WO2004035777A1 - Roteines et genes associes a la differenciation des cellules adipeuses - Google Patents

Roteines et genes associes a la differenciation des cellules adipeuses Download PDF

Info

Publication number
WO2004035777A1
WO2004035777A1 PCT/JP2003/013267 JP0313267W WO2004035777A1 WO 2004035777 A1 WO2004035777 A1 WO 2004035777A1 JP 0313267 W JP0313267 W JP 0313267W WO 2004035777 A1 WO2004035777 A1 WO 2004035777A1
Authority
WO
WIPO (PCT)
Prior art keywords
polynucleotide
protein
nucleotide sequence
seq
homology
Prior art date
Application number
PCT/JP2003/013267
Other languages
English (en)
Japanese (ja)
Inventor
Masayoshi Imagawa
Yuichi Oku
Original Assignee
Nissui Pharmaceutical Co., Ltd.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Nissui Pharmaceutical Co., Ltd. filed Critical Nissui Pharmaceutical Co., Ltd.
Publication of WO2004035777A1 publication Critical patent/WO2004035777A1/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/04—Anorexiants; Antiobesity agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/06—Antihyperlipidemics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/10—Antimycotics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/12—Antihypertensives
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • 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 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.
  • Patent Document 1 Japanese Patent Application Laid-Open No. H8-33333
  • Non-Patent Document 1 Biochemical and Biophysical Research
  • 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).
  • Predipocytes preadipocytes
  • Adipocytes adipocytes
  • 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
  • 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.
  • PPARa has been shown to be particularly important for adipocyte differentiation.
  • forcible expression of PPAR ⁇ in lipoblasts and preadipocytes differentiates into adipocytes.
  • PPARA plays an important role in adipocyte differentiation.
  • C / EBP also forms a family similar to PPAR, and recently, C / EBP ⁇ functions as a master regulator of adipocyte differentiation like PPARa. It is clear that there is.
  • 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 / ⁇ ⁇ ⁇ 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 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.
  • 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.
  • 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.
  • clone 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • Can be sorted by Hybridization methods are described, for example, in Molecular Cloning.
  • 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.
  • 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%.
  • 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.
  • yeast When yeast is used as the host, GAP DH Promoter, ADH promoter, PGK promoter, PH05 promoter and the like.
  • a human cytomegalovirus promoter 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.
  • Escherichia coli examples include Escherichia coli JM109 (ATCC53332, manufactured by Toyobo Co., Ltd.) and JM103 (Nucleic Acids Research, ( Nucleic Acids Research), vol. 9, 309 (19981)), ⁇ 12 ⁇ D ⁇ 1 [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' ⁇ Rekylura. Biology, Volume 41, Volume 4 59 (1 969)], C600 [Genetics, 39, 44 (1954)].
  • 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)].
  • yeast examples 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.
  • 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 TM cells derived from ia n_ eggs, cells derived from Mamestra brassicae, and cells derived from Estigmena acrea.
  • the virus is BmNPV
  • a silkworm-derived cell line Boombyx mori cell; BmN cell
  • Sf cells examples 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)].
  • animal cells examples 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.
  • Insect cells or insects can be transformed, for example, according to the method described in Bio / Technology, 6, 47-55 (1988).
  • 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.
  • a protein can be recovered from the culture medium or the transformant.
  • 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.
  • carbon sources include glucose, dextrin, soluble starch, and sucrose.
  • 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.
  • yeast, vitamins, growth promoting factors and the like may be added.
  • the pH of the medium is preferably about 5-8.
  • 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.
  • 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.
  • 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.
  • 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
  • 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 ⁇ 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.
  • 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
  • the pH is about 6-8.
  • Culture is usually about 30 ° C ⁇
  • the protein of the present invention can be produced in the cells of the transformant.
  • the protein of the present invention can be separated and purified from the culture by, for example, the following method.
  • 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).
  • 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.
  • a suitable protein modifying enzyme for example, trypsin, chymotrypsin, arginylendopeptidase, protein kinase, glycosidase and the like are used.
  • 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.
  • 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.
  • SEQ ID NO: 1 The total length of the human homologue predicted and sequenced in this way is the nucleotide sequence shown in 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 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/.
  • 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;
  • 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.
  • 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.
  • 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).
  • 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.
  • a plurality of polynucleotides including the polynucleotide of the present invention are bound on a substrate, and the method described above is used.
  • 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.
  • 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.
  • ABIPRISM 310 or PRISM 310 manufactured by Applied Biosystems is used. Detection by capillary electrophoresis using 0000 or PRISM 370 is also possible.
  • 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.
  • 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.
  • 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.
  • 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% ⁇ 90% to the nucleotide sequence represented by SEQ ID NO: 1.
  • a diagnostic agent comprising a polynucleotide comprising a nucleotide sequence having a homology of 95% or a polynucleotide complementary to the polynucleotide.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • an immunoassay is generally used.
  • 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.
  • 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.
  • 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.
  • the spleen cell which is an antibody-producing cell
  • the -antibody produced is purified by various methods, and the obtained antibody is bound to a solid phase.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the diagnostic agent for detecting the protein of the present invention described above examples include the diagnostic agent containing the protein of the present invention or a salt thereof.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • a drug comprising a polynucleotide or a polynucleotide complementary to the polynucleotide and comprising a polynucleotide having at least 10 consecutive nucleotide sequences.
  • 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.
  • 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
  • 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,
  • an oligonucleotide may be provided that forms a triple helix with the gene.
  • an oligonucleotide may be provided that forms a triple helix with the gene.
  • Lee et al. Nucleic Acids Res (1979) 6: 3073; Cooney et al., Science (1988) 241: 456; Dervan et al., Science (1991) 251: 1360.
  • the ligoma can be administered as such, or the relevant oligomer can be activated in vivo.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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
  • 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.
  • penetrants such as bile salts or fusidic acid or other surfactants.
  • 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.
  • the protein of the present invention used for treatment can also be produced in the subject to be treated.
  • 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.
  • 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
  • DMEM differentiation-inducing medium
  • 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
  • Dex 1 M Dexamethasone
  • DMEM differentiation promoting medium
  • FBS FBS
  • 5 ug / ml insulin 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.
  • TRIzol GIBC0 BRL
  • 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.
  • driver-mRNA 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.
  • 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.
  • 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.
  • 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.
  • DNA was amplified by PCR using two primers specific to the two adapter sites.
  • Tester cDNA hybridized with the driver cDNA was not amplified.
  • 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).
  • nucleotide R-5 ′ 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.
  • DNA fragment Su was labeled with [ ⁇ -32 ⁇ ] 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).
  • nucleotide RT was subcloned into a T vector (pBluescript KS +), and the nucleotide sequence was determined by DSQ-100 (Shimadzu Corporation).
  • 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.
  • 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.
  • a ligation reaction of 1 bp — 370 bp clone 104 polynucleotide to pBluescrit SK + vector (Xba i / EcoRV) was performed.
  • the ligation reaction mixture was used as a competent cell, and transformed into Escherichia coHDH5 ⁇ (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.
  • Example 4 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.
  • nucleotide sequence of the mouse clone 104 polynucleotide was 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.
  • 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 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 TM
  • Labeling was performed with [ ⁇ - 32 ⁇ ] 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.
  • 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- ⁇ . After a new change in the hybridization buffer, a heat-denatured probe was added, and hybridization was performed at 42 ° C- ⁇ . 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.
  • 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.
  • NIH-3T3 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.
  • GEBCO BRL total TRINA
  • 3T 3 —L 1 ATCC No. CCL-92.1.
  • 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).
  • FIG. 3 shows the results.
  • 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.
  • NIH-3T3 cells ATCC No.
  • CRL-1658 the same mouse embryo-derived fibroblast cell line as 3T3—L1
  • 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.
  • 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.
  • clone 104 human homologous polynucleotide 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.
  • 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.
  • 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.
  • 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.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Medicinal Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Public Health (AREA)
  • Animal Behavior & Ethology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Veterinary Medicine (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • General Chemical & Material Sciences (AREA)
  • Diabetes (AREA)
  • Hematology (AREA)
  • Molecular Biology (AREA)
  • Obesity (AREA)
  • Urology & Nephrology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Biochemistry (AREA)
  • Biomedical Technology (AREA)
  • Heart & Thoracic Surgery (AREA)
  • Cardiology (AREA)
  • Immunology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Emergency Medicine (AREA)
  • Pathology (AREA)
  • Physics & Mathematics (AREA)
  • Cell Biology (AREA)
  • Biotechnology (AREA)
  • Food Science & Technology (AREA)
  • Genetics & Genomics (AREA)
  • Biophysics (AREA)
  • Oncology (AREA)
  • Analytical Chemistry (AREA)
  • Communicable Diseases (AREA)
  • Microbiology (AREA)
  • Zoology (AREA)

Abstract

Gènes exprimés 6 heures après le déclenchement d'une différenciation des précurseurs de cellules adipeuses en cellules adipeuses, cette expression présentant une crête au bout de 3 heures. Polynucléotide contenant une séquence de base similaire à la séquence de base représenté par la séquence SEQ ID NO:1 ou SEQ ID NO:3 ou homologue de ladite séquence d'au moins 80 % ou polynucléotide complémentaire adu premier nucléotide. Protéine codée par un polynucléotide représenté par SEQ ID NO:1 ou SEQ ID NO:3 et contenant une équence amino acide semblable à celle d'une protéine représentée par SEQ ID NO:2 ou SEQ ID NO:4 sur la totalité de sa longueur de chaîne ou présentant une homologie d'au moins 80 % avec ladite séquence.
PCT/JP2003/013267 2002-10-18 2003-10-16 Roteines et genes associes a la differenciation des cellules adipeuses WO2004035777A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP2002304839A JP2004135605A (ja) 2002-10-18 2002-10-18 脂肪細胞分化関連遺伝子およびタンパク質
JP2002-304839 2002-10-18

Publications (1)

Publication Number Publication Date
WO2004035777A1 true WO2004035777A1 (fr) 2004-04-29

Family

ID=32105140

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP2003/013267 WO2004035777A1 (fr) 2002-10-18 2003-10-16 Roteines et genes associes a la differenciation des cellules adipeuses

Country Status (2)

Country Link
JP (1) JP2004135605A (fr)
WO (1) WO2004035777A1 (fr)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20110104666A1 (en) 2009-11-02 2011-05-05 Toshiya Matsubara Insulin resistance marker

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999055865A1 (fr) * 1998-04-29 1999-11-04 Genesis Research And Development Corporation Limited Polynucleotides isoles de cellules de la peau et leurs procedes d'utilisation
WO2001040466A2 (fr) * 1999-12-01 2001-06-07 Genentech, Inc. Polypeptides secretes et transmembranaires et acides nucleiques codant ces polypeptides

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999055865A1 (fr) * 1998-04-29 1999-11-04 Genesis Research And Development Corporation Limited Polynucleotides isoles de cellules de la peau et leurs procedes d'utilisation
WO2001040466A2 (fr) * 1999-12-01 2001-06-07 Genentech, Inc. Polypeptides secretes et transmembranaires et acides nucleiques codant ces polypeptides

Also Published As

Publication number Publication date
JP2004135605A (ja) 2004-05-13

Similar Documents

Publication Publication Date Title
US6262333B1 (en) Human genes and gene expression products
AU2023214237A1 (en) Modified polynucleotides for the production of biologics and proteins associated with human disease
CA2443617C (fr) Sequences repetees du gene ca125 et leurs utilisations dans des interventions diagnostiques et therapeutiques
US20020015950A1 (en) Atherosclerosis-associated genes
CZ20023567A3 (cs) Sloučeniny a způsoby pro terapii a diagnostiku karcinomu plic
JPH10513045A (ja) ヒトの胎児の脾臓で発現する新規なケモカイン、その産生と使用
CA2457301A1 (fr) Proteine, oligonucleotide anti-sens et anticorps de leptine porcine
US7309760B2 (en) Repeat sequences of the CA125 gene and their use for diagnostic and therapeutic interventions
US20040014169A1 (en) Novel G protein-coupled receptors
JP2003527091A (ja) 新規カルパインプロテアーゼ２６１７６およびその使用
CN1469926A (zh) 治疗和诊断肺癌的组合物和方法
WO2004035777A1 (fr) Roteines et genes associes a la differenciation des cellules adipeuses
EP0973887B1 (fr) Proteine leptine porcine, sequences d'acides nucleiques codant pour ladite proteine et utilisations
JP2002538766A (ja) ＨＲＰＣａ９およびＨＲＰＣａ１０の核酸およびポリペプチド
JP4504682B2 (ja) 脂肪細胞分化関連遺伝子およびタンパク質
JPH11501817A (ja) ヒト臍静脈内皮細胞で発現されるヒアルロン酸レセプタ
JP2002501744A (ja) Ｗｎｔ−１０ａ
MXPA03007067A (es) Acido nucleico expresado en el hipotalamo o tejido muscular en animales obesos.
JP2002520057A (ja) 新規な哺乳類ペプチダーゼのクローニング及び特性化
JP2003250550A (ja) 脂肪細胞分化関連遺伝子およびタンパク質
AU2001272982B2 (en) GP286 nucleic acids and polypeptides
JP4255094B2 (ja) ヒト破骨細胞由来遺伝子
CN108329372B (zh) N6-(2-羟乙基)腺苷及其衍生物在制备治疗痛风药物中的应用
EP1613769A1 (fr) Gene induit par l'insuline utilise comme cible therapeutique dans le diabete
JP2004500872A (ja) Ｇｐ３５４核酸およびポリペプチド

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): US

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IT LU MC NL PT SE SI SK TR

121 Ep: the epo has been informed by wipo that ep was designated in this application
122 Ep: pct application non-entry in european phase