WO2007049359A1 - Method of controlling glucose metabolism - Google Patents

Method of controlling glucose metabolism Download PDF

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Publication number
WO2007049359A1
WO2007049359A1 PCT/JP2005/020144 JP2005020144W WO2007049359A1 WO 2007049359 A1 WO2007049359 A1 WO 2007049359A1 JP 2005020144 W JP2005020144 W JP 2005020144W WO 2007049359 A1 WO2007049359 A1 WO 2007049359A1
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WIPO (PCT)
Prior art keywords
amino acid
acid sequence
test substance
protein
gene
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PCT/JP2005/020144
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French (fr)
Japanese (ja)
Inventor
Jun Wada
Original Assignee
Tanai, Takeo
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Application filed by Tanai, Takeo filed Critical Tanai, Takeo
Priority to PCT/JP2005/020144 priority Critical patent/WO2007049359A1/en
Publication of WO2007049359A1 publication Critical patent/WO2007049359A1/en

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/81—Protease inhibitors
    • C07K14/8107—Endopeptidase (E.C. 3.4.21-99) inhibitors
    • C07K14/811—Serine protease (E.C. 3.4.21) inhibitors
    • C07K14/8121—Serpins
    • 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
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • 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
    • G01N33/66—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood sugars, e.g. galactose
    • 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
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/04—Endocrine or metabolic disorders
    • G01N2800/042—Disorders of carbohydrate metabolism, e.g. diabetes, glucose metabolism

Definitions

  • the present invention relates to a therapeutic or prophylactic agent for a sugar metabolism-related disease, and more particularly to a protein having a glucose tolerance improving activity or an insulin response promoting activity.
  • Diabetes is a condition in which the homeostasis of glucose metabolism (glucose renewal and clearance) in the living body is broken, particularly a hyperglycemic condition.
  • the background of diabetes mellitus is the insufficiency and / or dysfunction of insulin, a blood glucose-regulating hormone, and treatment of diseases closely related to glucose metabolism (glucose metabolism-related diseases) by correcting or substituting insulin action. Or prevention becomes possible. So far, insulin has been known as an in-vivo substance (hormone) that improves glucose metabolism function, and insulin preparations have been used to improve glucose metabolism function. Yes.
  • the protein consisting of the amino acid sequence represented by SEQ ID NO: 2 is known to be a protein that is specifically expressed in the visceral adipose tissue of obese animals (see Non-Patent Document 1).
  • the function of the protein in vivo is unknown, and the relationship between sugar metabolism-related diseases has not been known at all.
  • Non-Patent Document 1 Hida K et al, J. Lipid Research, 41, l615-p l622 (2000)
  • the problem to be solved by the present invention is to provide a therapeutic or preventive agent for a sugar metabolism-related disease, specifically, a protein having a glucose tolerance improving activity and a ginsulin response promoting activity.
  • the protein comprising the amino acid sequence represented by SEQ ID NO: 2 has a glucose tolerance improving activity and an insulin responsiveness promoting activity, and is associated with glucose metabolism-related diseases.
  • the inventors have found that it can be a therapeutic agent or a preventive agent, and have completed the present invention.
  • a therapeutic agent for a disease related to sugar metabolism comprising a protein comprising any of the following amino acid sequences (hereinafter sometimes referred to as the present protein) or a fragment thereof as an active ingredient:
  • Preventive agent a protein comprising any of the following amino acid sequences (hereinafter sometimes referred to as the present protein) or a fragment thereof as an active ingredient:
  • a therapeutic or prophylactic agent for a sugar metabolism-related disease comprising as an active ingredient a gene encoding a protein comprising an amino acid sequence selected from the amino acid sequence group described in [1];
  • test substance promotes the expression of a protein consisting of an amino acid sequence selected from the amino acid sequence group described in [1] or a gene encoding the protein, is used as an index.
  • Glucose tolerance improving activity assay method
  • a reporter gene comprising a test substance and a reporter gene operatively linked to an expression control region of a gene encoding a protein comprising an amino acid sequence selected from the amino acid sequence group described in [1]
  • a therapeutic or prophylactic agent for a sugar metabolism-related disease comprising as an active ingredient a substance selected by the search method according to [1 0] or a pharmaceutically acceptable salt thereof;
  • a therapeutic or preventive agent for a sugar metabolism-related disease comprising as an active ingredient a substance that induces the expression of a protein comprising an amino acid sequence selected from the amino acid sequence group described in [1] or a function promoting substance 1 It is about.
  • a therapeutic or preventive agent for a glucose metabolism-related disease characterized by having a glucose tolerance-improving activity and ginsulin responsive promoting activity, a method for assaying glucose tolerance-improving activity, and a glucose metabolism-related disease using the assay It has become possible to provide a method for searching for therapeutic or preventive agents.
  • the first aspect of the present invention relates to a therapeutic or preventive agent for a sugar metabolism-related disease comprising the present protein or a fragment thereof as an active ingredient.
  • a protein consisting of any of the following amino acid sequences means (a) an amino acid sequence represented by SEQ ID NO: 2 and (b) an amino acid sequence represented by SEQ ID NO: 2.
  • An amino acid sequence in which one or more amino acids have been deleted, appended, inserted or substituted (c) represented by the 2nd 2nd amino acid to the 4th 4th 4th amino acid in the amino acid sequence represented by SEQ ID NO: 2
  • An amino acid sequence, () encoded by DNA having the nucleotide sequence shown by the nucleotide from the 2nd 8th to the 7th nucleotide in the nucleotide sequence shown by SEQ ID NO: 1 (G) 80% or more of sequence identity with DNA having the nucleotide sequence represented by nucleotide sequence represented
  • And (g) include, for example, the processing that the protein having the amino acid sequence represented by SEQ ID NO: 2 undergoes in the cell and the species difference of the organism from which the protein is derived. It includes naturally occurring mutations due to individual differences, organizational differences, and artificial amino acid mutations.
  • amino acid modification As a technique for artificially carrying out the “amino acid deletion, addition or substitution” in the above (b) (hereinafter sometimes referred to as amino acid modification as a whole), for example, the amino acid represented by SEQ ID NO: 2 is used.
  • a conventional site-directed mutagenesis is performed on the DNA that codes the sequence, and then this DNA is expressed by a conventional method.
  • a site-specific mutagenesis method for example, a method using an amber mutation (gapped, duplex method, Nucleic Acids Res., 12,9441-9456 (1984)
  • a mutagenesis primer is used as PCR methods that were used.
  • the number of amino acids modified as described above is at least one residue, specifically one or several, or more. The number of such modifications may be in a range in which the activity of promoting sugar metabolism of the protein can be found.
  • substitutions are particularly preferable.
  • the substitution is more preferably substitution with an amino acid having similar properties such as hydrophobicity, charge, pK, and steric features.
  • Such substitutions include, for example: 0 glycine, alanine; ii) valine, isoleucine, leucine; iii) aspartic acid, glutamic acid, asparagine, glutamine, IV) serine, threonine; V) lysine, arginine; vi) phenol Substitution within the group of lulanin and tyrosine.
  • amino acid sequence 1 represented by the 4th to 4th amino acids from the 2nd 2nd amino acid in the amino acid sequence represented by SEQ ID NO: 2 is represented by the sequence number 2 that is a secreted protein.
  • sequence identity refers to sequence identity and homology between two DNAs or two proteins.
  • the “sequence identity” is determined by comparing two sequences that are optimally aligned over the region of the sequence to be compared.
  • the DNA or protein to be compared may have an addition or deletion (for example, a gap) in the optimal alignment of the two sequences.
  • Such sequence identity can be calculated, for example, by creating an alignment using the ClustalW algorithm (Nucleic Acid Res 22 (22): 4673-4680 (l994)) using Vector NTI.
  • sequence identity in the present invention may be 80% or more, preferably 90% or more, and more preferably 95% or more.
  • hybridize under stringent conditions the hybridization used here is, for example, by Sambrook J., Frisch E, R, Maniatis T., Molecular Cloning No. 2 Edition (Molecular Cloning 2nd edition), Cold Spring Harbor Laboratory press
  • under stringent conditions means, for example, 6 x SSC (a solution containing 1.5 M NaCl and 0.15 M trisodium citrate is 10 XSSC), 45 ° C in a solution containing 50% formamide. After forming a hybrid in C, wash under 2 X SSC at 50 ° C (Molecular Biology, John Wiley & Sons, NY (1989), 6.3.1-6.3.6), etc. Can be mentioned.
  • the salt concentration in the washing step is, for example, from 50 ° C for 2 XSSC (low stringency conditions) to 50 ° C for 0, 2 X SSC ( ⁇ stringency conditions) You can select from.
  • the temperature in the washing step can be selected, for example, from room temperature (low stringency conditions) to 65 ° C. (high stringency conditions). It is also possible to change both salt concentration and temperature.
  • the above-described protein include a human protein consisting of the amino acid sequence shown by SEQ ID NO: 2, a rat homolog consisting of the amino acid sequence shown by SEQ ID NO: 4, and an amino acid shown by SEQ ID NO: 6. Mention may be made of mouse homozygous consisting of an acid sequence.
  • the fragment of the protein represents a peptide fragment consisting of an amino acid partial sequence of 15 to 100 residues, preferably 15 to 50 residues of the protein, and the sugar of the protein As long as hypermetabolic activity is retained, there is no particular limitation.
  • this gene for example, (I) the base sequence encoding the amino acid sequence represented by SEQ ID NO: 2, and (II) the amino acid sequence represented by SEQ ID NO: 2, one or more (III) in the amino acid sequence shown in SEQ ID NO: 2, from the amino terminus
  • the present protein is produced and obtained according to a usual genetic engineering method. In this way, the present protein can be prepared.
  • a plasmid that can express this gene in a host cell is prepared, introduced into a host cell, transformed, and further transformed by culturing the transformed host cell (transformant). You can get this protein from the culture.
  • the plasmid contains genetic information that can be replicated in the host cell, can grow independently, can be easily isolated and purified from the host cell, and can function in the host cell.
  • Preferred examples include those in which a gene encoding the present protein is introduced into an expression vector having a unique promoter and having a detectable potential.
  • Various expression vectors are commercially available. For example, expression vectors used for expression in E.
  • coli are expression vectors containing promoters such as lac, trp, and tac, which are commercially available from Falmacia, Takara Shuzo and others. Restriction enzymes used for introducing the gene encoding this protein into the expression vector are also commercially available from Takara Shuzo. If it is necessary to induce higher expression, a ribosome binding region may be linked upstream of the gene encoding this protein. Examples of the liposome-binding domain used include those described in reports by Guarente L, et al. (Cell 20, p543) and Taniguchi et al. (Genetics of Industrial Microorganisms, p202, Kodansha).
  • the vectors used for expression in mammalian cells are the SV 40 virus promoter, rhinoceros megalovirus promoter (CMV promoter), Raus Sarcoma Virus promoter — (RSV promoter),] 3 actin gene promoter, a P
  • CMV promoter rhinoceros megalovirus promoter
  • RSV promoter Raus Sarcoma Virus promoter —
  • 3 actin gene promoter a P
  • expression vectors containing a promoter such as a 2-gene promoter, which are obtained from Toyobo, Takara Shuzo, etc. It is sold.
  • host cells include prokaryotic or eukaryotic microorganism cells, insect cells, or mammalian cells.
  • E. coli and the like can be preferably mentioned.
  • the plasmid obtained as described above can be introduced into the host cell by an ordinary genetic engineering method.
  • the transformant can be cultured by a conventional method used for culturing microorganisms, insect cells or mammalian cells.
  • culture is performed in a medium containing an appropriate carbon source, nitrogen source, and micronutrients such as vitamins as appropriate.
  • the culture method may be any of solid culture and liquid culture, and preferred examples include liquid culture such as aeration-stirring culture.
  • This protein can be obtained by combining methods commonly used for isolation and purification of general proteins. For example, the transformants obtained by the above culture are collected by centrifugation or the like, and the transformants are crushed or dissolved, and if necessary, proteins are solubilized, ion exchange, hydrophobicity, gel filtration, etc. The steps using the various chromatographies described above may be purified singly or in combination. An operation for restoring the higher order structure of the purified protein may be further performed. For example, the transformant obtained by the above culture may be removed by centrifugation or the like, and the present protein may be purified from the culture supernatant in the same manner as described above. -
  • examples of the glucose metabolism-related diseases include diseases associated with abnormal glucose metabolism functions in the living body. Specifically, diseases such as diabetes, impaired glucose tolerance, and arteriosclerosis associated with sustained hyperglycemia. Examples include illness. That is, the sugar metabolism-related disease is not particularly limited as long as it is a disease having various pathologies caused by a decrease in insulin function or dysfunction.
  • a disease to which the therapeutic or preventive agent for a sugar metabolism-related disease of the present invention can be applied a disease state in which blood glucose homeostasis such as diabetes is broken can be mentioned.
  • the therapeutic or prophylactic agent for a sugar metabolism-related disease of the present invention is characterized by having a glucose tolerance enhancing activity, a ginsulin response promoting activity, that is, a glucose tolerance improving activity.
  • glucose tolerance enhancing activity is measured by performing OGTT (glucose tolerance test: a test that measures changes in blood glucose level and insulin level after loading a certain amount of sugar in mice). This means that the decrease in blood glucose level after glucose load is overcome in OGTT.
  • OGTT can be performed by the method described in Examples 9 and 10 of the present specification.
  • insulin responsiveness is measured by performing ITT (insulin tolerance test: measuring changes in blood glucose level after loading a mouse with a certain amount of insulin). It shows that ITT has overcome the decrease in blood glucose level after insulin loading.
  • I T T can be carried out by the method described in Examples 9 and 10 of the present specification.
  • the protein to be measured when the protein to be measured shows a blood glucose level comparable to that of the present protein used in Examples 9 and 10 of the present invention, the protein to be measured is “glucose tolerance enhancing activity” or “insulin It can be said that it shows responsiveness.
  • the “gene encoding the protein (hereinafter sometimes referred to as the present gene)” used in the first step is not particularly limited as long as it is a cell expressing the gene.
  • the endogenous gene of the present invention or the gene introduced into the cell as a foreign gene may be used, but the endogenous gene of the cell to be used is preferred.
  • this gene is preferably the gene of the animal species from which the cell is used.
  • derived animal species include rodent mammals such as rats, mice, and guinea pigs, dogs, monkeys, and humans.
  • Examples of the cells include cells isolated from animal tissues and organs, cells and tissues forming a group having the same function / morphology, and the like, and may be cells in any differentiation process. Les.
  • the compound used as the test substance is not particularly limited, and examples thereof include proteins, peptides, nucleic acids, inorganic compounds, and organic compounds prepared by natural or synthetic chemistry.
  • a test substance specifically, a peptide library of amino acids 3 to 50 residues, preferably 5 to 20 residues, or a combinatorial chemistry technique known to those skilled in the art was prepared.
  • a low molecular weight organic compound library having a molecular weight of 100-200, preferably 200-800 can be mentioned.
  • control cell in the second step represents a cell that can express the tree gene used in the first step and is not brought into contact with a test substance.
  • the conditions for bringing the test substance into contact with the cells are not particularly limited, but conditions that do not significantly change the culture conditions (temperature, pH, medium composition, etc.) can be adopted so that the cells do not die. I like it.
  • the concentration of the test substance to be contacted with a cell capable of expressing the gene encoding this protein is not particularly limited, and is usually about 0.1 ⁇ ⁇ to about 100 ⁇ ⁇ , preferably 1 Any value between ⁇ 5 and 50 ⁇ ⁇ ⁇ is acceptable.
  • the time for contacting the muscle cell or hepatocyte with the test substance is usually about 5 minutes to 30 minutes, preferably about 10 minutes to 20 minutes.
  • the test substance can be used by appropriately dissolving or suspending it in a solvent such as water, a buffer such as phosphate buffer or Tris buffer, ethanol, acetone, dimethyl sulfoxide, or a mixture thereof. [0 0 2 9]
  • Control cell not to be contacted with test substance refers to a cell that can be expressed in the gene used in the first step and is not in contact with the test substance. “When the test substance is not contacted” includes the case where the same amount of solvent (blank) as the test substance is added instead of the test substance, or the case where a negative control substance that does not affect the expression of this gene is added. It is.
  • the conditions for bringing the test substance into contact with the cells are not particularly limited, but it is preferable to employ conditions that do not significantly change the culture conditions (temperature, pH, medium composition, etc.) so that the cells do not die. .
  • Detection and quantification of the expression level of this gene can be carried out by a known method such as Northern plotting or RT-PCR using RNA prepared from the cells or a complementary polynucleotide transcribed therefrom. Specifically, by using a polynucleotide having at least 15 bases continuous in the base sequence of this gene and / or its complementary polynucleotide as a primer or a probe, the presence or absence of expression of the tree gene in RNA or Its expression level can be detected and measured.
  • Such a probe or primer is based on the base sequence of this gene, for example primer 3 (HYPERLINK http: "www.eenome.wi.mit.edu/cgi-bill/primer/primer3.cgihttp7 /www,genome.wi.mit.edu/cgi-bm/pnmer/prjmer3.cgi) or the vector NTKlnfomax)).
  • the primer or probe When using Northern blotting, the primer or probe is labeled with a radioactive isotope (32P, 33P, etc .: RI) or a fluorescent substance, and transferred to nylon membrane or the like according to a conventional method. After hybridization with RNA, the formed duplex of primer or probe (DNA or RNA) and rigid A is derived from the primer or probe label (RI or fluorescent substance). Illustrating a method of detecting and measuring with a radiation detector (BAS-1800II, manufactured by Fuji Film) or a fluorescence detector as a signal.
  • a radiation detector BAS-1800II, manufactured by Fuji Film
  • a fluorescence detector as a signal.
  • the probe is labeled according to the protocol, and is amplified with cell-derived RNA, and then the signal derived from the probe label is detected.
  • a method of detecting and measuring with a multi-bioimager STORM860 can also be used.
  • cDNA was prepared from cell-derived RNA according to a conventional method, and was prepared based on the sequence of this gene so that the target tree gene region could be amplified using this as a cage.
  • a pair of primers (a normal strand that binds to the above cDNA (-strand) and a reverse strand that binds to the + strand) are hybridized with this, and PCR is performed according to a conventional method. The method of taking out can be illustrated.
  • the amplified double-stranded DNA is detected by a method of detecting the labeled double-stranded DNA produced by performing the above PCR using a primer previously labeled with RI or a fluorescent substance.
  • a method may be used in which the double-stranded DNA is transferred to nylon membrane or the like according to a conventional method, and the labeled primer is used as a probe to hybridize with the primer.
  • the generated labeled double-stranded DNA product can be measured with an Agilent 2100 Bioanalyzer (manufactured by Yokogawa Analytical Systems).
  • an RT-PCR reaction solution is prepared according to the protocol using SYBR Green RT-PCR Reagents (Applied Biosystems) and reacted with ABI PRIME 7900 Sequence Detection System (Applied Biosystems). Objects can also be detected. [0 0 3 3]
  • the expression of this gene in the cells to which the test substance is added is 1.5 times or more, preferably 2 times or more, more preferably 3 times or more compared to the expression level in the control cells without adding the test substance,
  • the test substance can be selected as an expression inducer of this gene.
  • the present invention comprises the following steps (1) to (3), wherein the assay for glucose tolerance promoting activity is provided:
  • Examples of the “cell capable of expressing the present protein” include the same cells as the “cell capable of expressing the gene encoding the present protein (the present gene)” in 1) above.
  • Detection and quantification of the expression level of the protein can be quantified according to a known method such as Western plotting using an antibody recognizing the protein.
  • the Western Plot method uses an antibody that recognizes this protein as the primary antibody, and then labels it with a radioisotope such as 125 I, a fluorescent substance, or an enzyme such as horseradish peroxidase (HRP) as the secondary antibody.
  • a radioisotope such as 125 I
  • a fluorescent substance such as a fluorescent substance
  • HRP horseradish peroxidase
  • detection is performed according to the protocol using ECL Plus Western Blotting Detection System (manufactured by Amersham Pharmacia Biotech), and multi-biomeasuring STORM860 (Amersham (Falmacia Biotech).
  • the form of the antibody is not particularly limited, and it may be a polyclonal antibody using the protein as an immunizing antigen or a monoclonal antibody thereof, and at least among the amino acid sequences constituting the protein.
  • the antibody of the present invention also includes an antibody having an antigen binding property to a continuous polypeptide consisting of usually 8 amino acids, preferably 15 amino acids, more preferably 20 amino acids.
  • the present invention comprises a method for assaying glucose tolerance promoting activity, characterized by comprising the following steps (1) to (3): provide. Ie:
  • test substance is the same as described above.
  • “Expression control region of the gene encoding this protein (this gene)” usually refers to a range from several kb to several tens of kb upstream of the chromosomal gene.
  • 5'_race method 5 ' -RACE method
  • 5'full Race Core Kit (Takara Shuzo) etc.
  • (ii) Obtain 5' upstream region using Genome Walker Kit (Clontech) etc.
  • the upstream region can be identified by a method including a step of measuring promoter activity;
  • a reporter gene formed by linking the expression control region of this gene in a functional manner may be prepared by a method known to those skilled in the art. That is, it was extracted according to the usual genetic engineering techniques described in “Molecular Cloning laboratory Manual 2nd edition” (1989 Cold Spring Harbor Laboratory Press, “Current Protocols In Molecular Biology” (1987), John Wiley & Sons, Inc., etc.) The expression control region of this gene can be incorporated on a plasmid containing a reporter gene.
  • Reporter genes include dalcronidase (GUS), luciferase, chloramphenicol transferase (CAT), -galactosidase, and green fluorescent protein (GFP).
  • GUS dalcronidase
  • CAT chloramphenicol transferase
  • GFP green fluorescent protein
  • the prepared reporter gene operably linked to the expression control region of this gene is inserted into a vector that can be used in a cell into which the reporter gene is to be introduced, using ordinary genetic engineering techniques. Can be prepared and introduced into a suitable host cell. Transformed cells can be obtained by culturing in a medium under selection conditions according to the reporter gene.
  • a method of measuring the expression level of the reporter gene a method corresponding to each reporter gene may be used.
  • a luciferase gene when used as a reporter gene, the transformed cells are cultured for a few minutes, an extract of the cells is obtained, then the extract is reacted with luciferin and ATP to cause chemiluminescence, and the luminescence intensity Promoter activity can be detected by measuring.
  • a commercially available Luciferase reaction detection kit such as Pitsukagene Dual Kit (registered trademark; manufactured by Toyo Ink) can be used.
  • Contact with a test substance with a ⁇ cell capable of expressing this protein '' or ⁇ a cell containing a reporter gene that is operably linked to the expression control region of this gene '' is a condition that allows the cell to grow
  • a mammalian cell as a host
  • D-MEM a mammalian cell
  • OPTI-MEM OPTI-MEM
  • RPM to which mammal-derived serum such as urine fetal serum is appropriately added. It can be cultured in a commercially available medium such as 1 1 6 4 medium (manufactured by Gibco-BRL).
  • control cell not to be contacted with test substance means “cell capable of expressing a gene encoding this protein” and “this protein used in each first step” “Expressable cell” or “cell containing a reporter gene operably linked to the expression control region of this gene” refers to the cell when the test substance is not contacted.
  • the same amount of solvent (blank) as the test substance is added instead of the test substance, or the negative control that does not affect the expression of the gene, protein or reporter gene. This includes the case of adding oral substance.
  • the expression induction activity of the gene or the protein is determined. Can be selected. That is, the expression level of the gene, the protein or the reporter gene in cells to which the test substance is added is 1.2 times or more, preferably 1. If it is 5 times or more, more preferably 2 times or more, the test substance can be selected as an expression inducer (expression promoting substance) of this gene or protein.
  • the expression inducer of the present gene or protein selected as described above is also a glucose tolerance-promoting activity, that is, an activity to improve glucose tolerance, specifically, an insulin responsiveness promoting activity. It is a candidate for a therapeutic or prophylactic agent.
  • the present protein and its fragments, or the compounds found by the search method of the present invention are used as they are as pharmaceuticals as they are or as a known pharmaceutically acceptable carrier (I-arming agent, diluent, bulking agent, binder) And a lubricant, a flow aid, a disintegrant, a surfactant, and the like) and additives for the above can be mixed to prepare a pharmaceutical composition.
  • a pharmaceutically acceptable carrier I-arming agent, diluent, bulking agent, binder
  • a lubricant, a flow aid, a disintegrant, a surfactant, and the like additives for the above can be mixed to prepare a pharmaceutical composition.
  • the pharmaceutical composition is prepared in the form to be prepared (tablets, pills, capsules, powders, granules, syrups, emulsions, suspensions, etc .; injections, drops, external preparations, suppositories) Depending on the parenteral administration agent) etc., it can be administered systemically or locally, orally or parenterally. In the case of parenteral administration, intravenous administration, intradermal administration, subcutaneous administration, rectal administration, transdermal administration, etc. are possible.
  • the above-mentioned appropriate dosage form is an acceptable normal carrier, excipient, binder, stabilizer, diluent, etc. active ingredient (this protein, or a substance selected by the search method of the present invention or its pharmacologically. It can be produced by blending acceptable salts and the like. When used in an injection form, acceptable buffering agents, solubilizing agents, isotonic agents, and the like can be added.
  • the dosage varies depending on the type of active ingredient, administration route, administration subject or patient's age, weight, symptoms, etc., but cannot be generally specified.
  • Several mg to 2 g, preferably about 5 mg to several tens mg can be administered once to several times a day.
  • about 0.1 mg to about 50 O mg may be administered as an active ingredient in adults, and the daily dose can be administered once or divided into several times.
  • the active ingredient substance examples include the gene itself.
  • the gene it may be possible to incorporate the gene into a gene therapy vector and perform gene therapy.
  • the dosage and administration method of the gene therapy composition vary depending on the patient's weight, age, symptoms, etc., and can be appropriately selected by those skilled in the art.
  • the gene therapy is performed in the same manner as in this type of gene therapy.
  • mammals patients directly suffering from a glucose metabolism-related disease using this gene or a chemical modification thereof.
  • these genes are introduced into the target cells of the patient to control the expression of the target gene by the cells.
  • phosphorothioate for example, phosphorothioate, phosphorodithioate, anorequinolephospho triestenole, anorequinole phosphonate, anorequil phosphoamidate, etc.
  • phosphorothioate for example, phosphorothioate, phosphorodithioate, anorequinolephospho triestenole, anorequinole phosphonate, anorequil phosphoamidate, etc.
  • derivatives that can enhance intracellular stability (Alltisense RNA and DNA” published by WILEY-LISS, 1992, pp.1-50, J. Med. Chem. 36: 1923.1937, 1993) . These can be synthesized according to conventional methods.
  • This gene can be formulated using a stabilizer, a buffer, a solvent, etc. that are usually used for administration.
  • the polynucleotide used is preferably 100 bases or more, more preferably 300 bases or more, and even more preferably 500 bases or more.
  • This method also includes an in vivo method for introducing a gene into cells in a living body and an ex vivo method for introducing a gene into a cell once taken out of the body and returning the cell to the body (Nikkei Science, 1994). April issue, pages 20-45, monthly pharmacy, 36 (1), 23-48 (1994), experimental medicine extra, 12 (15), all pages (1994)).
  • the in vivo method is preferable, and there are a viral introduction method (method using a recombinant virus) and a non-viral introduction method (see the above-mentioned documents).
  • Examples of the method using the above recombinant virus include a retrovirus, an adenovirus, an adeno-associated virus, a herpes virus, a vaccinia virus, a poliovirus, a symvirus virus, etc. Introducing it into the body. Of these, methods using retroviruses, adenoviruses, adeno-related viruses, etc. are particularly preferred. Non-viral introduction methods include the ribosome method, the lipofectin method, etc., and the liponome method is particularly preferable. Other non-viral introduction methods include, for example, the microinjection method, the calcium phosphate method, and the electric mouth poling method.
  • the pharmaceutical composition for gene therapy comprises the present gene or a recombinant virus containing these genes and infected cells into which these viruses have been introduced as active ingredients.
  • the form of administration of the composition to a patient, the route of administration, and the like can be appropriately determined according to the disease and symptoms to be treated. For example, it can be administered into veins, arteries, subcutaneously, intramuscularly, etc. in an appropriate dosage form such as an injection, or can be directly administered and introduced into a disease target site of a patient.
  • the composition for gene therapy includes, for example, a form in which a viral vector containing this gene is embedded in a ribosome or a membrane fusion ribosome in addition to an administration form such as an injection containing this gene (sen Dirulus (HVJ). Ribosome etc.).
  • liposomal formulation forms include suspensions, freezing agents, centrifugal concentrated freezing agents, and the like.
  • the gene therapy composition can also be in the form of a cell culture infected with a virus introduced with a vector containing the gene.
  • the dosage of the active ingredient in these various forms of preparation can be appropriately adjusted depending on the degree of the disease to be treated, the age of the patient, the body weight, and the like. Usually, about 0.0001-100mg per patient adult, preferably about 0.001-lOmg once every few to several months
  • the retroviral titer can be selected from an amount range of about lxlO 3 pft lx lO iS pfu per kg patient body weight per day.
  • about lxlO 4 cells / body-1 ⁇ 10 cells / body may be administered.
  • Rat Adipose cDNA Marathon-Ready cDNA ( CLONTECH , Inc.) 1 mu L, SEQ ID NO: 7 at the indicated comprising the nucleotide sequence primer one 1 20 pmol, consisting of the nucleotide sequence represented by SEQ ID NO: 8 plies 2 pM, TaKaRa ⁇ -Taq polymerase (Takara Shuzo) 2U, TaKaRa ⁇ -Taq Polymerase attached 5 ⁇ L of buffer and TaKaRa ⁇ -Taq Polymerase attached dNTP mixture (2.5 ml) 4 ML 50 ⁇ L of the reaction solution was prepared.
  • PCR is performed under the conditions of 50 ° C incubation cycle consisting of 94 ° C for 30 seconds, 60 ° C for 30 seconds, 72 ° C for 1 minute, and finally 72 ° C for 5 minutes. It was. After PCR, a PCR product showing about 1.5 Kbp was recovered by agarose electrophoresis. Next, the recovered PCR product was subcloned into pT7-Blue vector (Novage 11), and E. coli JM109 strain competent senore (Toyobo) was transformed with the plasmid.
  • ThermO Sequenase II dye terminator kit (Amersham Pharmacia Biotech) and ABI3 73 DNA sequence reader (PE Applied Biosystems) were prepared using the plasmid containing the PCR product (about 1.5 Kbp) obtained in Example 1 as a saddle.
  • the nucleotide sequence of this gene derived from rat consisting of the sequence was determined.
  • Mouse Normal Adipose cDNA (BioChain) 1 ⁇ L, primer consisting of the nucleotide sequence shown in SEQ ID NO: 9 3 20pmoK primer consisting of the nucleotide sequence shown in SEQ ID NO: 10 4 20pmol, TaKaRa Ex-Taq polymerase (Takara Shuzo) 2U
  • a 50 ⁇ L reaction solution containing 5 ⁇ L of the buffer attached to TaKaRa Ka ⁇ -Taq polymerase and 4 L of dNTP mixture (2.5 mM) attached to TaKaRa ⁇ -Taq polymerase was prepared.
  • PCR is conducted at 94 ° C for 30 seconds, then at 65 ° C for 30 seconds, and then at 50 ° C for 2 minutes at 72 ° C for 50 minutes, and finally at 72 ° C for 5 minutes. It was done in After PCR, a PCR product showing about 1.2 kbp was recovered by agarose electrophoresis. The recovered PCR product was subcloned into pT7-Blue vector (Novagen), and then E. coli JMl09 strain competent cell (Toyobo) was transformed with the plasmid.
  • PCR is performed under the conditions of 50 ° C incubation cycle consisting of 94 ° C for 30 seconds, 65 ° C for 30 seconds, and 72 ° C for 2 minutes, and finally 72 ° C for 5 minutes. It was. After PCR, a PCR product showing about 1.2 kbp was recovered by agarose electrophoresis. The recovered PCR product was subcloned into pT7-Blue vector (Novagen), and E. coli JM109 strain competent cell (Toyobo) was transformed with the plasmid.
  • Plasmid 0.1 / zg containing the human gene derived from Example 6 obtained in Example 6, primer consisting of the base sequence shown in SEQ ID NO: 13 20 pmol, primer consisting of the base sequence shown in SEQ ID NO: 14 8 Prepare a 50 ⁇ L reaction solution containing 20 pmol, TaKaRa ⁇ -Taq polymerase (Takara Shuzo) 2 U, buffer attached to TaKaRa Ex-Taq polymerase and dNTP mixture (2.5 mM) attached to TaKaRa ⁇ -Taq polymerase. did.
  • PCR is performed at 94 ° C for 30 seconds, then at 65 ° C for 30 seconds, and then at 72 ° C for 2 minutes, repeated 50 times, and finally at 72 ° C for 5 minutes. It was conducted.
  • the PCR product was erased with restriction enzymes Ndel and BamHI, and the PCR product showing about 1.2 kbp was recovered by agarose electrophoresis.
  • the recovered PCR product was cloned into pET16b (Novagen), and transformed into E. coli BL2l (DE3) plysS (Novagen) according to the usual method.
  • Recombinant Escherichia coli 0iMO64-pET16b / BL21 prepared in Example 7 was added to 30 mL of LB medium (tryptone lw / v%, yeast extract 0.5 w / v%, NaCl O.5 w / v%) containing 50 ug / ml of ampicillin. After inoculation, the cells were cultured overnight at 37 ° C with shaking.
  • IPTG isopropyl 1 3-D-thiogalatatoviranoside
  • the cells are collected by centrifugation (800 ° Oxg, 5 minutes, 4 ° C), sonicated, and then centrifuged (120 ° O xg, 15 minutes, 4 ° C) was performed to obtain the supernatant.
  • the obtained supernatant lOmL was passed through a column packed with lmL affinity carrier lmL (Ni-NTA, manufactured by QIAGEN) bound with nickel.
  • Example 9 One group of 10 DIO (dietary obesity induced) mice (male) constructed by the method described in Example 9 was used. To this, a PBS buffer solution of the protein derived from human prepared in Example 8 was intraperitoneally administered for 2 weeks so that the pure amount of the test substance was 1 mg / kg-body weight ⁇ 2 times / day.
  • the glucose tolerance test and the insulin responsiveness test were performed by the following methods.
  • glucose tolerance test After performing long-term diet load shown in Example 9, 18 to 24 hours fasted animals glucose solution in Underwater feeding from the previous day (19 / kg, 10mL / k g ) was orally administered (disposable disposable syringe made of polypropylene equipped with a disposable metallic sonde). Before administration of glucose, 20, 60 and 120 minutes after glucose administration, about 50 L of blood was collected from the orbital venous plexus of the animal using a pilled cabilla, and Annice Sense 11 (Daikin Industries, Ltd.) was collected. The blood glucose level of whole blood was measured by the oxygen electrode method.
  • Insulin load test After the above OGTT, the test was conducted at intervals of about 2 weeks. Insurin solution before th 18-24 hours fasted animals under water consumption (0.35U / k g, lOmL / kg) intraperitoneal administration (polypropylene Day Supoza Bull syringe and the needle is mounted) the. The blood glucose level was measured by the above method (1) immediately before the insulin load, 30, 60 and 120 minutes later.
  • mice treated with coconut-derived protein showed a significant hypoglycemic effect 120 minutes after glucose administration compared to the phosphate buffer administration group (Table 1).
  • the group of mice treated with the human protein showed a significant hypoglycemic effect 60 and 90 minutes after glucose administration compared to the phosphate buffer group. It was recognized (Table 2). From these results, it was shown that glucose tolerance and insulin responsiveness were improved by administration of this protein in the obese group of mice (dietary obesity-induced mice).
  • Adenovirus is prepared using Adenovirus Expression Kit (Takara Shuzo).
  • the sense strand of mouse M0 64 gene prepared in Example 4 is inserted into the Swal site of the cosmid vector pAxCAwt (Takara Shuzo) to obtain pAxCAwt-mM064.
  • the obtained P AxCAwt-mM064 is infected with E. coli DH5a by in vitro packaging using I packaging kit GigapackXL (Stratagene). Infected E. coli is cultured in 50 ml 50 g / ml ampicillin-containing LB medium, and a large amount of cosmid DNA is prepared using Lambda DNA purification Kit (Toyobo).
  • the cells After completion of the culture, the cells are detached from the dish, and the recovered cell suspension and 293 cells are mixed and further cultured in 10% FCS-supplemented D-MEM medium (Takara Shuzo). When the cells are completely dead, freeze the culture medium with dry ice. After freezing and thawing 6 times, the supernatant obtained by centrifugation at 5000 rpm for 5 minutes is stored as a primary recombinant adenovirus solution (AxCAwt-mM064).
  • Recombinant adenovirus DM solution is obtained by dissolving in 50 ⁇ l of sterilized distilled water.
  • the obtained recombinant adenovirus DNA solution is cleaved with Xhol 5U (Takara Shuzo) to confirm the target fragment (0.48 Kbp).
  • this recombinant adenovirus DNA is subcultured by repeating the above procedure. In this way, a high titer recombinant virus is produced. Titer The measurement is performed by an existing method (Japanese Patent Laid-Open No. 7-298877).
  • Recombinant adenovirus prepared in the previous section so that the proportion of adenovirus dose is lxlOio PFU (10 mice per group) to 10-week-old male KKAy mice (special Claire) that are obese animals Is prepared by diluting with saline.
  • This aqueous solvent is administered to the DIO (dietary obesity-inducing) mouse prepared in Example 9, 0.2 ml per mouse using the syringe.
  • DIO mice dietary obesity-inducing mice
  • the mouse M064 gene prepared in Example 4 was transformed into a plasmid having a mouse aP2 promoter region (about 5.4 kb) (Shimomura, I et al, Genes & Develop memt, 12, 3182).
  • the resulting p Blue SK / maP 2 pro / mMO 64 / po 1 y A was digested with Hindill and Notl to obtain the mouse a P 2 promoter region, the mouse-derived gene and SV 40 E poly A Obtain about 5 ⁇ g of a fragment (approximately 7.1 kb) containing the protein (hereinafter referred to as this introduced gene).
  • This transgene was treated with lOmM squirrel monohydrochloride (pH 8.5), 1 mM EDT
  • This transgene can be prepared by adjusting to O.l g / ⁇ 1 with 2Na (pH 8.5).
  • Pronuclear fertilized eggs are obtained by in vitro fertilization.
  • the fertilized egg was treated with mWM medium (5.14 g / L Na Cl, 0.36 g / L KCL, 0.16 g / L to remove cumulus cells and the like.
  • the microphone mouth injection system consists of a micromanipulator (Leica) and an Hoffman inverted microscope (Fisse). Prepare the transgene preparation solution described in the previous section at about 500 to about 1000 copies / ⁇ 1, and use the supernatant obtained by centrifugation at 13,000 rpm 3 min as the transgene solution. .
  • the fertilized egg described in the previous section ii) is fixed with a support pipette, and the introduced transgene preparation solution in the injection pipette is used as the male pronucleus. Inject approximately 2 ⁇ 1.
  • incubate with mWM medium in the presence of 5% C02 at 37 ° C for 12-16 hours. Embryos that develop in the 2-cell stage are transferred to the oviduct of the temporary parent.
  • Offspring are obtained about 3 weeks later from female mice transplanted with fertilized eggs.
  • a part of the tail is cut out to extract chromosomal DNA, and the transgene-derived fragment is extracted into the extracted DNA.
  • Chromosomal DNA is prepared by conventional methods (e.g., Sam Brook J., Frisch EF, Maniatis T., Molecular Cloning 2nd edition), Cold Spring Harbor Laboratory press (Cold Spring Harbor Laboratory press). ) Etc.).
  • Chromosomal DNA extracted according to the method described in the previous section is cleaved with a restriction enzyme such as EcoR1, BamHl, HindiII, and subjected to agarose gel electrophoresis.
  • the gel after electrophoresis was washed with a solution containing 1.5 M Na CL and 0.15 M trisodium citrate (hereinafter referred to as 10 XSSC), and then commercially available nylon membrane Hy bobd-N (Amersham Blotting with Armavier).
  • the Southern blotting method is used to detect signals on the membrane in the same manner as described above, except that the chromosomal DNA of a mouse into which this transgene has not been introduced (hereinafter referred to as this wild type mouse) is used. And the wild type mouse signal, and the number of introduced copies of this gene is evaluated from the intensity of the signal detected only in the transformed mouse.
  • SEQ ID NO: 7 PCR primer
  • SEQ ID NO: 8 PCR primer
  • Sequence number 9 Primer for PCR SEQ ID NO: 10: PCR primer SEQ ID NO: 11: PCR primer SEQ ID NO: 12: PCR primer SEQ ID NO: 13: PCR primer SEQ ID NO: 14: PCR primer

Abstract

[PROBLEMS] To provide a remedy or a preventive for a glucose metabolism related disease and a method of searching for the same. [MEANS FOR SOLVING PROBLEMS] The remedy or preventive for a glucose metabolism related disease containing as an active ingredient a protein comprising any of the following amino acid sequences or a fragment thereof: <amino acid sequence group> (a) a specific amino acid sequence, (b) an amino acid sequence in which one or more amino acids are deleted, added, inserted or replaced in the amino acid sequence shown in a, (c) an amino acid sequence represented by amino acid numbers from 22 to 414 in the amino acid sequence shown in a, (d) an amino acid sequence in which a methionine residue is added to the amino terminus of the amino acid sequence shown in (c), (e) an amino acid sequence which has a sequence identity of 80% or higher to the amino acid sequence shown in a.

Description

明 細 書  Specification
【発明の名称】 糖代謝制御方法  Method for controlling sugar metabolism
【技術分野】  【Technical field】
【0 0 0 1】  [0 0 0 1]
本発明は、 糖代謝関連疾患の治療剤又は予防剤、 詳しくは、 耐糖能改善活性やインスリ ン応答性促進活性を有する蛋白質等に関する。  The present invention relates to a therapeutic or prophylactic agent for a sugar metabolism-related disease, and more particularly to a protein having a glucose tolerance improving activity or an insulin response promoting activity.
【背景技術】  [Background]
【0 0 0 2】  [0 0 0 2]
生体における糖代謝 (糖の新生及びクリアランス)の恒常性が破綻した状態、 特に高血 糖状態が持続した病態が糖尿病である。 糖尿病の病態の背景には、 血糖調節ホルモンであ るインスリンの分泌不全や作用不全があり、 インスリン作用を是正あるいは代替すること により、 糖代謝に密接に関連する疾患 (糖代謝関連疾患)の治療または予防が可能となる。 こ れまでに、 糖代謝機能を改善する生体内物質 (ホルモン）としてはィンスリンが知ら れており、 インスリン製剤が糖代謝機能の改善に用いられているが、 更に優れた薬剤が求 められている。  Diabetes is a condition in which the homeostasis of glucose metabolism (glucose renewal and clearance) in the living body is broken, particularly a hyperglycemic condition. The background of diabetes mellitus is the insufficiency and / or dysfunction of insulin, a blood glucose-regulating hormone, and treatment of diseases closely related to glucose metabolism (glucose metabolism-related diseases) by correcting or substituting insulin action. Or prevention becomes possible. So far, insulin has been known as an in-vivo substance (hormone) that improves glucose metabolism function, and insulin preparations have been used to improve glucose metabolism function. Yes.
一方、 配列番号 2で示されるアミノ酸配列からなる蛋白質は、 肥満動物の内臓脂肪組織 に特異的に発現する蛋白質であることが知られている (非特許文献 1を参照)。 しかしな がら、 前記蛋白質の生体内での機能は不明であり、 糖代謝関連疾患の関係についても全く 知られていなかった。 On the other hand, the protein consisting of the amino acid sequence represented by SEQ ID NO: 2 is known to be a protein that is specifically expressed in the visceral adipose tissue of obese animals (see Non-Patent Document 1). However, the function of the protein in vivo is unknown, and the relationship between sugar metabolism-related diseases has not been known at all.
【非特許文献 1】 Hida K et al, J. Lipid Research, 41， l615 - p l622 (2000) [Non-Patent Document 1] Hida K et al, J. Lipid Research, 41, l615-p l622 (2000)
【発明の開示】 DISCLOSURE OF THE INVENTION
【発明が解決しようとする課題】  [Problems to be solved by the invention]
【0 0 0 3】  [0 0 0 3]
本発明が解決すべき課題は、 糖代謝関連疾患の治療剤又は予防剤、 詳しくは、 耐糖能改 善活性ゃィンスリン応答性促進活性を有する蛋白質等を提供することにある。  The problem to be solved by the present invention is to provide a therapeutic or preventive agent for a sugar metabolism-related disease, specifically, a protein having a glucose tolerance improving activity and a ginsulin response promoting activity.
【課題を解決するための手段】  [Means for Solving the Problems]
【0 0 0 4】  [0 0 0 4]
本発明者らは、 かかる状況のもと鋭意検討した結果、 配列番号 2で示されるアミノ酸配 列からなる蛋白質が、 耐糖能改善活性やインスリン応答性促進活性を有し、 糖代謝関連疾 患の治療剤又は予防剤となり得ることを見出し、 本発明を完成するに至った.  As a result of intensive studies under these circumstances, the present inventors have found that the protein comprising the amino acid sequence represented by SEQ ID NO: 2 has a glucose tolerance improving activity and an insulin responsiveness promoting activity, and is associated with glucose metabolism-related diseases. The inventors have found that it can be a therapeutic agent or a preventive agent, and have completed the present invention.
【0 0 0 5】  [0 0 0 5]
即ち、 本発明は、  That is, the present invention
〔1〕 下記のいずれかのアミノ酸配列からなる蛋白質 (以下、 本明細書において、 本蛋 白質と称する場合がある。 ）又はそのフラグメントを有効成分として含有する、 糖代謝関 連疾患の治療剤又は予防剤：  [1] A therapeutic agent for a disease related to sugar metabolism, comprising a protein comprising any of the following amino acid sequences (hereinafter sometimes referred to as the present protein) or a fragment thereof as an active ingredient: Preventive agent:
<アミノ酸配列群 > <Amino acid sequence group>
(a)配列番号 2で示されるァミノ酸配列、  (a) the amino acid sequence represented by SEQ ID NO: 2,
(b)配列番号 2で示されるアミノ酸配列において、 1もしくは複数のアミノ酸が欠失、 付加、 揷入もしくは置換されたァミノ酸配列、  (b) in the amino acid sequence represented by SEQ ID NO: 2, an amino acid sequence in which one or more amino acids are deleted, added, inserted or substituted,
(c)配列番号 2で示されるアミノ酸配列における第 2 2番目のアミノ酸から第 4 1 4番 目のアミノ酸で示されるアミノ酸配列、  (c) the amino acid sequence represented by the 4th to 4th amino acids from the second 2nd amino acid in the amino acid sequence represented by SEQ ID NO: 2,
(d)前記 (c)のアミノ酸配列において、 そのアミノ末端にメチォニンが付加されたァ ミノ酸配列、  (d) in the amino acid sequence of (c), an amino acid sequence in which methionine is added to the amino terminus;
(e)配列番号 2で示されるアミノ酸配列と 8 0 %以上の配列同一性を有するアミノ酸配 列、  (e) an amino acid sequence having 80% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 2,
(f)配列番号 1で示される塩基配列における第 224番目のヌクレオチドから第 1 4 6 (f) From the 224th nucleotide to the 1 46 in the nucleotide sequence represented by SEQ ID NO: 1.
5番目までのヌクレオチドで示される塩基配列を有する DNAによりコードされるァミノ 酸配列、 Amino encoded by DNA having the base sequence shown by the 5th nucleotide Acid sequence,
(g)配列番号 1で示される塩基配列における第 2 2 4番目のヌクレオチドから第 1 4 6 (g) 2nd to 4th nucleotides to 1st to 4th 6 in the base sequence represented by SEQ ID NO: 1
5番目までのヌクレオチドで示される塩基配列を有する D N Aと 8 0 %以上の配列同一性 を有する塩基配列を有する D N Aによりコードされ、 かつ糖代謝 進活性を有するアミノ 酸配列、 An amino acid sequence encoded by a DNA having a nucleotide sequence having a nucleotide sequence of 80% or more and having a nucleotide sequence represented by nucleotides up to the fifth nucleotide, and having a sugar metabolizing activity;
(h)配列番号 1で示される塩基配列における第 1番目のヌクレオチドから第 1 2 4 2番 目までのヌクレオチドで示される塩基配列を有する D N Aに対し相補性を有する D N Aと、 ストリンジェン卜な条件下でハイブリダィズする D N Aによりコードされ、 かつ糖代謝 亢進活性を有するァミノ酸配列；  (h) DNA having complementarity to the DNA having the nucleotide sequence represented by nucleotides from the first nucleotide to the 1242th nucleotide in the nucleotide sequence represented by SEQ ID NO: 1, and stringent conditions An amino acid sequence encoded by DNA that hybridizes underneath and having a glucose metabolism-enhancing activity;
[2] 〔1〕 に記載のアミノ酸配列群から選択されるアミノ酸配列からなる蛋白質をコードす る遺伝子を有効成分として含有する、 糖代謝関連疾患の治療剤又は予防剤；  [2] A therapeutic or prophylactic agent for a sugar metabolism-related disease, comprising as an active ingredient a gene encoding a protein comprising an amino acid sequence selected from the amino acid sequence group described in [1];
〔3〕 耐糖能改善活性を有することを特徴とする、 〔1〕 又は 〔2〕 に記載の治療剤又 は予防剤；  [3] The therapeutic or prophylactic agent according to [1] or [2], characterized by having glucose tolerance improving activity;
〔4〕 耐糖能改善活性が、 インスリン応答性促進活性である、 〔3〕 に記載の治療剤又 は予防剤；  [4] The therapeutic or prophylactic agent according to [3], wherein the glucose tolerance improving activity is insulin responsiveness promoting activity;
〔5〕 被験物質が、 〔1〕 に記載のアミノ酸配列群から選択されるアミノ酸配列からな る蛋白質又は該蛋白質をコードする遺伝子の発現を促進するか否かを指標とする、 被験物 質の耐糖能改善活性の検定方法；  [5] Whether the test substance promotes the expression of a protein consisting of an amino acid sequence selected from the amino acid sequence group described in [1] or a gene encoding the protein, is used as an index. Glucose tolerance improving activity assay method;
〔6〕 下記の工程 (1)〜(3)を含むことを特徴とする、 〔5〕 に記載の耐糖能改善  [6] Improve glucose tolerance according to [5], comprising the following steps (1) to (3):
活性の検定方法： Activity assay method:
(1)被験物質と、 〔1〕 に記載のアミノ酸配列群から選択されるアミノ酸配列からな る蛋白質をコ一ドする遺伝子を発現可能な細胞とを接触させる第一工程、  (1) a first step of contacting a test substance with a cell capable of expressing a gene encoding a protein consisting of an amino acid sequence selected from the amino acid sequence group described in [1],
(2)被験物質を接触させた細胞における、 前記遺伝子の発現量を測定し、 該発現量を 被験物質に接触させない対照細胞における前記遺伝子の発現量と比較する第二工程、 及び (2) a second step of measuring the expression level of the gene in a cell contacted with the test substance and comparing the expression level with the expression level of the gene in a control cell not contacted with the test substance; and
(3)前記第二工程の比較結果に基づいて、 被験物質が前記遺伝子の発現量を増加させるか否 かを指標として被験物質の耐糖能改善活性を評価する第三工程； (3) Third step of evaluating the glucose tolerance improving activity of the test substance using as an index whether or not the test substance increases the expression level of the gene based on the comparison result of the second step;
[7] 下記の工程 (1)〜(3)を含むことを特徴とする、 〔5〕 に記載の耐糖能改善  [7] Improve glucose tolerance according to [5], comprising the following steps (1) to (3)
活性の検定方法： Activity assay method:
(1)被験物質と、 〔1〕 に記載のアミノ酸配列群から選択されるアミノ酸配列からなる 蛋白質を発現可能な細胞とを接触させる第一工程、  (1) a first step of contacting a test substance with a cell capable of expressing a protein comprising an amino acid sequence selected from the amino acid sequence group described in [1],
(2)被験物質を接触させた細胞における、 前記蛋白質の発現量を測定し、 該発現量を被 験物質に接触させない対照細胞における前記蛋白質の発現量と比較する第二工程、 及び (2) a second step of measuring the expression level of the protein in a cell contacted with a test substance and comparing the expression level with the expression level of the protein in a control cell not contacted with the test substance; and
(3)前記第二工程の比較結果に基づいて、 被験物質が前記蛋白質の発現量を増加させる か否かを指標として被験物質の耐糖能改善活性を評価する第三工程； (3) a third step of evaluating the glucose tolerance improving activity of the test substance using as an index whether or not the test substance increases the expression level of the protein based on the comparison result of the second step;
〔8〕 下記の工程 (1)〜(3)を含むことを特徴とする、 〔5〕 に記載の耐糖能改善  [8] Improve glucose tolerance according to [5], comprising the following steps (1) to (3):
活性の検定方法： Activity assay method:
(1)被験物質と、 〔1〕 に記載のアミノ酸配列群から選択されるアミノ酸配列からな る蛋白質をコードする遺伝子の発現制御領域を機能可能な形で連結されてなるレポ一ター 遺伝子を含有する細胞とを接触させる第一工程、  (1) A reporter gene comprising a test substance and a reporter gene operatively linked to an expression control region of a gene encoding a protein comprising an amino acid sequence selected from the amino acid sequence group described in [1] The first step of contacting the cells to be
(2)被験物質を接触させた細胞における、 前記レポータ一遺伝子の発現量を測定し、 該発現量を被験物質に接触させない対照細胞における前記遺伝子の発現量と比較する第二 工程、 及び  (2) a second step of measuring an expression level of the reporter gene in a cell contacted with a test substance, and comparing the expression level with an expression level of the gene in a control cell not contacted with the test substance; and
(3)前記第二工程の比較結果に基づレ、て、 前記レポ一タ一遺伝子の発現量を増加させる か否かを指標として被験物質の耐糖能改善活性を評価する第三工程：  (3) Third step of evaluating the glucose tolerance improving activity of the test substance using as an index whether or not to increase the expression level of the reporter gene based on the comparison result of the second step:
〔9〕 耐糖能改善活性が、 インスリン応答性促進活性である、 〔5〕〜〔8〕 のいずれ かに記載の検定方法；  [9] The assay method according to any one of [5] to [8], wherein the glucose tolerance improving activity is insulin responsiveness promoting activity;
〔 1 0〕 〔5〕〜〔8〕 のいずれかに記載の検定方法により測定された、 被験物質の耐 糖能改善活性を指標として、 糖代謝関連疾患の治療剤又は予防剤の候補物質を選別するこ とを特徴とする、 糖代謝関連疾患の治療剤又は予防剤の探索方法； [10] Resistance of the test substance measured by the assay method described in any of [5] to [8] A method for searching for a therapeutic or prophylactic agent for a sugar metabolism-related disease, comprising selecting candidate substances for the therapeutic or prophylactic agent for a glucose metabolism-related disease using the glucose capacity improving activity as an index;
〔1 1〕 〔1 0〕 に記載の探索方法により選抜された物質またはその薬学的に許容され る塩を有効成分して含有する、 糖代謝関連疾患の治療剤又は予防剤；  [1 1] A therapeutic or prophylactic agent for a sugar metabolism-related disease, comprising as an active ingredient a substance selected by the search method according to [1 0] or a pharmaceutically acceptable salt thereof;
〔1 2〕 〔1〕 に記載のアミノ酸配列群から選択されるアミノ酸配列からなる蛋白質の 発現誘導物質、 又は機能促進物質を有効成分として含有する、 糖代謝関連疾患の治療剤又 は予防剤 1に関するものである。  [1 2] A therapeutic or preventive agent for a sugar metabolism-related disease, comprising as an active ingredient a substance that induces the expression of a protein comprising an amino acid sequence selected from the amino acid sequence group described in [1] or a function promoting substance 1 It is about.
【発明の効果】  【The invention's effect】
【0 0 0 6】  [0 0 0 6]
本発明により、 耐糖能改善活性ゃィンスリン応答性促進活性を有することを特徴とする 糖代謝関連疾患の治療剤もしくは予防剤、 耐糖能改善活性の検定方法、 及び当該検定方法 を用いる糖代謝関連疾患の治療剤もしくは予防剤の探索方法等を提供することが可能にな つた。  According to the present invention, a therapeutic or preventive agent for a glucose metabolism-related disease characterized by having a glucose tolerance-improving activity and ginsulin responsive promoting activity, a method for assaying glucose tolerance-improving activity, and a glucose metabolism-related disease using the assay It has become possible to provide a method for searching for therapeutic or preventive agents.
【発明を実施するための最良の形態】  BEST MODE FOR CARRYING OUT THE INVENTION
【0 0 0 7】  [0 0 0 7]
以下に本発明を詳細に説明する。  The present invention is described in detail below.
(1)治療剤又は予防剤 (1) Therapeutic or preventive agent
本発明の第一の態様は、 前記本蛋白質又はそのフラグメントを有効成分として含有する 糖代謝関連疾患の治療剤又は予防剤に関する。 The first aspect of the present invention relates to a therapeutic or preventive agent for a sugar metabolism-related disease comprising the present protein or a fragment thereof as an active ingredient.
本明細書において、 「下記のいずれかのアミノ酸配列からなる蛋白質」 （本蛋白質)と は、 （a)配列番号 2で示されるァミノ酸配列、 （b)配列番号 2で示されるアミノ酸配 列において、 1もしくは複数のアミノ酸が欠失、 付カロ、 挿入もしくは置換されたアミノ酸 配列、 （c)配列番号 2で示されるアミノ酸配列における第 2 2番目のアミノ酸から第 4 1 4番目のアミノ酸で示されるアミノ酸配列、 （d)前記 (c)のアミノ酸配列において 、 そのアミノ末端にメチォニンが付加されたアミノ酸配列、 （e)配列番号 2で示される ァミノ酸配列と 8 0 %以上の配列同一性を有するァミノ酸配列、 （ )配列番号 1で示さ れる塩基配列における第 2 8 7番目のヌクレオチドカゝら第 1 4 6 5番目までのヌクレオチ ドで示される塩基配列を有する DNAによりコードされるアミノ酸配列、 （g)配列番号 1で示される塩基配列における第 2 8 7番目のヌクレオチドから第 1 6 4 5番目までのヌ クレオチドで示される塩基配列を有する DNAと 8 0 %以上の配列同一性を有する塩基配 列を有する D N Aによりコードされるアミノ酸配列、 及び (h)配列番号 1で示される塩 基配列における第 2 8 7番目のヌクレオチドカゝら第 1 4 6 5番目までのヌクレオチドで示 される塩基配列を有する D N Aに対し相補性を有する DNAと、 ストリンジェン卜な条件 下でハイプリダイズする D N Aによりコードされるアミノ酸配列からなり、 かつ糖代謝亢 進活性を有する蛋白質である。 In the present specification, “a protein consisting of any of the following amino acid sequences” (the present protein) means (a) an amino acid sequence represented by SEQ ID NO: 2 and (b) an amino acid sequence represented by SEQ ID NO: 2. An amino acid sequence in which one or more amino acids have been deleted, appended, inserted or substituted, (c) represented by the 2nd 2nd amino acid to the 4th 4th 4th amino acid in the amino acid sequence represented by SEQ ID NO: 2 An amino acid sequence, (d) in the amino acid sequence of (c), an amino acid sequence in which methionine is added to the amino terminus, (e) having 80% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 2 An amino acid sequence, () encoded by DNA having the nucleotide sequence shown by the nucleotide from the 2nd 8th to the 7th nucleotide in the nucleotide sequence shown by SEQ ID NO: 1 (G) 80% or more of sequence identity with DNA having the nucleotide sequence represented by nucleotides 2 8 7 to 1 6 4 5 in the nucleotide sequence represented by SEQ ID NO: 1 An amino acid sequence encoded by a DNA having a nucleotide sequence having a nucleotide sequence, and (h) the nucleotide sequence from the 2nd 8th to the 7th nucleotide in the base sequence represented by SEQ ID NO: 1 A protein having a complementary activity to a DNA having a base sequence and an amino acid sequence encoded by DNA that is hybridized under stringent conditions and having a glucose metabolism-promoting activity.
【0 0 0 8】  [0 0 0 8]
ここで、 前記 (b)におけるアミノ酸の欠失、 付加、 挿入もしくは置換」 や前記 (e  Here, amino acid deletion, addition, insertion or substitution in (b) above or (e
)及び (g)にある 「80%以上の配列同一性」 には、 例えば、 配列番号 2で示されるァ ミノ酸配列を有する蛋白質が細胞内で受けるプロセシング、 該蛋白質が由来する生物の種 差、 個体差、 組織問の差異等により天然に生じる変異や、 人為的なアミノ酸の変異等が含 まれる。 ) And (g) include, for example, the processing that the protein having the amino acid sequence represented by SEQ ID NO: 2 undergoes in the cell and the species difference of the organism from which the protein is derived. It includes naturally occurring mutations due to individual differences, organizational differences, and artificial amino acid mutations.
前記 (b)における 「アミノ酸の欠失、 付加もしくは置換」 （以下、 総じてアミノ酸の 改変と記すこともある。 ）を人為的に行う場合の手法としては、 例えば、 配列番号 2で示 されるアミノ酸配列をコ一ドする D N Aに対しで慣用の部位特異的変異導入を施し、 その 後この D N Aを常法により発現させる手法が挙げられる。 ここで部位特異的変異導入法と しては、例えば、 アンバー変異を利用する方法 (ギャップド，デュプレックス法、 Nucleic Ac ids Res., 12,9441-9456(1984)), 変異導入用プライマ一を用いた PCRによる方法等が挙 げられる。 前記で改変されるアミノ酸の数については、 少なくとも 1残基、 具体的には 1若しくは 数個、 又はそれ以上である。 かかる改変の数は、 当該蛋白質の糖代謝充進活性を見出すこ とのできる範囲であれば良い。 As a technique for artificially carrying out the “amino acid deletion, addition or substitution” in the above (b) (hereinafter sometimes referred to as amino acid modification as a whole), for example, the amino acid represented by SEQ ID NO: 2 is used. For example, a conventional site-directed mutagenesis is performed on the DNA that codes the sequence, and then this DNA is expressed by a conventional method. Here, as a site-specific mutagenesis method, for example, a method using an amber mutation (gapped, duplex method, Nucleic Acids Res., 12,9441-9456 (1984)), a mutagenesis primer is used. PCR methods that were used. The number of amino acids modified as described above is at least one residue, specifically one or several, or more. The number of such modifications may be in a range in which the activity of promoting sugar metabolism of the protein can be found.
また前記欠失、 付加又は置換のうち、 特にアミノ酸の置換に係る改変が好ましい。 当該 置換は、 疎水性、 電荷、 p K、 立体構造上における特徴等の類似した性質を有するァミノ 酸への置換がより好ましい。 このような置換としては、 例えば、 0グリシン、 ァラニン； ii)バリン、 イソロイシン、 ロイシン； iii)ァスパラギン酸、 グルタミン酸、 ァスパラギ ン、 グルタミン、 IV)セリン、 スレオニン； V)リジン、 アルギニン; vi)フエ二ルァラニン 、 チロシンのグループ内での置換が挙げられる。 Among the deletions, additions or substitutions, modifications relating to amino acid substitution are particularly preferable. The substitution is more preferably substitution with an amino acid having similar properties such as hydrophobicity, charge, pK, and steric features. Such substitutions include, for example: 0 glycine, alanine; ii) valine, isoleucine, leucine; iii) aspartic acid, glutamic acid, asparagine, glutamine, IV) serine, threonine; V) lysine, arginine; vi) phenol Substitution within the group of lulanin and tyrosine.
【0 0 0 9】  [0 0 0 9]
前記 (c)における、 「配列番号 2で示されるアミノ酸配列における第 2 2番目のアミ ノ酸から第 4 1 4番目のアミノ酸で示されるアミノ酸配列 1とは、 分泌蛋白質である配列 番号 2で示されるアミノ酸配列からなる蛋白質において、 シグナルぺプチド部分に相当す る第 1番目のアミノ酸から第 2 1番目のアミノ酸で示される部分アミノ酸配列を除いたァ ミノ酸配列に相当する。 In the above (c), “the amino acid sequence 1 represented by the 4th to 4th amino acids from the 2nd 2nd amino acid in the amino acid sequence represented by SEQ ID NO: 2” is represented by the sequence number 2 that is a secreted protein. The amino acid sequence of the first amino acid corresponding to the signal peptide portion minus the partial amino acid sequence represented by the second first amino acid.
【0 0 1 0】  [0 0 1 0]
本発明において 「配列同一性」 とは、 2つの D N A又は 2つの蛋白質間の配列の同一性 及び相同性をいう。 前記 「配列同一性」 は、 比較対象の配列の領域にわたって、 最適な状 態にアラインメントされた 2つの配列を比較することにより決定される。 ここで、 比較対 象の D N A又は蛋白質は、 2つの配列の最適なアラインメントにおいて、 付加又は欠失（ 例えばギャップ等)を有していてもよい。 このような配列同一性に関しては、 例えば、 Ve ctor NTIを用いて、 ClustalWアルゴリズム (Nucleic Acid Res„22(22):4673-4680(l994) を利用してアラインメントを作成することにより算出することができる。 尚、 配列同一性 は、 配列解析ソフト、 具体的には Vector NTI、 GENETYX-MACや公共のデータベースで 提供される解析ツールを用いて測定される。 前記公共データベースは、 例えば、 ホームべ ージァドレス http=〃www.ddbj.nig.ac.jpにおいて、 一般的に利用可能である。  In the present invention, “sequence identity” refers to sequence identity and homology between two DNAs or two proteins. The “sequence identity” is determined by comparing two sequences that are optimally aligned over the region of the sequence to be compared. Here, the DNA or protein to be compared may have an addition or deletion (for example, a gap) in the optimal alignment of the two sequences. Such sequence identity can be calculated, for example, by creating an alignment using the ClustalW algorithm (Nucleic Acid Res 22 (22): 4673-4680 (l994)) using Vector NTI. Note that sequence identity is measured using sequence analysis software, specifically Vector NTI, GENETYX-MAC, or analysis tools provided by public databases, such as home page dressing. Generally available at http = 〃www.ddbj.nig.ac.jp.
本発明における配列同一性は、 8 0 %以上であればよいが、 好ましくは 9 0 %以上、 よ り好ましくは 9 5 %以上である。 The sequence identity in the present invention may be 80% or more, preferably 90% or more, and more preferably 95% or more.
【0 0 1 1】  [0 0 1 1]
前記 (h)における 「ス トリンジェントな条件下でハイブリダイズする」 に関して、 こ こで使用されるハイブリダイゼーションは、 例えば、 Sambrook J.,Frisch E,R, Mania tis T.著、 モレキュラークローニング第 2版 (Molecular Cloning 2nd edition), コーノレ ドスプリングノヽーノくーラボラ トリ一発 ί (Cold Spring Harbor Laboratory press  Regarding “hybridize under stringent conditions” in (h) above, the hybridization used here is, for example, by Sambrook J., Frisch E, R, Maniatis T., Molecular Cloning No. 2 Edition (Molecular Cloning 2nd edition), Cold Spring Harbor Laboratory press
)等に記載される通常の方法に準じて行うことができる。 また 「ストリンジェントな条件 下」 とは、 例えば、 6 xSSC (1.5M NaCl、 0.15 Mクェン酸三ナトリウムを含む 溶液を 1 0 XSSCとする）、 5 0 %フオルムアミ ドを含む溶液中で 4 5 °Cにてハイプリッド を形成させた後、 2 X SSCで 5 0 °Cにて洗浄するような条件 (Molecular Biology, John Wi ley & Sons,N.Y.(1989),6.3.1-6.3.6)等を挙げることができる。 洗浄ステップにお ける塩濃度は、例えば、 2 XSSCで 5 0 °Cの条件 (低ストリンジエンシーな条件)から 0 , 2 X SSCで 5 0 °Cまでの条件 (髙ストリンジエンシーな条件)から選択することができ る。 洗浄ステップにおける温度は、 例えば、 室温 (低ストリンジエンシーな条件)から 6 5 °C (高ストリンジエンシーな条件)から選択することができる。 また、 塩濃度と温度の両方を変 えることもできる。 ) And the like. In addition, “under stringent conditions” means, for example, 6 x SSC (a solution containing 1.5 M NaCl and 0.15 M trisodium citrate is 10 XSSC), 45 ° C in a solution containing 50% formamide. After forming a hybrid in C, wash under 2 X SSC at 50 ° C (Molecular Biology, John Wiley & Sons, NY (1989), 6.3.1-6.3.6), etc. Can be mentioned. The salt concentration in the washing step is, for example, from 50 ° C for 2 XSSC (low stringency conditions) to 50 ° C for 0, 2 X SSC (髙 stringency conditions) You can select from. The temperature in the washing step can be selected, for example, from room temperature (low stringency conditions) to 65 ° C. (high stringency conditions). It is also possible to change both salt concentration and temperature.
【0 0 1 2】  [0 0 1 2]
上述の本蛋白質の具体例として、 配列番号 2で示されるアミノ酸配列からなるヒ ト型蛋 白質の他、 配列番号 4で示されるアミノ酸配列からなるラッ卜型ホモログや、 配列番号 6 で示されるァミノ酸配列からなるマウス型ホモ口グを挙げることができる。  Specific examples of the above-described protein include a human protein consisting of the amino acid sequence shown by SEQ ID NO: 2, a rat homolog consisting of the amino acid sequence shown by SEQ ID NO: 4, and an amino acid shown by SEQ ID NO: 6. Mention may be made of mouse homozygous consisting of an acid sequence.
【0 0 1 3】 本明細書において、 本蛋白質のフラグメントとは、 前記本蛋白質の 1 5〜1 0 0残基、 好ましくは 1 5〜5 0残基のアミノ酸部分配列からなるぺプチドフラグメントを表し、 本 蛋白質の糖代謝亢進活性が保持されている限り特に限定はなレ、。 [0 0 1 3] In the present specification, the fragment of the protein represents a peptide fragment consisting of an amino acid partial sequence of 15 to 100 residues, preferably 15 to 50 residues of the protein, and the sugar of the protein As long as hypermetabolic activity is retained, there is no particular limitation.
【0 0 1 4】  [0 0 1 4]
本蛋白質の調製方法について以下に説明する。  The method for preparing this protein will be described below.
まず、 本蛋白質をコードする遺伝子 (本遺伝子)、 例えば、 （I)配列番号 2で示され るアミノ酸配列をコードする塩基配列、 （II)配列番号 2で示されるアミノ酸配列におい て、 1もしくは複数のアミノ酸が欠失、 付加もしくは置換されたアミノ酸配列をコードす る塩基配列、 （III)配列番号 2で示されるアミノ酸配列において、 そのアミノ末端からFirst, in the gene encoding this protein (this gene), for example, (I) the base sequence encoding the amino acid sequence represented by SEQ ID NO: 2, and (II) the amino acid sequence represented by SEQ ID NO: 2, one or more (III) in the amino acid sequence shown in SEQ ID NO: 2, from the amino terminus
2 1個のアミノ酸を欠失させた部分アミノ酸配列をコードする塩基配歹 ϋ (即ち、 配列番号2 Base sequence encoding a partial amino acid sequence with one amino acid deleted 欠 失 (i.e., SEQ ID NO:
2で示されるアミノ酸配列における第 2 2番目力ゝら第 4 1 4番目までのアミノ酸で示され るァミノ酸配列をコードする塩基配列)、 （IV)前記 (III)のァミノ酸配列において、 そのアミノ末端にメチォニンが付加されたアミノ酸配列をコードする塩基配列、 （V)配 列番号 2で示されるアミノ酸配列と 8 0 %以上の配列同一性を有するアミノ酸配列をコー ドする塩基配列、 （VI)配列番号 1で示される塩基配列、 （VII)配列番号 2で示される 塩基配列における第 2 8 7番目のヌクレオチドカ ら第 1 4 6 5番目までのヌクレオチドで 示される塩基配列、 （VIII)配列番号 1で示される塩基配列における第 2 8 7番目のヌク レオチドカゝら第 1 4 6 5番目までのヌクレオチドで示される塩基配列を有する D N Aと 8 0 %以上の配列同一性を有する塩基配列、 又は (IX)配列番号 1で示される塩基配列におけ る第 2 8 7番目のヌクレオチドから第 1 4 6 5番目までのヌクレオチドで示される塩基配 列を有する D N Αに対し相補性を有する D N Aと、 ストリンジェントな条件下でハイブリ ダイズする D N Aの塩基配列等の塩基配列を有する遺伝子を、 通常の遺伝子工学的方法 (例 えば、 Sambrook J.,Frisch E.F., Maniatis T.著、モレキュラークローニング第 2版 (Molecula r Cloning 2nd edition), コールドスプリングハーバーラボラトリ一発行 (Cold (IV) the amino acid sequence represented by the amino acid sequence represented by amino acids from the 2nd second force to the 4th to 4th amino acids in the amino acid sequence represented by (2), (IV) in the amino acid sequence of (III), (V) a base sequence encoding an amino acid sequence having methionine added to the amino terminus, (V) a base sequence encoding an amino acid sequence having 80% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 2, (VI ) A base sequence represented by SEQ ID NO: 1, (VII) a base sequence represented by nucleotides from the 2nd 87th nucleotide to the 1st 4th 6th 5th nucleotide in the base sequence represented by SEQ ID NO: 2, (VIII) sequence A nucleotide sequence having 80% or more sequence identity with DNA having the nucleotide sequence shown by nucleotides up to the 2nd 8 7th nucleotide in the nucleotide sequence shown by the number 1 to the 1st 4 6th nucleotide, or (IX) SEQ ID NO DNA having a nucleotide sequence represented by nucleotides from the 2nd 7th nucleotide to the 1st 4th 6th 5th nucleotide in the nucleotide sequence represented by 1 and a stringent condition A gene having a base sequence such as a DNA base sequence that hybridizes with a conventional genetic engineering method (e.g., Sambrook J., Frisch EF, Maniatis T., Molecular Cloning 2nd edition ), Cold Spring Harbor Laboratory Issuance (Cold
Spring Harbor Laboratory press)等に記載されている方法)に準じて取得する。 次いで、 得 られた本遺伝子を用いることにより、 通常の遺伝子工学的方法に準じて本蛋白質を製 造 ·取得する。 このようにして本蛋白質を調製することができる。 Acquired according to the method described in Spring Harbor Laboratory press). Next, by using the obtained present gene, the present protein is produced and obtained according to a usual genetic engineering method. In this way, the present protein can be prepared.
【0 0 1 5】  [0 0 1 5]
例えば、 本遺伝子が宿主細胞中で発現できるようなプラスミ ドを作製し、 これを宿主細 胞に導入して形質転換し、 さらに形質転換された宿主細胞 (形質転換体)を培養すること で得られる培養物から本蛋白質を取得すればよレ、。 上記プラスミ ドとしては、 例えば、 宿 主細胞中で複製可能な遺伝情報を含み、 自立的に増殖できるものであって、 宿主細胞から の単離 ·精製が容易であり、 宿主細胞中で機能可能なプロモータ一を有し、 検出可能なマ 一力一をもつ発現ベクターに、 本蛋白質をコードする遺伝子が導入されたものを好ましく 挙げることができる。 尚、 発現べクタ一としては、 各種のものが市販されている。 例えば 、 大腸菌での発現に使用される発現べクタ一は、 lac、 trp、 tac などのプロモー ターを含む発現ベクターであって、 これらはフアルマシア社、 宝酒造等から市販されてい る。 当該発現べクタ一に本蛋白質をコードする遺伝子を導入するために用いられる制限酵 素も宝酒造等から市販されている。 さらなる高発現を導くことが必要な場合には、 本蛋白 質をコ一ドする遺伝子の上流にリボゾーム結合領域を連結してもよい。 用いられるリポゾ ーム結合領域としては、 Guarente L,ら (Cell 20, p543)や谷口ら（Genetics oflndust rial Microorganisms, p202,講談社)による報告に記載されたものを挙げることができ る。  For example, a plasmid that can express this gene in a host cell is prepared, introduced into a host cell, transformed, and further transformed by culturing the transformed host cell (transformant). You can get this protein from the culture. For example, the plasmid contains genetic information that can be replicated in the host cell, can grow independently, can be easily isolated and purified from the host cell, and can function in the host cell. Preferred examples include those in which a gene encoding the present protein is introduced into an expression vector having a unique promoter and having a detectable potential. Various expression vectors are commercially available. For example, expression vectors used for expression in E. coli are expression vectors containing promoters such as lac, trp, and tac, which are commercially available from Falmacia, Takara Shuzo and others. Restriction enzymes used for introducing the gene encoding this protein into the expression vector are also commercially available from Takara Shuzo. If it is necessary to induce higher expression, a ribosome binding region may be linked upstream of the gene encoding this protein. Examples of the liposome-binding domain used include those described in reports by Guarente L, et al. (Cell 20, p543) and Taniguchi et al. (Genetics of Industrial Microorganisms, p202, Kodansha).
【0 0 1 6】  [0 0 1 6]
哺乳動物細胞での発現に使用されるべクタ—は、 S V 4 0ウィルスプロモーター、 サイ 卜メガロウイルスプロモーター（C M Vプロモータ一)、 Raus Sarcoma Virusプロモータ — (R S Vプロモーター)、 ]3ァクチン遺伝子プロモーター、 a P 2遺伝子プロモーター 等のプロモーターを含む発現ベクターであって、 これらは、 東洋紡社、 宝酒造社等から巿 販されている。 The vectors used for expression in mammalian cells are the SV 40 virus promoter, rhinoceros megalovirus promoter (CMV promoter), Raus Sarcoma Virus promoter — (RSV promoter),] 3 actin gene promoter, a P These are expression vectors containing a promoter such as a 2-gene promoter, which are obtained from Toyobo, Takara Shuzo, etc. It is sold.
【0 0 1 7】  [0 0 1 7]
宿主細胞としては、 原核生物もしくは真核生物である微生物細胞、 昆虫細胞又は哺乳動 物細胞等を挙げることができる。 例えば、 本蛋白質の大量調製が容易になるという観点で は、 大腸菌等を好ましく挙げることができる。  Examples of host cells include prokaryotic or eukaryotic microorganism cells, insect cells, or mammalian cells. For example, from the viewpoint of facilitating the large-scale preparation of the present protein, E. coli and the like can be preferably mentioned.
前記のようにして得られたプラスミ ドは、 通常の遺伝子工学的方法により前記宿主細胞 に導入することができる。 The plasmid obtained as described above can be introduced into the host cell by an ordinary genetic engineering method.
【0 0 1 8】  [0 0 1 8]
形質転換体の培養は、 微生物培養、 昆虫細胞もしくは哺乳動物細胞の培養に使用される 通常の方法によって行うことができる。 例えば大腸菌の場合、 適当な炭素源、 窒素源およ びビタミン等の微量栄養物を適宜含む培地中で培養を行う。 培養方法としては、 固体培養 、 液体培養のいずれの方法でもよく、 好ましくは、 通気撹絆培養法等の液体培養を挙げる ことができる。  The transformant can be cultured by a conventional method used for culturing microorganisms, insect cells or mammalian cells. For example, in the case of Escherichia coli, culture is performed in a medium containing an appropriate carbon source, nitrogen source, and micronutrients such as vitamins as appropriate. The culture method may be any of solid culture and liquid culture, and preferred examples include liquid culture such as aeration-stirring culture.
【0 0 1 9】  [0 0 1 9]
本蛋白質の取得は、 一般の蛋白質の単離 ·精製に通常使用される方法を組み合わせて実 施すればよい。 例えば、 前記の培養により得られた形質転換体を遠心分離等で集め、 該形 質転換体を破砕または溶解せしめ、 必要であれば蛋白質の可溶化を行い、 イオン交換、 疎 水、 ゲルろ過等の各種クロマトグラフィーを用いた工程を単独で、 若しくは組み合わせる ことにより精製すればよい。 精製された蛋白質の高次構造を復元する操作をさらに行って もよい。 また、 例えば、 前記の培養により得られた形質転換体を遠心分離などで除去し、 培養上清から本蛋白質を前記と同様にして精製してもよい。 - This protein can be obtained by combining methods commonly used for isolation and purification of general proteins. For example, the transformants obtained by the above culture are collected by centrifugation or the like, and the transformants are crushed or dissolved, and if necessary, proteins are solubilized, ion exchange, hydrophobicity, gel filtration, etc. The steps using the various chromatographies described above may be purified singly or in combination. An operation for restoring the higher order structure of the purified protein may be further performed. For example, the transformant obtained by the above culture may be removed by centrifugation or the like, and the present protein may be purified from the culture supernatant in the same manner as described above. -
【0 0 2 0】 [0 0 2 0]
本明細書において、 糖代謝関連疾患としては、 生体における糖代謝機能の異常を伴う疾 患が挙げられ、 具体的には、 糖尿病、 耐糖能異常、 高血糖の持続に伴う動脈硬化症等の疾 患が挙げられる。 すなわち、 糖代謝関連疾患は、 インスリ ンの機能低下または機能不全に よって生ずる種々の病態を有する疾患であれば特に限定は無い。 本発明の糖代謝関連疾患 の治療剤又は予防剤を適用可能な疾患として、 好ましくは、 糖尿病などの血糖の恒常性が 破綻した病態をあげることができる。 本発明の糖代謝関連疾患の治療剤又は予防剤は、 耐 糖能亢進活性ゃィンスリン応答性促進活性、 すなわち耐糖能改善活性を有することを特徴 とするものである。  In this specification, examples of the glucose metabolism-related diseases include diseases associated with abnormal glucose metabolism functions in the living body. Specifically, diseases such as diabetes, impaired glucose tolerance, and arteriosclerosis associated with sustained hyperglycemia. Examples include illness. That is, the sugar metabolism-related disease is not particularly limited as long as it is a disease having various pathologies caused by a decrease in insulin function or dysfunction. As a disease to which the therapeutic or preventive agent for a sugar metabolism-related disease of the present invention can be applied, a disease state in which blood glucose homeostasis such as diabetes is broken can be mentioned. The therapeutic or prophylactic agent for a sugar metabolism-related disease of the present invention is characterized by having a glucose tolerance enhancing activity, a ginsulin response promoting activity, that is, a glucose tolerance improving activity.
【0 0 2 1】  [0 0 2 1]
本明細書において 「耐糖能亢進活性」 とは、 O G T T (糖負荷試験：マウスに一定量の 糖を負荷した後の血糖値やインスリン値の変動を測定する試験)を実施することによって 測定することができ、 O G T Tにおいて糖負荷後の血糖値の低下が克進されていることを 表す。 具体的には、 本明細書実施例 9及び 1 0に記載の方法で O G T Tを実施することが できる。  In this specification, “glucose tolerance enhancing activity” is measured by performing OGTT (glucose tolerance test: a test that measures changes in blood glucose level and insulin level after loading a certain amount of sugar in mice). This means that the decrease in blood glucose level after glucose load is overcome in OGTT. Specifically, OGTT can be performed by the method described in Examples 9 and 10 of the present specification.
本明細書において、 「インスリ ン応答性」 とは、 I T T (インスリン負荷試験:マウス に一定量のィンスリンを負荷した後の血糖値ゃィンスリン値の変動を測定する）を実施す ることによって測定することができ、 I T Tにおいてインスリン負荷後の血糖値の低下が 克進されていることを表す。 具体的には、 本明細書実施例 9及び 1 0に記載の方法で I T Tを実施することができる。 In this specification, “insulin responsiveness” is measured by performing ITT (insulin tolerance test: measuring changes in blood glucose level after loading a mouse with a certain amount of insulin). It shows that ITT has overcome the decrease in blood glucose level after insulin loading. Specifically, I T T can be carried out by the method described in Examples 9 and 10 of the present specification.
前記 O G T T及び I T Tにおいて、 測定対象のタンパク質が、 本発明実施例 9及び 1 0で 用いられる本タンパク質と同程度の血糖値を示す場合に、 測定対象のタンパク質は 「耐糖 能亢進活性」 や 「インスリン応答性」 を示すということができる。 In the above OGTT and ITT, when the protein to be measured shows a blood glucose level comparable to that of the present protein used in Examples 9 and 10 of the present invention, the protein to be measured is “glucose tolerance enhancing activity” or “insulin It can be said that it shows responsiveness.
【0 0 2 2】  [0 0 2 2]
(II)検定方法 (II) Testing method
本発明の第二の態様は、 被験物質が本蛋白質又は本蛋白質をコードする遺伝子 (本遺伝 子)の発現を促進するか否かを指標とする、 被験物質の耐糖能改善活性の検定方法に関す る。 According to a second aspect of the present invention, there is provided a method for assaying a glucose tolerance improving activity of a test substance using as an index whether or not the test substance promotes the expression of the present protein or a gene encoding the present protein (the present gene). about The
(II- 1)本蛋白質をコードする遺伝子の発現量を指標とする方法  (II-1) Method using the expression level of the gene encoding this protein as an index
すなわち、 下記の工程 (1)~(3)： That is, the following steps (1) to (3):
(1)被験物質と、 本蛋白質をコードする遺伝子を発現可能な細胞とを接触させる第一 工程、  (1) a first step of contacting a test substance with a cell capable of expressing a gene encoding this protein;
(2)被験物質を接触させた細胞における、 前記遺伝子の発現量を測定し、 該発現量を 被験物質を接触させない対照細胞における前記遺伝子の発現量と比較する第二工程、 及び (2) a second step of measuring the expression level of the gene in a cell contacted with a test substance and comparing the expression level with the expression level of the gene in a control cell not contacted with the test substance; and
(3)前記第二工程の比較結果に基づいて、 被験物質が前記遺伝子の発現量を増加させ るか否かを指標として被験物質の耐糖能改善活性を評価する第三工程、 (3) Based on the comparison result of the second step, the third step of evaluating the glucose tolerance improving activity of the test substance using as an index whether or not the test substance increases the expression level of the gene,
を有する。 Have
【0 0 2 3】  [0 0 2 3]
前記第一工程において用いられる 「本蛋白質をコードする遺伝子 (以下、 本遺伝子と称 する場合がある)」 としては、 本遺伝子を発現している細胞であれば特に限定は無く、 使 用する細胞の内在性の本遺伝子、 外来遺伝子として細胞に導入された本遺伝子のいずれで も良いが、 使用する細胞の内在性の本遺伝子が好ましい。 外来遺伝子として導入する場合 、 本遺伝子は用いられる細胞の由来動物種の本遺伝子であることが好ましい。  The “gene encoding the protein (hereinafter sometimes referred to as the present gene)” used in the first step is not particularly limited as long as it is a cell expressing the gene. The endogenous gene of the present invention or the gene introduced into the cell as a foreign gene may be used, but the endogenous gene of the cell to be used is preferred. When introduced as a foreign gene, this gene is preferably the gene of the animal species from which the cell is used.
具体的には、 脂肪組織由来細胞、 本遺伝子発現ベクターを導入されてなる形質転換細胞 等が挙げられる。 由来動物種としては、 ラット、 マウス、 モルモッ ト等のげつ歯類哺乳動 物、 ィヌ、 サル、 ヒ ト等が挙げられる。 Specific examples include adipose tissue-derived cells and transformed cells into which this gene expression vector has been introduced. Examples of derived animal species include rodent mammals such as rats, mice, and guinea pigs, dogs, monkeys, and humans.
【0 0 2 4】  [0 0 2 4]
前記細胞としては、 動物の組織や臓器から分離された細胞や、 同一の機能 ·形態を持つ 集団を形成している細胞 且織)等も含まれ、 いかなる分化過程にある細胞であつてもよ レ、。  Examples of the cells include cells isolated from animal tissues and organs, cells and tissues forming a group having the same function / morphology, and the like, and may be cells in any differentiation process. Les.
【0 0 2 5】  [0 0 2 5]
被験物質として用いられる化合物には特に限定は無く、 蛋白質、 ペプチド、 核酸、 無機 化合物、 天然もしくは合成化学的に調製された有機化合物等が挙げられる。 被験物質とし て、 具体的には、 アミノ酸 3〜 5 0残基、 好ましくは 5〜 2 0残基のペプチドライブラリ 一や、 当業者に公知のコンビナトリァルケミストリ一の技術を用いて調製された分子量 1 0 0〜 2 0 0 0、 好ましくは 2 0 0〜 8 0 0の低分子有機化合物ライブラリ一を挙げるこ とができる。  The compound used as the test substance is not particularly limited, and examples thereof include proteins, peptides, nucleic acids, inorganic compounds, and organic compounds prepared by natural or synthetic chemistry. As a test substance, specifically, a peptide library of amino acids 3 to 50 residues, preferably 5 to 20 residues, or a combinatorial chemistry technique known to those skilled in the art was prepared. A low molecular weight organic compound library having a molecular weight of 100-200, preferably 200-800 can be mentioned.
【0 0 2 6】  [0 0 2 6]
前記の第二工程における 「対照細胞」 とは前記第一工程で用いられる木遺伝子を発現可 能な細胞において、 被験物質を接触させない場合の当該細胞を表す。 「被験物質を接触さ せない場合」 には、 被験物質の代わりに被験物質と同量の溶媒 (ブランク）を添加する場 合や、 本遺伝子の発現に影響を与えないネガティブコントロール物質を添加する場合も含 まれる。  The “control cell” in the second step represents a cell that can express the tree gene used in the first step and is not brought into contact with a test substance. When not contacting the test substance, add the same amount of solvent (blank) as the test substance instead of the test substance, or add a negative control substance that does not affect the expression of this gene. Also included.
【0 0 2 7】  [0 0 2 7]
また、 被験物質と細胞とを接触させる条件は、 特に制限されないが、 該細胞が死なない ように、 その培養条件 (温度、 p H、 培地組成など)を大きく変化させない条件を採用するこ とが好ましレ、。  The conditions for bringing the test substance into contact with the cells are not particularly limited, but conditions that do not significantly change the culture conditions (temperature, pH, medium composition, etc.) can be adopted so that the cells do not die. I like it.
【0 0 2 8】  [0 0 2 8]
本蛋白質をコ一ドする遺伝子を発現可能な細胞と接触させる被験物質の濃度としては、 特に限定は無く、 通常約 0 . 1 μ Μ〜約 1 0 0 μ Μであればよく、 好ましくは 1 μ Μ〜 5 0 μ Μであればよい。 筋肉細胞もしくは肝細胞と被験物質とを接触させる時間は、 通常 5 分間〜 3 0分間程度あり、 好ましくは 1 0分間〜 2 0分間程度である。 被験物質は適宜、 水、 リン酸バッファーもしくはトリスバッファ一等のバッファー、 エタノール、 ァセトン、 ジメチルスルホキシドもしくはこれらの混合物などの溶媒に溶解又は懸濁して用いること ができる。 【0 0 2 9】 The concentration of the test substance to be contacted with a cell capable of expressing the gene encoding this protein is not particularly limited, and is usually about 0.1 μ μ to about 100 μ μ, preferably 1 Any value between μ 5 and 50 μ よ い is acceptable. The time for contacting the muscle cell or hepatocyte with the test substance is usually about 5 minutes to 30 minutes, preferably about 10 minutes to 20 minutes. The test substance can be used by appropriately dissolving or suspending it in a solvent such as water, a buffer such as phosphate buffer or Tris buffer, ethanol, acetone, dimethyl sulfoxide, or a mixture thereof. [0 0 2 9]
「被験物質を接触させない対照細胞」 としては、 第一工程で用いられる本遺伝子を発現 可能な細胞において、 被験物質を接触させない場合の当該細胞を表す。 「被験物質を接触 させない場合」 には、 被験物質の代わりに被験物質と同量の溶媒 (ブランク）を添加する 場合や、 本遺伝子の発現に影響を与えないネガティブコントロール物質を添加する場合も 含まれる。  “Control cell not to be contacted with test substance” refers to a cell that can be expressed in the gene used in the first step and is not in contact with the test substance. “When the test substance is not contacted” includes the case where the same amount of solvent (blank) as the test substance is added instead of the test substance, or the case where a negative control substance that does not affect the expression of this gene is added. It is.
また、 被験物質と細胞とを接触させる条件は、 特に制限されないが、 該細胞が死なない ように、 その培養条件 (温度、 p H、 培地組成など〉 を大きく変化させない条件を採用する ことが好ましい。 The conditions for bringing the test substance into contact with the cells are not particularly limited, but it is preferable to employ conditions that do not significantly change the culture conditions (temperature, pH, medium composition, etc.) so that the cells do not die. .
【0 0 3 0】  [0 0 3 0]
本遺伝子の発現レベルの検出及び定量は、 前記細胞から調製した R N A又はそれから転写 された相補的なポリヌクレオチドを用いて、 ノーザンプロット法、 RT-PCR法など公知の方 法で実施できる。 具体的には、 本遺伝子の塩基配列において連続する少なくとも 1 5塩基 を有するポリヌクレオチド及び/又はその相補的なポリヌクレオチドをプライマーまたはプ ローブとして用いることによって、 R N A中の木遺伝子の発現の有無やその発現レベルを 検出、 測定することができる。 そのようなプローブもしくはプライマーは、 本遺伝子の塩 基 配 列 を も と に 、 例 え ば primer 3 (HYPERLINK http: " www.eenome.wi.mit.edu/cgi-bil l/primer/primer3.cgihttp7/www,genome.wi.mit.edu/cgi-b m/pnmer/prjmer3.cgi)あるいはベクター NTKlnfomax社製）を利用して設計することがで さる。  Detection and quantification of the expression level of this gene can be carried out by a known method such as Northern plotting or RT-PCR using RNA prepared from the cells or a complementary polynucleotide transcribed therefrom. Specifically, by using a polynucleotide having at least 15 bases continuous in the base sequence of this gene and / or its complementary polynucleotide as a primer or a probe, the presence or absence of expression of the tree gene in RNA or Its expression level can be detected and measured. Such a probe or primer is based on the base sequence of this gene, for example primer 3 (HYPERLINK http: "www.eenome.wi.mit.edu/cgi-bill/primer/primer3.cgihttp7 /www,genome.wi.mit.edu/cgi-bm/pnmer/prjmer3.cgi) or the vector NTKlnfomax)).
【0 0 3 1】  [0 0 3 1]
ノーザンブロット法を利用する場合、 前記プライマ一もしくはプローブを放射性同位元 素 (32P、 33Pなど： RI)や蛍光物質などで標識し、 それを、 常法に従ってナイロンメンブ レン等にトランスファーした細胞由来の RNAとハイブリダィズさせた後、形成された前記 プライマ一もしくはプロ一ブ (DNAまたは RNA)と剛 Aとの二重鎖を、前記プライマーもし くはプロープの標識物 (RI若しくは蛍光物質)に由来するシグナルとして放射線検出器（ BAS-1800II, 富士フィルム社製)または蛍光検出器で検出、 測定する方法を例示すること 力、でさる。 また、 AlkPhos Direct Labelling and Detection System(Amersham Pharamcia Biotech社製)を用いて、 該プロトコールに従って前記プローブを標識し、 細胞由来の RNA とノ、ィプリダイズさせた後、 前記プローブの標識物に由来するシグナルをマルチバイオイ メージャー STORM860(Amersham Pharmacia Biotech社製)で検出、 測定する方法を使用 することもできる。  When using Northern blotting, the primer or probe is labeled with a radioactive isotope (32P, 33P, etc .: RI) or a fluorescent substance, and transferred to nylon membrane or the like according to a conventional method. After hybridization with RNA, the formed duplex of primer or probe (DNA or RNA) and rigid A is derived from the primer or probe label (RI or fluorescent substance). Illustrating a method of detecting and measuring with a radiation detector (BAS-1800II, manufactured by Fuji Film) or a fluorescence detector as a signal. In addition, using the AlkPhos Direct Labeling and Detection System (manufactured by Amersham Pharamcia Biotech), the probe is labeled according to the protocol, and is amplified with cell-derived RNA, and then the signal derived from the probe label is detected. A method of detecting and measuring with a multi-bioimager STORM860 (manufactured by Amersham Pharmacia Biotech) can also be used.
【0 0 3 2】  [0 0 3 2]
RT-PCR法を利用する場合は、 細胞由来の RNAから常法に従って cDNAを調製して、 これを鎵型として標的の木遺伝子の領域が増幅できるように、 本遺伝子の配列に基づき調 製した一対のプライマー (上記 c D N A (—鎖)に結合する正鎖、 +鎖に結合する逆鎖)をこれ とハイブリダィズさせて、 常法に従って P C R法を行い、 得られた増幅二本鎖 D N Aを検 出する方法を例示することができる。 なお、 増幅された二本鎖 D N Aの検出は、 上記 P C Rを予め RIや蛍光物質で標識しておいたプライマ一を用いて行うことによって産生される 標識二本鎖 D N Aを検出する方法、 産生された二本鎖 D N Aを常法に従ってナイロンメン プレン等にトランスファ一させて、 標識した前記プライマ一をプローブとして使用してこ れとハイブリダィズさせて検出する方法などを用いることができる。 なお、 生成された標 識ニ本鎖 D N A産物はアジレント 2100バイオアナライザ (横河アナリティカルシステムズ 社製)などで測定することができる。 また、 SYBR Green RT-PCR Reagents(Applied Bios ystems 社製）で該プロ トコ一ルに従って RT-PCR反応液を調製し、 ABI PRIME 7900 Sequence Detection System(Applied Biosystems社製)で反応させて、 該反応物を検出す ることもできる。 【0 0 3 3】 When using the RT-PCR method, cDNA was prepared from cell-derived RNA according to a conventional method, and was prepared based on the sequence of this gene so that the target tree gene region could be amplified using this as a cage. A pair of primers (a normal strand that binds to the above cDNA (-strand) and a reverse strand that binds to the + strand) are hybridized with this, and PCR is performed according to a conventional method. The method of taking out can be illustrated. The amplified double-stranded DNA is detected by a method of detecting the labeled double-stranded DNA produced by performing the above PCR using a primer previously labeled with RI or a fluorescent substance. For example, a method may be used in which the double-stranded DNA is transferred to nylon membrane or the like according to a conventional method, and the labeled primer is used as a probe to hybridize with the primer. The generated labeled double-stranded DNA product can be measured with an Agilent 2100 Bioanalyzer (manufactured by Yokogawa Analytical Systems). In addition, an RT-PCR reaction solution is prepared according to the protocol using SYBR Green RT-PCR Reagents (Applied Biosystems) and reacted with ABI PRIME 7900 Sequence Detection System (Applied Biosystems). Objects can also be detected. [0 0 3 3]
被験物質を添加した細胞における本遺伝子の発現が被験物質を添加しなレ、対照細胞での 発現量と比較して 1.5倍以上、 好ましくは 2倍以上、 更に好ましくは 3倍以上であれば、 該被験物質は本遺伝子の発現誘導物質として選択することができる。  If the expression of this gene in the cells to which the test substance is added is 1.5 times or more, preferably 2 times or more, more preferably 3 times or more compared to the expression level in the control cells without adding the test substance, The test substance can be selected as an expression inducer of this gene.
【0 0 3 4】  [0 0 3 4]
11-2)本蛋白質の発現量を指標として用いる検定方法  11-2) Assay method using the expression level of this protein as an index
本発明は、 下記の工程 (1)〜(3)を含むことを特徴とする、 耐糖能促進活性の検定 The present invention comprises the following steps (1) to (3), wherein the assay for glucose tolerance promoting activity is provided:
方法を提供する。 すなわち： Provide a method. Ie:
(1)被験物質と、 本蛋白質を発現可能な細胞とを接触させる第一工程、  (1) a first step of contacting a test substance with a cell capable of expressing the protein;
(2)被験物質を接触させた細胞における、 前記蛋白質の発現量を測定し、 該発現量を被 験物質に接触させない対照細胞における前記蛋白質の発現量と比較する第二工程、 及び (2) a second step of measuring the expression level of the protein in a cell contacted with a test substance and comparing the expression level with the expression level of the protein in a control cell not contacted with the test substance; and
(3)前記第二工程の比較結果に基づいて、 被験物質が前記蛋白質の発現量を増加させる か否かを指標として被験物質のィンスリン様活性を評価する第三工程。 (3) A third step of evaluating the insulin-like activity of the test substance using as an index whether or not the test substance increases the expression level of the protein based on the comparison result of the second step.
ここで、 被験物質としては前記と 1同じものが挙げられる。 また、 「本蛋白質を発現可能 な細胞」 としては、 前記 1)における 「本蛋白質をコードする遺伝子 (本遺伝子)を発現 可能な細胞」 と同じものが挙げられる。 Here, the same test substances as those mentioned above can be cited. Examples of the “cell capable of expressing the present protein” include the same cells as the “cell capable of expressing the gene encoding the present protein (the present gene)” in 1) above.
【0 0 3 5】  [0 0 3 5]
本蛋白質の発現レベルの検出及び定量は、 本蛋白質を認識する抗体を用いたウェスタン プロット法等の公知方法に従って定量できる。 ウェスタンプロッ卜法は、 一次抗体として 本蛋白質を認識する抗体を用いた後、二次抗体として 125 Iなどの放射性同位元素、蛍光物 質、 ホースラディッシュペルォキシダーゼ (HRP)等の酵素等で標識した一次抗体に結合 する抗体を用いて標識し、 これら標識物質由来のシグナルを放射線測定器 (BA I - 1800II: 富士フィルム社製など)、 蛍光検出器などで測定することによって実施できる。 また、 一 次抗体として本蛋白質を認識する抗体を用いた後、 ECL Plus Western Blotting Detection System (アマシャムフアルマシアバイオテク社製)を利用して該プロ トコールに従って検出 し、 マルチバイオメージャー STORM860(アマシャム.フアルマシアバイオテク社製 )で測定することもできる。  Detection and quantification of the expression level of the protein can be quantified according to a known method such as Western plotting using an antibody recognizing the protein. The Western Plot method uses an antibody that recognizes this protein as the primary antibody, and then labels it with a radioisotope such as 125 I, a fluorescent substance, or an enzyme such as horseradish peroxidase (HRP) as the secondary antibody. This can be carried out by labeling with an antibody that binds to the primary antibody, and measuring the signal derived from these labeling substances with a radiation measuring instrument (BA I-1800II: manufactured by Fuji Film Co., Ltd.) or a fluorescence detector. In addition, after using an antibody that recognizes this protein as a primary antibody, detection is performed according to the protocol using ECL Plus Western Blotting Detection System (manufactured by Amersham Pharmacia Biotech), and multi-biomeasuring STORM860 (Amersham (Falmacia Biotech).
【0 0 3 6】  [0 0 3 6]
抗体は、 その形態に特に制限はなく、 前記本蛋白質を免疫抗原とするポリクローナル抗 体であっても、 またそのモノクローナル抗体であってもよく、 さらには本蛋白質を構成す るアミノ酸配列のうち少なくとも連続する、 通常 8アミノ酸、 好ましくは 1 5アミノ酸、 より好ましくは 2 0アミノ酸からなるポリぺプチドに対して抗原結合性を有する抗体も、 本発明の抗体に含まれる。  The form of the antibody is not particularly limited, and it may be a polyclonal antibody using the protein as an immunizing antigen or a monoclonal antibody thereof, and at least among the amino acid sequences constituting the protein. The antibody of the present invention also includes an antibody having an antigen binding property to a continuous polypeptide consisting of usually 8 amino acids, preferably 15 amino acids, more preferably 20 amino acids.
【0 0 3 7】  [0 0 3 7]
これらの抗体の製造方法は、 すでに周知であり、 本発明の抗体もこれらの常法に従って 製造 Tるこ とがでさる (Current protocols in Molecular Biology edit. Ausubel et al.(l987)Publish, John Wiley and Sons. Sectionll. l2〜11.13)。  Methods for producing these antibodies are already well known, and the antibodies of the present invention can also be produced according to these conventional methods (Current protocols in Molecular Biology edit. Ausubel et al. (L987) Publish, John Wiley). and Sons. Sectionll. l2-11.13).
【0 0 3 8】  [0 0 3 8]
Π-3)本遺伝子の発現制御領域を用いたレポーター遺伝子ァッセィを用いる検定方法 本発明は、 下記の工程（1 )〜（3 )を含むことを特徴とする、 耐糖能促進活性の検定 方法を提供する。 すなわち：  Π-3) Assay method using reporter gene assay using expression control region of this gene The present invention comprises a method for assaying glucose tolerance promoting activity, characterized by comprising the following steps (1) to (3): provide. Ie:
(1)被験物質と、 本蛋白質をコ一ドする遺伝子の発現制御領域を機能可能な形で連結 されてなるレポータ一遺伝子を含有する細胞とを接触させる第一工程、  (1) a first step of bringing a test substance into contact with a cell containing a reporter gene that is operably linked to an expression control region of a gene encoding this protein;
(2)被験物質を接触させた細胞における、 前記レポ一タ一遺伝子の発現量を測定し、 該発現量を被験物質に接触させない対照細胞における前記遺伝子の発現量と比較する第二 工程、 及び  (2) a second step of measuring an expression level of the reporter gene in a cell contacted with a test substance and comparing the expression level with an expression level of the gene in a control cell not contacted with the test substance; and
(3)前記第二工程の比較結果に基づいて、 前記レポータ一遺伝子の発現量を増加させるか否 かを指標として被験物質のィンスリン様活性を評価する第三工程。 ここで、 被験物質は、 前記と同じものが挙げられる。 (3) A third step of evaluating the insulin-like activity of the test substance using as an index whether or not to increase the expression level of the reporter gene based on the comparison result of the second step. Here, the test substance is the same as described above.
「本蛋白質をコードする遺伝子 (本遺伝子)の発現制御領域」 とは、 通常、 当該染色体 遺伝子の上流数 k bから数十 k bの範囲を指し、 例えば、 （i)5'_レース法 (5'-RACE 法) (例えば、 5'full Race Core Kit (宝酒造社製)等を用いて実施されうる)、 オリゴ  “Expression control region of the gene encoding this protein (this gene)” usually refers to a range from several kb to several tens of kb upstream of the chromosomal gene. For example, (i) 5'_race method (5 ' -RACE method) (for example, it can be carried out using 5'full Race Core Kit (Takara Shuzo) etc.), oligo
キャップ法、 S1プライマ一マッピング等の通常の方法により、 5'末端を決定するステツ プ； (ii)Genome Walker Kit (クローンテック社製)等を用いて 5'·上流領域を取得し、 得られた上流領域について、 プロモーター活性を測定するステップ;、 を含む手法等によ り同定することが出来る。 Step for determining the 5 'end by a conventional method such as cap method or S1 primer mapping; (ii) Obtain 5' upstream region using Genome Walker Kit (Clontech) etc. The upstream region can be identified by a method including a step of measuring promoter activity;
【0 0 3 9】  [0 0 3 9]
本遺伝子の発現制御領域を機能可能な形で連結されてなるレポーター遺伝子は、 当業者 に公知の方法で調製すればよい。 すなわち、 「Molecular Cloning laboratory Manual 2nd editionj (1989入 Cold Spring Harbor Laboratory Press, 「Current Protocols In Molecular Biology」 （1987)，John Wiley & Sons, Inc.等に記載される通常の遺伝子工学的 手法に従って切り出された本遺伝子の発現調節領域を、 レポーター遺伝子を含むプラスミ ド上に組み込むことができる。  A reporter gene formed by linking the expression control region of this gene in a functional manner may be prepared by a method known to those skilled in the art. That is, it was extracted according to the usual genetic engineering techniques described in “Molecular Cloning laboratory Manual 2nd edition” (1989 Cold Spring Harbor Laboratory Press, “Current Protocols In Molecular Biology” (1987), John Wiley & Sons, Inc., etc.) The expression control region of this gene can be incorporated on a plasmid containing a reporter gene.
【0 0 4 0】  [0 0 4 0]
レポーター遺伝子としては、 ダルクロニダーゼ (GUS)、 ルシフェラーゼ、 クロラムフ ェニコールトランスァセチラーゼ (CAT)、 -ガラク トシダーゼ及びグリーン蛍光タンパ ク質 (GFP)等が挙げられる。  Reporter genes include dalcronidase (GUS), luciferase, chloramphenicol transferase (CAT), -galactosidase, and green fluorescent protein (GFP).
調製した本遺伝子の発現制御領域を機能可能な形で連結されてなるレポーター遺伝子を、 通常の遺伝子工学的手法を用いて、 当該レポータ一遺伝子を導入する細胞において使用 可能なベクターに挿入し、プラスミ ドを作製し、適当な宿主細胞へ導入することができる。 レポーター遺伝子に応じた選抜条件の培地で培養することにより、 形質転換細胞を得る ことができる。  The prepared reporter gene operably linked to the expression control region of this gene is inserted into a vector that can be used in a cell into which the reporter gene is to be introduced, using ordinary genetic engineering techniques. Can be prepared and introduced into a suitable host cell. Transformed cells can be obtained by culturing in a medium under selection conditions according to the reporter gene.
【0 0 4 1】  [0 0 4 1]
また、 レポーター遺伝子の発現量を測定する方法としては、 個々のレポ一ター遺伝子に 応じた方法を利用すればよレ、。 例えば、 レポーター遺伝子としてルシフェラーゼ遺伝子を 用いる場合には、 前記形質転換細胞を数目間培養後、 当該細胞の抽出物を得、 次いで当該 抽出物をルシフェリンおよび A T Pと反応させて化学発光させ、 その発光強度を測定する ことによりプロモーター活性を検出することができる。 この際、 ピツカジーンデュアルキ ット (登録商標;東洋インキ製)等の市販のルシフェラ一ゼ反応検出キットを用いること ができる。  In addition, as a method of measuring the expression level of the reporter gene, a method corresponding to each reporter gene may be used. For example, when a luciferase gene is used as a reporter gene, the transformed cells are cultured for a few minutes, an extract of the cells is obtained, then the extract is reacted with luciferin and ATP to cause chemiluminescence, and the luminescence intensity Promoter activity can be detected by measuring. In this case, a commercially available Luciferase reaction detection kit such as Pitsukagene Dual Kit (registered trademark; manufactured by Toyo Ink) can be used.
【0 0 4 2】  [0 0 4 2]
上記 Π- 1)〜Π-3)において、 「本蛋白質をコードする遺伝子を発現可能な細胞」、  In the above Π-1) to Π-3), “a cell capable of expressing a gene encoding this protein”,
「本蛋白質を発現可能な細胞」 又は 「本遺伝子の発現制御領域を機能可能な形で連結され てなるレポーター遺伝子を含有する細胞」 と、 被験物質との接触は、 当該細胞が成育可能 な条件で培養しながら行えばよく、 例えば、 哺乳動物細胞を宿主とする本発明形質転換細 胞の場合、 適宜ゥシ胎児血清等の哺乳動物由来の血清を添加した D— M E M、 O P T I - M E M, R P M 1 1 6 4培地 (G i b c o— B R L製)等の市販の培地中で培養できる。  Contact with a test substance with a `` cell capable of expressing this protein '' or `` a cell containing a reporter gene that is operably linked to the expression control region of this gene '' is a condition that allows the cell to grow For example, in the case of the transformed cell of the present invention using a mammalian cell as a host, D-MEM, OPTI-MEM, RPM to which mammal-derived serum such as urine fetal serum is appropriately added. It can be cultured in a commercially available medium such as 1 1 6 4 medium (manufactured by Gibco-BRL).
【0 0 4 3】  [0 0 4 3]
上記 ΙΙ- 1)〜ΙΙ-3)において、 「被験物質に接触させない対照細胞」 とは、 各第一ェ 程で用いられる 「本蛋白質をコードする遺伝子を発現可能な細胞」、 「本蛋白質を発現可 能な細胞」 又は 「本遺伝子の発現制御領域を機能可能な形で連結されてなるレポーター遺 伝子を含有する細胞」 において、 被験物質を接触させない場合の当該細胞を表す。 r被験 物質を接触させない場合」 には、 被験物質の代わりに被験物質と同量の溶媒 (ブランク） を添加する場合や、 本遺伝子、 本蛋白質もしくはレポーター遺伝子の発現に影響を与えな いネガティブコント口ール物質を添加する場合も含まれる。 【0 0 4 4】 In the above-mentioned) -1) to ΙΙ-3), “control cell not to be contacted with test substance” means “cell capable of expressing a gene encoding this protein” and “this protein used in each first step” “Expressable cell” or “cell containing a reporter gene operably linked to the expression control region of this gene” refers to the cell when the test substance is not contacted. When the test substance is not contacted, the same amount of solvent (blank) as the test substance is added instead of the test substance, or the negative control that does not affect the expression of the gene, protein or reporter gene. This includes the case of adding oral substance. [0 0 4 4]
上記 Π-1)〜ΙΙ-3)において、 各第一工程及び第二工程で測定した本遺伝子、 本蛋白 質又はレポ一ター遺伝子の発現レベルに基づき、 本遺伝子もしくは本蛋白質の発現誘導活 性を有する被験物質を選択することができる。 すなわち、 被験物質を添加した細胞におけ る本遺伝子、 本蛋白質又はレポ一タ一遺伝子の発現が被験物質を添加しない対照細胞での 発現量と比較して 1 . 2倍以上、 好ましくは 1 . 5倍以上、 更に好ましくは 2倍以上であ れば、 該被験物質は本遺伝子もしくは本蛋白質の発現誘導物質 (発現促進物質)として選 択することができる。  Based on the expression level of the gene, the protein or the reporter gene measured in each of the first step and the second step in the above Π-1) to ΙΙ-3), the expression induction activity of the gene or the protein is determined. Can be selected. That is, the expression level of the gene, the protein or the reporter gene in cells to which the test substance is added is 1.2 times or more, preferably 1. If it is 5 times or more, more preferably 2 times or more, the test substance can be selected as an expression inducer (expression promoting substance) of this gene or protein.
上記のように選択される本遺伝子もしくは本蛋白質の発現誘導物質もまた、 耐糖能促准 活性、 即ち耐糖能を改善する活性、 詳しくはインスリン応答性促進活性を有する本発明の 糖代謝関連疾患の治療剤又は予防剤の候補物質である。 The expression inducer of the present gene or protein selected as described above is also a glucose tolerance-promoting activity, that is, an activity to improve glucose tolerance, specifically, an insulin responsiveness promoting activity. It is a candidate for a therapeutic or prophylactic agent.
【0 0 4 5】  [0 0 4 5]
本蛋白質及びそのフラグメント、 あるいは本発明探索方法で見出される化合物は、 これ らを医薬品として用いるにあたり、 そのままもしくは公知の薬学的に許容される担体 (I武 形剤、 希釈剤、 増量剤、 結合剤、 滑沢剤、 流動助剤、 崩壊剤、 界面活性剤等などが含まれ る)や 用の添加剤などと混合して医薬組成物として調製することができる。 当該医薬組 成物は、 調製する形態 (錠剤、 丸剤、 カプセル剤、 散剤、 穎粒剤、 シロップ剤、 乳剤、 懸 濁液などの経口投与剤;注射剤、 点滴剤、 外用剤、 坐剤などの非経口投与剤)等に応じて 、 全身的にまたは局所的に、 経口投与または非経口投与することができる。 非経口投与す る場合には、 静脈投与、 皮内投与、 皮下投与、 直腸投与、 経皮投与すること等が可能であ る。  The present protein and its fragments, or the compounds found by the search method of the present invention, are used as they are as pharmaceuticals as they are or as a known pharmaceutically acceptable carrier (I-arming agent, diluent, bulking agent, binder) And a lubricant, a flow aid, a disintegrant, a surfactant, and the like) and additives for the above can be mixed to prepare a pharmaceutical composition. The pharmaceutical composition is prepared in the form to be prepared (tablets, pills, capsules, powders, granules, syrups, emulsions, suspensions, etc .; injections, drops, external preparations, suppositories) Depending on the parenteral administration agent) etc., it can be administered systemically or locally, orally or parenterally. In the case of parenteral administration, intravenous administration, intradermal administration, subcutaneous administration, rectal administration, transdermal administration, etc. are possible.
【0 0 4 6】  [0 0 4 6]
前記の適当な投与剤型は許容される通常の担体、 賦型剤、 結合剤、 安定剤、 希釈剤等に 有効成分 (本蛋白質、 又は本発明探索方法により選抜される物質またはその薬学的に許容 される塩等)を配合することにより製造することができる。 また注射剤型で用いる場合に は、 許容される緩衝剤、 溶解補助剤、 等張剤等を添加することもできる。  The above-mentioned appropriate dosage form is an acceptable normal carrier, excipient, binder, stabilizer, diluent, etc. active ingredient (this protein, or a substance selected by the search method of the present invention or its pharmacologically. It can be produced by blending acceptable salts and the like. When used in an injection form, acceptable buffering agents, solubilizing agents, isotonic agents, and the like can be added.
また投与量は、 有効成分の種類、 投与経路、 投与対象または患者の年齢、 体重、 症状な どによって異なり一概に規定できないが、 通常、 経口の場合には成人で 1日あたり有効成 分量として、 数 m g〜 2 g程度、 好ましくは 5 m g〜数十 m g程度を、 1日 1〜数回にわ けて投与することができる。 注射の場合には成人で有効成分量として約 0 . 1 m g〜約 5 0 O m gを投与すればよく、 1日の投与量を 1回または数回に分けて投与することができ る。 The dosage varies depending on the type of active ingredient, administration route, administration subject or patient's age, weight, symptoms, etc., but cannot be generally specified. Several mg to 2 g, preferably about 5 mg to several tens mg can be administered once to several times a day. In the case of injection, about 0.1 mg to about 50 O mg may be administered as an active ingredient in adults, and the daily dose can be administered once or divided into several times.
【0 0 4 7】  [0 0 4 7]
上記有効成分物質として、 本遺伝子そのものを挙げることができる。 この場合は、 本遺 伝子を遺伝子治療用ベクターに組込み、 遺伝子治療を行うことも考えられる。 これらの場 合も、 遺伝子治療用組成物の投与量、 投与方法は患者の体重、 年齢、 症状などにより変動 し、 当業者であれば適宜選択することが可能である。  Examples of the active ingredient substance include the gene itself. In this case, it may be possible to incorporate the gene into a gene therapy vector and perform gene therapy. Also in these cases, the dosage and administration method of the gene therapy composition vary depending on the patient's weight, age, symptoms, etc., and can be appropriately selected by those skilled in the art.
【0 0 4 8】  [0 0 4 8]
上記遺伝子治療にっき詳述すれば、 該遺伝子治療は、 通常のこの種の遺伝子治療と同様 にして、 例えば本遺伝子またはそれらの化学的修飾体を直接糖代謝関連疾患に罹患した哺 乳動物 (患者)の体内に投与することにより目的遺伝子の発現を制御する方法、 もしくは これらの遺伝子を患者の標的細胞に導入することにより該細胞による目的遺伝子の発現を 制御する方法により実施できる。  In detail, the gene therapy is performed in the same manner as in this type of gene therapy. For example, mammals (patients) directly suffering from a glucose metabolism-related disease using this gene or a chemical modification thereof. ) In the body, or by introducing these genes into the target cells of the patient to control the expression of the target gene by the cells.
【0 0 4 9】  [0 0 4 9]
ここで前記化学修飾体としては、 例えばホスホロチォエート、 ホスホロジチォェ一ト、 ァノレキノレホスホ トリエステノレ、 ァノレキノレホスホナート、 ァノレキルホスホアミデートなどの 、 細胞内への移行性または細胞内での安定性を高め得る誘導体 ("Alltisense RNA and DNA "WILEY-LISS刊、 1992年、 pp.1-50, J. Med.Chem. 36:1923.1937,1993)が含まれる 。 これらは常法に従い合成することができる。 Here, as the chemical modification, for example, phosphorothioate, phosphorodithioate, anorequinolephospho triestenole, anorequinole phosphonate, anorequil phosphoamidate, etc. Includes derivatives that can enhance intracellular stability ("Alltisense RNA and DNA" published by WILEY-LISS, 1992, pp.1-50, J. Med. Chem. 36: 1923.1937, 1993) . These can be synthesized according to conventional methods.
本遺伝子は、 その投与に当たり、 通常慣用される安定化剤、 緩衝液、 溶媒などを用いて 製剤化され得る。 This gene can be formulated using a stabilizer, a buffer, a solvent, etc. that are usually used for administration.
【0 0 5 0】  [0 0 5 0]
本遺伝子を患者の標的細胞に導入する方法において、 用いられるポリヌクレオチドは、 好ましくは 100塩基以上、 より好ましくは 300塩基以上、 さらに好ましくは 500塩基以上 の  In the method of introducing this gene into a target cell of a patient, the polynucleotide used is preferably 100 bases or more, more preferably 300 bases or more, and even more preferably 500 bases or more.
長さを有するものとすればよい。 また、 この方法は、 生体内の細胞に遺伝子を導入する in vivo法および一旦体外に取り出した細胞に遺伝子を導入し、 該細胞を体内に戻す ex vivo 法を包含する（目経サイエンス， 1994年 4月号， 20-45頁、 月刊薬事, 36(1)， 23-48 (1994 )、 実験医学増刊， 12 (15), 全頁 （1994)など参照)。 この内では in vivo法が好ましく、 これには、 ウィルス的導入法 (組換えウィルスを用いる方法）と非ウィルス的導入法がある (前記各文献参照)。 What is necessary is just to have length. This method also includes an in vivo method for introducing a gene into cells in a living body and an ex vivo method for introducing a gene into a cell once taken out of the body and returning the cell to the body (Nikkei Science, 1994). April issue, pages 20-45, monthly pharmacy, 36 (1), 23-48 (1994), experimental medicine extra, 12 (15), all pages (1994)). Among these, the in vivo method is preferable, and there are a viral introduction method (method using a recombinant virus) and a non-viral introduction method (see the above-mentioned documents).
【0 0 5 1】  [0 0 5 1]
上記組換えウィルスを用いる方法としては、 例えばレトロウイルス、 アデノウイルス、 アデノ関連ウィルス、 ヘルぺスウィルス、 ワクシニアウィルス、 ポリオウイルス、 シンビ スウィルスなどのウイルスゲノムに本遺伝子のポリヌクレオチドを組込んで生体内に導入 する方法が挙げられる。 この中では、 レトロウイルス、 アデノウイルス、 アデノ関連ウイ ルスなどを用いる方法が特に好ましい。 非ウィルス的導入法としては、 リボソーム法、 リ ポフエクチン法などが挙げられ、 特にリポノーム法が好ましい。 他の非ウィルス的導入法 としては、 例えばマイクロインジェクション法、 リン酸カルシウム法、 エレク ト口ポレー ション法なども挙げられる。  Examples of the method using the above recombinant virus include a retrovirus, an adenovirus, an adeno-associated virus, a herpes virus, a vaccinia virus, a poliovirus, a symvirus virus, etc. Introducing it into the body. Of these, methods using retroviruses, adenoviruses, adeno-related viruses, etc. are particularly preferred. Non-viral introduction methods include the ribosome method, the lipofectin method, etc., and the liponome method is particularly preferable. Other non-viral introduction methods include, for example, the microinjection method, the calcium phosphate method, and the electric mouth poling method.
【0 0 5 2】  [0 0 5 2]
遺伝子治療用製剤組成物は、 本遺伝子又はこれらを含む組換えウィルスおよびこれらゥ ィルスが導入された感染細胞などを有効成分とするものである。 該組成物の患者への投与 形態、 投与経路などは、 治療目的とする疾患、 症状などに応じて適宜決定できる。 例えば 注射剤などの適当な投与形態で、 静脈、 動脈、 皮下、 筋肉内などに投与することができ、 また患者の疾患対象部位に直接投与、 導入することもできる。 in vivo法を採用する場合 、 遺伝子治療用組成物は、 本遺伝子を含む注射剤などの投与形態の他に、 例えば本遺伝子 を含有するウィルスベクターをリボソームまたは膜融合リボソームに包埋した形態 (セン ダイゥィルス (HVJ).リボソームなど)とすることができる。 これらのリポソ一ム製剤形態 には、 懸濁剤、 凍結剤、 遠心分離濃縮凍結剤などが含まれる。 また、 遺伝子治療用組成物 は、 本遺伝子を含有するベクターを導入されたウイルスで感染された細胞培養液の形態と することもできる。 これら各種形態の製剤中の有効成分の投与量は、 治療目的である疾患 の程度、 患者の年齢、 体重などにより適宜調節することができる。 通常、 患者成人 1入当 たり約 0.0001-100mg、好ましくは約 0.001-lOmgが数目ないし数力月に 1回投与される量 と  The pharmaceutical composition for gene therapy comprises the present gene or a recombinant virus containing these genes and infected cells into which these viruses have been introduced as active ingredients. The form of administration of the composition to a patient, the route of administration, and the like can be appropriately determined according to the disease and symptoms to be treated. For example, it can be administered into veins, arteries, subcutaneously, intramuscularly, etc. in an appropriate dosage form such as an injection, or can be directly administered and introduced into a disease target site of a patient. In the case of adopting the in vivo method, the composition for gene therapy includes, for example, a form in which a viral vector containing this gene is embedded in a ribosome or a membrane fusion ribosome in addition to an administration form such as an injection containing this gene (sen Dirulus (HVJ). Ribosome etc.). These liposomal formulation forms include suspensions, freezing agents, centrifugal concentrated freezing agents, and the like. The gene therapy composition can also be in the form of a cell culture infected with a virus introduced with a vector containing the gene. The dosage of the active ingredient in these various forms of preparation can be appropriately adjusted depending on the degree of the disease to be treated, the age of the patient, the body weight, and the like. Usually, about 0.0001-100mg per patient adult, preferably about 0.001-lOmg once every few to several months
すればよい。 本遺伝子を含むレトロウイルスベクターの場合は、 レトロウイルス力価とし て、 1 日患者体重 lkg当たり約 lxlO3 pft l x lO iS pfuとなる量範囲から選ぶことができ る。 本遺伝子を導入した細胞の場合は、 lxlO 4細胞/ body- 1x10 細胞/ body程度を投与す ればよい。 do it. In the case of a retroviral vector containing this gene, the retroviral titer can be selected from an amount range of about lxlO 3 pft lx lO iS pfu per kg patient body weight per day. In the case of cells into which this gene has been introduced, about lxlO 4 cells / body-1 × 10 cells / body may be administered.
【0 0 5 3】  [0 0 5 3]
以下に本発明を実施例で説明するが、 本発明はこれらに限定されるものではない。 【実施例 1]  EXAMPLES The present invention will be described below with reference to examples, but the present invention is not limited to these examples. [Example 1]
【0 0 5 4】  [0 0 5 4]
(ラット由来の本遺伝子のクローニング）  (Cloning of this gene from rat)
Rat Adipose cDNA Marathon-Ready cDNA (CLONTECH社) 1 μ L、配列番号 7で示され た塩基配列からなるプライマ一 1 20pmol、 配列番号 8で示される塩基配列からなるプライ マー 2 20pmol、 TaKaRa Εχ-Taqポリメラーゼ (宝酒造社) 2U、 TaKaRa Εχ-Taqポリメラ一 ゼ添付のノく ッファー 5 μ L 及び TaKaRa Εχ-Taq ポリ メ ラーゼ添付の dNTP mixture(2.5ml)4 M Lを含む 50 μ Lの反応液を調製した。 P C Rは、 まず 94°Cで 30秒間、 次いで 60°Cで 30秒間、 更に 72°Cで 1分間からなる保温サイクルを 50回繰り返し、 最後 に 72°Cで 5分間保温する条件にて行われた。 P C R後、 ァガロース電気泳動で約 1 . 5 Kbp を示す PCR産物を回収した。 次いで回収された P C R産物を pT7-Blue vector (Novage 11 社)にサブクローニングした後、当該プラスミ ドで E.coliJM109株コンビテントセノレ (東洋紡 社)を形質転換した。形質転換された細胞を 50 μ g/mLアンピシリン含有 LB培地 lOOmLで 培養することにより得られる培養菌体から QIAGEN Plasmid Maxi kit(QIAGEN社)を用 いて分離 ·精製することにより、 ラット由来の本遺伝子 (rM064)を含むプラスミ ドを得た。 【実施例 2】 Rat Adipose cDNA Marathon-Ready cDNA ( CLONTECH , Inc.) 1 mu L, SEQ ID NO: 7 at the indicated comprising the nucleotide sequence primer one 1 20 pmol, consisting of the nucleotide sequence represented by SEQ ID NO: 8 plies 2 pM, TaKaRa Εχ-Taq polymerase (Takara Shuzo) 2U, TaKaRa Εχ-Taq Polymerase attached 5 μL of buffer and TaKaRa Εχ-Taq Polymerase attached dNTP mixture (2.5 ml) 4 ML 50 μL of the reaction solution was prepared. PCR is performed under the conditions of 50 ° C incubation cycle consisting of 94 ° C for 30 seconds, 60 ° C for 30 seconds, 72 ° C for 1 minute, and finally 72 ° C for 5 minutes. It was. After PCR, a PCR product showing about 1.5 Kbp was recovered by agarose electrophoresis. Next, the recovered PCR product was subcloned into pT7-Blue vector (Novage 11), and E. coli JM109 strain competent senore (Toyobo) was transformed with the plasmid. By isolating and purifying the transformed cells from cultured cells obtained by culturing transformed cells in lOOmL of LB medium containing 50 μg / mL ampicillin using the QIAGEN Plasmid Maxi kit (QIAGEN) A plasmid containing (rM064) was obtained. [Example 2]
【0 0 5 5】  [0 0 5 5]
(ラット由来の本遺伝子 (rM064)の塩基配列の決定）  (Determination of the nucleotide sequence of this rat-derived gene (rM064))
実施例 1で得られた PCR産物 (約 1 . 5 Kbp)を含むプラスミ ドを鎊型として、 Therm 0 Sequenase IIダイ ·ターミネ一ターキット (Amersham Pharmacia Biotech社)及び ABI3 73DNA配列読み取り装置 (PE Applied Biosystems社)を用いて、 サンガーの方法 〔F.Sang er,S.Nicklen,A.R.Couison 、 Proceedings or National Academy of science U.S.A,(19 77),74, 5463-5467] により、 配列番号 3で示される塩基配列からなるラット由来の本遺 伝子の塩基配列を決定した。 ThermO Sequenase II dye terminator kit (Amersham Pharmacia Biotech) and ABI3 73 DNA sequence reader (PE Applied Biosystems) were prepared using the plasmid containing the PCR product (about 1.5 Kbp) obtained in Example 1 as a saddle. The base represented by SEQ ID NO: 3 by the method of Sanger [F. Sanger, S. Nicklen, ARCouison, Proceedings or National Academy of science USA, (1977), 74, 5463-5467]. The nucleotide sequence of this gene derived from rat consisting of the sequence was determined.
【実施例 3】  [Example 3]
【0 0 5 6】  [0 0 5 6]
(マウス由来の本遺伝子のクローニング）  (Cloning of this gene from mouse)
Mouse Normal Adipose cDNA(BioChain社) 1 μ L、 配列番号 9で示される塩基配列から なるプライマー 3 20pmoK 配列番号 1 0で示される塩基配列からなるプライマー 4 20pmol、 TaKaRa Ex-Taqポリメラーゼ (宝酒造社) 2U、 TaKaRa Εχ-Taqポリメラーゼ添付 のバッファ一 5 μ L及び TaKaRa Εχ-Taqポリメラ一ゼ添付の dNTP mixture(2.5mM)4 L を含む 50 μ Lの反応液を調製した。 PCRは、まず 94°Cで 30秒間、次いで 65°Cで 30秒間、 更に 72°Cで 2分問からなる保温サイクルを 50回繰り返し、 最後に 72°Cで 5分問保温する 条 f牛にて行われた。 P C R後、 ァガロース電気泳動で約 1.2kbpを示す PCR産物を回収 した。 回収された PCR産物を pT7-Blue vector (Nova gen社)にサブクローニングした後、 当該ブラスミ ドで E.coliJMl09株コンピテントセル (東洋紡社)を形質転換した。形質転換さ れた細胞を 50 μ g/mLアンピシリン含有 LB培地 lOOmLで培養することにより得られる培 養菌体から QIAGENPlasmidMaxikit(QIAGEN社)を用いて分離 ·精製することにより、 マウス由来の本遺伝子 (mM064 )の塩基配列を含むプラスミ ドを得た。  Mouse Normal Adipose cDNA (BioChain) 1 μL, primer consisting of the nucleotide sequence shown in SEQ ID NO: 9 3 20pmoK primer consisting of the nucleotide sequence shown in SEQ ID NO: 10 4 20pmol, TaKaRa Ex-Taq polymerase (Takara Shuzo) 2U A 50 μL reaction solution containing 5 μL of the buffer attached to TaKaRa Kaχ-Taq polymerase and 4 L of dNTP mixture (2.5 mM) attached to TaKaRa Εχ-Taq polymerase was prepared. PCR is conducted at 94 ° C for 30 seconds, then at 65 ° C for 30 seconds, and then at 50 ° C for 2 minutes at 72 ° C for 50 minutes, and finally at 72 ° C for 5 minutes. It was done in After PCR, a PCR product showing about 1.2 kbp was recovered by agarose electrophoresis. The recovered PCR product was subcloned into pT7-Blue vector (Novagen), and then E. coli JMl09 strain competent cell (Toyobo) was transformed with the plasmid. By isolating and purifying the transformed cells from cultured cells obtained by culturing the transformed cells in lOOmL LB medium containing 50 μg / mL ampicillin using QIAGENPlasmidMaxikit (QIAGEN), this gene derived from mouse ( A plasmid containing the base sequence of mM064) was obtained.
【実施例 4】  [Example 4]
【0 0 5 7】  [0 0 5 7]
(マウス由来の本遺伝子の塩基配列の決定）  (Determination of the nucleotide sequence of this mouse-derived gene)
実施例 3で得られた PCR産物 (約 1 . 5 Kbp)を含むプラスミ ドを踌型として、 Thermo S equenase IIダイ'ターミネータ一キット（Amersham Pharmacia Biotech社)及び ABI373D NA配列読み取り装置 (PE Applied Biosystems社)を用いて、 サンガーの方法 〔F.Sanger， S.Nicklen'A.R'Coulson著、 Proceedings of National Academy of Science U.S.A.(1977) ,74,5463-5467] により、 配列番号 5で示される塩基配列からなるマウス由来の本遺伝 子 (mM064)の塩基配列を決定した。 Using the plasmid containing the PCR product (about 1.5 Kbp) obtained in Example 3 as a saddle, Thermo Sequenase II dye terminator kit (Amersham Pharmacia Biotech) and ABI373D NA sequence reader (PE Applied Biosystems) By the method of Sanger [F. Sanger, S. Nicklen'A.R'Coulson, Proceedings of National Academy of Science USA (1977), 74,5463-5467]. The base sequence of this mouse-derived gene (mM064) consisting of the base sequence was determined.
【実施例 5】  [Example 5]
【0 0 5 8】  [0 0 5 8]
(ヒ ト由来の本遺伝子のクローニング）  (Cloning of this gene derived from human)
Human Adipocyte Marathon-Ready cDNA(CLONTECH ¾) l μ L, 配列番号 1 1で示さ れる塩基配列からなるプライマー 5 20pmol、 配列番号 1 2で示される塩基配列からなる プライマー 6 20pmol、 TaKaRa Εχ-Taqポリメラーゼ (宝酒造社) 2U、 TaKaRa Εχ-Taqポ リメラーゼ添付のバッファー 5 μ L 及ぴ TaKaRa Εχ-Taq ポリメラ一ゼ添付の dNTP mixture(2.5mM)4 M Lを含む 50 Lの反応液を調製した。 PCRは、 まず 94°Cで 30秒間、 次いで 65°Cで 30秒間、 更に 72°Cで 2分間からなる保温サイクルを 50回繰り返し、 最後 に 72°Cで 5分間保温する条件にて行われた。 PCR後、 ァガロース電気泳動で約 1.2kbpを 示す PCR産物を回収した。回収された PCR産物を pT7-Blue vector(Novagen社)にサブク ローニングした後、当該プラスミ ドで E.coliJM109株コンビテントセル (東洋紡社)を形質転 換した。 形質転換された細胞を 50 μ g/mLアンピシリン含有 LB培地 lOOmLで培養するこ とにより得られる培養菌体から QIAGENPlasmid Maxikit(QIAGEN社)を用いて分離 ·精 製することにより、 ヒ ト由来の本遺伝子（h MO 6 4 )の塩基配列を含むプラスミ ドを得た。 【実施例 6】 Human Adipocyte Marathon-Ready cDNA (CLONTECH ¾) l μL, Primer 5 20 pmol consisting of the base sequence shown in SEQ ID NO: 1 1, consisting of the base sequence shown in SEQ ID NO: 12 Primer 6 20 pmol, TaKaRa Εχ-Taq polymerase (Takara Shuzo) 2 U, TaKaRa Εχ-Taq Polymerase attached buffer 5 μL and TaKaRa Εχ-Taq Polymerase containing dNTP mixture (2.5 mM) 4 ML The reaction solution was prepared. PCR is performed under the conditions of 50 ° C incubation cycle consisting of 94 ° C for 30 seconds, 65 ° C for 30 seconds, and 72 ° C for 2 minutes, and finally 72 ° C for 5 minutes. It was. After PCR, a PCR product showing about 1.2 kbp was recovered by agarose electrophoresis. The recovered PCR product was subcloned into pT7-Blue vector (Novagen), and E. coli JM109 strain competent cell (Toyobo) was transformed with the plasmid. By separating and purifying the transformed cells using QIAGENPlasmid Maxikit (QIAGEN) from the cultured cells obtained by culturing the transformed cells in lOOmL of LB medium containing 50 μg / mL ampicillin. A plasmid containing the base sequence of the gene (hMO64) was obtained. [Example 6]
【0 0 5 9】  [0 0 5 9]
(ヒ ト由来の本遺伝子の塩基配列の決定）  (Determination of the nucleotide sequence of this gene derived from human)
実施例 5で得られた PCR産物 (約 1 . 5 Kbp)を含むプラスミ ドを錄型として、 Thermo Sequenasellダイ ·ターミネータ一キット（Amersham Pharmacia Biotech社)及び ABI373 DNA配列読み取り装置 (PE Applied Biosystems社)を用いて、 サンガーの方法 [： F.Sanger ，S.Nicklen，A.R.Coulson著、 Proceedings of Nationa 1 Academy of Science U.S.A.(1977),74, 5463-5467] により、 配列番号 1で示される塩基配列からなるヒ ト由来の本遺伝子 の塩基配列を決定した。  Using the plasmid containing the PCR product (about 1.5 Kbp) obtained in Example 5 as a saddle, Thermo Sequenasell dye terminator kit (Amersham Pharmacia Biotech) and ABI373 DNA sequence reader (PE Applied Biosystems) From the nucleotide sequence represented by SEQ ID NO: 1 according to the method of Sanger [: F. Sanger, S. Nicklen, ARCoulson, Proceedings of Nation 1 Academy of Science USA (1977), 74, 5463-5467] The base sequence of this gene derived from human was determined.
【実施例 8】  [Example 8]
【0 0 6 0】  [0 0 6 0]
(ヒ ト由来の本遺伝子の大腸菌発現用ベクター構築）  (Construction of E. coli expression vector for this gene derived from human)
実施例 6で得られたヒ ト由来の本遺伝子を含むプラスミ ド 0.1 /z g、 配列番号 1 3で示さ れる塩基配列からなるプライマー 7 20pmol、 配列番号 1 4で示される塩基配列からなる プライマ一 8 20pmol、 TaKaRa Εχ-Taqポリメラーゼ (宝酒造社) 2U、 TaKaRa Ex-Taqポ リメラ一ゼ添付のバッファー 及び TaKaRa Εχ-Taq ポリメラーゼ添付の dNTP mixture(2.5mM)4 Lを含む 50 μ Lの反応液を調製した。 PCRは、 まず 94°Cで 30秒間、 次いで 65°Cで 30秒問、 更に 72°Cで 2分問からなる保温サイクルを 50回繰り返し、 最後 に 72°Cで 5分間保温する条件にて行われた。 PCR産物を制限酵素 Ndelと BamHIにて消 化し、 ァガロース電気泳動で約 1.2kbpを示す PCR産物を回収した。 回収された PCR産物 を pET16b (Novagen 社製）へクローニングしたのち、 通常の方法に従って、 大腸菌 BL2l(DE3)plysS(Novagen社製)に形質転換した。  Plasmid 0.1 / zg containing the human gene derived from Example 6 obtained in Example 6, primer consisting of the base sequence shown in SEQ ID NO: 13 20 pmol, primer consisting of the base sequence shown in SEQ ID NO: 14 8 Prepare a 50 μL reaction solution containing 20 pmol, TaKaRa Εχ-Taq polymerase (Takara Shuzo) 2 U, buffer attached to TaKaRa Ex-Taq polymerase and dNTP mixture (2.5 mM) attached to TaKaRa Εχ-Taq polymerase. did. PCR is performed at 94 ° C for 30 seconds, then at 65 ° C for 30 seconds, and then at 72 ° C for 2 minutes, repeated 50 times, and finally at 72 ° C for 5 minutes. It was conducted. The PCR product was erased with restriction enzymes Ndel and BamHI, and the PCR product showing about 1.2 kbp was recovered by agarose electrophoresis. The recovered PCR product was cloned into pET16b (Novagen), and transformed into E. coli BL2l (DE3) plysS (Novagen) according to the usual method.
【実施例 8】  [Example 8]
【0 0 6 1】  [0 0 6 1]
(ヒ ト由来の本タンパクの精製）  (Purification of the human protein)
実施例 7で作成した組換え体大腸菌 0iMO64-pET16b/BL21)をアンピシリン 50ug/ml を 含む LB培地（トリプトン lw/v%、 酵母エキス 0.5w/v%、 NaCl O.5w/v%)30mLに接種後、 37°Cで一晩振とう培養した。 培養後の培養液 15mLをアンピシリン 50ug/mlを含む LB培 地（トリプトン lw/v%、 酵母エキス 0.5w/v%、 NaCl O.5w/v%)500mLに接種後、 37°Cで 2 時問培養後、 IPTG (ィソプロピル一 ]3— D—チォガラタ トビラノシド)を終濃度 0.5mMに なるように添加し、 37°Cでさらに 4時間培養した。 遠心分離 ( 8 0 0 O xg, 5分間， 4°C)によ り菌体を回収し、 菌体を超音波破砕した後、 遠心分離（1 2 0 0 O xg, 15 分問， 4°C)を行 いその上清を得た。 得られた上清 lOmL をニッケルを結合させたァフィ二ティ担体 lmL(Ni-NTA、 QIAGEN社製)を充填したカラムに通した。 25mMイミダゾールを含む洗 浄液 (25mMィミダゾール、 50mM Tris pH8.0)で力ラムを洗浄した後、 400mMィミダゾ一 ルを含む溶出液 (400mMィミダゾール、 50mM Tris pH8.0)で溶出させ、 ァフィ二ティ精製 M064タンパクを得た。 ァフィ二ティ精製タンパク M064を 20mM Tris pH7.0溶液にバ ッファ一置換後、 陽イオン交換クロマトカラム (S-Sepharose、 アマシャムフアルマシア社 製)に吸着させ、 20mM Tris pH7.0、 1M NaCLにより塩濃度勾配を上げることにより溶出 させ、 最終精製 hM064タンパクを得た。 Recombinant Escherichia coli 0iMO64-pET16b / BL21 prepared in Example 7 was added to 30 mL of LB medium (tryptone lw / v%, yeast extract 0.5 w / v%, NaCl O.5 w / v%) containing 50 ug / ml of ampicillin. After inoculation, the cells were cultured overnight at 37 ° C with shaking. Inoculate 15 mL of the culture solution after culturing to 500 mL of LB medium (tryptone lw / v%, yeast extract 0.5 w / v%, NaCl O.5 w / v%) containing ampicillin 50 ug / ml, then at 37 ° C for 2 hours After incubation, IPTG (isopropyl 1) 3-D-thiogalatatoviranoside) was added to a final concentration of 0.5 mM, and the mixture was further cultured at 37 ° C for 4 hours. The cells are collected by centrifugation (800 ° Oxg, 5 minutes, 4 ° C), sonicated, and then centrifuged (120 ° O xg, 15 minutes, 4 ° C) was performed to obtain the supernatant. The obtained supernatant lOmL was passed through a column packed with lmL affinity carrier lmL (Ni-NTA, manufactured by QIAGEN) bound with nickel. After washing the power ram with a wash solution containing 25 mM imidazole (25 mM imidazole, 50 mM Tris pH 8.0), eluting with an eluent containing 400 mM imidazole (400 mM imidazole, 50 mM Tris pH 8.0) Purified M064 protein was obtained. Affinity-purified protein M064 was buffer-substituted with 20 mM Tris pH 7.0 solution, and then a cation exchange chromatography column (S-Sepharose, Amersham Almasia) And then eluted by raising the salt concentration gradient with 20 mM Tris pH 7.0, 1 M NaCL to obtain the final purified hM064 protein.
【実施例 9】  [Example 9]
【 0 0 6 2】  [0 0 6 2]
(D I〇(食餌性肥満誘導)マウスの作成）  (DIO creation (dietary obesity induction) mice)
ICRマウス個体について、 それぞれを体重平均値が同程度になるように普通食群 (Std食 群)と高脂肪 ·高庶糖食群 (H H食群)に分け、 約 1 0週齢以降からは 8週問、 普通食（ D12328(Research Diets社製)）もしくは、 高脂肪食 (Dl2331(Research Diets社製))を自 由に摂取させた。 飼育する際の環境条件は室温 20〜26° (：、 湿度 40〜70%、 明暗各 12時間 ( 照明:午前 6時〜午後 6時)、 換気回数 12回/時に維持された飼育室にてステンレス製六連 ケージ (W:750 X D;100 X H:100mm)内での個別飼育を実施した。  Individual ICR mice are divided into a normal diet group (Std diet group) and a high-fat / high-sugar diet group (HH diet group) so that the average weight values are about the same. Weekly, regular diet (D12328 (Research Diets)) or high fat diet (Dl2331 (Research Diets)) was freely taken. Environmental conditions for breeding are room temperature 20-26 ° (:, humidity 40-70%, light and darkness 12 hours each (lighting: 6 am-6 pm), in a breeding room maintained at 12 / hour ventilation Individual breeding was performed in a stainless steel six-cage cage (W: 750 XD; 100 XH: 100 mm).
【実施例 1 0】  [Example 10]
【0 0 6 3】  [0 0 6 3]
(ヒ ト由来本タンパクの肥満動物への投与実験一 1)  (Experimental experiment on administration of this protein derived from humans to obese animals 1)
実施例 9に記載される方法により構築された DIO (食餌性肥満誘導)マゥス (雄性） 1 0匹を 1群とした。 これに、 実施例 8で調製されたヒ ト由来本タンパクの PBS緩衝溶液を 、 被験物質純分で lmg/kg—体重 x2回/日になるように、 2週問腹腔内投与した。  One group of 10 DIO (dietary obesity induced) mice (male) constructed by the method described in Example 9 was used. To this, a PBS buffer solution of the protein derived from human prepared in Example 8 was intraperitoneally administered for 2 weeks so that the pure amount of the test substance was 1 mg / kg-body weight × 2 times / day.
投与 2週間後に耐糖能試験およびィンスリン応答性試験を実施した。 Two weeks after the administration, a glucose tolerance test and an insulin responsiveness test were performed.
耐糖能試験およびィンスリン応答性試験については以下の方法で行った。 The glucose tolerance test and the insulin responsiveness test were performed by the following methods.
(1)糖負荷試験 (OGTT):実施例 9に示した長期間食餌負荷を実施した後、 前日から摂 水下で 18〜24時間絶食した動物にグルコース溶液 (19/kg、 10mL/kg)を経口投与 (ディス ポーザブル金属性ゾンデを装着したポリプロピレン製デイスポーザブル注射筒）した。 グ ルコース投与前、 グルコース投与後 20、 60および 120分後に動物の眼窩静脈叢からへパリ ン加キヤビラリ一を用いて、 約 50 L血液を採取し、 アン卜センス 11(ダイキン工業株式 会社)を用いて酸素電極法にて全血の血糖値を測定した。 (1) glucose tolerance test (OGTT): After performing long-term diet load shown in Example 9, 18 to 24 hours fasted animals glucose solution in Underwater feeding from the previous day (19 / kg, 10mL / k g ) Was orally administered (disposable disposable syringe made of polypropylene equipped with a disposable metallic sonde). Before administration of glucose, 20, 60 and 120 minutes after glucose administration, about 50 L of blood was collected from the orbital venous plexus of the animal using a pilled cabilla, and Annice Sense 11 (Daikin Industries, Ltd.) was collected. The blood glucose level of whole blood was measured by the oxygen electrode method.
(2)ィンスリン負荷試験 (ιρΙΤΤ):上記 OGTTを実施後、 2週間程度間隔をおいて実施し た。 前目から摂水下で 18〜24時間絶食した動物にィンスリン溶液 (0.35U/kg、 lOmL/kg) を腹腔内投与 (注射針を装着したポリプロピレン製デイスポーザブル注射筒）した。 ィン スリン負荷直前、 30、 60および 120分後に上記 (1)の方法にて血糖値を測定した。 (2) Insulin load test (ιρΙΤΤ): After the above OGTT, the test was conducted at intervals of about 2 weeks. Insurin solution before th 18-24 hours fasted animals under water consumption (0.35U / k g, lOmL / kg) intraperitoneal administration (polypropylene Day Supoza Bull syringe and the needle is mounted) the. The blood glucose level was measured by the above method (1) immediately before the insulin load, 30, 60 and 120 minutes later.
(結果)  (Result)
糖負荷試験ではヒ 卜由来の本タンパクの投与マウス群はりん酸緩衝液投与群に比して、 グルコース投与 120分後に有意な血糖降下作用が認められた (表 1)。 また、 インスリン 負荷試験を実施し同様に比較したところ、 ヒ ト由来の本タンパクの投与マウス群はりん酸 緩衝液投与群に比して、 グルコース投与 60分および 90分後に有意な血糖降下作用が認 められた (表 2)。 これらの結果から、 肥満マウス群 (食餌性肥満誘導マウス)に対して、 本蛋 白質の投与により耐糖能およびィンスリン応答性が改善することが示された。  In the glucose tolerance test, mice treated with coconut-derived protein showed a significant hypoglycemic effect 120 minutes after glucose administration compared to the phosphate buffer administration group (Table 1). In addition, when an insulin tolerance test was performed and compared in the same manner, the group of mice treated with the human protein showed a significant hypoglycemic effect 60 and 90 minutes after glucose administration compared to the phosphate buffer group. It was recognized (Table 2). From these results, it was shown that glucose tolerance and insulin responsiveness were improved by administration of this protein in the obese group of mice (dietary obesity-induced mice).
【 0 0 6 4】  [0 0 6 4]
l ¾ 1 J  l ¾ 1 J
Figure imgf000016_0001
Figure imgf000016_0001
p <0.05リん酸緩衝液投与群 【 0 0 6 5】 p <0.05 phosphate buffer administration group [0 0 6 5]
l表 2】  l Table 2]
Figure imgf000017_0001
Figure imgf000017_0001
P<0 05りん酸锾衝液投与群  P <0 05 phosphate phosphate group
【実施例 1 1】  [Example 1 1]
【 0 0 6 6】  [0 0 6 6]
(肥満動物への本遺伝子の投与実験）  (Experimental administration of this gene to obese animals)
肥満動物への本遺伝子の投与はアデノウイルスにより行うことができる。 アデノウィル スの作製は Adenovirus Expression Kit (宝酒造社)により行う。  Administration of this gene to obese animals can be performed by adenovirus. Adenovirus is prepared using Adenovirus Expression Kit (Takara Shuzo).
i)コスミ ドベクターへの本遺伝子の揷入 i) Insertion of this gene into the cosmid vector
実施例 4で調製されたマウス M0 6 4遺伝子のセンス鎖をコスミ ドベクター pAxCAwt (宝 酒造社)の Swa l部位に揷入し、 pAxCAwt-mM064 を得る。 得られた PAxCAwt-mM064 を; Iパッケージングキット GigapackXL(Stratagene社)を用いてインビトロパッケージン グを行い大腸菌 DH5 aに感染させる。 感染させた大腸菌を、 50ml 50 g/mlアンピシリ ン含有 LB培地で培養し、 Lambda DNA purification Kit (東洋紡社)によりコスミ ド DNA を大量調製する。 The sense strand of mouse M0 64 gene prepared in Example 4 is inserted into the Swal site of the cosmid vector pAxCAwt (Takara Shuzo) to obtain pAxCAwt-mM064. The obtained P AxCAwt-mM064 is infected with E. coli DH5a by in vitro packaging using I packaging kit GigapackXL (Stratagene). Infected E. coli is cultured in 50 ml 50 g / ml ampicillin-containing LB medium, and a large amount of cosmid DNA is prepared using Lambda DNA purification Kit (Toyobo).
ii)組換えアデノウィルスの作製 ii) Production of recombinant adenovirus
293細胞 (宝酒造社)を 10%FCS添加 D-MEM培地 (宝酒造社)で 37°C、 5%二酸化炭素下で 培養する。 i)で調製された PAxCAwt-mM0648 μ gと制限酵素処理済み DNA-TPC (宝酒造） 5 μ 1とを混合し、 当該混合物を用いてリン酸カルシウム法にて 293細胞にコトランスフエ クシヨンを行う。当該細胞をそのまま 18時間培養後、培地を 10%FCS添加 D-MEM培地 (宝 酒造社)と交換し、 さらに 12時間培養する。 培養終了後、 後細胞をディッシュから剥がすこ とにより、回収された細胞懸濁液と 293細胞を混ぜてさらに 10%FCS添カ卩 D-MEM培地 (宝 酒造社)にて培養をつづけ、 293細胞が完全に死滅した時点で培養液をドライアイスにて急 凍する。 凍結融解 6回後、 5000rpm5分間遠心することにより得られた上清を 1次組換え アデノウィルス液 (AxCAwt-mM064)として保存する。 Incubate 293 cells (Takara Shuzo) in D-MEM medium (Takara Shuzo) with 10% FCS at 37 ° C and 5% carbon dioxide. Mix 648 μg of P AxCAwt-mM0648 prepared in i) with 5 μ 1 of DNA-TPC (Takara Shuzo) that has been treated with restriction enzyme, and perform cotransfusion on 293 cells using the mixture by the calcium phosphate method. The cells are cultured as they are for 18 hours, and then the medium is replaced with 10% FCS-added D-MEM medium (Takara Shuzo) and further cultured for 12 hours. After completion of the culture, the cells are detached from the dish, and the recovered cell suspension and 293 cells are mixed and further cultured in 10% FCS-supplemented D-MEM medium (Takara Shuzo). When the cells are completely dead, freeze the culture medium with dry ice. After freezing and thawing 6 times, the supernatant obtained by centrifugation at 5000 rpm for 5 minutes is stored as a primary recombinant adenovirus solution (AxCAwt-mM064).
iii)組換えァデノウィルスの確認および力価測定 iii) Confirmation and titration of recombinant adenovirus
293細胞 (宝酒造社)に前項で調製された 1次組換えウィルス液を 10 μ 1加え、 これに 5%F CS添加 D MEM培地 (宝酒造社)を加えた後、 当該混合物を 37°C、 5%二酸化炭素下で 1時 間培養する。 さらに当該混合物に 5%FCS添加 D瑚 EM培地 (宝酒造社)を加えた後、 これ を 37°C、 5%二酸化炭素下で培養する。 3 日間培養後、 ウィルス感染させた 293細胞 (宝酒 造社)の培養液を回収し、 これをドライアイスにて急凍する。 凍結融解 6回後、 5000rl)m5 分問遠心することにより得られた上淸を 2 次組換えアデノウイルス液として保存する。 一 方、 前記操作により同時に得られた沈殿 (細胞)も回収し、 ドライアイスにて急凍する。 回収 された細胞に 10xTNE(500mM トリスー塩酸 (pH=7.5)、 1M NaCl、 lOOmM EDTA)40 μ 1 及び proteinaseK(20mg πύ)4 μ 1、 滅菌蒸留水 356 μ 1を加え、 当該混合液を Voltexでよ く懸濁した。 得られた懸濁物に、 10%8Ο34 μ 1をカ卩え、 これを 50°Cで 1時間保温する。 そ の後、 フエノール/クロ口ホルム/イソァミルアルコーノレ (二ツボンジーン社)処理後、 ェタノ ール沈殿を行い、 得られた DNAペレッ トを風乾させた後、 20 g/ml RNaseA (二ツボンジ ーン社)を含む滅菌蒸留水 50 μ 1に溶解することにより、組換えアデノウィルス DM溶液を 得る。 得られた組換えアデノウイルス DNA溶液を Xhol 5U (宝酒造社)にて切断し、 目的の 断片 (0.48 Kbp)を確認する。 続いてこの組換えアデノウィルス DNAを前記操作を繰り返す ことにより継代培養する。 このようにしてし、 高力価組換えウィルスを作製する。 力価の 測定は既存の方法 (特開平 7-298877)により測定する。 Add 10 μl of the primary recombinant virus solution prepared in the previous section to 293 cells (Takara Shuzo), add 5% F CS-added D MEM medium (Takara Shuzo), add the mixture to 37 ° C, Incubate for 1 hour in 5% carbon dioxide. Furthermore, 5% FCS-added D-EM medium (Takara Shuzo) is added to the mixture, and this is cultured at 37 ° C under 5% carbon dioxide. After culturing for 3 days, collect the culture solution of virus-infected 293 cells (Takara Shuzo) and freeze it with dry ice. After freezing and thawing six times, store the upper eyelid obtained by centrifugation at 5000 rl) m5 for 2 minutes as a secondary recombinant adenovirus solution. On the other hand, the precipitate (cells) obtained at the same time by the above operation is also collected and rapidly frozen with dry ice. Add 10xTNE (500 mM Tris-HCl (pH = 7.5), 1 M NaCl, lOOmM EDTA) 40 μ 1 and proteinase K (20 mg πύ) 4 μ 1 and sterilized distilled water 356 μ 1 to the collected cells. Suspended well. Add 10% 8-34 μ 1 to the resulting suspension and incubate at 50 ° C for 1 hour. Then, after treatment with phenol / chloroform / isoamyl alcohole (Nitsubon Gene), ethanol precipitation was performed, and the resulting DNA pellet was air-dried, followed by 20 g / ml RNaseA (Nibonbonji). Recombinant adenovirus DM solution is obtained by dissolving in 50 μl of sterilized distilled water. The obtained recombinant adenovirus DNA solution is cleaved with Xhol 5U (Takara Shuzo) to confirm the target fragment (0.48 Kbp). Subsequently, this recombinant adenovirus DNA is subcultured by repeating the above procedure. In this way, a high titer recombinant virus is produced. Titer The measurement is performed by an existing method (Japanese Patent Laid-Open No. 7-298877).
iv)肥満動物への本遺伝子の投与 iv) Administration of this gene to obese animals
肥満動物である 10週齢の雄性の KKAyマウス（目本クレア)に対して、 アデノウィル ス投与量 lxlOio PFU(1群 10匹)の割合になるように、 前項により作製された組換えアデ ノウィルスを生理食塩水で希釈することにより調製する。 本水溶剤を実施例 9で作成した DIO (食餌性肥満誘導)マウスに、 マウス 1匹当たり 0.2mlずつシリンジを用いて腹腔內に 投与する。 投与 2週間後に被験肥満動物である DIOマウスに対して耐糖能試験およびィン スリン応答性試験を実施する。  Recombinant adenovirus prepared in the previous section so that the proportion of adenovirus dose is lxlOio PFU (10 mice per group) to 10-week-old male KKAy mice (special Claire) that are obese animals Is prepared by diluting with saline. This aqueous solvent is administered to the DIO (dietary obesity-inducing) mouse prepared in Example 9, 0.2 ml per mouse using the syringe. Two weeks after administration, DIO mice, which are test obese animals, are subjected to glucose tolerance test and insulin responsiveness test.
【実施例 12】  [Example 12]
【0067】  [0067]
(形質転換非ヒ ト動物の作製:形質転換マゥスの作製）  (Production of transformed non-human animals: Production of transformed mice)
i)導入遺伝子断片の調製 i) Preparation of transgene fragment
実施例 4で調製されたマウス M064遺伝子を、 マウス aP2プロモータ一領域 (約 5. 4kb)を有するプラスミ ド (Shimomura, I et al,， Genes & Develop memt, 12， 3182  The mouse M064 gene prepared in Example 4 was transformed into a plasmid having a mouse aP2 promoter region (about 5.4 kb) (Shimomura, I et al, Genes & Develop memt, 12, 3182).
(1998))に挿入することにより、 プラスミ ド p B 1 u e S K/maP 2 p r o/mMO (1998)) to insert the plasmid p B 1 u e S K / maP 2 p ro / mMO
64/p o 1 y Aを得る。  Get 64 / p o 1 y A.
得られた p B l u e SK/maP 2 p r o/mMO 64/p o 1 y Aを H i n dill及び N o t lで消化することにより、 マウス a P 2プロモーター領域、 マウス由来の本遺伝子 及び S V 40 Eポリ Aを含む断片 (約 7.1 kb)を約 5μ g取得する (以下、 本導入遺 伝子と記す。 ）。 本導入遺伝子を lOmM卜リス一塩酸（pH8.5)、 1 mM EDT  The resulting p Blue SK / maP 2 pro / mMO 64 / po 1 y A was digested with Hindill and Notl to obtain the mouse a P 2 promoter region, the mouse-derived gene and SV 40 E poly A Obtain about 5 μg of a fragment (approximately 7.1 kb) containing the protein (hereinafter referred to as this introduced gene). This transgene was treated with lOmM squirrel monohydrochloride (pH 8.5), 1 mM EDT
A · 2Na(pH8.5)で O.l g/μ 1となるように調製することで本導入遺伝子 This transgene can be prepared by adjusting to O.l g / μ 1 with 2Na (pH 8.5).
調製液を得る。 Obtain the preparation.
11)受精卵の採取 11) Collection of fertilized eggs
C 57 B L/6 J系の雌性及び雄性のマウス（8週齢以上)を用い、 5単位の妊馬血清 性ゴナドトロピンとヒ ト繊毛性ゴナドトロピンとの両者の腹腔内投与により過剰排卵を惹 起し、 体外受精によって前核期受精卵を得る。 該受精卵は、 卵丘細胞などを除去するため に、 mWM培地（5.14g/L Na C l、 0.36 g/L KCL、 0. 16 g/L  C 57 BL / 6 J strains of female and male mice (8 weeks of age or older) induced hyperovulation by intraperitoneal administration of 5 units of pregnant mare serum gonadotropin and human ciliary gonadotropin. Pronuclear fertilized eggs are obtained by in vitro fertilization. The fertilized egg was treated with mWM medium (5.14 g / L Na Cl, 0.36 g / L KCL, 0.16 g / L to remove cumulus cells and the like.
KH2 P04、 0.53 g/L 乳酸 Ca ' 5H2〇、 0. 29 g/L Mg S O 4/7 HH 20、 0. 1 9g/L NaHC03、 37mg/L EDTA/2Na、 1 g/L グル コース、 3. 7ml/L 乳酸 Na、 35 mgLピルビン酸 N a、 3 g/L ゥシ血  KH2 P04, 0.53 g / L Lactic acid Ca '5H20, 0.29 g / L MgSO 4/7 HH 20, 0.19 g / L NaHC03, 37 mg / L EDTA / 2Na, 1 g / L Glucose, 3 7ml / L Na lactate, 35mgL pyruvate Na, 3g / L
清アルブミン、 80 mg/L ペニシリン、 50 mg/L ス トレプトマイシン、 5 mg /L フヱノールレツド)で洗浄する。 Wash with clean albumin, 80 mg / L penicillin, 50 mg / L streptomycin, 5 mg / L phenol red).
iii)受精卵への本導入遺伝子の注入 iii) Injection of this transgene into fertilized eggs
マイク口インジェクションシステムは、 マイクロマニピュレーター装置（ライカ製）と ホフマン倒立型顕微鏡 (ッァィス製)から構成される。 前項に記載される本導入遺伝子調 製液を約 500〜約 1000コピー/ μ 1に調製し、 13, 000 r pmX 3分間遠心分 離することにより得られた上清を導入本遺伝子溶液とする。 マイクロマニピュレータ一の インジェクションチャンバ一内で、 前項 ii)に記載される受精卵を支持ピぺットで固定 し、 注入用ピぺットに入れた前記の本導入遺伝子調製液を雄性前核に約 2 μ 1注入する。 注入後、 mWM培地で 5%C02存在下、 37 °Cで 12〜 16時問培養する。 2細胞期に 発生した胚を仮親の卵管に移植する。  The microphone mouth injection system consists of a micromanipulator (Leica) and an Hoffman inverted microscope (Fisse). Prepare the transgene preparation solution described in the previous section at about 500 to about 1000 copies / μ1, and use the supernatant obtained by centrifugation at 13,000 rpm 3 min as the transgene solution. . In the injection chamber of the micromanipulator, the fertilized egg described in the previous section ii) is fixed with a support pipette, and the introduced transgene preparation solution in the injection pipette is used as the male pronucleus. Inject approximately 2 μ1. After the injection, incubate with mWM medium in the presence of 5% C02 at 37 ° C for 12-16 hours. Embryos that develop in the 2-cell stage are transferred to the oviduct of the temporary parent.
iv)受精卵の仮親への移植 iv) Transplantation of fertilized eggs to temporary parents
前項 iii)に記載される胚を、 予め、 移植用に準備した同系の偽妊娠状態の雌性マウ スの左右の卵管に 1匹あたり 20〜25個注入する。  20-25 embryos per mouse are injected into the left and right oviducts of a syngeneic pseudopregnant female mouse prepared for transplantation in advance.
V)形質転換マウスの選抜 V) Selection of transformed mice
受精卵を移植した雌マウスから、 約 3週間後に仔を得る。 仔が 6週齢程度になったら尾 の一部を切り取って染色体 DNAを抽出し、 抽出された DNA中に本導入遺伝子由来の断 片が存在するかについて PCR法により調べる。 染色体 DNAは通常の方法 (例えば、 Sa mbrook J.,Frisch E.F.,Maniatis T.著、 モレキュラークローニング第 2版 (Molecular Cloning 2nd edition), コールドスプリングハーバ一ラボラ卜リ一発行 (Cold S pring Harbor Laboratory press)等に記載されている方法)により抽出する。 得られた目 染色体 DNA5n gを銃型として、 配列番号 1 5と配列番号 16に示される塩基配列から なる DNA各 1 Opmolをプライマーとして、 ExTaqポリメラーゼ 2. 5ユニット (宝酒造製)を用いて、 94°Cで 2分問、 58°Cで 2分間、 72 °Cで 2分間の保温を 1サ ィクルとしてこれを 1サイクル、 次に 94°Cで 1分間、 58°Cで 1分問、 72°Cで 1分間 の保温を 1サイクルとしてこれを 55サイクル、 最後に 72 °Cで 5分間保温の条件で PC R反応を行う。 当該反応により得られる PCR反応産物をァガロースゲル電気泳動に供す る。 II的断片が確認された個体を、 染色体中に本導入遺伝子が揷入された形質転換マウス (以下、 本形質転換マウスと記す。 ）であると判断する。 Offspring are obtained about 3 weeks later from female mice transplanted with fertilized eggs. When the pup is about 6 weeks old, a part of the tail is cut out to extract chromosomal DNA, and the transgene-derived fragment is extracted into the extracted DNA. Check for presence of fragments by PCR. Chromosomal DNA is prepared by conventional methods (e.g., Sam Brook J., Frisch EF, Maniatis T., Molecular Cloning 2nd edition), Cold Spring Harbor Laboratory press (Cold Spring Harbor Laboratory press). ) Etc.). Using 5 ng of the obtained chromosome DNA as a gun, and using ExTaq polymerase 2.5 units (manufactured by Takara Shuzo Co., Ltd.) using ExTaq polymerase 2.5 units (manufactured by Takara Shuzo Co.) 2 cycles at ° C, 2 minutes at 58 ° C, 2 minutes at 72 ° C for 1 cycle, then one cycle, then 94 ° C for 1 minute, 1 minute at 58 ° C, 72 Incubate for 1 minute at 1 ° C for 55 minutes, and finally perform a PCR reaction at 72 ° C for 5 minutes. The PCR reaction product obtained by the reaction is subjected to agarose gel electrophoresis. Individuals with confirmed II-like fragments are judged to be transformed mice in which this transgene is inserted into the chromosome (hereinafter referred to as this transformed mouse).
V1)本導入遺伝子コピー数の解析 V 1 ) Analysis of copy number of this transgene
次に、 本形質転換マウスにおける本導入遺伝子のコピー数について、 通常のサザンプロ ッティング法 (例えば、 Sambrook J., Frisch E.F., ManiatisT.著、 モレキュラークロ 一二ング第 2版 (Molecular Cloning 2nd edition), コ一ルドスプリングハ一バー ラボラトリー発行 (Cold Spring Harbor Laboratory press)等に記載されている方法） により調べる。 前項で得られたプラスミ ド p B 1 u e SK/m a P 2 p r o/mMO 64 /p o 1 yAを B amH Iで消化する。 得られる約 0. 7 Kbの断片を市販の放射能標 識キット (Ra n d om P r i me L a b e l i n g K i t, ベーリンガ一マンノ、 ィム製)を用いて添付のプロトコールに従って 32P標識し、 検出プローブとして使用する (以下、 本検出プローブと記す。 ；)。 本検出プローブは 100°Cで 10分加熱した後に急 冷して使用する。 前項に記載される方法に従って抽出した染色体 DNAを E c oR 1、 B amHl、 H i n d i I Iなどの制限酵素で切断し、 ァガロースゲル電気泳動する。 電気 泳動後のゲルを 1. 5M Na CL、 0. 1 5 Mクェン酸三ナトリウムを含む溶液 (以 下、 10 X S S Cと記す。 ）で洗浄した後、 市販のナイロンメンブラン Hy b o b d—N (アマシャムフアルマシア製)にキヤビラリ一法でブロッティングする。 該メンブランを ポリエチレン袋に入れ、 変性サケ精子 DNAを 1 O O^g/m l となるように添加した D I G EASY Hy b溶液 (ベーリンガ一マンハイム製〉 を加えて、 50°Cで 2時間保 温する。 袋內の溶液を除去し、 新たに、 変性サケ精子 DN Aを 100 i g/m 1 となるように 添加した D I G EASY Hy b溶液 2〜3m 1を注入する。 さらに、 本検出プローブ 1 m 1以下を添加し、 50 °Cで約 15時間保温する。 保温後、 メンブランを取り出し て、 0. 1%ラウリル硫酸ナトリウム (以下、 SDSと記す。 ）を含む、 10X S SCを 5倍に希釈した溶液中で室温下 15分間保温する。 メンブランを取り出し、 新たな 10 X S SCを 5倍に希釈した溶液中で保温する操作をさらに 1〜2回繰り返す。 さらに、 0. 1%SDSを含む、 10 X S S Cを 100倍に希釈した溶液中で、 50〜 68° (：、 30分 間保温する。 メンブランを取り出し、 余分な水分を除去した後、 イメージングプレート（ BAS 3— 2040。 富士フィルム製)を露光させる。 露光後のイメージングプレートを イメージアナライザー (フジフィルム製)で分析し、 メンブラン上のシグナルを検出する。 本導入遺伝子を導入しないマウス (以下、 本野生型マウスと記す。 ）の染色体 DNAを用いる こと以外は前記と同様にサザンプロッティング法を行い、 メンブレン上のシグナルを検出 する。 本形質転換マウスと本野生型マウスのシグナルを比較し、 本形質転換マウスにのみ 検出されるシグナルの強度から、 本遺伝子の導入コピー数の多少を評価する。  Next, regarding the copy number of this transgene in this transformed mouse, the usual Southern plotting method (e.g., Sambrook J., Frisch EF, Maniatis T., Molecular Cloning 2nd edition), Check by the method published in Cold Spring Harbor Laboratory press). The plasmid pB1ueSK / maP2pro / mMO64 / po1yA obtained in the previous section is digested with BamHI. The obtained approximately 0.7 Kb fragment was labeled with 32P using a commercially available radiolabeling kit (Random Prime Labeling Kit, Boehringer Manno, Im) according to the attached protocol. (Hereinafter referred to as the present detection probe;). This detection probe is heated at 100 ° C for 10 minutes and then cooled before use. Chromosomal DNA extracted according to the method described in the previous section is cleaved with a restriction enzyme such as EcoR1, BamHl, HindiII, and subjected to agarose gel electrophoresis. The gel after electrophoresis was washed with a solution containing 1.5 M Na CL and 0.15 M trisodium citrate (hereinafter referred to as 10 XSSC), and then commercially available nylon membrane Hy bobd-N (Amersham Blotting with Armavier). Place the membrane in a polyethylene bag, add DIG EASY Hy b solution (manufactured by Boehringer Mannheim) with denatured salmon sperm DNA added to 1 OO ^ g / ml, and incubate at 50 ° C for 2 hours. Remove the bag solution and inject 2 to 3 ml of DIG EASY Hyb solution to which denatured salmon sperm DN A is added to a concentration of 100 ig / m 1. In addition, this detection probe is less than 1 ml And incubate for about 15 hours at 50 ° C. After incubation, remove the membrane and dilute 10X S SC 5 times containing 0.1% sodium lauryl sulfate (hereinafter referred to as SDS). Incubate for 15 minutes at room temperature Remove the membrane and incubate in a solution diluted with 10-fold fresh 10 XS SC one or two more times, and 0.1 XSSC containing 10 XSSC Incubate the membrane in a 100-fold diluted solution at 50 to 68 ° (:, 30 minutes. Remove and remove excess water, then expose the imaging plate (BAS 3-2040, manufactured by Fuji Film) Analyze the exposed imaging plate with an image analyzer (manufactured by Fuji Film) to detect the signal on the membrane The Southern blotting method is used to detect signals on the membrane in the same manner as described above, except that the chromosomal DNA of a mouse into which this transgene has not been introduced (hereinafter referred to as this wild type mouse) is used. And the wild type mouse signal, and the number of introduced copies of this gene is evaluated from the intensity of the signal detected only in the transformed mouse.
【配列表フリーテキスト】  [Sequence Listing Free Text]
【0068】  [0068]
配列番号 7 ： PCR用プライマー SEQ ID NO: 7: PCR primer
配列番号 8 ： PCR用プライマー SEQ ID NO: 8: PCR primer
配列番号 9 ： PCR用プライマー 配列番号 10： PCR用プライマ一 配列番号 11:PCR用プライマ一 配列番号 12:PCR用プライマー 配列番号 13:PCR用プライマ一 配列番号 14:PCR用プライマー Sequence number 9: Primer for PCR SEQ ID NO: 10: PCR primer SEQ ID NO: 11: PCR primer SEQ ID NO: 12: PCR primer SEQ ID NO: 13: PCR primer SEQ ID NO: 14: PCR primer

Claims

請 求 の 範 囲 The scope of the claims
【請求項 1】 [Claim 1]
下記のいずれかのアミノ酸配列からなる蛋白質又はそのフラグメントを有効成分として 含有する、 糖代謝関連疾患の治療剤又は予防剤： A therapeutic or prophylactic agent for sugar metabolism-related diseases, comprising as an active ingredient a protein comprising any of the following amino acid sequences or a fragment thereof:
〈アミノ酸配列群 >  <Amino acid sequence group>
(a)配列番号 2で示されるァミノ酸配列、  (a) the amino acid sequence represented by SEQ ID NO: 2,
(b)配列番号 2で示されるァミノ酸配列において、 1もしくは複数のァミノ酸が欠失、 付加、 揷入もしくは置換されたァミノ酸配列、  (b) in the amino acid sequence represented by SEQ ID NO: 2, in which one or more amino acids are deleted, added, inserted or substituted,
(c)配列番号 2で示されるァミノ酸配列における第 22番目のアミノ酸から第 414番 目のァミノ酸で示されるアミノ酸配列、  (c) the amino acid sequence represented by the 22nd to 414th amino acids in the amino acid sequence represented by SEQ ID NO: 2,
(d)前記 (c)のアミノ酸配列において、 そのアミノ末端にメチォニンが付加されたァ ミノ酸配列、  (d) in the amino acid sequence of (c), an amino acid sequence in which methionine is added to the amino terminus;
(e)配列番号 2で示されるァミノ酸配列と 80%以上の配列同一性を有するァミノ酸配 列、  (e) an amino acid sequence having 80% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 2,
(£)配列番号 1で示される塩基配列における第 224番目のヌクレオチドから第 146 (£) From the 224th nucleotide to the 146th nucleotide in the base sequence represented by SEQ ID NO: 1.
5番目までのヌクレオチドで示される塩基配列を有する DNAによりコードされるァミノ 酸配列、 An amino acid sequence encoded by DNA having a base sequence represented by the 5th nucleotide;
(g)配列番号 1で示される塩基配列における第 224番目のヌクレオチドから第 146 (g) from the 224th nucleotide to the 146th nucleotide in the nucleotide sequence represented by SEQ ID NO: 1
5番目までのヌクレオチドで示される塩基配列を有する DNAと 80%以上の配列同一性 を有する塩基配列を有する DNAによりコードされ、 かつ糖代謝亢進活性を有するアミノ 酸配列、 An amino acid sequence encoded by a DNA having a base sequence represented by nucleotides up to the 5th nucleotide and having a base sequence having a sequence identity of 80% or more and having a glucose metabolism-enhancing activity;
(h)配列番号 1で示される塩基配列における第 224番目のヌクレオチドから第 146 (h) from the 224th nucleotide to the 146th nucleotide in the nucleotide sequence represented by SEQ ID NO: 1.
5番目までのヌクレオチドで示される塩基配列を有する DNAに対し相補性を有する DN Aと、 ストリンジヱントな条件下でハイブリダィズする DNAによりコードされ、 かつ糖 代謝亢進活性を有するァミノ酸配列。 An amino acid sequence encoded by DNA complementary to DNA having a base sequence represented by nucleotides up to the fifth nucleotide and DNA hybridized under stringent conditions and having a glucose metabolism-enhancing activity.
【請求項 2】  [Claim 2]
請求項 1に記載のアミノ酸配列群から選択されるアミノ酸配列からなる蛋白質をコード する遺伝子を有効成分として含有する、 糖代謝関連疾患の治療剤又は予防剤。 A therapeutic or prophylactic agent for a sugar metabolism-related disease, comprising as an active ingredient a gene encoding a protein comprising an amino acid sequence selected from the amino acid sequence group according to claim 1.
【請求項 3】  [Claim 3]
耐糖能改善活性を有することを特徴とする、 請求項 1又は 2に記載の治療剤又は予防剤 The therapeutic or prophylactic agent according to claim 1 or 2, which has glucose tolerance improving activity.
【請求項 4】 [Claim 4]
耐糖能改善活性が、 インスリン応答性促進活性である、 請求項 3に記載の治療剤又は予 防剤。 The therapeutic agent or preventive agent according to claim 3, wherein the glucose tolerance improving activity is an insulin responsive promoting activity.
【請求項 5】  [Claim 5]
被験物質が、 請求項 1に記載のァミノ酸配列鮮から選択されるアミノ酸配列からなる蛋 白質又は該蛋白質をコードする遺伝子の発現を促進するか否かを指標とする、 被験物質の 耐糖能改善活性の検定方法。 Improvement of glucose tolerance of a test substance using as an index whether or not the test substance promotes the expression of a protein comprising an amino acid sequence selected from the amino acid sequence of claim 1 or a gene encoding the protein Activity assay method.
【請求項 6】 も  [Claim 6]
下記の工程 (1)〜(3)を含むことを特徴とする、 請求項 5に記載の耐糖能改善活性 の検定方法： The method for assaying glucose tolerance improving activity according to claim 5, comprising the following steps (1) to (3):
(1)被験物質と、 請求項 1に記載のァミノ酸配列群から選択されるアミノ酸配列から なる蛋白質をコードする遺伝子を発現可能な細胞とを接触させる第一工程、  (1) a first step of contacting a test substance with a cell capable of expressing a gene encoding a protein comprising an amino acid sequence selected from the amino acid sequence group according to claim 1,
(2)被験物質を接触させた細胞における、 前記遺伝子の発現量を測定し、 該発現量を 被験物質に接触させなレ、対照細胞における前記遺伝子の発現量と比較する第二工程、 及び (2) a second step of measuring the expression level of the gene in the cell contacted with the test substance, and comparing the expression level with the expression level of the gene in the control cell without contacting the test substance with the test substance; and
(3)前記第二工程の比較結果に基づいて、 被験物質が前記遺伝子の発現量を増加させ るか否かを指標として被験物質の耐糖能改善活性を評価する第三工程。 (3) A third step of evaluating the glucose tolerance improving activity of the test substance using as an index whether or not the test substance increases the expression level of the gene based on the comparison result of the second step.
【請求項 7】 [Claim 7]
下記の工程 (1)〜(3)を含むことを特徴とする、 請求項 5に記載の耐糖能改善活性 の検定方法： The method for assaying glucose tolerance improving activity according to claim 5, comprising the following steps (1) to (3):
(1)被験物質と、 請求項 1に記載のアミノ酸配列群から選択されるアミノ酸配列からな る蛋白質を発現可能な細胞とを接触させる第一工程、  (1) a first step of contacting a test substance with a cell capable of expressing a protein comprising an amino acid sequence selected from the amino acid sequence group according to claim 1.
(2)被験物質を接触させた細胞における、 前記蛋白質の発現量を測定し、 該発現量を被 験物質に接触させない対照細胞における前記蛋白質の発現量と比較する第二工程、 及び (2) a second step of measuring the expression level of the protein in a cell contacted with a test substance and comparing the expression level with the expression level of the protein in a control cell not contacted with the test substance; and
(3)前記第二工程の比較結果に基づいて、 被験物質が前記蛋白質の発現量を増加させる か否かを指標として被験物質の耐糖能改善活性を評価する第三工程。 (3) A third step of evaluating the glucose tolerance improving activity of the test substance using as an index whether or not the test substance increases the expression level of the protein based on the comparison result of the second step.
【請求項 8】  [Claim 8]
下記の工程 (1)〜(3)を含むことを特徴とする、 請求項 5に記載の耐糖能改善活性 の検定方法： The method for assaying glucose tolerance improving activity according to claim 5, comprising the following steps (1) to (3):
(！)被験物質と、 請求項 1に記載のアミノ酸配列群から選択されるァミノ酸配列から なる蛋白質をコ一ドする遺伝子の発現制御領域を機能可能な形で連結されてなるレポータ 一遺伝子を含有する細胞とを接触させる第一工程、  (!) A reporter gene that is operably linked to a test substance and an expression control region of a gene that encodes a protein comprising an amino acid sequence selected from the amino acid sequence group according to claim 1. A first step of contacting the contained cells,
(2)被験物質を接触させた細胞における、 前記レポーター遺伝子の発現量を測定し、 該発現量を被験物質に接触させない対照細胞における前記遺伝子の発現量と比較する第二 工程、 及び  (2) a second step of measuring the expression level of the reporter gene in a cell contacted with the test substance and comparing the expression level with the expression level of the gene in a control cell not contacted with the test substance; and
(3)前記第二工程の比較結果に基づいて、 前記レポ一タ一遺伝子の発現量を増加させ るか否かを指標として被験物質の耐搪能改善活性を評価する第三工程。  (3) A third step of evaluating the tolerance improvement activity of the test substance using as an index whether or not to increase the expression level of the reporter gene based on the comparison result of the second step.
【請求項 9】  [Claim 9]
耐糖能改善活性が、 インスリン応答性促進活性である、 請求項 5〜 8のいずれかに記載 の検定方法。 The assay method according to claim 5, wherein the glucose tolerance improving activity is insulin responsiveness promoting activity.
【請求項 1 0】  [Claim 1 0]
請求項 5〜 8のいずれかに記載の検定方法により測定された、 被験物質の耐糖能改善活 性を指標として、 糖代謝関連疾患の治療剤又は予防剤の候補物質を選別することを特徴と する、 糖代謝関連疾患の治療剤又は予防剤の探索方法。 A candidate for a therapeutic or prophylactic agent for a glucose metabolism-related disease is selected using as an index the activity to improve glucose tolerance of the test substance measured by the assay method according to any one of claims 5 to 8. A method for searching for a therapeutic or prophylactic agent for a sugar metabolism-related disease.
【請求項 1 1】  [Claim 1 1]
請求項 1 0に記載の探索方法により選抜された物質またはその薬学的に許容される塩を 有効成分して含有する、 糖代謝関連疾患の治療剤又は予防剤。 An agent for treating or preventing a sugar metabolism-related disease, comprising as an active ingredient a substance selected by the search method according to claim 10 or a pharmaceutically acceptable salt thereof.
【請求項 1 2】  [Claim 1 2]
請求項 1に記載のアミノ酸配列群から選択されるアミノ酸配列からなる蛋白質の発現誘 導物質を有効成分として含有する、 糖代謝関連疾患の治療剤又は予防剤。 A therapeutic or preventive agent for a sugar metabolism-related disease, comprising as an active ingredient a protein expression inducer comprising an amino acid sequence selected from the amino acid sequence group according to claim 1.
PCT/JP2005/020144 2005-10-26 2005-10-26 Method of controlling glucose metabolism WO2007049359A1 (en)

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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2005298389A (en) * 2004-04-09 2005-10-27 Sumitomo Chemical Co Ltd Therapeutic agent or prophylactic agent for sugar metabolism-related disease

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2005298389A (en) * 2004-04-09 2005-10-27 Sumitomo Chemical Co Ltd Therapeutic agent or prophylactic agent for sugar metabolism-related disease

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
HIDA K. ET AL.: "Identification of genes specifically expressed in the accumulated visceral adipose tissue of OLETF rats", JOURNAL OF LIPID RESEARCH, vol. 41, no. 10, 2000, pages 1615 - 1622, XP002293069 *
HIDA K. ET AL.: "Visceral adipose tissue-derived serine protease inhibitor: A unique insulin-ensitizing adipocytokine in obesity", PROC. NATL. ACAD. SCI. USA, vol. 102, no. 30, 2005, pages 10610 - 10615, XP002375792 *
WADA J. ET AL.: "Adipocytokine to Shikkan Naizo Shibo Tokuiteki Adipocytokine no Cloning", ENDOCRINOLOGY & DIABETOLOGY, vol. 14, no. 4, 28 April 2002 (2002-04-28), pages 388 - 393, XP003011626 *

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