'Connor, J. Neurochem (1991) 56: 560. Alternatively, an oligonucleotide may be provided that forms a triple helix with the gene. For example, Lee et al., Nucleic Acids Res (1979) 6: 3073; Cooney et al., Science (1988) 241: 456; Dervan et al., Science (1991) 251: 1360. These The ligoma can be administered as such, or the relevant oligomer can be activated in vivo.

 Alternatively, gene therapy may be used to effect the intracellular production of the polynucleotides of the present invention by cells in the subject to be treated. For example, as described above, the polynucleotide of the present invention may be processed and put into a replication-defective retrovirus vector to activate it. The retroviral expression construct is then isolated and packaged transduced with a retroviral plasmid containing RNA encoding the protein of the present invention. After introduction into cells, the packaging cells may then produce infectious virus particles containing the polynucleotide of interest. These producer cells may be administered to a subject to be treated and the cells may be processed and processed in vivo to activate the protein in vivo. The methods of gene therapy are described in Human Molecular Genetics, T. Strachan and A.P.Read.BIOS Scientific Publishers Ltd (1986), Chapter 20, Gene The rapy and other Molecular Genetic-based Therapeutic.

 Approaches (and references therein) are applicable.

A protein or a protein having at least 80%, 85%, 90% or 95% homology to the entire length of the protein represented by SEQ ID NO: 2 A drug can be prepared by incorporating an immunologically specific antibody against a peptide consisting of a part of the amino acid sequence constituting the above. In addition, a protein or a protein having homology of at least 80%, 85%, 90% or 95% to the full length of the protein represented by SEQ ID NO: 4 is used. A medicament can be prepared by incorporating an antibody that is immunologically specific for a peptide consisting of a part of the amino acid sequence that constitutes it. Administration of such an antibody can be expected to reduce adipocyte differentiation activity. Prescription and administration

 In a method for treating a subject requiring enhanced activation of the protein of the present invention, the soluble form of the protein of the present invention, or a peptide constituting a part of the protein, or A therapeutically effective amount of a salt thereof, and an antibody, agonist, antagonist peptide, or a small molecule thereof against the protein or a peptide constituting a part of the protein is administered to a medicament. May be administered to the subject to be treated.

 Such formulations comprise a therapeutically effective amount of the above substances, or salts thereof, and a pharmaceutically acceptable carrier or excipient. Such carriers include, but are not limited to, saline, buffered saline, dextrose, water, glycerol, ethanol, and mixtures thereof. The formulation should be appropriate for the route of administration and is well known to those skilled in the art. The invention further relates to pharmaceutical packs and kits comprising one or more containers filled with one or more of the above-mentioned components of the invention.

 The proteins and other compounds of the present invention may be used alone or in combination with other compounds, such as therapeutic compounds. Preferred forms of systemic administration of the pharmaceutical composition include injection, typically by intravenous injection. Other injection routes can be used, such as subcutaneous, intramuscular or intraperitoneal. Another means for systemic administration involves transmucosal or transdermal administration using penetrants such as bile salts or fusidic acid or other surfactants. In addition, if an enteric formulation or a capsule formulation is formulated successfully, oral administration is also possible. Administration of these compounds may be topical or in the form of salves, pastas, gels and the like.

The required dosage range depends on the peptide, route of administration, nature of the formulation, Depends on the nature of the individual's symptoms and the judgment of the attending physician. Suitable doses range from 0.1 to 100 g / kg body weight of the subject. Nevertheless, the required dosage will likely vary widely given the various efficiencies of the different compounds used and the different routes of administration. For example, oral administration may require a higher dose than intravenous injection. Variations on these dosages can be made using standard routine experimentation for optimization that is well understood in the art.

 In the above treatment methods, often referred to as "gene therapy", the protein of the present invention used for treatment can also be produced in the subject to be treated. Thus, for example, by using a retrovirus plasmid vector, cells derived from the subject to be treated may be processed ex vivo using a polynucleotide such as DNA or RNA encoding a protein. The treated cells are then introduced into a treatment subject. BRIEF DESCRIPTION OF THE FIGURES

 FIG. 1 is a schematic diagram of the clone 104 mouse cDNA clone.

 Figure 2 shows that the mouse is specifically expressed in adipocyte differentiation, and that the mouse mouth 104 polynucleotide is expressed within 6 hours after peaking at 3 hours after induction of adipocyte differentiation. This is an autoradiogram showing that

Figure 3 shows the autoradiograms of the 3T3-L1 and NIH-3T3 telogens during the telogen and growth phases at 0 and 3 hours after the initiation of differentiation induction. It is. Example 1 Confirmation of differentiation into fat cells

Mouse 3 T 3- L 1 cells (ATCCN o. CCL- 9 2. 1 .) , And basal medium (DMEM, AO 11 g / ral KM, 10% Calf Serum) 5% C 0 2 and have use of, 3 7 The cells were cultured in a collagen type II dish (manufactured by FALCON) at ° C. After 2 days after the cells reach confluence, after the cells have entered the quiescent phase, the medium is changed to a differentiation-inducing medium (DMEM, 40 ifg / ml KM, 10% FBS, 0.5 mM 1-methyl-3-isobutyl). Xanthin (hereinafter referred to as IBMX), 10 wg / m 1 insulin, 1 M Dexamethasone (hereinafter referred to as Dex). Was changed to a differentiation promoting medium (DMEM, 40 g / ml KM, 10% FBS, 5 ug / ml insulin) The medium was replaced with a differentiation promoting medium every two days. It began to contain fat droplets, and after one week it was confirmed that it had differentiated into mature adipocytes.

 Example 2

 Preparation of mRNA

 Before the induction of adipocyte differentiation and 3 hours after the initiation of differentiation induction, TRIzol (GIBC0 BRL) was used from mouse 3T3-L1 cells (ATC CN o. CCL-92.1. The total amount of RNA was prepared according to the instruction manual attached to the company. From this total amount of RNA, mRNA was prepared using 01igotex-dT30 (manufactured by Daiichi Kagaku) according to the attached instruction manual.

 Example 3

 Isolation of clones with increased expression three hours after induction of adipocyte differentiation by PCR-select cDNA Subtraction

 Known PCR select c DNA subtraction kit (CL0NTECH

Laboratories) according to the following i)-vii) steps. i) Synthesis of tester cDNA and driver cDNA The mRNA before the induction of adipocyte differentiation was defined as driver-mRNA, and the mRNA 3 hours after induction of differentiation was defined as tester mRNA. Single-stranded cDNA was synthesized from these driver mRNA and tester mRNA, and then double-stranded driver- 1 cDNA and tester- 1 cDNA were synthesized.

 ii) Cleavage of double-stranded cDNA by restriction enzyme Rsal

 Each of the double-stranded driver-cDNA and tester-cDNA obtained in the above step i) was cut with a restriction enzyme Rsai to produce blunt-ended (bluntend) fragments. ii ii) Adapter ligation —— The cleaved tester cDNA obtained in step ii) is divided into two groups, one adapter ligating the adapter 1 and the other a adapter 2 Was ligated.

 iv) First noise hybridization

 Hybridization was separately performed between the two types of testers cDNA ligated to the adapter obtained in the above step iii) and an excess amount of driver cDNA.

 V) Second noise distillation

 Further, an excess amount of the driver cDNA and the two types of test cDNA obtained by hybridizing the driver obtained in the above-mentioned step (V) are combined and hybridized. Was.

 vi) First PCR

 The hybridized tester cDNA obtained in the above step iv) was used to carry out the PCR method using a primer common to the two kinds of adapters to amplify the DNA.

 vii) Second PCR

Finally, DNA was amplified by PCR using two primers specific to the two adapter sites. Tester cDNA hybridized with the driver cDNA was not amplified. On the other hand, only the polynucleotides (genes) whose activation was increased within 6 hours as a peak at the third hour of differentiation induction, ie, at the third hour of differentiation induction, were amplified.

 Example 4

 Subcloning of PCR product and mouse clone 104

 FIG. 1 shows a schematic diagram of the cloning of clone 104 mouse cDNA. The -S'e-.c 0 nd obtained by the subtraction method of Example 3 above was subjected to phenol extraction and CIAA extraction of the reaction solution containing the amplified polynucleotide after PCR. Then, ethanol precipitation was performed, the DNA pellet was dissolved in sterilized water 171, and 10 mM Tris-HCl (pH 7.5),

The adapter was reacted at 37 ° C overnight under the conditions of 10 mM MgCl ₂ and 1 mM DTT, and the adapter was cut. After electrophoresis on a 0.7% agarose gel, purification was performed using DE81 (What at man) to obtain a DNA fragment Su (see FIG. 1).

 Designing primers based on the information of the DNA fragment Su,

As a result of 5′-RACE, a 3.2 kbp band was obtained. This was recovered and called nucleotide R-5 ′ (see FIG. 1). The nucleotide R-5 ′ was subcloned into a T vector (pBluescript KS +), and the base sequence was determined using DSQ-100 (Shimadzu Corporation). Furthermore, a primer for 5'-RACE was designed again on the 5, side of nucleotide R-5, and 5, -RACE was performed. As a result, a 1.2 kbp band was obtained. This was recovered and called nucleotide R-5, '(see Figure 1). The nucleotide sequence of this DNA fragment was determined in the same manner. On the 3 'side, the DNA fragment Su was labeled with [α-32Ρ] d CTP (manufactured by Amershara Pharmacia biotech), and this was used as a probe to perform cDNA lively screening. Positive phages were isolated. DNA is prepared from this positive phage, digested with EcoRI restriction enzyme, electrophoresed on a 0.7% agarose gel, and DE81 (

 The purified DNA was purified using Wattman), subcloned into the EcoRi site of pBluescript KS +, and the base sequence of 1.6 kbp was determined by DSQ-100 (Shimadzu Corporation). This was read as nucleotide Sc-. (See Fig. 1).

 Next, when a search was performed against the nucleotide sequence using the nucleotide Sc, a matching nucleotide sequence was registered in the EST. Based on this nucleotide sequence, a primer was designed, and RT- PCR was performed. As a result, a band of about 1.5 kbp was obtained, which was recovered and called nucleotide RT (see FIG. 1). The nucleotide RT was subcloned into a T vector (pBluescript KS +), and the nucleotide sequence was determined by DSQ-100 (Shimadzu Corporation).

 As described above, as a result of performing 5, -RACE, cDNA library screening and RT-PCR for the purpose of isolating the full-length mouse clone 104 cDNA, a 6812 bp clone was obtained. Polynucleotide was obtained. The polynucleotide is referred to as clone 104 polynucleotide (sometimes referred to simply as clone 104). The nucleotide sequence of the polynucleotide is shown in SEQ ID NO: 1. The translation initiation codon was assumed to be the translation start codon at the ATG of the 61st base appearing at the 5th and 6th side in the in-frame. As a result, mouse clone 104 polynucleotide was expected to be a gene encoding 127 amino acids. The amino acid sequence of mouse clone 104 is shown in SEQ ID NO: 1.

The QRF is included in the mouse clone 104 cDNA. Was 61 bp-368 lbp. To generate a plasmid containing this region, a ligation reaction of 1 bp — 370 bp clone 104 polynucleotide to pBluescrit SK + vector (Xba i / EcoRV) was performed. Was. The ligation reaction mixture was used as a competent cell, and transformed into Escherichia coHDH5α (ATCC 5386.8, manufactured by Toyobo Co., Ltd.) by the heat shock method to obtain subcloned mouse clones. A plasmid of 104 full-length ORF was obtained. The transformant produced in Example 4 was transferred to the International Depositary Authority, the National Institute of Advanced Industrial Science and Technology, Patent Organism Depositary Sender (Tsukuba-Higashi 1-1, Ibaraki, Japan 1 Central No. 6). FERMBP has been deposited as 084991. The transformant was originally deposited with the International Depositary on August 7, 2002 (Original Deposit Date) as FERMP-18970, Was transferred to a deposit under the Budapest Treaty on September 18, 2008.

 Comparison with existing nucleotide sequences and amino acid sequences

 For the determined nucleotide sequence of the mouse clone 104 polynucleotide, a homology search was performed by BLASTN on each database of GenBank, EMBL, DDBJ and PDB. As a result, the nucleotide sequence of the clone 104 polynucleotide is not identical to the known polynucleotides registered, and is particularly important when preadipocytes differentiate into adipocytes. As a polynucleotide whose expression is abnormally increased, it was found to be a novel substance.

In addition, the amino acid sequence of mouse clone 104 protein predicted from the nucleotide sequence of mouse clone 104 polynucleotide was stored in the respective databases of GenBank CDS translations. PDB, SwissProt, PIR, and PRF. A homology search was performed with BLASTP. As a result, the amino acid sequence of mouse clone 104 protein was registered. It has no identity with known known proteins, and was found to be a novel protein as a protein whose expression is specifically increased when preadipocytes differentiate into adipocytes.

 Example 5

 Recovery of inserts and preparation of probes

 In Example 4, a plasmid containing the DNA fragment Su obtained by the subtraction method was prepared by the SDS method according to the method described in Molecular Cloning, and this plasmid was prepared. After digestion with restriction enzymes Xbal and Hindlll, electrophoresis was performed on a 1.0% agarose gel, and a band corresponding to the insert was recovered using DE81 (Whatman). 50 to 100 ng of DNA fragment from BcaBEST ™

Labeling was performed with [α- ³²社] dCTP (manufactured by Amersham Pharmacia biotech) using a Labeling kit (manufactured by TaKaRa) to obtain a labeled probe. The obtained labeled probe was passed through a column using Sephadex G-50 (manufactured by Amersham Pharmacia biotech) to prepare a ³² P-labeled probe.

 Example 6

3Z3-Plot analysis of expression changes in early stage of 3T3-L1 differentiation Mouse 3T before (0 hour) and 0.5, 1, 3, 6, 12, 24 hours after initiation of adipocyte differentiation induction 3 — L 1 cells (ATCCN o. CCL-9 2. 1.) Were electrophoresed on a 1% denaturing gel (2% formaldehyde, 1 x Mops, 1% agarose). After the gel was treated with 50 iiiM NaOH for 25 minutes (denatured in alkaline) and 200 raM NaOAc (pH 4.0) for 40 minutes (neutralization), the RNA was treated with HybondNI (Amersham Pharmacia biotech). The transfer was performed for 12 hours or more, and the transfer was performed using 20 X SSC The transfer file was transferred to 50 n \ M NaOH for 5 minutes. After the treatment, the plate was washed with 2 XSSC and dried at 80 ° (: 2 hours. After that, it was fixed by irradiation with UV.

 Hybridization buffer (5 X SSPE, 50%

Prehybridization was performed with formaldehyde, 5X Denhalt's, 0.1% SDS, 20 g / ml salmonsperm DNA) at 42 ° C- 晚. After a new change in the hybridization buffer, a heat-denatured probe was added, and hybridization was performed at 42 ° C- 晚. The filter is then washed with the primary wash solution (2X SSPE 0.1% SDS) for 10 minutes at room temperature, and then with the primary wash solution for 55 minutes at -55-65 ° C. 65 using secondary wash solution (1X SSPE 0.1% SDS). After washing with a tertiary washing solution (0.5 X SSP E 0.1% SDS) for 15 minutes at 65 ° C for 10 minutes, autoradiography was performed. The results obtained are shown in Figure 2.

 According to the autoradiogram in Fig. 2, the labeled probe prepared from clone 104 did not react with total RNA prepared from cells before the initiation of adipocyte differentiation induction (0 hour), but did not initiate differentiation induction. It was found that the cells reacted particularly strongly at the hour, and that the cells at the 2nd and 6th hours also reacted slightly. This indicates that clone 104 is a sequence whose expression is specifically increased in the early stages of adipocyte differentiation, particularly within 6 hours after the peak at 3 hours after the initiation of adipocyte differentiation induction. Was. Example 7

 Northern blot analysis of expression comparison between telogen and proliferative phase of 3T3—L1 and NIH-3T3

3 T3-L1 cells (ATCC No. CCL-92.1.) (The property of differentiation to mature cells during induction of differentiation in resting cells, and maturation even after induction of differentiation during growth phase It does not differentiate into fat cells.) And NIH-3T3 cells (ATCC No. CR-1658) (which has the property of not differentiating into mature adipocytes even after induction of differentiation) are cultured in a basal medium, and the cells are 70-80% At the time of confluence (proliferating cells), change the medium to a differentiation-inducing medium, and use total TRINA (GIBCO BRL) from the cells before differentiation induction and 3 hours after the initiation of differentiation induction, for total RNA. Collected. On the other hand, 3T 3 —L 1 (ATCC No. CCL-92.1.) And NIH—3T 3 cells (ATCC No. CRL-1658) (cells in resting phase) by the method described in Example 1 above. Differentiation induction was performed, and total RNA was recovered from the cells 3 hours after the induction of differentiation and pre-differentiation-induction using TRIzol (GIBCO BRL).

 Northern blot analysis was performed by the method described in Example 6 using the collected total RNA. Figure 3 shows the results. According to the autoradiogram of FIG. 3, clone 104 showed particularly strong expression 3 hours after induction of differentiation in the telogen of 3T3-L1 cells (ATCC No. CCL-92.1.). You can see that it is rising. 3T3-L1 cells (ATCC No. CCL-92.1.) Differentiate into adipocytes when a differentiation inducer is added in the quiescent state, but do not differentiate in the proliferative state. Yes. Also, NIH-3T3 cells (ATCC No. CRL-1658), the same mouse embryo-derived fibroblast cell line as 3T3—L1, are differentiation-inducing agents in either the growth phase or the telogen phase. It has the property that it does not bind to fat cells even if it is added. These results indicate that clone 104 is strongly expressed only under conditions that differentiate into adipocytes, suggesting that clone 104 may be deeply involved in adipocyte differentiation. Suggestive.

 Example 8

Identification of human homologs i) Prediction of human homologue sequence using NCBI Genome Sequencing (Human Genome Database)

 The full-length sequence of the clone 104 mouse determined in the above Example 4 was obtained by using NCBI Genome Sequencing® BLAST the Human genome (http: //www.ncbi.nlra. By homology search, a sequence with high homology was found on chromosome 3. The sequence was composed of a total of 25 exons. The gt-ag group was conserved between all exons and introns, and from these facts it was possible to predict the full-length sequence of the human homolog.

 The present inventors have already reported clone 24 (Japanese Patent Application No. 2001- 3 7 4 7 8).

5) and clone 1558 (Japanese Patent Application No. 2002-0487042) predict a human homologous sequence by the same method. The results at this time clearly show that the predicted sequence shows almost 100% homology with the sequence actually determined by RT-PCR cloning and sequencing. From this, the predicted sequence of clone 104 human homologue can be used as the clone 104 human homologue nucleotide sequence.

 The full-length nucleic acid sequence of the clone 104 human homolog polynucleotide determined as described above is shown in SEQ ID NO: 3.

 In addition, the agreement rate between the clone 104 polynucleotide of the mouse shown in SEQ ID NO: 1 and the human homologue shown in SEQ ID NO: 3 was 56678 /

6 894 = 82.4%.

In clone 104 human homologous polynucleotide, the ATG at the 58th base corresponding to the amino acid defined as the initial methionine in mouse clone 104 was assumed to be the translation initiation codon. As a result, clone 104 human homologous polynucleotides had 124 amino acids. It is predicted to be a gene encoding a protein consisting of amino acids, and the amino acid sequence of the protein is shown in SEQ ID NO: 4. Industrial applicability

 The mouse 104 polynucleotide of the present invention and the mouse clone 104 protein deduced from the clone 104 polynucleotide initiate adipocyte differentiation induction. Polynucleotides and proteins extracted from cells within 6 hours after the peak at 3 hours later, which are novel. In addition, human protein 104-polynucleotide and the clone 104 protein estimated from the clone 104-polynucleotide are as follows: As in the case of mice, it is estimated that cells are extracted from cells within 6 hours with the peak at 3 hours after the initiation of induction of adipose cell differentiation. These polynucleotides and proteins are extremely useful substances for elucidating the mechanism of obesity, appropriately grasping the progress of obesity, and using it for prevention or treatment of obesity.

 Clone 104 polynucleotide of the present invention, clone 104 protein or peptide deduced from the clone 104 polynucleotide, and antagonis against these peptides Oragonist is useful for the treatment and diagnosis of lifestyle-related diseases such as diabetes, hypertension, and arteriosclerosis, including obesity.

 Of the clone 104 polynucleotide of the present invention or the polynucleotide complementary to the polynucleotide, the polynucleotide having at least 10 consecutive nucleotide sequences is the following. It is useful for treating and diagnosing diabetes, including obesity, hypertension, and atherosclerosis.

Claims

The scope of the claims

1. A polynucleotide encoding a protein associated with adipocyte differentiation and expressed within 6 hours after the peak at 3 hours after the initiation of adipocyte differentiation induction, represented by SEQ ID NO: 1. A polynucleotide comprising a nucleotide sequence having at least 80% homology to the same or at least 80% homology to the nucleotide sequence, or a polynucleotide complementary to the polynucleotide.

 2.-Adipocyte-related to adipocyte differentiation-3 hours after initiation of differentiation induction-Polynucleotide encoding a protein expressed within 6 hours after peak-3 A polynucleotide containing a nucleotide sequence having homology of at least 80% to the nucleotide sequence No. 61 to 36581 of the nucleotide sequence represented by 1, or the polynucleotide; Complementary polynucleotide.

 3. The polynucleotide according to claim 1 or 2, wherein the polynucleotide is DNA or RNA.

 4. A protein that is expressed within 6 hours after peaking at 3 hours after the initiation of adipocyte differentiation induction, and is the same or less than the full length of the protein represented by SEQ ID NO: 2. A protein that promotes adipocyte differentiation with homology of 80%.

 5. A recombinant vector containing the polynucleotide according to claim 1 or 2, or a polynucleotide complementary to the polynucleotide.

6. A transformant carrying the recombinant vector according to claim 5.

7. An antibody immunologically specific for the protein of claim 4.

8. Antagonize or agonize the protein of claim 4 A method for identifying a compound that

 (a) contacting a candidate compound with cells expressing the protein of claim 4 or cells responsive to the protein of claim 4 (b) observing binding or stimulation or inhibition of a functional response Or a method of comparing the ability of a cell contacted with a candidate compound with respect to the activity of the protein according to claim 4 with a cell of the same type not contacted.

 9. An agonist defined by the method of claim 8. '.

10. An angelic gonist identified by the method of claim 8.

11. A method comprising culturing the transformant according to claim 6 under conditions sufficient for the production of the protein according to claim 4, and recovering the protein from the medium or the transformant. A method for producing protein or its salt.

 12. A drug containing the polynucleotide according to claim 1 or 2, or a polynucleotide complementary to the polynucleotide.

 13. A polynucleotide containing a nucleotide sequence having at least 80% homology to the nucleotide sequence represented by SEQ ID NO: 1, or a polynucleotide containing the same. A medicament comprising a polynucleotide having a sequence of at least 10 consecutive nucleotides among complementary polynucleotides.

 14. A polynucleotide or a polynucleotide comprising a nucleotide sequence having at least 80% homology to the nucleotide sequence Nos. 61 to 3681 of the nucleotide sequence represented by SEQ ID NO: 1 A pharmaceutical comprising a polynucleotide having a sequence of at least 10 contiguous nucleotides among polynucleotides complementary to the nucleotide.

15. The polynucleotide according to claim 1 or 2, or the polynucleotide. A diagnostic agent containing a polynucleotide that is complementary to the nucleotide.

 16. A polynucleotide containing a nucleotide sequence having at least 80% homology to the nucleotide sequence represented by SEQ ID NO: 1, or a polynucleotide containing the same. A diagnostic agent comprising a polynucleotide having at least 10 consecutive nucleotide sequences among complementary polynucleotides.

 17. A polynucleotide or a polynucleotide comprising a nucleotide sequence having at least 80% homology to nucleotide numbers 61 to 3681 of the nucleotide sequence represented by SEQ ID NO: 1. Complementary to a polynucleotide. A diagnostic agent comprising a polynucleotide having at least 10 contiguous base sequences among the polynucleotides.

 18. A drug containing the protein according to claim 4 or a salt thereof.

 19. A medicament comprising a peptide consisting of at least 5 consecutive amino acids of the protein according to claim 4 or a salt thereof.

 20. A diagnostic drug containing the protein according to claim 4 or a salt thereof.

 21. A diagnostic agent comprising a peptide consisting of at least five consecutive amino acids of the protein according to claim 4 or a salt thereof.

 1. A drug containing the antibody according to claim 7.

 23. A diagnostic agent comprising the antibody according to claim 7.

 24. A medicament containing the agonist according to claim 9.

 25. A diagnostic agent comprising the agonist according to claim 9.

26. A medicament containing the fan gonist according to claim 10. 27. A diagnostic agent comprising the antagonist according to claim 10. 28. Claims for the treatment of lifestyle-related diseases selected from obesity, hypertension, hyperlipidemia, diabetes, and heart disease and stroke due to arteriosclerosis. Terms 1 2, 1, 3, 14, 14, 18, 19,

22. The medicament according to 2, 24 or 26.

 29. A diagnostic agent for lifestyle-related diseases selected from obesity, hypertension, hyperlipidemia, diabetes, and heart disease and stroke due to arteriosclerosis. , twenty one,

23, 25 or 27.

 30. A polynucleotide encoding a protein involved in adipocyte differentiation, which is at least 80% identical or at least 80% homologous to the nucleotide sequence of SEQ ID NO: 3. Or a polynucleotide complementary to the polynucleotide, which comprises a base having the formula: 31. A polynucleotide encoding a protein involved in adipocyte differentiation, which is identical or less than nucleotides 58 to 3669 of the nucleotide sequence represented by SEQ ID NO: 3 A polynucleotide comprising a nucleotide sequence having homology of 80% or a polynucleotide complementary to the polynucleotide.

 32. A polynucleotide, wherein the polynucleotide according to claim 30 or 31 is DNA or RNA.

 33. A protein that promotes adipocyte differentiation and has at least 80% homology to the full length of the protein represented by SEQ ID NO: 4.

 34. A method for identifying a compound that agonizes or agonizes a protein according to claim 33,

 (a) contacting a candidate compound with a cell that expresses the protein of claim 33 or a cell that responds to the protein of claim 33;

(b) observing stimulation or inhibition of binding or a functional response; or alternatively, the protein of claim 33, wherein the cell has been contacted with a candidate compound. A method comprising comparing the ability for quality activity to a non-contacting allogeneic cell.

 35. An agonist identified by the method of claim 34. 36. Antagonist identified by the method of claim 34

37. A recombinant vector containing the polynucleotide according to claim 30 or 31.

 38. A transformant carrying the recombinant vector according to claim 37

39. An antibody immunologically specific for the protein of claim 33.

 40.Cultivating the transformant according to claim 38 under conditions sufficient to produce the protein according to claim 33, and recovering protein or a salt thereof from the medium or the transformant. A method for producing a protein, characterized in that:

 41. A medicament containing the polynucleotide according to claim 30 or 31.

 42. A polynucleotide comprising a nucleotide sequence having at least 80% homology to the nucleotide sequence represented by SEQ ID NO: 3, or a polynucleotide complementary to the polynucleotide. Includes polynucleotides having at least 10 contiguous nucleotides among nucleotides

43. A polynucleotide or a polynucleotide comprising a nucleotide sequence having the same or at least 80% homology to nucleotide numbers 58 to 3669 of the nucleotide sequence represented by SEQ ID NO: 3 A drug containing a polynucleotide having a continuous sequence of at least 10 nucleotides among polynucleotides complementary to the nucleotide.

44. A diagnostic agent comprising the polynucleotide according to claim 30 or 31.

 45. A polynucleotide containing a nucleotide sequence having at least 80% homology to the nucleotide sequence represented by SEQ ID NO: 3, or a polynucleotide containing the same. A diagnostic agent comprising a polynucleotide having at least 10 consecutive nucleotide sequences among complementary polynucleotides.

 46.Has the same or at least 80% homology to nucleotides Nos. 58 to 3669 of the nucleotide sequence represented by SEQ ID NO: 3 A diagnostic agent comprising a polynucleotide having a nucleotide sequence of at least 10 contiguous, out of a nucleotide or a polynucleotide complementary to the polynucleotide.

 47. It contains the protein of claim 33 or a salt thereof.

48. A medicament comprising a peptide comprising at least 5 consecutive amino acids of the protein according to claim 33 or a salt thereof.

 49. A diagnostic agent comprising the protein of claim 33 or a salt thereof.

 50. A diagnostic agent comprising a peptide consisting of at least five consecutive amino acids of the protein according to claim 33 or a salt thereof.

 51. A drug containing the antibody according to claim 39.

 52. A diagnostic agent comprising the antibody according to claim 39.

 53. A medicine containing the agonist according to claim 35.

 54. A diagnostic agent comprising the agonist according to claim 35.

 55. A medicament comprising the antagonist according to claim 36.

56. A diagnostic agent comprising the antagonist of claim 36.

5 7. For obesity, hypertension, hyperlipidemia, diabetes, and arteriosclerosis The drug according to claim 41, 42, 43, 47, 48, 51, 53 or 55, which is a therapeutic drug for lifestyle-related diseases selected from diseases and strokes.

 58. A diagnostic drug for lifestyle-related diseases selected from obesity, hypertension, hyperlipidemia, diabetes mellitus, and heart disease and stroke due to arteriosclerosis. , 50, 52, 54 or 56.

 59. Regarding the polynucleotide represented by SEQ ID NO: 3, a method using a nan0 chip, a method for determining the polynucleotide sequence, a method using a DNA chip or a DNA array, and using a mass spectrometer A method for examining nucleotide polymorphisms, which comprises applying a method selected from methods and methods using primer extension.