WO2005033700A1 - Methode de criblage d'un agent ameliorant la resistance a l'insuline - Google Patents

Methode de criblage d'un agent ameliorant la resistance a l'insuline Download PDF

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Publication number
WO2005033700A1
WO2005033700A1 PCT/JP2004/014802 JP2004014802W WO2005033700A1 WO 2005033700 A1 WO2005033700 A1 WO 2005033700A1 JP 2004014802 W JP2004014802 W JP 2004014802W WO 2005033700 A1 WO2005033700 A1 WO 2005033700A1
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present
amino acid
receptor
salt
protein
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PCT/JP2004/014802
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English (en)
Japanese (ja)
Inventor
Hideaki Tojo
Hiroyuki Sumi
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Takeda Pharmaceutical Company Limited
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Publication of WO2005033700A1 publication Critical patent/WO2005033700A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/06—Antihyperlipidemics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P5/00—Drugs for disorders of the endocrine system
    • A61P5/48—Drugs for disorders of the endocrine system of the pancreatic hormones
    • A61P5/50—Drugs for disorders of the endocrine system of the pancreatic hormones for increasing or potentiating the activity of insulin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/12—Antihypertensives
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value

Definitions

  • the present invention relates to an insulin resistance improving agent, a diabetes preventive / therapeutic agent, and the like, and the like.
  • Insulin resistance is a condition in which the sensitivity of insulin to tissues decreases, and in type II diabetes in particular, it is a major etiological factor involved in the onset and progression of diabetes in addition to insulin secretion deficiency. In general, insulin resistance is considered to be closely related to obesity, since many diabetic patients with obesity generally exhibit insulin resistance. Furthermore, it is known that insulin resistance is observed not only in diabetes but also in diseases caused by abnormal lipid metabolism such as arteriosclerosis (Saltiel, AR, Cell, vol. 104, pp. 517-529, 2001). .
  • the mechanism of action of insulin resistance remains largely unknown, but one idea suggests that it is similar to the mechanism of inflammation at the animal level or at the experimental level using cultured cell lines.
  • LPS lipopolysaccharide
  • the offspring develop obesity and insulin resistance after growth (Nilsson et al, Endocrinol, 142, 262-2630, 2001).
  • TNF- a which is one of the inflammatory site power-ins, is produced and secreted more in obese adipocytes and inhibits the insulin action (Hotamisligil GS et al., Science, Vol.
  • thiazolidine derivatives which are insulin ameliorating drugs, have anti-inflammatory activity
  • ob / ob mice which are obese model mice, have been altered in the expression of several proteins involved in the inflammatory response, including LPS-binding protein (Soukas, A. et al, Genes Develop, 14 Vol. 963-980, 2000).
  • Triggering receptor expressed on myeloid cells 2 Is a single transmembrane membrane protein belonging to the immunoglobulin superfamily found as a homologue protein of ⁇ -1 which is thought to be involved in non-patent literature 1 (Non-Patent Document 1 Bauchon, A. et al., J. Immunol., 164, 4991-4995, 2000, Daws MR et al, Eur J Immunol, 31, 783-791, 2001).
  • TREM-l is abundantly expressed in neutrophils and monocytes, and its expression has the effect of inflammation by promoting secretion of lipopolysaccharide-induced TNF- a and interleukin-lj8. In addition, inhibiting this can suppress the acute inflammatory response in mice
  • TREM-2 like TREM-1, signals by interacting with DAP12
  • DAP12 Daws MR et al, Eur J Immunol, Vol. 31, pp. 783-791, 2001
  • CCR7 CC chemokine receptor 7
  • LPS lipopolysaccharide
  • LTA lipoticoic acid
  • dextran sulfate dextran sulfate
  • the present inventors have already a model animal exhibiting obesity and insulin resistance KKA y mice (Nishimura, ⁇ ⁇ , Exp Animal , 18 Certificates, 147 - 157 p., 1969) in adipose cells, the expression TREM-2 has been found among the upregulated genes, and it has been reported that suppression of TREM-2 function improves diabetes (Patent Document 1: Japanese Patent Application No. 2003-144204). Disclosure of the invention
  • gandarioside is a ligand of TREM-2.Based on this finding, the present inventors have further studied and found that The invention has been completed.
  • the present invention (1) using (a) a protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1 or a partial peptide thereof or a salt thereof, and (b) using a glycoconjugate A method for screening a compound or a salt thereof that changes the binding property between the protein or a salt thereof and the complex bran,
  • glycoconjugate is 3'-sialyl lactose, 5, -sialyl ratatoose, sialyl oleylis X, sialyl lewis A, sialyl lacto-N-tetraose a, sialyl ratato-N-tetraose b, sialyl ratato-N-
  • the screening method according to any one of the above (1) to (5), wherein the screening method is tetraose c or disialyl lacto-N-tetraose.
  • (10) (a) a protein or a salt thereof having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1; and (b) a complex saccharide.
  • a preventive / therapeutic agent for hypoglycemia comprising a complex carbohydrate
  • an insulin sensitizer comprising the compound of the above (21) or a salt thereof
  • FIG. 1 shows the results of quantification of the fluorescent label GM3 bound to mTREM2-Fc.
  • the garden shows the amount of binding to Protein G agarose to which mTREM2-Fc was bound, and the mouth shows the amount of binding to Protein G agarose to which mTREM2_Fc was not immobilized.
  • the horizontal axis shows the amount of the fluorescent label GM3 added, and the vertical axis shows the fluorescence intensity of GM3 contained in the eluate.
  • FIG. 2 is a diagram showing the results of quantification of the fluorescent label GM3 bound to mTREM1-Fc, mTREM2-Fc, hTREM1-Fc and hTREM2-Fc.
  • the mouth indicates the amount bound to mTREMl-Fc
  • the garden indicates mT
  • the amount of binding to REM2-Fc, ⁇ indicates the amount of binding to hTREM1-Fc, and indicates the amount of binding to hTREM2-Fc.
  • the horizontal axis indicates the amount of the fluorescent label GM3 added, and the vertical axis indicates the fluorescence intensity of the fluorescent GM3 contained in the eluate.
  • a protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1 (hereinafter sometimes referred to as the receptor of the present invention, the protein of the present invention, or the protein used in the present invention)
  • are cells of human warm-blooded animals eg, guinea pigs, rats, mice, chickens, egrets, stags, hidges, horses, monkeys, etc.
  • Organ cells eg, hepatocytes, spleen cells, nerve cells, glial cells, knees
  • Organ cells bone marrow cells, mesangial cells, Langerhans cells, epidermal cells, epithelial cells, goblet cells, endothelial cells, smooth muscle cells, fibroblasts, fiber cells, muscle cells, fat cells, immune cells (eg, macrophages, T cells, B cells, natural killer cells, mast cells, neutrophils, basophils, eosinophils, monocytes), megakaryocytes,
  • the amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 1 includes, for example, about 60% or more, preferably about 70% or more, of the amino acid sequence represented by SEQ ID NO: 1.
  • the amino acid sequence preferably has about 80% or more, preferably about 90% or more, and preferably about 95% or more homology.
  • Examples of the protein containing an amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 1 include, for example, a protein containing the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1 above. However, a protein having substantially the same activity as the protein having the amino acid sequence represented by SEQ ID NO: 1 is preferred. Examples of the protein containing the amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 1 include a protein containing the amino acid sequence represented by SEQ ID NO: 2, and the like.
  • substantially equivalent activities include, for example, signal transduction activity (eg, intracellular signal transduction activity of the protein of the present invention (preferably TREM-2)), ligand binding activity
  • binding activity between the protein of the present invention preferably TREM-2
  • a ligand or a small molecule etc.
  • the properties are qualitatively (eg, physiologically or pharmacologically) equivalent. Therefore, signal transduction activity, ligand binding activity, etc. are equivalent (eg, about 0.01 to 100 times, preferably about 0.1 to 10 times, more preferably 0.5 to 2 times). However, the quantitative factors such as the degree of these activities and the molecular weight of the protein may be different.
  • the signal transduction activity and the ligand binding activity can be measured according to a method known per se, for example, the method described in J. Exp. Med. 194, .1111-1112, 2001 or a method analogous thereto. it can.
  • the signal of the protein (preferably TREM-2) of the present invention causes, for example, phosphorylation of TREM-2 or DAP12 (DNAX adapter protein 12), activates ERK (extracellular signal-related protein), and induces inflammatory cytokines ( Eg, TNF-secretion).
  • the above-mentioned signal transduction activity is, for example, as compared to the protein-expressing cells of the present invention (eg, TREM-2 expressing animal cells), as necessary, (a) microbial cell lysate, microbial culture supernatant, eukaryotic cell lysate, A solution containing a ligand such as eukaryotic cell culture supernatant, (b) the ligand itself, ( c ) a substance having the same binding activity as the natural ligand to the protein of the present invention, or (d) a protein of the present invention (eg, By adding an antibody that activates TREM-2), (1) the amount of phosphorylated ERK produced, (2) the amount of extracellular TNF-a produced or (3) the phosphorylated TREM- Measure the yield of 2.
  • the protein-expressing cells of the present invention eg, TREM-2 expressing animal cells
  • ligand a complex saccharide described later is used.
  • the amount of phosphorylated ERK or TNF-a produced can be measured by a known method (eg, Western blotting, EIA, etc.) using an anti-phosphorylated ERK antibody or an anti-TNF-A antibody.
  • the amount of phosphorylated TREM-2 produced was measured using an anti-TREM-2 antibody and an anti-phosphorylated tyrosine antibody, and the amount of phosphorylated DAP12 was produced using an anti-DAP12 antibody and an anti-phosphorylated antibody.
  • a known method eg, immunoprecipitation method, Western blot method, etc.
  • Immunoprecipitation of TREM-2 or DAP12 is performed by expressing each protein in animal cells as a recombinant protein tagged with, for example, FLAG, His, V5, myc, HA, etc., and using each anti-tag antibody. You can do it too.
  • the ligand binding activity can be measured using the protein (preferably TREM-2) of the present invention and a ligand, for example, by an immunoprecipitation method, a protein affinity purification method, a yeast two-hybrid method, or the like.
  • the receptor of the present invention includes (1) (i) one or two or more amino acids in the amino acid sequence represented by SEQ ID NO: 1 (for example, about 1 to 100, preferably 1 to 100). About 30 amino acids, preferably about 1 to 10 amino acids, and more preferably about 1 to 5 amino acids, and (ii) 1 amino acid sequence represented by SEQ ID NO: 1. Also Has two or more amino acids (for example, 1 to: about L00 ', preferably about 1 to 30, preferably about 1 to 10, and more preferably about 1 to 5).
  • Amino acid sequence (iii) one or more amino acid sequences represented by SEQ ID NO: 1 (eg, 1 to: about L00, preferably about 1 to 30, preferably 1 to 1) An amino acid sequence having about 0, more preferably a number (1 to 5) amino acids inserted, and (iv) one or more amino acids in the amino acid sequence represented by SEQ ID NO: 1 (for example, 1 to 1) An amino acid sequence in which about 100, preferably about 1 to 30, preferably about 1 to 10, and more preferably a number (1 to 5) of amino acids have been substituted with another amino acid, or V) so-called muteins, such as proteins containing amino acid sequences combining them; (2) (i) SEQ ID NO: 2 1 or 2 or more in the amino acid sequence (for example, about 1 to 100, preferably about 1 to 30, preferably about 1 to 10, and more preferably about 1 to 5) (Ii) 1 or 2 or more amino acids in the amino acid sequence represented by SEQ ID NO: 2 (for example, about 1 to 100, preferably about 1
  • amino acids in the amino acid sequence represented by SEQ ID NO: 2 for example, about 1 to 100, preferably about 1 to 30, preferably about 1 to 10, more preferably (Iv) one or two or more amino acids in the amino acid sequence represented by SEQ ID NO: 2 (for example, about 1 to 100 amino acids, preferably (1 to 5) amino acids) Is an amino acid sequence in which about 1 to 30 amino acids, preferably about 1 to 10 amino acids, and more preferably several (1 to 5) amino acids have been substituted with other amino acids; or
  • the position of the insertion, deletion or substitution is not particularly limited.
  • receptor of the present invention examples include, for example, a protein containing the amino acid sequence represented by SEQ ID NO: 1 (human TREM-2) and a protein containing the amino acid sequence represented by SEQ ID NO: 2 ( Mouse TREM-2).
  • Partial peptide of the receptor of the present invention (hereinafter sometimes referred to as the partial peptide of the present invention) ) May be any partial peptide that can be used in a screening method for a drug or the like described below.
  • the protein molecule of the present invention is exposed outside the cell membrane. Sites having substantially the same ligand binding activity and the like are used.
  • the partial peptide of the present invention lacks one or more (preferably about 1 to 10, preferably 1 to 5) amino acids in its amino acid sequence, Alternatively, one or more (preferably about 1 to 20, more preferably about 1 to 10, and more preferably about 1 to 5) amino acids are added to the amino acid sequence. Or 1 or 2 or more (preferably: about! 20, more preferably about 1 to 10, and more preferably a number (1 to 5)) of amino acids in the amino acid sequence. Or one or more (preferably about 1 to 10, more preferably several, and more preferably about 1 to 5) amino acids in the amino acid sequence are replaced with other amino acids. May be.
  • the partial peptide of the present invention also contains the extracellular domain of the receptor of the present invention.
  • the extracellular domain include a peptide having the 14th to 16th amino acid sequence or the 19th to 17th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 1; And a peptide having an amino acid sequence at the 14th to 170th amino acid sequence or at the 19th to 170th amino acid sequence.
  • the left end is the N-terminus (amino terminus) and the right end is the C-terminus (carboxyl terminus) according to the convention of peptide labeling.
  • C-terminal carboxy (-C00H), carboxylate (- C00-), amide (-C0NH 2) or ester - may be a (C00R).
  • R in the ester e.g., methyl, Echiru, n- propyl, alkyl such as isopropyl or n- heptyl, for example, cyclopentyl, C 3 _ 8 cycloalkyl such as cyclohexyl, for example, phenyl, - a Nafuchinore which C 6 _ 12 Ariru Piparoiruo, for example, benzyl, which are widely used as well, an ester for oral administration of C 7 _ 14 Ararukiru such phenylene Lou al kills or ⁇ - naphthylmethyl etc. ⁇ - Nafuchiru alkyl such phenethyl Xymethyl is used.
  • the receptor of the present invention and the partial peptide of the present invention have carboxy (or carboxylate) other than at the C-terminus
  • those in which carboxy is amidated or esterified also include those of the present invention. It is included in the partial peptide of the present invention.
  • the ester in this case, for example, the above-mentioned C-terminal ester and the like are used.
  • the receptor of the present invention and the partial peptide of the present invention include an N-terminal amino acid residue (eg, a methionine residue).
  • the amino group is protected by a protecting group (for example, alkyl such as formyl or acetyl, etc.), the glutamine residue at the N-terminus generated by cleavage in vivo is pyroglutamine-oxidized,
  • a protecting group for example, alkyl such as formyl or acetyl, etc.
  • the glutamine residue at the N-terminus generated by cleavage in vivo is pyroglutamine-oxidized
  • Substituents on the side chains of amino acids for example, _0H, -SH, amino group, imidazole group, indole group, guanidino group, etc.
  • suitable protecting groups for example, C w alkynyl such as formyl, acetyl, etc.
  • complex proteins such as so-called glycoproteins to which sugar chains are bound.
  • salts with physiologically acceptable acids eg, inorganic acids, organic acids
  • bases eg, alkali metal salts
  • physiologically acceptable acids eg, inorganic acids, organic acids
  • bases eg, alkali metal salts
  • Physiologically acceptable acid addition salts are preferred.
  • Such salts include, for example, salts with inorganic acids (eg, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid) or organic acids (eg, acetic acid, formic acid, propionic acid, fumaric acid, maleic acid) And succinic acid, tartaric acid, citric acid, malic acid, oxalic acid, benzoic acid, methanesulfonic acid and benzenesulfonic acid.
  • inorganic acids eg, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid
  • organic acids eg, acetic acid, formic acid, propionic acid, fumaric acid, maleic
  • the glycoconjugate (hereinafter sometimes abbreviated as the ligand of the present invention) may be any as long as it specifically binds to the receptor of the present invention.
  • the sugar chain portion of the complex carbohydrate specifically binds to the receptor of the present invention
  • the sugar chain of the complex carbohydrate is A chain portion is also included in the ligand of the present invention.
  • the dissociation constant of the binding to the receptor of the present invention is 10 nM or less, preferably 2 M or less, more preferably 1 M or less, particularly preferably 200 nM or less, and most preferably 100 nM or less. nM or less.
  • Examples of the ligand of the present invention include gandarioside (eg, GM1, GM2, GM3, GD3, GDla, etc.), sialyloligosugar (eg, 3, -sialylratose, 5′-sialylratathose, sialylluis X, sialylluis A, sialyllac) G-N-tetraoose 3, sialyl lacto-N-tetraose b, sialyl ratato-N-tetraose c, disialyl lacto-N-tetraose, etc., lipopolysaccharide (LPS) (eg, LPS from Escherichia coli, LPS from Pseudomonas aeruginosa, etc.) Lipotic acid (LTA) (eg, LTA derived from Staphylococcus aureus, LTA derived from Bacillus subtilis),
  • the ligand of the present invention includes (a) a partial structure constituting ganglioside, sialyloligosaccharide, lipopolysaccharide, lipoticoic acid or dextran sulfate, and (b)
  • Preferred ligands of the present invention include, for example, gandariosides (eg, GM1, GM2, GM3, GD3, GDla, etc.), sialyloligosaccharides (eg, 3, -sialylactose, 5, -sialyllatase, sialyl Louis X, sialyl Lewis A, sialyl lactone) G-N-tetraose a, sialyl lacto-N-tetraose b, sialyl lacto-N-tetraose 0, disialyl lacto-N-tetraose, etc.). More preferably, it is GM3.
  • sialyloligosaccharides eg, 3, -sialylactose, 5, -sialyllatase, sialyl Louis X, sialyl Lewis A, sialyl lactone
  • Labeled glycoconjugates are also included in the ligand of the present invention.
  • the labeling substance a radioactive isotope (e.g., [125 1], [131 1], [], [14 C], [32 P], [33 P] and [35 S]), fluorescent substances [e.g. , Cyanine fluorescent dye (eg, Cy2,
  • Cy3, Cy5, Cy5.5, Cy7 (manufactured by Amersham Bioscience), fluorescamine, fluorescein isothiocyanate, NBD (7-nitrobenz-2-oxa-1,3-diazol), etc.), Enzymes (eg, ⁇ -galactosidase, mono-dalcosidase, alkaline phosphatase, peroxidase, malate dehydrogenase, etc.), luminescent substances (eg, noreminol, luminol derivatives, luciferin, lucigenin, etc.), biotin And lanthanide elements. Among them, a fluorescent substance is preferable. More NBD is preferred.
  • the labeled ligand is a gandarioside labeled with a fluorescent substance, and more preferably GM3 labeled with NBD.
  • the receptor of the present invention and the partial peptide of the present invention can be produced from the above-mentioned cells or tissues of humans or warm-blooded animals by a known method for purifying polypeptides, It can also be produced by culturing a transformant transformed with DNA encoding the polypeptide. Further, it can be produced according to the peptide synthesis method. For example, Genomics, 56, 12-21, 1999, Biochim.
  • the human or mammalian tissues or cells are homogenized and then extracted with an acid or the like, and the extract is subjected to reverse phase chromatography, ion exchange chromatography, etc. Purification and isolation can be performed by combining the above chromatography.
  • a commercially available resin for synthesizing a polypeptide can be usually used.
  • a resin include chloromethyl resin, hydroxymethyl resin, benzhydrylamine resin, aminomethyl resin, 4-benzyloxybenzyl alcohol resin, 4-methylbenzylhydramine resin, PAM resin, and the like.
  • an amino acid appropriately protected with a monoamino group and a side chain functional group can be synthesized according to the sequence of the target polypeptide. Is then condensed on a resin according to various condensation methods known per se. At the end of the reaction, the polypeptide is cleaved from the resin, and at the same time, various protecting groups are removed.In addition, an intramolecular disulfide bond formation reaction is carried out in a highly diluted solution to obtain the desired polypeptide, receptor, partial peptide or their amino acids.
  • the protected Amino acids c above can be used a variety of activation reagents that can be used in polypeptide synthesis, in particular, it is Karupojiimi earth.
  • Karposi Examples of imids include DCC, ⁇ , ' ⁇ '-diisopropyl carbodiimide, and ⁇ -ethyl ⁇ , 1- (3-dimethylaminoprolyl) carbodiimide.
  • Protected amino acids can be added directly to the resin along with an antioxidant (eg, ⁇ B t, HOOB t), or the protected amino acid can be pre-activated as a symmetric anhydride or HOB t ester or HO ⁇ B t ester. After that, it can be added to the resin.
  • the solvent used for activating the protected amino acid or condensing with the resin can be appropriately selected from solvents known to be usable for the polypeptide condensation reaction.
  • acid amides such as N, N-dimethylformamide, N, N-dimethylacetamide, N-methylvirolidone, halogenated hydrocarbons such as methylene chloride, methylform, and trifluoromethyl Alcohols such as ethanol, sulfoxides such as dimethyl sulfoxide, ethers such as pyridine, dioxane and tetrahydrofuran; nitriles such as acetonitrile and propionitrile; esters such as methyl acetate and ethyl acetate; A mixture or the like is used.
  • the reaction temperature is appropriately selected from a range known to be usable for the polypeptide bond formation reaction, and is usually appropriately selected from a range of about 120 ° C to 50 ° C.
  • the activated amino acid derivative is usually used in a 1.5 to 4-fold excess.
  • Examples of the protecting group for the starting amino group include Z, Boc, t-pentyloxycarbo-nore, isoborninoleoxycarbonyl, 4-methoxybenzyloxycanolebonyl, C11Z, Br-Z
  • adamantyl oxycarbonyl, trifluoroacetinole, phthaloynole, honoleminole, 2-butenophenorelesnorefueinole, difuinore phosphinochioil, Fmoc and the like can be used.
  • the carboxyl group is, for example, alkyl esterified (for example, methyl, ethyl, propynole, petitnole, t-butynole, cyclopentynole, cyclohexynole, cyclohexyl).
  • Linear, branched or cyclic alkyl esterification such as tyl, cyclooctyl, 2-adamantyl, etc., aralkyl esterification (eg, benzyl ester, 4-nitrobenzinoleestenole, 4-methoxypentinole) Estenole, 4-methylbenzene ester, benzhydryl ester), phenacyl ester, benzyloxycarbonyl hydrazide, t-butoxycarbol hydrazide, trityl hydrazide and the like.
  • aralkyl esterification eg, benzyl ester, 4-nitrobenzinoleestenole, 4-methoxypentinole
  • Estenole 4-methylbenzene ester, benzhydryl ester
  • phenacyl ester benzyloxycarbonyl hydrazide
  • t-butoxycarbol hydrazide trityl hydrazide
  • the hydroxyl group of serine can be protected, for example, by esterification or etherification.
  • a group suitable for the esterification for example, a group derived from carbonic acid such as a lower (Cw) alkanol group such as an acetyl group, an aroyl group such as a benzoyl group, a benzyloxycarbonyl group, and an ethoxycarbonyl group can be used. It is possible.
  • Examples of a group suitable for ethereal dan include a benzyl group, a tetrahydrovinyl group, a t_butyl group, and the like.
  • the protecting group of the phenolic hydroxyl group of tyrosine for example, B zl, C l 2 _
  • Examples of the protecting group for imidazole of histidine include Tos, 4-methoxy-12,3,6-trimethylbenzenesulfonyl, DNP, benzyloxymethyl,
  • Examples of the activated carboxyl groups of the raw material include, for example, corresponding acid anhydrides, azides, and activated decay esters [alcohols (eg, pentachlorophenol, 2,4,5-trichlorophenol, 2 , 4-dinitrophenol, cyanomethinorea, phenolic phenol, HONB, N-hydroxysuccinimide, N-hydroxyphthalimid, HOB tester).
  • alcohols eg, pentachlorophenol, 2,4,5-trichlorophenol, 2 , 4-dinitrophenol, cyanomethinorea, phenolic phenol, HONB, N-hydroxysuccinimide, N-hydroxyphthalimid, HOB tester.
  • As the activated amino group of the raw material for example, a corresponding phosphoric amide is used.
  • Methods for removing (eliminating) protecting groups include, for example, catalytic reduction in a hydrogen stream in the presence of a catalyst such as Pd-black or Pd-carbon, or hydrogen fluoride anhydride or methanesulfonic acid.
  • a catalyst such as Pd-black or Pd-carbon, or hydrogen fluoride anhydride or methanesulfonic acid.
  • Reduction by pum is also used.
  • the elimination reaction by the above-mentioned acid treatment is generally carried out at a temperature of about 120 ° C to 40 ° C.
  • the acid treatment for example, anisol, phenanol, thioanisole, methacrylone, paracrezonole, dimethinoresolefide, It is effective to add a force-thione scavenger such as 1,4-butanedithiol, 1,2-ethanedithiol and the like.
  • a force-thione scavenger such as 1,4-butanedithiol, 1,2-ethanedithiol and the like.
  • the 2,4-dinitrophenyl group used as an imidazole protecting group of histidine is removed by thiophenol treatment, and the formyl group used as an indole protecting group of tributofan is treated with the above 1,1,1-ethanedithiol, 1,4-butanedithiol.
  • alkali treatment with dilute sodium hydroxide solution, dilute ammonia, etc.
  • the protection of the functional group which should not be involved in the reaction of the raw materials, the protecting group, the elimination of the protective group, the activation of the functional group involved in the reaction, and the like can be appropriately selected from known groups or known means.
  • a peptide (polypeptide) chain is desired on the amino group side. Then, a polypeptide is obtained by removing only the protecting group of the N-terminal monoamino group of the peptide chain and a polypeptide from which only the protecting group of the C-terminal carboxyl group is removed.
  • the two polypeptides are condensed in a mixed solvent as described above. The details of the condensation reaction are the same as described above.
  • all the protecting groups are removed by the above-mentioned method to obtain a desired crude polypeptide. This crude polypeptide is purified by various known purification means, and the main fraction is freeze-dried to obtain the desired receptor or an amide of the partial peptide.
  • an ester of the receptor or partial peptide of the present invention or a salt thereof for example, after condensing a monocarboxylic group of the carboxy terminal amino acid with a desired alcohol to form an amino acid ester, the receptor or a portion thereof is obtained.
  • an ester form of the desired receptor or a partial peptide thereof can be obtained.
  • the receptor or partial peptide of the present invention can be obtained by a known method for synthesizing a peptide.
  • a partial peptide of the receptor can be produced by cleaving the receptor with an appropriate peptidase.
  • a method for synthesizing a peptide for example, any of a solid phase synthesis method and a liquid phase synthesis method may be used. That is, a partial peptide or amino acid capable of constituting the receptor or partial peptide of the present invention is condensed with the remaining portion, and when the product has a protecting group, the protecting group is eliminated to remove the desired peptide. Can be manufactured. Examples of the known condensation method and elimination of the protecting group include the methods described in the following (i) to (V).
  • the receptor or partial peptide of the present invention can be purified and isolated by combining crystals and the like.
  • the receptor or partial peptide obtained by the above method is a free form, it can be converted into an appropriate salt by a known method or a method analogous thereto. It can be converted to a free form or another salt by a known method or a method analogous thereto.
  • the polynucleotide encoding the receptor or partial peptide of the present invention may be any polynucleotide containing the above-described nucleotide sequence encoding the receptor or partial peptide of the present invention. Good. Of these, DNA is preferable, and the DNA may be any of genomic DNA, genomic DNA library, cDNA derived from the above-described cells and tissues, cDNA library derived from the above-described cells and tissues, and synthetic DNA.
  • the vectors used for the library are pateriophage, plasmid, And phagemid. Alternatively, it can be directly amplified by reverse transcriptase polymerase chain reaction (hereinafter abbreviated as RT-PCR method) using a preparation of a total RNA or mRNA fraction from the cells and tissues described above.
  • RT-PCR method reverse transcriptase polymerase chain reaction
  • Examples of the DNA encoding the receptor of the present invention include a DNA containing the nucleotide sequence represented by SEQ ID NO: 3 or SEQ ID NO: 4, or the nucleotide sequence represented by SEQ ID NO: 3 or SEQ ID NO: 4 Encodes a receptor having a nucleotide sequence that hybridizes under high stringent conditions to a protein having substantially the same activity as a protein having the amino acid sequence represented by SEQ ID NO: 3 or SEQ ID NO: 4. Any DNA may be used.
  • Examples of the DNA that can hybridize with the nucleotide sequence represented by SEQ ID NO: 3 or SEQ ID NO: 4 under high stringency conditions include, for example, the nucleotide sequence represented by SEQ ID NO: 3 or SEQ ID NO: 4 DNA containing a base sequence having a homology of 60% or more, preferably about 70% or more, preferably about 80% or more, preferably about 90% or more, and preferably about 95% or more. Is used.
  • Hybridization is carried out by a method known per se or a method analogous thereto, for example, by the method of Molecular Cloning 2nd Edition (J. Sambrook et al., Cold Spring
  • the high stringent conditions include, for example, a sodium concentration of about 19 to 40 mM, preferably about 19 to 20 mM, and a temperature of about 50 to 70 ° C, preferably about 60 ° C.
  • the condition of ⁇ 65 ° C is shown.
  • the case where the sodium concentration is about 19 mM and the temperature is about 65 is most preferable.
  • examples of the DNA encoding the receptor containing the amino acid sequence represented by SEQ ID NO: include a DNA containing the base sequence represented by SEQ ID NO: 3, and the like.
  • DNA encoding the receptor containing the amino acid sequence represented by 2 DNA containing the base sequence represented by SEQ ID NO: 4 or the like is used.
  • the DNA encoding the partial peptide of the present invention may be any DNA containing a base sequence encoding the partial peptide of the receptor of the present invention.
  • any of genomic DNA, genomic DNA library, cDNA derived from the above-described cells and tissues, cDNA library derived from the above-described cells and tissues, and synthetic DNA may be used.
  • a DNA having a partial base sequence of a DNA having the base sequence represented by SEQ ID NO: 3 or SEQ ID NO: 4, or a base sequence represented by SEQ ID NO: 3 or SEQ ID NO: 4 Encoding a receptor having a nucleotide sequence that hybridizes under high stringent conditions and having substantially the same activity as a protein containing the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2 DNA having a partial nucleotide sequence of DNA to be used is used.
  • the DNA hybridizable to the nucleotide sequence represented by SEQ ID NO: 3 or SEQ ID NO: 4 has the same significance as described above.
  • the polynucleotide (eg, DNA) encoding the receptor or partial peptide of the present invention may be labeled by a method known per se. Labeling substances include radioisotopes, fluorescent substances (eg, fluorescein, etc.), luminescent substances, enzymes, biotin, lanthanide elements, and the like.
  • a PCR method known per se using a synthetic DNA primer having a partial nucleotide sequence of the receptor or partial peptide of the present invention is used.
  • Hybridization with DNA amplified or incorporated into an appropriate vector, labeled with a DNA fragment encoding a part or all of the receptor or partial peptide of the present invention or with synthetic DNA can be sorted out.
  • Hypridease Chillon methods are described, for example, Molecuiar Cloning 2 nd Edition (J. Sambrook et al., Cold Spring Harbor Lab. Press, 1989) can be carried out according to the method described in. When a commercially available library is used, the procedure can be performed according to the method described in the attached instruction manual.
  • the DNA encoding the cloned receptor can be used as it is depending on the purpose, or digested with a restriction enzyme or added with a linker as desired.
  • the DNA may have ATG as a translation initiation codon at the 5 'end and TAA, TGA or TAG as a translation stop codon at the 3' end. These translation initiation codon and translation termination codon can be added using an appropriate synthetic DNA adapter;
  • the expression vector for the receptor or partial peptide of the present invention includes, for example, (a) cutting out a DNA fragment of interest from DNA encoding the receptor or partial peptide of the present invention, and (b) the DNA fragment Is ligated downstream of the promoter in an appropriate expression vector.
  • Plasmids derived from E. coli eg, pBR322, pBR325, pUC12, pUC13
  • Bacillus subtilis-derived plasmids eg, pUB110, pTP5, pC194
  • Plasmid derived from yeast eg, ⁇ pSH19, pSH15
  • Bacteriophage such as ⁇ phage, animal viruses such as retroinoles, vaccinia virus, baculovirus, etc., pAlll, pXTl, p RcZCMV, pRc / RSV, pcDNAI / Neo and the like are used.
  • the promoter used in the present invention may be any promoter as long as it is appropriate for the host used for gene expression.
  • SRa promoter when animal cells are used as host, SRa promoter, SV40 promoter, HIV 'LTR promoter, CMV promoter, HSV-TK promoter and the like can be mentioned.
  • CMV site's megalovirus
  • SRCK SRCK promoter
  • the host is Escherichia, the trp promoter, lac promoter, recA promoter, LPL promoter, lpp promoter, T7 promoter, etc .; if the host is Bacillus, SP ⁇ 1 promoter, SPO
  • yeast such as 2 promoters, penP promoters, etc., PHO5 promoter, PGK promoter, GAP promoter, ADH promoter and the like are preferable.
  • a polyhedrin promoter, a P10 promoter and the like are preferable.
  • the expression vector may further contain, if desired, an enhancer, a splicing signal, a polyA addition signal, a selection marker, an SV40 replication origin (hereinafter sometimes abbreviated as SV40 ori), and the like.
  • an enhancer for example, dihydrofolate reductase (hereinafter sometimes abbreviated as dh fr) gene [methotrexate (MTX) resistance], ampicillin phosphorus resistant gene (hereinafter sometimes abbreviated as Amp r), Neomycin resistance gene (hereinafter sometimes abbreviated as Neo T , G418 resistance) and the like.
  • dhfr gene is used as a selection marker using Chinese hamster cells deficient in the dhfr gene
  • the target gene can be selected using a thymidine-free medium.
  • a signal sequence suitable for the host is added to the N-terminal side of the receptor of the present invention. If the host is a genus Escherichia, a Pho A signal sequence, an Omp A signal sequence, etc., if the host is a Bacillus genus, an ⁇ -amylase. Signal sequence, a subtilisin signal sequence, etc. If the host is yeast, MFa signal sequence, SUC2 signal sequence, etc.If the host is an animal cell, insulin signal sequence, Hi-interferon signal sequence, antibody molecule, signal sequence, etc. Are available respectively.
  • a transformant can be produced using the thus-constructed DNA-containing vector encoding the receptor or partial peptide of the present invention.
  • Escherichia bacteria for example, Escherichia bacteria, Bacillus bacteria, yeast, insect cells, insects, animal cells, and the like are used.
  • Specific examples of the genus Escherichia include, for example, Escherichia coli.
  • Bacillus bacteria include, for example, Bacillus.
  • subtilis MI 114 (Gene, 24, 255 983)], 207-21 [Journal of Biochemistry, 95, 87 (1984)] and the like.
  • yeast examples include, for example, Saccharomyces cerevisiae AH 22, AH 22 R ", NA87-11A, DKD-5D, 20B-12, Schizosaccharomyces pombe NC YC 1913, NCYC 2036 And Pichia pastoris K # 71.
  • insect cells for example, when the virus is Ac NPV, Spodoptera frugiperda cell lines (Spodoptera frugiperda cells), MG1 cells derived from the midgut of Trichoplusia, and High cells derived from eggs of Trichoplusia ni Five TM cells, cells derived from Mamestra brassicae or cells derived from Estigmena acrea are used.
  • a silkworm-derived cell line Boombyx raori N cell; BmN cell
  • Sf cell include Sf9 cell (ATCC CRL1711), Sf21 cell (Vaughn, J.L., et al., In Vivo).
  • insects for example, silkworm larvae are used [Maeda et al., Nature, Vol. 315, 592 (1985)].
  • animal cells examples include monkey cells COS-7 (COS 7), Vero, Chinese hamster cells CHO (hereinafter abbreviated as CH ⁇ cells), dh fr gene-deficient Chinese hamster cells CHO (hereinafter CHO ( dhfr-) cells), mouse L cells, mouse AtT-20, mouse myeloma cells, rat GH3, and human FL cells.
  • the yeast can be transformed according to the method described in, for example, Methods in Enzymology, Vol. 194, 182-187 (1991), Proc. Natl. Acad. Sci. USA, Vol. 75, 1929 (1978). Can be.
  • Transformation of insect cells or insects can be performed, for example, according to the method described in Bio / Technology, 6, 47-55 (1988).
  • Transformation of animal cells can be performed, for example, according to the method described in Cell Engineering Separate Volume 8 New Cell Engineering Experimental Protocol. 263-267 (1995) (published by Shujunsha), Virology, 52, 456 (1973). Can do it.
  • a liquid medium is suitable as a medium used for the culturing, and a carbon medium necessary for the growth of the transformant is contained therein.
  • the carbon source include glucose, dextrin, soluble starch, and sucrose.
  • examples of the nitrogen source include ammonium salts, nitrates, corn chip liquor, peptone, potato zein, meat extract, soybean meal, and potato extract.
  • Inorganic or organic substances such as liquids and inorganic substances include, for example, calcium chloride, sodium dihydrogen phosphate, magnesium chloride and the like.
  • yeast extract, vitamins, growth promoting factors and the like may be added.
  • the pH of the medium is preferably about 5-8.
  • an M9 medium containing glucose and casamino acids As a medium for cultivating a bacterium belonging to the genus Escherichia, for example, an M9 medium containing glucose and casamino acids [Miller, Journal of Experiments in
  • an agent such as 3-indolylacrylic acid can be added to make the promoter work efficiently.
  • the cultivation is usually carried out at about 15 to 43 ° C for about 3 to 24 hours, and if necessary, aeration and stirring can be applied.
  • the cultivation is usually carried out at about 30 to 40 ° C for about 6 to 24 hours, and if necessary, aeration and stirring may be added.
  • the host is yeast
  • Burkholder's minimum medium Bostian, KL et al., Proc. Natl. Acad. Sci. USA, 77, 4505 (1980)
  • SD medium containing 0.5% casamino acid Bitter, GA et al., Proc. Natl. Acad. Sci. USA, 81, 5330 (1984)
  • the pH of the medium is adjusted to about 5-8.
  • the cultivation is usually performed at about 20 ° C to 35 ° C for about 24 to 72 hours, and aeration and stirring are added as necessary.
  • the medium used is 10% serum serum immobilized in Grace's Insect Medium (Grace, TCC, Nature, 195, 788 (1962)). And the like to which additives such as the above are appropriately added are used.
  • the pH of the medium is preferably adjusted to about 6.2 to 6.4. Culture is usually performed at about 27 ° C for about 3 to 5 days, and aeration and agitation are added as necessary.
  • a MEM medium containing about 5 to 20% fetal bovine serum for example, a MEM medium containing about 5 to 20% fetal bovine serum [Science, 122 vol., 501 (1952)], a DMEM medium [ Virology, 8 volumes, 396 (1959)], RPMI 1640 medium [The Journal of the American Medical Association 199 volumes, 519 (1967)], 199 medium
  • the pH is about 6-8.
  • Cultivation is usually carried out at about 30 ° C to 40 ° C for about 15 to 60 hours, and aeration and stirring are added as necessary.
  • the receptor or partial peptide of the present invention can be produced in the cells, cell membrane, or extracellular cells of the transformant.
  • the receptor or partial peptide of the present invention can be separated and purified from the above culture by, for example, the following method.
  • the receptor or partial peptide of the present invention When extracting the receptor or partial peptide of the present invention from cultured cells or cells, after culturing, cells or cells are collected by a known method, and the cells or cells are suspended in an appropriate buffer, and then subjected to ultrasound, Cells or by lysozyme and / or freeze-thaw After the cells are disrupted, a method of obtaining a crude polypeptide extract by centrifugation or filtration is used as appropriate.
  • the buffer may contain a protein denaturant such as urea or guanidine hydrochloride, or a surfactant such as Triton X-100 TM.
  • the receptor or partial peptide contained in the culture supernatant or the extract obtained in this way can be purified by appropriately combining known separation and purification methods.
  • These known separation and purification methods mainly include methods utilizing solubility such as salting out and solvent precipitation, dialysis, ultrafiltration, gel filtration, and SDS-polyacrylamide gel electrophoresis.
  • Method using difference in molecular weight method using charge difference such as ion exchange chromatography, method using specific affinity such as affinity chromatography, reverse phase high-performance liquid chromatography
  • a method utilizing a difference in hydrophobicity such as a method of isoelectric point electrophoresis, or a method utilizing a difference in isoelectric point, such as isoelectric focusing electrophoresis, may be used.
  • the thus obtained receptor or partial peptide When the thus obtained receptor or partial peptide is obtained as a free form, it can be converted into a salt by a method known per se or a method analogous thereto, and conversely, when the receptor or a partial peptide is obtained as a salt, It can be converted into a free form or another salt by a known method or a method analogous thereto.
  • Receptors or partial peptides produced by recombinants can be arbitrarily modified or polypeptides can be partially removed before or after purification by the action of an appropriate protein-modifying enzyme.
  • an appropriate protein-modifying enzyme for example, trypsin, chymotrypsin, arginyl endopeptidase, protein kinase, glycosidase and the like are used.
  • a commercially available product can be used as it is, or can be extracted or produced according to a method known per se or a method analogous thereto.
  • an antibody against a protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1 or a partial peptide thereof or a salt thereof (hereinafter, may be simply referred to as the antibody of the present invention) Is an antibody against the receptor of the present invention.
  • Any antibody that can be recognized may be a polyclonal antibody or a monoclonal antibody.
  • the antibody against the receptor of the present invention include an antibody that inactivates signal transduction of a receptor, an antibody that activates signal transduction of a receptor, and the like.
  • An antibody against the receptor of the present invention can be produced according to a known method for producing an antibody or antiserum using the receptor of the present invention as an antigen.
  • the receptor of the present invention is administered to a warm-blooded animal itself or together with a carrier or diluent at a site where antibody production is possible upon administration.
  • Complete Freund's adjuvant / incomplete Freund's adjuvant may be administered in order to enhance the antibody-producing ability upon administration. Administration is usually performed once every 2 to 6 weeks, for a total of about 2 to 10 times.
  • warm-blooded animals to be used include monkeys, rabbits, rabbits, dogs, guinea pigs, mice, rats, sheep, goats, and chickens, and mice and rats are preferably used.
  • a warm-blooded animal immunized with an antigen for example, an individual with an antibody titer is selected from a mouse, and the spleen or lymph node is collected 2 to 5 days after the final immunization and included in them.
  • an individual with an antibody titer is selected from a mouse, and the spleen or lymph node is collected 2 to 5 days after the final immunization and included in them.
  • a monoclonal antibody-producing hybridoma can be prepared.
  • the antibody titer in the antiserum can be measured, for example, by reacting the labeled polypeptide described below with the antiserum, and then measuring the activity of the labeling agent bound to the antibody.
  • the fusion operation can be performed according to a known method, for example, the method of Koehler and Milstein [Nature, 256, 495 (1975)].
  • the fusion promoter include polyethylene glycol (PEG) and Sendai virus, and PEG is preferably used.
  • PEG polyethylene glycol
  • myeloma cells include myeloma cells of warm-blooded animals such as NS_1, P3U1, SP2Z0, and AP-1, and P3U1 is preferably used.
  • the preferred ratio between the number of antibody-producing cells (spleen cells) used and the number of myeloma cells used is about 1: 1 to 20: 1, and PEG (preferably PEG1000 to PEG6000) Is added at a concentration of about 10 to 80%, and efficient cell fusion is performed by incubating at 20 to 40 ° (preferably at 30 to 37 ° C for 1 to 10 minutes).
  • PEG preferably PEG1000 to PEG6000
  • a variety of methods can be used to screen monoclonal antibody-producing hybridomas. For example, hybridoma culture supernatants can be applied to a solid phase (eg, microplate) on which polypeptide (protein) antigen is adsorbed directly or together with a carrier.
  • an anti-immunoglobulin antibody (anti-mouse immunoglobulin antibody is used if the cells used for cell fusion are mice) or peptide A, labeled with a radioactive substance or enzyme, and then add the solid phase.
  • an anti-immunoglobulin antibody anti-mouse immunoglobulin antibody is used if the cells used for cell fusion are mice
  • peptide A labeled with a radioactive substance or enzyme
  • Monoclonal antibodies can be selected according to known methods or modifications thereof. Usually, it can be performed in an animal cell culture medium supplemented with HAT (hypoxanthine, aminopterin, thymidine).
  • HAT hyperxanthine, aminopterin, thymidine
  • any medium can be used as long as it can grow a hybridoma. For example: !! 20%, preferably RPMI 1640 medium containing 10 to 20% fetal bovine serum, GIT medium containing 1 to 10% fetal bovine serum (Wako Pure Chemical Industries, Ltd. ) Or a serum-free medium for hybridoma cultivation (SFM-101, Nissui Pharmaceutical Co., Ltd.).
  • the culturing temperature is usually 20 to 40 ° C, preferably about 37 ° C.
  • the culturing time is usually 5 days to 3 weeks, preferably 1 week to 2 weeks.
  • the culture can be usually performed under 5% carbon dioxide gas.
  • the antibody titer of the hybridoma culture supernatant can be measured in the same manner as the measurement of the antibody titer in the antiserum described above.
  • Monoclonal antibodies can be separated and purified by known methods, for example, immunoglobulin separation and purification methods (eg, salting out method, alcohol precipitation method, isoelectric point precipitation method, electrophoresis method, ion exchanger (eg, DEAE)).
  • immunoglobulin separation and purification methods eg, salting out method, alcohol precipitation method, isoelectric point precipitation method, electrophoresis method, ion exchanger (eg, DEAE)
  • Adsorption / desorption method, ultracentrifugation method, gel filtration method, antigen-bound solid phase or specific purification method in which the antibody is collected using an active adsorbent such as protein A or protein G and the bond is dissociated to obtain the antibody) Can be done.
  • an active adsorbent such as protein A or protein G and the bond is dissociated to obtain the antibody
  • the polyclonal antibody of the present invention can be produced according to a known method or a method analogous thereto.
  • a immunizing antigen polypeptide antigen itself or a complex thereof with a carrier protein is formed, and a warm-blooded animal is immunized in the same manner as in the above-described method for producing a monoclonal antibody.
  • the antibody can be produced by collecting an antibody-containing substance against the antibody and separating and purifying the antibody.
  • the type of the carrier protein and the mixing ratio of the carrier and the hapten are determined by the antibody against the hapten immunized by cross-linking the carrier.
  • any kind may be cross-linked at any ratio.
  • serum albumin, thyroglobulin, hemocyanin, etc. in a weight ratio of about 0.1 to 2 per hapten per hapten.
  • a method of pulling force at 0, preferably about 1 to 5 is used.
  • Various condensing agents can be used for force coupling between the hapten and the carrier.
  • daltaraldehyde ⁇ carbodiimide, a maleimide active ester, an active ester reagent containing a thiol group or a dithioviridyl group, or the like is used.
  • the condensation product is administered to a warm-blooded animal itself or together with a carrier or diluent at a site where antibody production is possible.
  • Complete Freund's adjuvant / incomplete Freund's adjuvant may be administered in order to enhance the antibody-producing ability upon administration. The administration is usually made once every about 2 to 6 weeks, for a total of about 3 to 10 times.
  • the polyclonal antibody can be collected from the blood, ascites, etc., preferably from the blood, of the warm-blooded animal immunized by the above method.
  • the measurement of the polyclonal antibody titer in the antiserum can be performed in the same manner as the measurement of the antibody titer in the antiserum described above.
  • the separation and purification of the polyclonal antibody can be performed according to the same method for separation and purification of immunoglobulin as in the above-described separation and purification of the monoclonal antibody.
  • SEQ ID NO: 1 Complementary or substantially complementary to a polynucleotide (eg, DNA) encoding a protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1 or a partial peptide thereof or a salt thereof
  • a polynucleotide eg, DNA
  • Any polynucleotide (antisense polynucleotide) which has a nucleotide sequence complementary to or substantially complementary to a nucleotide or a part thereof and has an action capable of suppressing the expression of the polynucleotide. It may be.
  • DNA a polynucleotide encoding the receptor of the present invention
  • these DNAs may be abbreviated as the DNA of the present invention.
  • Antisense DNA having complementary base sequence or part thereof
  • antisense DNAs having a nucleotide sequence complementary to or substantially complementary to the DNA of the present invention or a part thereof, Any antisense DNA may be used as long as it has an effect of suppressing the expression of the DNA.
  • the nucleotide sequence substantially complementary to the DNA of the present invention is, for example, about the entire nucleotide sequence or a partial nucleotide sequence of the nucleotide sequence complementary to the DNA of the present invention (that is, the complementary strand of the DNA of the present invention).
  • the complementary sequence of the base sequence of the portion encoding the N-terminal part of the receptor of the present invention is approximately 70%. % Or more, preferably about 80% or more, more preferably about 90% or more, and most preferably about 95% or more.
  • These antisense DNAs can be produced using a known DNA synthesizer or the like.
  • nucleotide sequence complementary to or substantially complementary to the nucleotide sequence of DNA having the nucleotide sequence represented by SEQ ID NO: 3 or SEQ ID NO: 4, or an antisense having a portion thereof A polynucleotide, an antisense polynucleotide having a nucleotide sequence complementary to or substantially complementary to the nucleotide sequence of DNA having the nucleotide sequence represented by SEQ ID NO: 3 or SEQ ID NO: 4, or a portion thereof And so on.
  • an antisense polynucleotide having a base sequence complementary to the base sequence of DNA having the base sequence represented by SEQ ID NO: 3 or SEQ ID NO: 4, or a part thereof SEQ ID NO: 3 or SEQ ID NO: : Has a base sequence complementary to the base sequence of DNA having the base sequence represented by 4, or a part thereof Antisense polynucleotides and the like.
  • An antisense polynucleotide is usually composed of about 10 to 40 bases, preferably about 15 to 30 bases.
  • the phosphate residues (phosphates) of each nucleotide constituting the antisense DNA are, for example, chemically modified phosphate residues such as phosphorothioate, methylphosphonate, and phosphorodithionate. May be substituted.
  • These antisense polynucleotides can be produced using a known DNA synthesizer or the like.
  • the ligand of the present invention has a DAP12 phosphorylation promoting activity, an ERK phosphorylation promoting activity, an insulin signaling suppression activity, a TNF ct production promoting activity, a glucose uptake inhibiting activity, and the like.
  • the binding between the receptor of the present invention and the ligand of the present invention can be improved.
  • peptides, proteins, antibodies, non-peptide compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts, serum, etc. or salts thereof are efficiently screened. be able to.
  • the compound or a salt thereof includes (i) cell stimulating activity via the receptor of the present invention (eg, arachidonic acid release, acetylcholine release, intracellular Ca 2+ release, intracellular cAMP generation, intracellular Inhibition of cAMP production, intracellular cGMP production, inositol phosphate production, cell membrane potential fluctuation, intracellular protein phosphorylation, activation of c_fos, decrease in pH, GTP7S binding activity, cAMP dependent Activating protein kinase, c GMP-dependent protein kinase, phospholipid-dependent protein kinase, microtubule-associated protein kinase (MAP kinase), etc.
  • cell stimulating activity via the receptor of the present invention eg, arachidonic acid release, acetylcholine release, intracellular Ca 2+ release, intracellular cAMP generation, intracellular Inhibition of cAMP production, intracellular cGMP production, inositol phosphat
  • a comparison is made between (i) the case where the receptor of the present invention is brought into contact with the ligand of the present invention and (ii) the case where the receptor of the present invention is brought into contact with the ligand of the present invention and a test compound.
  • Perform The comparison is performed, for example, by measuring the binding amount of the ligand of the present invention to the receptor of the present invention, cell stimulating activity, and the like.
  • screening method of the present invention include, for example,
  • the method comprises measuring and comparing the amount of the ligand of the present invention bound to the cell or the membrane fraction when the ligand is brought into contact with the cell or the membrane fraction of the cell.
  • the ligand of the present invention is a labeled ligand; a receptor binding assay system such as the above-mentioned (a) to (c) screen-Jung method;
  • the receptor of the present invention a membrane fraction of an organ of a human or a warm-blooded animal is preferably used.
  • the receptor of the present invention and the like which is expressed in large amounts using a recombinant, is suitable for screening.
  • the above-described method for producing the receptor of the present invention and the like are used.
  • the preparation method described later when cells containing the receptor of the present invention or the cell membrane fraction are used, the preparation method described later may be followed.
  • the cell When a cell containing the receptor of the present invention is used, the cell may be immobilized with datalaldehyde, formalin, or the like.
  • the immobilization method can be performed according to a method known per se.
  • the cell containing the receptor of the present invention refers to a host cell expressing the receptor of the present invention.
  • the host cell include the aforementioned Escherichia coli, Bacillus subtilis, yeast, insect cells, animal cells, and the like. .
  • the manufacturing method is the same as described above.
  • the membrane fraction refers to a fraction containing a large amount of cell membrane obtained by a method known per se after cell framing.
  • Methods for crushing cells include crushing cells with a Potter-Elvehjem homogenizer, Waring Plender ⁇ Polytron.
  • fractionation by centrifugal force such as fractionation centrifugation and density gradient centrifugation, is mainly used.
  • the cell lysate may be reduced at low speed (500 rpm to 3000 rpm). Centrifuge for about 1 minute to 10 minutes (usually about 1 to 10 minutes)
  • the membrane fraction is rich in the expressed receptor of the present invention and membrane components such as cell-derived phospholipids and membrane proteins.
  • the amount of the receptor of the present invention in cells or in the membrane fraction containing the receptor of the main invention is preferably from 1 0 3 to 1 0 8 molecules per cell, 1 0 5-1 0 7 molecules It is preferred that The higher the expression level, the higher the ligand binding activity (specific activity) per membrane fraction, which not only enables the construction of a highly sensitive screening system, but also enables the measurement of a large number of samples in the same port. become.
  • the receptor fraction of the present invention and the ligand of the present invention are used.
  • the receptor fraction of the present invention a naturally occurring receptor fraction of the present invention or a recombinant receptor fraction of the present invention having an activity equivalent thereto is desirable.
  • equivalent activity refers to equivalent ligand binding activity and the like.
  • the labeled ligand e.g., a radioactive isotope (e.g., [125 1], [131 1], [3 ⁇ 4], [14 C], [32 P], [33 P] and [3 3 ⁇ 4]), Fluorescent substances [eg, cyanine fluorescent dyes (eg, Cy2, Cy3, Cy5, Cy5.5, Cy7 (manufactured by Amersham Biosciences), etc.), fluorescamine, phnoleorescene isothiocynate, NBD, etc.], enzymes (eg, , / 3-galactosidase, 1-dalcosidase, al-force phosphatase, peroxidase, malate dehydrogenase, etc., luminous substances (eg, luminol, luminol derivatives, luciferin, lucigenin, etc.), piotin, lanthanide
  • a ligand labeled with an element or the like can be used.
  • a compound of a compound that changes the binding between the ligand of the present invention and the receptor of the present invention is used.
  • the buffer may be any buffer such as a phosphate buffer having a pH of 4 to 10 (preferably pH 6 to 8) or a buffer of Tris-monohydrochloride, which does not inhibit the binding between the ligand and the receptor.
  • a buffer for the purpose of reducing non-specific binding, CHAPS, Tween-80 TM (Kaoichi Atlas), digitonin, dexcholate, etc. Can be added to the buffer.
  • a protease inhibitor such as PMSF, leptin, E-64 (manufactured by Peptide Research Laboratories) and pepstatin can be added.
  • a fixed amount 5000 to 500,000 cpm
  • 10 to 1 M 10 to 7 M of the test compound are allowed to coexist.
  • a reaction tube containing a large excess of the unlabeled ligand of the present invention is also prepared to determine the non-specific binding amount (NSB).
  • the reaction is carried out at 0 ° C to 50 ° C, preferably at 4 ° C to 37 ° C for 20 minutes to 24 hours, preferably 30 minutes to 3 hours.
  • the mixture is filtered through a glass fiber filter or the like, washed with an appropriate amount of the same buffer, and the radioactivity remaining on the glass fiber filter is measured with a liquid scintillation counter or a ⁇ _counter.
  • the specific binding amount (B-NSB) is 50%, for example. % Or less of the test compound can be selected as a candidate substance having a competitive inhibition ability.
  • compounds that bind to the receptor of the present invention can be screened by utilizing the surface plasmon sensor technology.
  • the receptor of the present invention is immobilized on the surface of a sensor chip of Biacore 3000 (Biacore)
  • the surface of the chip surface when a test compound dissolved in phosphate buffer solution (PBS) or the like is flowed.
  • a test compound that binds to the receptor of the present invention is selected by measuring the change in the plasmon.
  • a test compound that gives a measured value of change in surface plasmon of 5 or more resonance units is selected as a substance having a binding property to the receptor of the present invention.
  • the increase or decrease in fluorescence intensity and fluorescence polarization can be measured to screen for a compound that binds to the receptor of the present invention.
  • the cell-stimulating activity via the receptor of the present invention for example, arachidonic acid release, acetylcholine release, intracellular Ca 2+ release, intracellular CAM P production, inhibition of intracellular cAMP production, intracellular cGMP production, inositol phosphate production, fluctuation of cell membrane potential, phosphorylation of intracellular protein, activation of c-fos, decrease of pH, GTPTS binding activity, c AM P dependency Activities that promote or inhibit protein kinase activation, c GMP-dependent protein kinase activation, phospholipid-dependent protein kinase activation, microtubule-associated protein kinase (MAP kinase) activation, etc.
  • MAP kinase microtubule-associated protein kinase
  • cells containing the receptor of the present invention are cultured in a multiwell plate or the like.c When performing screening, replace with a fresh medium or an appropriate buffer that is not toxic to cells. After adding the test compound and incubating for a certain period of time, extract the cells or collect the supernatant, and quantitate the product produced according to each method. If the production of a substance (for example, arachidonic acid) as an indicator of cell stimulating activity is difficult to be assayed by a degrading enzyme contained in a cell, an inhibitor for the degrading enzyme may be added to perform the assay. In addition, activities such as inhibition of cAMP production can be detected as an activity of inhibiting production of cells whose basic production has been increased by forskolin or the like.
  • a substance for example, arachidonic acid
  • cells expressing the appropriate receptor of the present invention are required.
  • the above-mentioned cell lines expressing the receptor of the present invention and the like are desirable.
  • Test compounds include, for example, peptides, proteins, antibodies, non-peptidic compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts, and serum.
  • a compound that alters the binding between the ligand of the present invention and the receptor of the present invention is screened by measuring the stimulating activity of the ligand of the present invention on the cells expressing the receptor of the present invention. be able to.
  • the case where the ligand of the present invention is brought into contact with the receptor of the present invention and the DAP12 co-expressing cell and the case where the ligand of the present invention and the test compound are brought into contact with the receptor of the present invention and the DAP12 co-expressing cell Measure the degree of DAP12 phosphate in
  • a compound that changes the binding property between the ligand of the present invention and the receptor of the present invention is screened.
  • the degree of DAP12 phosphoric acid is measured by a known method, for example, a Western blot method using an anti-phosphorylated tyrosine antibody.
  • the receptor of the present invention and VAP-tagged DAP12 co-expression are produced by a known method.
  • the cells were seeded at 5xl0 4 cell / well in 24-well plates, and cultured for 48 hours.
  • the ligand of the present invention and a test compound are added, and the cells are cultured for 10 minutes.
  • Cells are lysed with a cell lysate [30 mM Tris-HCl (pH 7.4), 150 mM NaCl, 10 mM EDTA, 1% NP-40, 50 mM NaF, 1 mM Na-Vanadate], and then sonicated. Crush the membrane.
  • the cell lysate is immunoprecipitated with an anti-V5 antibody according to a known method, and the immunoprecipitated sample is analyzed by Western blotting.
  • DAP12 total protein in a sample is detected by a densitometer using an anti-V5 antibody (Invitrogen).
  • the degree of phosphorylation of DAP12 when cells were stimulated only with the ligand of the present invention was compared with the degree of phosphorylation of MP12 when the test compound and ⁇ : ligand of the invention were added to the cells.
  • a test compound that prevents the enhanced phosphorylation of DAP12 by the ligand is selected as an antagonist.
  • the production of intracellular cAMP is suppressed by stimulation with the ligand of the present invention.
  • the stimulatory activity of the ligand of the present invention on the cell expressing the receptor of the present invention is measured to screen for a compound that alters the binding between the ligand of the present invention and the receptor of the present invention. Can be.
  • the ligand of the present invention when the ligand of the present invention is brought into contact with the receptor-expressing cell of the present invention in the presence of a substance that increases the amount of intracellular cAMP, the ligand of the present invention and the test compound express the receptor of the present invention.
  • Compounds that alter the binding between the ligand of the present invention and the receptor of the present invention are screened by measuring and comparing the inhibitory activity of the cells on the production of intracellular cAMP when brought into contact with the cells.
  • Substances that increase the amount of intracellular cAMP include, for example, forskolin, calcito Nin and the like are used. +
  • the amount of cAMP produced in the cells expressing the receptor of the present invention was determined using an anti-cAMP antibody obtained by immunizing mice, rats, rabbits, goats, rabbits, etc., and [ 125 1] -labeled cAMP (both commercially available products). Can be measured by RIA system or EIA system combining anti-cAMP antibody and labeled cAMP. Moreover, the anti-cAMP antibody, SPA to use the beads and [125 1] -labeled cAMP scintillant containing fixed using such antibodies to such animal IgG used for protein A or an anti-cAMP antibody production (Scintillation Proximity Assay) is also possible (using a kit from Amersham-Pharmacia Biotech).
  • a test compound showing an activity of inhibiting the cAMP production-suppressing activity of the receptor-expressing cell of the present invention by the ligand of the present invention can be selected as a candidate substance having a competitive inhibition ability.
  • a compound showing agonist activity can be screened by bringing only the test compound into contact with the receptor-expressing cell of the present invention and examining the cAMP production inhibitory activity.
  • Cells expressing the receptor of the present invention are seeded on a 24-well plate at 5 ⁇ 10 4 cells / well, and cultured for 48 hours. Wash the cells with Hanks buffer ( ⁇ 7.4) containing 0.2raM 3-isobutyl-methylxanthine, 0.05% BSA and 20mM HEPES (hereinafter abbreviated as reaction buffer). Then, add 0.5 ml of reaction buffer and incubate for 30 minutes in the incubator.
  • Hanks buffer ⁇ 7.4
  • reaction buffer containing 0.2raM 3-isobutyl-methylxanthine, 0.05% BSA and 20mM HEPES
  • reaction buffer 0.25 ml of the reaction buffer was added to the cells, and 1 ⁇ L of the ligand of the present invention or 1 ⁇ L of the ligand of the present invention and the test compound were added thereto. ⁇ ⁇ Add 0.25 ml of reaction buffer containing forskolin to the cells, and incubate at 37 ° C for 24 minutes. Stop the reaction by adding ⁇ of 20% perchloric acid, then place on ice for 1 hour to extract intracellular cAMP. Measure the amount of cAMP in the extract using the cAMP EIA kit (Amersham Pharmacia Biotech).
  • a test compound for the cAMP production inhibitory activity of the ligand of the present invention Calculate the effect of A test compound that inhibits the activity of the ligside of the present invention and has a cAMP production activity of, for example, 50% or more can be selected as a candidate substance having a competitive inhibition ability.
  • the receptor-expressing cells of the present invention showing the property of increasing the intracellular cAMP level upon stimulation of the ligand of the present invention
  • the case where the ligand of the present invention is brought into contact with the receptor-expressing cells of the present invention is compared with
  • the ligand of the present invention and a test compound are brought into contact with a cell that expresses the receptor of the present invention
  • the activity of the cell for promoting intracellular cAMP production is measured and compared.
  • Compounds that alter the binding to the receptor can be screened.
  • a test compound showing an activity of inhibiting the cAMP production promoting activity of the receptor-expressing cell of the present invention by the ligand of the present invention can be selected as a candidate substance having a competitive inhibition ability.
  • a compound exhibiting agonist activity can be screened by bringing only the test compound into contact with the receptor-expressing cell of the present invention and examining cAMP production promoting activity.
  • the cAMP production promoting activity can be measured by adding the ligand of the present invention or the ligand of the present invention to cells expressing the receptor of the present invention (eg, animal cells such as CH0 cells) without adding forskolin in the above-described screening method.
  • CAMP produced by adding the compound is quantified and measured by the method described above.
  • the binding between the ligand of the present invention and the receptor of the present invention is changed by measuring the stimulating activity of the ligand of the present invention on cells expressing the receptor of the present invention using the CRE-reporter gene vector. Compounds to be screened.
  • DNA containing a CRE (cAMP response element) is inserted upstream of the reporter gene of the vector to obtain a CRE-reporter gene vector.
  • a CRE cAMP response element
  • stimulation accompanied by an increase in cAMP induces CRE-mediated reporter gene expression and subsequent production of a reporter gene gene product (protein). Induce.
  • protein reporter gene gene product
  • the ligand of the present invention is brought into contact with the receptor-expressing cell of the present invention into which a CRE-reporter gene vector has been introduced in the presence of a substance that increases the amount of intracellular cAMP.
  • the compound is brought into contact with a CRE-reporter-gene vector-transfected receptor-expressing cell of the present invention, the enzymatic activity of the reporter gene protein is measured and compared to obtain the ligand of the present invention and the ligand of the present invention. Screen for compounds that alter the binding to the receptor.
  • a substance that increases the amount of intracellular cAMP for example, phonorescoline, calcitonin, and the like are used.
  • the vector for example, Pitka Gene Basic Vector, Pikken Gene Enhancer Vector (Toyo Ink Manufacturing Co., Ltd.) and the like are used.
  • the DNA containing the CRE, the reporter gene of the vector for example ⁇ the multiple cloning site of the luciferase gene on stream, CRE-in reporter gene vector to £ method, enzyme reporter monogenic protein by the ligand of the present invention
  • a test compound that restores activity suppression can be selected as a candidate substance capable of competitive inhibition.
  • agonists can be screened by bringing only the test compound into contact with cells expressing the receptor of the present invention and measuring the same suppression of the amount of luminescence increased by forskolin stimulation as with the ligand of the present invention.
  • the cells expressing the receptor of the present invention into which the CRE-reporter gene (luciferase) has been introduced are seeded on a 24-well plate at 5 ⁇ 10 3 cells / well, and cultured for 48 hours. Wash the cells with Hank's buffer ( ⁇ 7.4) containing 0.2raM 3-isobutyl-methylxanthine, 0.05% BSA and 20mM HEPES (hereinafter abbreviated as reaction buffer). Then add 0.5 ml of reaction buffer and keep incubator for 30 minutes. After removing the reaction buffer and adding 0.25 ml of the reaction buffer to the cells, add 2 ⁇ l of the ligand of the present invention or 1 ⁇ l of the ligand of the present invention and the test compound.
  • Hank's buffer ⁇ 7.4
  • reaction buffer containing 0.2raM 3-isobutyl-methylxanthine, 0.05% BSA and 20mM HEPES
  • the ligand of the present invention suppresses an increase in luminescence by luciferase based on forskolin stimulation.
  • a compound that restores the inhibition can be selected as a candidate substance having competitive inhibitory ability.
  • reporter genes include alkaline phosphatase, chloramphenicol transferase (chloramphenicol)
  • a gene such as acetyltransferase) or j3-galactosidase may be used.
  • the enzyme activity of these reporter gene proteins is measured according to a known method or using a commercially available measurement kit.
  • Alkaline phosphatase activity can be measured using, for example, Lumi-Phos 530 manufactured by Wako Pure Chemical
  • chloramphen-chol 'acetyltransferase activity can be measured using, for example, FAST CAT chrolamphenicol Acetyltransferase Assay KiT manufactured by Wako Pure Chemical.
  • the receptor-expressing cells of the present invention release TNF o; outside the cells upon stimulation with the ligand of the present invention.
  • TNF a when the ligand of the present invention is brought into contact with the receptor-expressing cell of the present invention and when the ligand and the test compound of the present invention are brought into contact with the receptor-expressing cell of the present invention.
  • the compounds that change the binding between the ligand of the present invention and the receptor of the present invention are screened by measuring and comparing the release activities of the compounds.
  • a test compound that inhibits the activity of releasing the TNF by the ligand of the present invention can be selected as a candidate substance having a competitive inhibition ability.
  • the agonist activity was also determined by contacting the test compound alone with the receptor-expressing cells of the present invention and examining the TNF-releasing activity of the receptor-expressing cells of the present invention by a known method. Screening of compounds.
  • the receptor-expressing cells of the present invention were seeded at 5xl0 4 C ell / we ll in 24-well plates, 24 hr culture Yogo, Rigando of the present invention the ligand or a final concentration of 10 / M of the present invention at a final concentration of 10 M And test compounds are added to each well. After incubation at 37 ° C for 10 minutes, the cells were lysed with cell lysate (30 mM Tris-HCl (pH 7.4), 150 mM NaCl, 10 mM EDTA, 1% NP-40, 50 mM NaF, 1 mM Na-Vanadate ] And disintegrate the membrane by sonication.
  • the amount of TNFa contained in the cell lysate is measured according to the known EIA method.
  • the amount of TNFa is 0%, and the reaction buffer contains 10 / M of the ligand of the present invention.
  • test compound having a TNFa releasing activity of, for example, 50% or less can be selected as a candidate substance capable of competitive inhibition.
  • the Ca concentration in the cell is increased by the stimulation of the ligand of the present invention.
  • the intracellular in the case where the ligand of the present invention is brought into contact with the receptor-expressing cell of the present invention and the case where the ligand of the present invention and the test compound are brought into contact with the receptor-expressing cell of the present invention By measuring and comparing the calcium concentration increasing activity, a compound that changes the binding property between the ligand of the present invention and the receptor of the present invention is screened. The measurement is performed according to a known method.
  • a test compound that suppresses an increase in intracellular calcium concentration due to the ligand of the present invention can be selected as a candidate substance having a competitive inhibition ability.
  • agonists can be screened by measuring the increase in fluorescence intensity due to the addition of the test compound alone.
  • the receptor-expressing cell of the present invention was seeded on a sterilized microscope cover glass, and two days later, Replace the culture solution with HBSS in which 4raM Fura-2 AM (Dojindo Laboratories) is suspended, and leave at room temperature for 2 hours and 30 minutes. After washing with HBSS, place the cover glass on the cuvette, add the ligand of the present invention or the ligand of the present invention and the test compound, and measure the increase in the ratio of the fluorescence intensity at 505 nm at the excitation wavelengths of 340 nm and 380 nm by fluorescence measurement. Measure with a tester and compare.
  • FLIPR Molecular Devices
  • Fluo-3 AM manufactured by Dojindo Laboratories
  • the supernatant is washed several times by centrifugation, and the cells are transferred to a 96-well plate. Sow. Set in a FLIPR device, add the ligand of the present invention or the ligand of the present invention and a test compound in the same manner as in the case of Fura-2, measure the increase in the ratio of fluorescence intensity with a fluorimeter, and compare. I do.
  • a gene eg, aequorin
  • a gene protein eg, aequorin or the like
  • becomes Ca-binding and emits light it is also possible to screen for a compound that alters the binding between the ligand of the present invention and the receptor of the present invention.
  • a receptor-expressing cell of the present invention in which a gene for a protein that emits light by increasing intracellular Ca ions, is seeded on a 96-well plate, and the ligand of the present invention or the ligand of the present invention, Add the test compound, measure the increase in the ratio of fluorescence intensity with a fluorimeter, and compare.
  • a test compound that suppresses an increase in fluorescence intensity due to the ligand of the present invention can be selected as a candidate substance having a competitive inhibitory ability.
  • the intracellular inositol triphosphate concentration increases.
  • a compound that changes the binding property between the ligand of the present invention and the receptor of the present invention is screened by utilizing the activity of the ligand of the present invention to produce intracellular inositol triphosphate in the cells expressing the receptor of the present invention. be able to.
  • the ligand of the present invention when the ligand of the present invention is brought into contact with the receptor-expressing cell of the present invention in the presence of labeled inositol, By measuring and comparing the inositol triphosphate productivity when the cells are brought into contact with the receptor-expressing cells of the present invention, compounds that alter the binding between the ligand of the present invention and the receptor of the present invention are screened. The measurement is performed according to a known method. In this method, a test compound that suppresses inositol triphosphate-producing activity can be selected as a candidate substance capable of competitive inhibition.
  • agonists can be screened by bringing only the test compound into contact with the receptor-expressing cell of the present invention and measuring the increase in inositol triphosphate production.
  • the cells expressing the receptor of the present invention are seeded on a 24-well plate and cultured for one day. After that, the cells are cultured in a medium supplemented with myo- [2- 3 H] inositol (2.5 ⁇ Ci / well) for 1 day, and the cells are thoroughly washed with a medium free of radioactive inositol. After adding the ligand of the present invention or the ligand of the present invention and the test compound, 10% perchloric acid is added to stop the reaction. 1. neutralized with 5M hydroxide force Riumu and 60 mM HEPES solution and then passed through a column packed with AGlx8 resin (Bio- Rad) in 0.
  • the radioactivity eluted with 1 M ammonium formate and 0.1 M formic acid is measured using a liquid scintillation counter.
  • the radioactivity when the ligand of the present invention is not added is 0%, and the radioactivity when the ligand of the present invention is added is 100%, and the ligand of the present invention and the receptor of the present invention are the test compounds. Calculate the effect on binding.
  • test compound having an inositol triphosphate-producing activity of, for example, 50% or less can be selected as a candidate substance having an antagonistic ability.
  • the binding between the ligand of the present invention and the receptor of the present invention is changed by measuring the stimulating activity of the ligand of the present invention on cells expressing the receptor of the present invention using the TRE-reporter gene vector. Compounds to be screened.
  • DNA containing a TRE is inserted into the vector upstream of the reporter gene to obtain a TRE-reporter gene vector.
  • TPA response element DNA containing a TRE (TPA response element) is inserted into the vector upstream of the reporter gene to obtain a TRE-reporter gene vector.
  • stimulation accompanied by an increase in intracellular calcium concentration is caused by TRE-mediated reporter gene expression and subsequent reporter gene expression.
  • the ligand of the present invention when the ligand of the present invention is brought into contact with a TRE-reporter-gene vector-introduced receptor-expressing cell, the ligand of the present invention and a test compound are expressed in a TRE-reporter-gene vector.
  • a compound that changes the binding between the ligand of the present invention and the receptor of the present invention by measuring and comparing the enzymatic activity of the reporter gene protein when the cell is brought into contact with the cells expressing the receptor of the present invention. Screen.
  • the vector for example, Pitka Gene Basic Vector, Pitka Gene Enhancer Vector-1 (Toyo Ink Mfg. Co., Ltd.) and the like are used.
  • the DNA containing the TRE, the reporter gene of the vector for example ⁇ the multiple cloning site of the luciferase gene on stream, TRE-in c the method of the reporter gene vector, the enzymatic activity of the reporter gene protein by the ligand of the present invention
  • the test compound to be suppressed can be selected as a candidate substance having competitive inhibitory ability.
  • agonist screening is performed by bringing only the test compound into contact with the TRE-reporter gene vector-transfected receptor-expressing cell of the present invention and measuring the increase in luminescence in the same manner as the ligand of the present invention. You can also.
  • the TRE-reporter gene (luciferase) -introduced receptor-expressing cells of the present invention are seeded on a 24-well plate at 5 ⁇ 10 3 cells / well and cultured for 48 hours. After washing the cells with Hanks buffer (pH 7.4) containing 0.05% BSA and 20 mM HEPES, add ⁇ ligand of the present invention or ⁇ ligand of the present invention and a test compound, and add at 37 ° C. And react for 60 minutes.
  • Hanks buffer pH 7.4
  • BSA Hanks buffer
  • ⁇ ligand of the present invention or ⁇ ligand of the present invention and a test compound add at 37 ° C. And react for 60 minutes.
  • Cell lysing agent for Pitka Gene Toyo Ink Manufacturing
  • a luminescent substrate (Toyo Ink Mfg. Co., Ltd.) to the solution. Luminescence from Luciferase is measured with a noreminometer, liquid scintillation counter or top counter.
  • a noreminometer liquid scintillation counter or top counter.
  • the increase in intracellular calcium by the ligand of the present invention increases the amount of luminescence by luciferase.
  • a compound that suppresses this increase can be selected as a candidate substance capable of competitive inhibition.
  • Reporter genes include, for example, alkaline phosphatase, chloramphenicol transferase, and chloramphenicol.
  • acetyltransferase -A gene such as galactosidase may be used.
  • the enzyme activity of these reporter gene proteins is measured according to a known method or using a commercially available measurement kit. Alkaline phosphatase activity was measured using, for example, Lumi-Phos 530 manufactured by Wako Pure Chemical, and chloramphenicolone acetyltransferase activity was measured using, for example, FAST CAT chrolamphenicol Acetyltransferase Assay KiT manufactured by Wako Pure Chemical. The activity is measured using, for example, Aurora Gal-XE manufactured by Wako Pure Chemical Industries.
  • the MAP kinase is activated by the stimulation of the ligand of the present invention, and the cells expressing the receptor of the present invention proliferate. Utilizing this reaction, the stimulatory activity of the ligand of the present invention on the cells expressing the receptor of the present invention is measured to screen for a compound that changes the binding between the ligand of the present invention and the receptor of the present invention.
  • the t specifically capable of, the ligand of the present invention and when contacted with receptor-expressing cells of the present invention, a ligand and a test compound of the present invention, brought into contact with receptor-expressing cells of the present invention
  • a compound that changes the binding between the ligand of the present invention and the receptor of the present invention is screened.
  • the growth of the receptor-expressing cells of the present invention may be measured, for example, by measuring MAP kinase activity, thymidine uptake activity, cell number, and the like.
  • MAP kinase activity after adding the ligand of the present invention or the ligand of the present invention and a test compound to the receptor-expressing cells of the present invention, an anti-MAP kinase antibody was used from a cell lysate. After obtaining the MAP kinase fractions by immunoprecipitation, a known method, for example, manufactured by Wako pure Chemical Industries, Ltd. MAP of kinase Assay Kit and ⁇ - [32 P] - using ATP to measure MAP kinase activity, and compared.
  • the cells expressing the receptor of the present invention were seeded on a 24-well plate, cultured, and added with the ligand of the present invention or the ligand and the test compound of the present invention, and then thymidine labeled with radioactivity (eg, [methyl-II] -thymidine), then lyse the cells and measure the thymidine uptake activity by counting the radioactivity of the thymidine incorporated into the cells with a liquid scintillation counter. I do.
  • radioactivity eg, [methyl-II] -thymidine
  • the cells expressing the receptor of the present invention were seeded on a 24-well plate, cultured, supplemented with the ligand of the present invention or the ligand and the test compound of the present invention, and then treated with MTT (3- (4 , 5-dimethyto 2-thiazolyl)-2, 5-diphenyl-2H-tetrazolium bromide).
  • MTT 3- (4 , 5-dimethyto 2-thiazolyl)-2, 5-diphenyl-2H-tetrazolium bromide.
  • MTT formazan in which MTT has been changed by being taken into cells is dissolved in an aqueous solution of isopropanol acidified with hydrochloric acid, and then measured by absorption at 570 nm for comparison.
  • a test compound that suppresses the growth of the receptor-expressing cell of the present invention can be selected as a candidate substance having a competitive inhibition ability.
  • agonists can be screened by bringing only the test compound into contact with the receptor-expressing cell of the present invention and measuring the same cell proliferation activity as the ligand of the present invention.
  • the receptor-expressing cells of the present invention are seeded at 5,000 cells / well in a 24-well plate, and cultured for 1 day ( then in a serum-free medium for 2 days).
  • the ligand of the present invention or the ligand of the present invention and a test compound to the cells and culture for 24 hours, and then add [methyl-3 ⁇ 4] -thymidine at 0.015 MBq / ml.
  • wash the cells with PBS add methanol and leave for 10 minutes, then add 5% trichloroacetic acid and leave for 15 minutes to fix the cells. Wash the cells four times with distilled water 0.3 Lyse the cells with 3N sodium hydroxide solution, and measure the radioactivity in the lysate with a liquid scintillation counter.
  • a test compound that suppresses an increase in radioactivity when the ligand of the present invention is added can be selected as a candidate substance capable of competitive inhibition.
  • a compound that alters the binding between the ligand of the present invention and the receptor of the present invention is screened by measuring the stimulating activity of the ligand of the present invention on the cells expressing the receptor of the present invention. be able to.
  • ERK1 / 2 phosphorylation in the case of contacting the ligand of the present invention with the receptor-expressing cell of the present invention and in the case of contacting the ligand of the present invention and a test compound with the receptor-expressing cell of the present invention.
  • a compound that changes the binding between the ligand of the present invention and the receptor of the present invention is screened.
  • the degree of phosphorylation of ERK1 / 2 is measured by a known method, for example, Western blotting using an anti-phosphorylated ERK antibody.
  • Cells expressing the receptor of the present invention are prepared by a known method (the cells are seeded on a 24-well plate at 5 ⁇ 10 4 cells / well, and cultured for 48 hours. Add the ligand of the invention and the test compound, and incubate for 10 minutes Cell lysate [30 mM Tris-HCl (pH 7.4), 150 mM NaCl, 10 mM EDTA, 1% NP-40, 50 mM NaF , 1 was dissolved in mM Na-vanadate], further source - Keshiyon by ⁇ Re the t cells broken ⁇ to Yabu ⁇ a film by a known method, were immunoprecipitated with anti-V5 antibody, immunoprecipitated samples ⁇ We stamp lot Detect the total ERK1 / 2 protein in the sample by a known method using a densitometer using an anti-ERK1 / 2 antibody (Sigma) while using an anti-phosphorylated ERK antibody (Sigma).
  • the cells expressing the receptor of the present invention react with the ligand of the present invention to change extracellular pH. Utilizing this reaction, the stimulatory activity of the ligand of the present invention on the cells expressing the receptor of the present invention is measured, whereby the binding between the ligand of the present invention and the receptor of the present invention is measured. Compounds that alter compatibility can be screened.
  • the cells obtained when the ligand of the present invention was brought into contact with the receptor-expressing cell of the present invention and the cells obtained when the ligand and the test compound were brought into contact with the receptor-expressing cell of the present invention.
  • a compound that alters the binding between the ligand of the present invention and the receptor of the present invention is screened by measuring and comparing the change in external pH. The extracellular pH change is measured, for example, using a Cytosensor device (Molecular Devices).
  • a test compound that suppresses the extracellular pH change caused by the ligand of the present invention can be selected as a candidate substance capable of competitive inhibition.
  • agonists can be screened by bringing only the test compound into contact with the receptor-expressing cell of the present invention and measuring the extracellular pH change as in the ligand of the present invention.
  • the receptor-expressing cell of the present invention is cultured overnight in a capsule for a Cytosensor device, set in a chamber of the device, and RPMI1640 medium containing 0.1% BSA (molecular device) for about 2 hours until the extracellular pH is stabilized. Perfusion). After the pH has stabilized, the cells of the present invention or a medium containing a ligand of the present invention and a test compound are perfused onto the cells. The pH change of the medium caused by perfusion is measured and compared.
  • a compound that suppresses the extracellular pH change caused by the ligand of the present invention can be selected as a candidate substance having a competitive inhibition ability.
  • the receptor of the present invention (eg, TREM-2) is prepared by combining the extracellular domain of the receptor of the present invention (eg, TREM-2) and the fusion protein expression protein of CDSzeta with the NFAT-reporter gene. 2)
  • the signal can be detected by replacing the signal with the CD3 signal. Utilizing this reaction, the stimulatory activity of the ligand of the present invention on the cells expressing the receptor of the present invention is measured to screen for a compound that alters the binding between the ligand of the present invention and the receptor of the present invention. can do.
  • a receptor of the present invention eg, TREM-2
  • an extracellular domain of a receptor of the present invention eg, TREM-2
  • an expression vector of a fusion protein of CD3zeta and an NFAT-reporter gene eg, luciferase
  • a receptor of the present invention eg, luciferase
  • TREM-22 Activation of CD3zeta induces NFAT-mediated reporter gene expression and subsequent production of reporter gene gene products (proteins). That is, by measuring the enzymatic activity of the reporter gene protein, the signal intensity of the receptor of the present invention (eg, TREM-2) of the NFAT-reporter gene vector-introduced cells can be detected.
  • the ligand of the present invention is introduced into the expression cell into which the extracellular domain of the receptor of the present invention (eg, TREM-2) and the CD3zeta fusion protein expression vector and the NFAT-reporter gene have been introduced.
  • the ligand of the present invention and the test compound are introduced into the cells in which the extracellular domain of the receptor of the present invention (eg, TREM-2) and the fusion protein expression vector of CD3zeta and the NFAT-reporter gene have been introduced.
  • TREM-2 extracellular domain of the receptor of the present invention
  • a compound that changes the binding between the ligand of the present invention and the receptor of the present invention eg, TREM-2 is screened. I do.
  • Receptor of the present invention (Example, TREM- 2) of the extracellular domain and CD3zeta fusion protein expression vector and NFAT- reporter gene (luciferase) and introduced expression cells of at 5xl0 4 cell / well in 24-well plates Seed and incubate for 48 hours.
  • reaction buffer 1 Except for the reaction buffer 1, 0.25 ml of the reaction buffer was added to the cells, and then 1 ⁇ of the ligand of the present invention or 1 ⁇ of the ligand of the present invention and the test compound were added. Add 25 ml of application buffer to the cells and incubate at 37 ° C for 10 minutes. Lyse the cells with a cell lysing agent for Pitka Gene (Toyo Ink Mfg. Co., Ltd.) and add a luminescent substrate (Toyo Ink Mfg. Co., Ltd.) to the lysate. Luminescence from luciferase is measured with a luminometer, liquid scintillation counter or top counter.
  • a cell lysing agent for Pitka Gene Toyo Ink Mfg. Co., Ltd.
  • luminescent substrate Toyo Ink Mfg. Co., Ltd.
  • the amount of luminescence by luciferase in the case where only the ligand of the present invention is added and the case where 1 ⁇ M of the ligand of the present invention and the test compound are added are measured and compared.
  • the receptor gene RNA of the present invention is injected into Xenopus oocytes and stimulated with the ligand of the present invention, the cellular calcium concentration increases, and calcium-activated chloride current is generated. This can be considered as a change in the membrane potential (even when there is a change in the K ion concentration gradient).
  • the ligand of the present invention when the ligand of the present invention is brought into contact with the receptor gene RNA-introduced Xenopus laevis oocytes, the ligand of the present invention and the test compound are introduced into the receptor gene of the present invention.
  • the ligand of the present invention and the test compound are introduced into the receptor gene of the present invention.
  • a compound that changes the binding between the ligand of the present invention and the receptor of the present invention is screened.
  • a test compound that suppresses a change in cell membrane potential can be selected as a candidate substance having a competitive inhibition ability.
  • the receptor gene mRNA of the present invention may be prepared from a tissue or a cell, or may be transcribed from a plasmid in vitro.
  • the receptor gene mRNA of the present invention was cultured in MBS solution at 20 ° C. for 3 days, and this was placed in the cavity of a voltage clamp device in which Ringer solution was flowing, and the potential was measured. Insert the glass microelectrode for fixation and the glass microelectrode for potential measurement into the cell, and place the (-) electrode outside the cell. When the potential is stabilized, a change in potential is recorded by flowing a Ringer solution containing the ligand of the present invention or the ligand of the present invention and a test compound.
  • test compound was determined by comparing the change in the cell membrane potential of the oocyte of the receptor gene of the present invention into which RNA of the present invention was transfected with that of the Ringer solution containing only the ligand of the present invention. Can be measured.
  • a compound that suppresses the change in cell membrane potential can be selected as a candidate substance having a competitive inhibition ability.
  • a screening kit for a compound or a salt thereof that alters the binding property between the ligand of the present invention and the receptor of the present invention includes a receptor of the present invention or a cell containing the receptor of the present invention or a membrane fraction of the cell, And a ligand of the invention.
  • screening kit of the present invention examples include the following. 1. Screening reagent
  • the solution may be sterilized by filtration through a 0.45 / zm filter and stored at 4 ° C, or may be prepared at use.
  • CHO cells expressing the receptor of the present invention 1 2-well plates and passaged 5 X 1 0 5 cells / well, cultured for 2 days at 3 7 ° C, 5% C 0 2, 9 5% air What you did.
  • a radioisotope such as [], [ 125 1], [ 14 C], [ 32 P], [ 33 P], [ 35 S] in a suitable solvent or buffer Up to 4 ° C
  • the ligand of the present invention is dissolved to a concentration of 1 mM in PBS containing 0.1% ⁇ serum albumin (manufactured by Sigma) and stored at 120 ° C.
  • a compound or a salt thereof obtained by using the screening method or the screening kit of the present invention is a compound that alters the binding between the ligand of the present invention and the receptor of the present invention or the activity of the receptor of the present invention.
  • a compound that promotes or inhibits specifically, (i) a compound having a cell stimulating activity via the receptor of the present invention or a salt thereof (receptor agonist of the present invention); and (ii) a compound having the stimulating activity.
  • a compound that promotes the binding strength between the receptor of the present invention and the ligand of the present invention (iv) a binding force between the receptor of the present invention and the ligand of the present invention.
  • Such compounds include peptides, proteins, antibodies, Examples include compounds selected from non-peptide compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts, and serum.These compounds may be novel compounds. It may be a known compound.
  • salt of the compound those similar to the aforementioned salts of the receptor of the present invention are used.
  • the method of evaluating whether the receptor agonist of the present invention is an antagonist or an antagonist may be, for example, according to the following (i) or (ii).
  • a compound having a cell stimulating activity or a salt thereof is the receptor agonist (agonist) of the present invention, and a compound having no such activity or a salt thereof is a receptor antagonist (antagonist) of the present invention:
  • test compound is brought into contact with a cell containing the receptor of the present invention, and the cell stimulating activity of the receptor of the present invention is measured.
  • the compound having a cell stimulating activity or a salt thereof is the receptor agonist of the present invention.
  • the ligand of the present invention has a DAP12 phosphorylation promoting activity, an ERK phosphorylation promoting activity, an insulin signaling inhibitory activity, a TNF production promoting activity, a glucose uptake inhibiting activity, and the like.
  • the receptor agonist of the present invention has the physiological activity of the ligand of the present invention.
  • DAP12 phosphorylation promoting activity ERK phosphorylation promoting activity
  • insulin signaling inhibitory activity TNF a production promoting activity
  • glucose uptake inhibitory activity etc.
  • it is useful as an agent for preventing or treating hypoglycemia.
  • Physiological activity possessed by the ligand of the present invention eg, DAP12 phosphorylation promoting activity, ERK phosphorylation promoting activity, insulin signaling inhibition activity, TNF o; production promoting activity, glucose uptake inhibiting activity, etc.
  • safe and low toxic drugs such as insulin resistance improver, impaired bran tolerance, diabetes, obesity, hyperlipidemia, arteriosclerosis, prevention of hypertension or heart disease ⁇ Useful as a therapeutic agent.
  • the compound that promotes the binding strength between the receptor of the present invention and the ligand of the present invention is useful as a safe and low-toxicity drug, for example, an agent for preventing or treating hypoglycemia.
  • Compounds that inhibit the binding between the receptor of the present invention and the ligand of the present invention are safe and low-toxic drugs, such as insulin resistance improvers, impaired glucose tolerance, diabetes, obesity, hyperlipidemia, and arteries. It is useful as a preventive and therapeutic agent for sclerosis, hypertension or heart disease.
  • Compounds or salts thereof obtained using the screening method or screening kit of the present invention include, for example, peptides, proteins, antibodies, non-peptide compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, animal tissues A compound selected from the group consisting of extract, serum and the like, which compound alters the binding between the receptor of the present invention and the ligand of the present invention, and a compound which promotes or inhibits the activity or function of the receptor of the present invention And compounds that promote or inhibit (increase or decrease the expression level) the expression of the receptor gene of the present invention.
  • salt of the compound those similar to the aforementioned salts of the receptor of the present invention are used.
  • the compound or a salt thereof can be used, for example, as a sugar-coated tablet, capsule, elixir, microcapsule, etc., orally, or with water or another pharmaceutically acceptable liquid. It can be used parenterally in the form of injectable preparations such as sterile solutions or suspensions.
  • injectable preparations such as sterile solutions or suspensions.
  • the compound or a salt thereof is combined with a physiologically acceptable carrier, flavoring agent, excipient, vehicle, preservative, stabilizer, binder and the like. It can be manufactured by mixing in the unit dosage form generally required for the practice of preparations. The amount of active ingredient in these preparations is such that a suitable dosage in the specified range can be obtained.
  • Excipients that can be incorporated into tablets, forceps, etc. include, for example, binders such as gelatin, corn starch, tragacanth, gum arabic, excipients such as crystalline cellulose, corn starch, gelatin, alginic acid
  • binders such as gelatin, corn starch, tragacanth, gum arabic
  • excipients such as crystalline cellulose, corn starch, gelatin, alginic acid
  • swelling agents such as sucrose, lubricating agents such as magnesium stearate, sweetening agents such as sucrose, lactose or saccharin, and flavoring agents such as peppermint, cocoa oil or cherry are used.
  • a liquid carrier such as oils and fats can be further contained in the above-mentioned type of material.
  • Sterile compositions for injection are formulated according to standard pharmaceutical practice, such as dissolving or suspending the active substance in vehicles such as water for injection, and naturally occurring vegetable oils such as sesame oil and coconut oil. Can
  • aqueous solutions for injection include physiological saline, isotonic solutions containing glucose and other adjuvants (eg, D-sorbitol, D-mannitol, sodium chloride, etc.).
  • Agents for example, alcohols (eg, ethanol), polyalcohols (eg, propylene glycol, polyethylene glycol, etc.), nonionic surfactants (eg, polysorbate 80 TM, HCO-50, etc.) You can use it together.
  • the oily liquid include sesame oil and soybean oil, which may be used in combination with solubilizing agents such as benzyl benzoate and benzyl alcohol.
  • the compound or a salt thereof is treated with a buffer (eg, phosphate buffer, sodium acetate buffer, etc.), a soothing agent (eg, benzalkonium chloride, procaine hydrochloride, etc.), a stabilizer. Serum albumin, polyethylene glycol, etc.), preservatives (eg, benzyl alcohol, phenol, etc.), antioxidants and the like.
  • a buffer eg, phosphate buffer, sodium acetate buffer, etc.
  • a soothing agent eg, benzalkonium chloride, procaine hydrochloride, etc.
  • a stabilizer e.g, benzyl alcohol, phenol, etc.
  • the prepared injection solution is usually filled in an appropriate sample.
  • the preparations obtained in this way are safe and low toxic, and can be used, for example, in humans or warm-blooded animals (eg, mice, rats, puppies, sheep, pigs, puppies, pumas, birds, cats, dogs, Monkeys, chimpanzees, etc.). There is a difference depending on the dose of the compound or its salt, its action, target disease, subject of administration, route of administration and the like.
  • a diabetic patient per 6 kg of body weight
  • the compound when administered parenterally, for example, when the receptor antagonist of the present invention is administered to a diabetic patient (body weight: 60 kg) in the form of an injection, the compound can be administered in an amount of about 0.01 to about 0.01 per day. It is convenient to administer 30 mg, preferably about 0.:!2 Omg, more preferably about 0.1-1 Omg, by intravenous injection. In the case of other animals, the dose can be administered per 60 kg body weight.
  • the antibody of the present invention can specifically recognize the receptor of the present invention, it can be used for quantification of the receptor of the present invention in a test solution, particularly for quantification by a sandwich immunoassay. .
  • a method for quantifying the receptor of the present invention in a test solution comprising:
  • the activity of the labeling agent on the insolubilized carrier is measured.
  • a method for quantifying the receptor of the present invention in a test solution is provided.
  • one antibody may be an antibody that recognizes the N-terminal of the receptor of the present invention, and the other antibody may be an antibody that reacts with the C-terminal of the receptor of the present invention. desirable.
  • the receptor of the present invention can be quantified using a monoclonal antibody against the receptor of the present invention, and can also be detected by tissue staining or the like.
  • the antibody molecule itself may be used, or the F (ab ′) 2 , Fab ′, or Fab fraction of the antibody molecule may be used.
  • the method for quantifying the receptor of the present invention using the antibody of the present invention is not particularly limited, and may be an antibody, an antigen, or an antibody corresponding to the amount of antigen (eg, the amount of polypeptide) in the test solution. Any method that detects the amount of the complex by chemical or physical means and calculates this from a standard curve prepared using a standard solution containing a known amount of antigen can be used. Good. For example, a nephelometry, a competition method, an immunometric method, and a sandwich method are preferably used, and in terms of sensitivity and specificity, it is particularly preferable to use a sandwich method described later.
  • a labeling agent used in a measurement method using a labeling substance for example, a radioisotope, an enzyme, a fluorescent substance, a luminescent substance and the like are used. Radioisotopes, if example embodiment, [125 1], [131 1], [], are used like [14 c].
  • the above-mentioned enzymes those which are stable and have a large specific activity are preferable, and for example, ⁇ -galatatosidase, ⁇ -glucosidase, lipophosphatase, and c. Oxidase, malate dehydrogenase and the like are used.
  • the fluorescent substance for example, fluorescamine, fluorescein isothiocyanate and the like are used.
  • the luminescent substance for example, luminol, luminol derivative, luciferin, lucigenin and the like are used.
  • a biotin-avidin system can be used for binding the antibody or antigen to the labeling agent.
  • the carrier include insoluble polysaccharides such as agarose, dextran, and cellulose; synthetic resins such as polystyrene, polyacrylamide, and silicon; and glass.
  • the test solution is reacted with the insolubilized monoclonal antibody of the present invention (primary reaction), and further reacted with another labeled monoclonal antibody of the present invention (secondary reaction).
  • primary reaction the insolubilized monoclonal antibody of the present invention
  • secondary reaction another labeled monoclonal antibody of the present invention
  • the primary reaction and the secondary reaction may be performed in the reverse order, or may be performed simultaneously or at staggered times.
  • the labeling agent and the method of insolubilization can be in accordance with those described above. It is also used as an antibody for solid phase or an antibody for labeling in immunoassay by the sandwich method.
  • One type of antibody is not necessarily required, and a mixture of two or more types of antibodies may be used for the purpose of improving measurement sensitivity and the like.
  • the monoclonal antibody of the present invention used in the primary reaction and the secondary reaction is preferably an antibody having a different site to which the receptor of the present invention binds.
  • the antibody used in the primary reaction and the secondary reaction is, for example, when the antibody used in the secondary reaction recognizes the C-terminal of the receptor of the present invention, the antibody used in the primary reaction is Preferably, an antibody which recognizes other than the C-terminal, for example, the N-terminal, is used.
  • the monoclonal antibody of the present invention can be used in a measurement system other than the sandwich method, for example, a competition method, an immunometric method, or a nephrometry.
  • a competition method after the antigen in the test solution and the labeled antigen are allowed to react competitively with the antibody, the unreacted labeled antigen (F) and the labeled antigen (B) bound to the antibody are separated. (B / F separation)
  • the labeling amount of either B or F is measured, and the amount of antigen in the test solution is quantified.
  • a soluble antibody is used as the antibody
  • BZF separation is performed using polyethylene glycol
  • a liquid phase method using a second antibody against the antibody or an immobilized antibody is used as the first antibody.
  • an immobilization method using a soluble first antibody and an immobilized antibody as the second antibody is used.
  • the antigen in the test solution and the immobilized antigen are subjected to a competitive reaction with a certain amount of labeled antibody, and then the solid phase and the liquid phase are separated. After reacting the antigen with an excess amount of the labeled antibody, the immobilized antigen is added to bind the unreacted labeled antibody to the solid phase, and then the solid phase and the liquid phase are separated. Next, the amount of the label in either phase is measured to determine the amount of the antigen in the test solution.
  • nephelometry the amount of insoluble sediment resulting from an antigen-antibody reaction in a gel or in a solution is measured. Even when the amount of antigen in the test solution is small and only a small amount of sediment is obtained, laser nephrometry utilizing laser scattering is preferably used.
  • the receptor of the present invention can be quantified with good sensitivity by using the antibody of the present invention.
  • an increase in the concentration of the receptor of the present invention is detected by quantifying the concentration of the receptor of the present invention using the antibody of the present invention, for example, insulin resistance, impaired glucose tolerance, etc. It can be diagnosed as a disease such as diabetes, obesity, hyperlipidemia, arteriosclerosis, hypertension or heart disease, or is likely to be affected in the future. In addition, when a decrease in the concentration of the receptor of the present invention is detected, it can be diagnosed that the disease is, for example, a disease such as hypoglycemia, or that the disease is likely to be caused in the future.
  • the antibody of the present invention can be used for detecting the receptor of the present invention present in a subject such as a body fluid or a tissue.
  • preparation of an antibody column used for purifying the receptor of the present invention, detection of the receptor of the present invention in each fraction during purification, analysis of the behavior of the receptor of the present invention in test cells, etc. can be used for
  • An agent for preventing and treating hypoglycemia comprising the ligand of the present invention
  • the ligand of the present invention has MP12 phosphorylation promoting activity, ERK phosphorylation promoting activity, insulin signaling inhibition activity, TNF ⁇ production promoting activity, glucose uptake inhibitory activity, and the like. Therefore, when the ligand of the present invention is abnormal or defective, for example, hypoglycemia develops.
  • the ligand of the present invention can be used as a safe medicament such as an agent for preventing or treating hypoglycemia and the like.
  • the ligand of the present invention When used as the above-mentioned therapeutic / prophylactic agent, it should be purified to at least 90%, preferably 95% or more, more preferably 98% or more, and still more preferably 99% or more. Is preferred.
  • the ligand of the present invention can be used, for example, in the form of tablets, capsules, elixirs, microcapsenoles, and the like, which are sugar-coated as required, or with water or other pharmaceutically acceptable liquids. It can be used parenterally in the form of injections, such as sterile solutions or suspensions.
  • the ligand of the present invention is mixed with physiologically acceptable carriers, flavors, excipients, vehicles, preservatives, stabilizers, binders, and the like in a unit dosage form generally required for the practice of pharmaceutical preparations. It can be manufactured by The amount of the active ingredient in these preparations is such that a suitable dosage in the specified range can be obtained.
  • Additives that can be incorporated into tablets, capsules, etc. include, for example, binders such as gelatin, corn starch, tragacanth, gum arabic, excipients such as crystalline cellulose, corn starch, gelatin, alginic acid Lubricating agents such as magnesium stearate, sweetening agents such as sucrose, lactose or saccharin, and flavoring agents such as peppermint, cocoa oil or cherry.
  • a liquid carrier such as oils and fats can be further contained in the above-mentioned type of material.
  • Sterile compositions for injection can be formulated according to normal pharmaceutical practice, such as dissolving or suspending the active substance in vehicles such as water for injection, and naturally occurring vegetable oils such as sesame oil and coconut oil. it can.
  • Aqueous liquids for injection include, for example, saline, isotonic solutions containing dextrose and other auxiliaries (eg, D-sorbitol, D-mannitol, sodium salt, etc.)
  • Suitable solubilizers for example, alcohols (eg, ethanol, etc.), polyalcohols (eg, propylene glycol, polyethylene glycol, etc.), nonionic surfactants (eg, Polysorbate 80 TM, HC0 -50 etc.).
  • oily liquor include sesame oil and soybean oil, and may be used in combination with solubilizers such as benzyl benzoate and benzyl alcohol.
  • buffers eg, phosphate buffer, sodium acetate buffer, etc.
  • soothing agents eg, benzalkonium chloride, proforce hydrochloride, etc.
  • stabilizers eg, human serum albumin, polyethylene glycol, etc.
  • Preservatives eg, benzyl alcohol, phenol, etc.
  • antioxidants eg, antioxidants and the like.
  • the prepared injection is usually filled in an appropriate ampoule.
  • the preparations obtained in this way are safe and have low toxicity, for example, warm-blooded animals (e.g. humans, rats, mice, guinea pigs, egrets, birds, higgies, pigs, pests, pomas, cats, animals Dogs, monkeys, chimpanzees, etc.).
  • warm-blooded animals e.g. humans, rats, mice, guinea pigs, egrets, birds, higgies, pigs, pests, pomas, cats, animals Dogs, monkeys, chimpanzees, etc.
  • the dose of the ligand of the present invention varies depending on the target disease, the subject of administration, the administration route, and the like.
  • the ligand of the present invention is orally administered for the treatment of hypoglycemia, it is generally required for an adult (body weight 60 kg).
  • the ligand is administered in an amount of about 0.1 to 100 mg, preferably about 1.0 to 50 mg, more preferably about 1.0 to 20 mg per day.
  • the single dose of the ligand varies depending on the administration subject, target disease, and the like.
  • the ligand of the present invention is formed in the form of an injection for the treatment of hypoglycemia.
  • the dose When administered to a human (with a body weight of 60 kg), about 0.01 to 30 mg, preferably about 0.:! To 20 mg, more preferably about 0.1 to 10 mg of the ligand per day is injected into the affected area. It is more convenient to administer. In the case of other animals, the dose can be administered in terms of weight per 60 kg.
  • the “compound or salt thereof that inhibits the activity of the ligand of the present invention on the protein of the present invention” is a compound or a salt thereof that inhibits the activating effect of the ligand of the present invention on the protein of the present invention. Any one may be used, for example, peptide, protein, antibody, non-peptide compound, synthetic compound, fermentation product, cell extract, plant extract, animal tissue extract, serum and the like. When the compound or a salt thereof is used as the above agent, it may be used in the same manner as in the above [1].
  • a prophylactic / therapeutic agent for hypoglycemia comprising a compound or a salt thereof that promotes the activating effect of the ligand of the present invention on the protein of the present invention.
  • the “compound or salt thereof that promotes the activating action of the ligand of the present invention on the protein of the present invention” includes a compound or a salt thereof that promotes the activating action of the ligand of the present invention on the protein of the present invention. Any of them may be used, for example, peptides. Proteins, antibodies, non-peptide compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts, serum, and the like. When the compound or a salt thereof is used as the above agent, it may be used in the same manner as in the above [1].
  • bases, amino acids, and the like are indicated by abbreviations based on the abbreviations by the IUPAC-IUB Commission on Biochemical Nomenclature or conventional abbreviations in the relevant field.
  • optical isomer of an amino acid the L-form is indicated unless otherwise specified.
  • Y Thymine (T) or cytosine (C)
  • M Aden (A) or cytosine (C)
  • B Guanine (G), Guanine (G) or Thymine (T)
  • V Adenine (A), guanine (G) or cytosine (C)
  • N Aden (A), guanine (G), cytosine (C) or thymine (T) or unknown or other salt
  • RNA ribonucleic acid
  • mRNA messenger ribonucleic acid
  • DIEA Disopropylethylamine G 1 y or G: Glycine ''
  • H is or H
  • sequence numbers in the sequence listing in the present specification indicate the following sequences.
  • the amino acid sequence of human TREM-2 is shown.
  • [SEQ ID NO: 13] 7 shows the nucleotide sequence of a primer used in Reference Example 6.
  • Example 7 shows the nucleotide sequence of a primer used in Reference Example 6.
  • Epididymal adipose tissue was removed from KKA y mice (14 weeks old, os) and C57BL / 6 mice (14 weeks old, os) used as controls, and homogenized in IS0GEN reagent (Wako Pure Chemical Industries). After chilling, total RA was extracted from each tissue by black-mouth extraction and isopropanol precipitation, and poly (A) + RNA was further purified by Oligotex dT30 (Takara Bio).
  • cDNA is synthesized by Reverse Transcriptase (Superscript RTII; Invitrogen), and KKA y mouse adipose tissue is synthesized using PCR-Select cDNA Subtraction Kit (Clontech).
  • the cDNA group whose expression level was increased or decreased was selectively amplified as a PCR fragment.
  • the amplified cDNA group was ligated to pT7Blue-T, and cloned by transforming Escherichia coli DH5 ⁇ ( each insertion sequence was vector sequence M13 primer ⁇ 7 (SEQ ID NO: 7).
  • the TREM-2 full-length coding region cDNA was cloned from human adipocyte and mouse 3T3-L1 adipocyte cDNA libraries by PCR to confirm the expression fluctuation of TREM-2 and to obtain experimental materials for functional analysis.
  • the reaction was performed using the Advantage-2 cDNA PCR Kit (Clontech) at 35 cycles of 98 ° C for 20 seconds and 68 ° C for 1 minute and 30 seconds.
  • the reaction was performed using Pfu Turbo DNA polymerase (Stratagene) at 35 cycles of 95 ° C for 20 seconds, 65 ° C for 40 seconds, and 72 ° C for 1 minute.
  • TREM-2 isolated from human adipocytes was consistent with known human TREM-2 (AF213457).
  • mouse TREM-2 has three known cDNA sequences, TREM-2a (AY024348), TREM-2b (AY024349), and TREM_2c (AF213458), and TREM-2 obtained from 3T3-L1 adipocytes. All of the 6 clones were consistent with TREM-2a.
  • Reference example 3
  • RT-PCR was performed on RNA extracted from epididymal adipose tissue of KKA y mice (14 weeks old, os) and C57BL / 6 mice (14 weeks old, os).
  • 0.5 ig of total RNA extracted from each tissue was synthesized with AMV (Avian Myeloblastosis Virus) reverse transcriptase (TakaraPio) using 01igo-dT-adapter primer (TakaraVio) and cDNA was synthesized.
  • PCR was performed using primers (SEQ ID NO: 9 and SEQ ID NO: 10) for TREM-2 closing.
  • the reaction was performed using Advan1: age_2 cDNA PCR Kit (Clontech) at 98 ° C for 20 seconds and 68 ° C for 1 minute and 30 seconds in 25 cycles.
  • Advan1 age_2 cDNA PCR Kit (Clontech)
  • the expression level of TREM-2 in the adipose tissue of the KKA y mouse was similar to the expression level of TREM-2 in the C57BL / 6 mouse, similar to the result of the microarray in Reference Example 1. A difference of more than 5 times could be confirmed.
  • a heron was immunized with a peptide composed of the 133rd Leu to the 147th Ser of the mouse TREM-2 sequence [SEQ ID NO: 2] as an antigen to obtain an anti-TREM-2 polyclonal antibody.
  • KKA y mice and C57BL / 6 mice epididymis adipose tissue using excised each 2 0 mu ⁇
  • the antibody obtained by homogenizing the Toko filtrate was analyzed by Western plotting, TREM-2 from the KKA y mice tissues Band was detected, but not detected in the tissue derived from C57BL / 6 mouse.
  • RNA was extracted from epididymal adipose tissue, mesenteric adipose tissue, skeletal muscle, liver, testis, spleen, brain, and kidney of KKA y mice and C57BL / 6 mice, respectively, and the RT-PCR method described in Reference Example 3 was used. Examination of the distribution of TREM-2 expression tissues revealed that KKA y mice had remarkable expression in epididymal fat tissue and mesenteric fat tissue. Meanwhile, these fat groups Almost no expression was observed in tissues other than tissues.
  • the copy number of TREM-2 mRNA per 1 ng of total RNA was measured by quantitative RT-PCR.
  • C RT-PCR was performed using SYBR Green RT-PCR reagent kit (Applied Biosystems) according to the attached protocol.
  • Automatic detection ⁇ Quantification system Quantification was performed using ABI PRISM 7700 (Applied Biosystems).
  • the following primers were used for quantitative RT-PCR of mouse TREM-2.
  • the following primers were used for quantitative RT-PCR of human TREM-2.
  • the following primers were used for quantitative RT-PCR of mouse TNF- ⁇ .
  • TREM-2 expression was quantified by the quantitative RT-PCR method described in Reference Example 6 (1), KKA y In the epididymal adipose tissue and mesenteric adipose tissue of the mouse, the expression level was increased 10 times or more compared to the C57BL / 6 mouse. Furthermore, TREM-2 mRNA expression in epididymal adipose tissue of KKA y mice at 7, 14, and 28 weeks of age was measured in the same manner, and the expression increased in proportion to the increase in blood glucose with age. . At this time, TNF is an indicator of insulin resistance properties - mRNA expression level of a is increased similarly to TREM- 2, TREM - 2 and TNF - correlation coefficient of the expression of a (R 2 value) 0. 8802.
  • Total RNA was extracted from subcutaneous adipose tissue from a human diabetic patient, and the amount of TREM-2 mRNA contained in the total RNA was compared with that from a non-diabetic patient by the quantitative RT-PCR method described in Reference Example 6 (1). did.
  • TREM-2 extracellular domain (hereinafter abbreviated as Sol TREM-2) neutralizing TREM2 ligand
  • Sol TREM-2 recombines at the N-terminus of the polypeptide consisting of 147 amino acids from Ala at position 18 to Glu at position 164, which corresponds to the extracellular domain in the mouse TREM-2 sequence (SEQ ID NO: 2).
  • One having four amino acids (Gly-Ser-His-Met) added at the time of constructing the protein expression vector was prepared as an E. coli recombinant protein.
  • a total of 400 zg Sol TREM of KKA y mice (14 weeks old, male) at lOO ⁇ ug 3 days before, 2 days before, 1 day before glucose tolerance test and 4 times each on the day of glucose tolerance test -2 was administered intraperitoneally. After administration of Sol TREM-2, glucose was loaded by intraperitoneally administering 1 g / kg of glucose to each mouse, and blood glucose levels were measured up to 2 hours after glucose loading.
  • mice treated with Sol TREM-2 showed a remarkable decrease in blood glucose 30 minutes after the glucose load compared to the control mice. A decrease in blood glucose of about 100 rag / dL was observed from 60 minutes to 120 minutes.
  • the mice administered Sol TREM-2 showed approximately a lower value than control mice. A significant decrease in AUC value of 70% was observed.
  • the mRNA expression level of TNF- ⁇ in epididymal white adipose tissue after Sol TREM2 administration was reduced to 50% or less as a result of measurement by the quantitative RT-PCR method described in Reference Example 6 (1). Furthermore, by increasing the number of administrations to 8 times from 7 days before the glucose tolerance test to the day of the glucose tolerance test, the blood glucose lowering effect became more remarkable, and the fasting blood glucose level of the group without Sol TREM-2 was reduced to about In contrast to 250 mg / dL, the fasting blood glucose level of the Sol TREM-2 administration group decreased to almost a normal level of about 140 mg / dL.
  • TREM-2 is the amino acid sequence of mouse TREM-2 (SEQ ID NO: 2), which is the polypeptide N consisting of 147 amino acids from Ala at the 18th position to Glu at the 164th position corresponding to the extracellular domain.
  • -A His-Tag ligated end was expressed in E. coli under the control of the T7 promoter.
  • the target protein was purified from the cell lysate using a nickel chelate column (Bulmacier) (hereinafter abbreviated as N-His-TREM2).
  • GM3 used was Hytest (# 8G16-4h).
  • the GM3 content of the eluate increased to about 0.3, 1.0 and 1.6 at the absorbance at 492 nm.
  • the absorbance at 492 nm when GM3 was not added was below the detection limit.
  • TREM-2 and LPS bind, that TREM-2 and LPS binding are inhibited by GM3, and that GM3, which inhibits the binding of TREM-2 and LPS, competes with LPS for TREM- 2 was shown to be bound.
  • Example 2 Competitive inhibition of gangliosides (GMl, GM2, GM3, GD3), lipoteichoic acid, and dextran sulfate for binding of TREM-2 and LPS
  • the TREM2 protein used in the binding test was a polypeptide of the 147 amino acids from the 18th Ala to the 164th Glu corresponding to the extracellular domain in the amino acid sequence of mouse TREM-2 (SEQ ID NO: 2).
  • -A human immunoglobulin Fc fragment ligated to the end was expressed as a secreted protein in animal cells (mouse myeloma cells) under the control of the CMV promoter.
  • the target protein was purified from the culture using Protein A column (Pharmacia) (hereinafter abbreviated as mTREM2-Fc).
  • GM3 and 0_ (acetamido-3, 5-dideoxy-D-glycero- -D-galacto-2-nonulopyranosylonic acid)-(2 ⁇ 3)-0_ ⁇ -D-galactopyranosyl- (1 ⁇ 4) -0 - ⁇ -D- glucopyranosyl- (1 ⁇ 1)-(2S, 3R, 4E)-2-N- (7-nitrobenz-2-oxa-1, 3-diazol-4 "yl) aminooctanamido-4-octadecene- l, 3-diol (Peptide Laboratories, Inc .; hereinafter abbreviated as fluorescent label GM3) was used.
  • secondary selection is performed by selecting a substance that retains the antagonist activity using DAP12 phosphorylation as an index.
  • GM3 and a test compound are added to TREM-2 and VAP-tagged DAP12 co-expressing CH0 cells prepared by a known method, and cultured for 10 minutes. Cells are lysed with a cell lysate [30 mM Tris-HCl (pH 7.4), 150 mM NaCl, 10 mM EDTA, 1% NP-40, 50 mM NaF, 1 mM Na-Vanadate], and then sonicated. Stele the membrane.
  • the cell lysate is immunoprecipitated with an anti-V5 antibody, and the immunoprecipitated sample is analyzed by Western blotting.
  • DAP12 total protein in the sample is detected with a densitometer using an anti-V5 antibody (invitrogen).
  • C) Detect DAP12 phosphorylated with an oxidized tyrosine antibody (Sigma) by the same method.
  • C) Compare the two to detect the degree of tyrosine phosphorylation per DAP12 protein. The degree of phosphorylation of DAP12 when cells were stimulated with GM3 alone without the addition of the test compound was compared with the degree of phosphorylation of DAP12 when the test compound and GM3 were added.
  • the test compound that interferes is secondarily selected as an antagonist.
  • the second-selected test compound is administered to a diabetes model mouse such as a KKA y mouse, and a blood glucose level, a glucose tolerance test, a blood insulin level, a blood lipid level, etc. are measured to determine whether insulin resistance is improved. Confirm.
  • a diabetes model mouse such as a KKA y mouse
  • a blood glucose level, a glucose tolerance test, a blood insulin level, a blood lipid level, etc. are measured to determine whether insulin resistance is improved.
  • the human TREM-2 protein used in the binding test consists of 154 amino acids from the 14th Glu to the 167th Glu corresponding to the extracellular domain in the amino acid sequence of human TREM-2 (SEQ ID NO: 1).
  • Human immunoglobulin Fc fragment conjugated to the C-terminus of the polypeptide was expressed as a secreted protein in animal cells (mouse myeloma cells) under the control of the CMV promoter.
  • the target protein was purified from the culture using a protein A column (Pharmacia) (hereinafter abbreviated as hTREM2-Fc).
  • the mouse TREM-2 protein used in Example 3 was used.
  • Human TREM-1 protein is Recombinant Human TREM-1 / Fc Chimera (R & D System; hereinafter abbreviated as hTREMl-Fc), and mouse TREM-1 protein is Recombinant Mouse TRE-l / Fc Chimera (R & D System; mTREMl-Fc).
  • step 8 each protein and the fluorescent label GM3 were eluted from Protein G agarose. For each eluate, the fluorescence intensity at 535 nm with respect to the excitation light at 485 nm was measured, and the fluorescent label GM3 bound to each protein was quantified.
  • mice TREM- 2 Oyopi person TREM- 2 it forces s Wachikararu having substantially binding force to the equivalent GM3.
  • the protein eg, human TREM-2, mouse TREM-2, etc.
  • glycoconjugates preferably, gandarioside, more preferably, GM3
  • gandarioside preferably, GM3
  • GM3 glycoconjugates

Abstract

L'invention concerne une méthode destinée au criblage d'un agent améliorant la résistance à l'insuline. L'invention concerne plus précisément une méthode qui consiste à utiliser (a) une protéine ayant une séquence d'acides aminés identique ou sensiblement identique à la séquence d'acides aminés représentée par la séquence SEQ ID NO :1, sa séquence peptidique partielle ou son sel et (b) un complexe glucidique capable de se fixer de manière spécifique à ladite protéine, pour cribler un composé ou son sel qui modifie les propriétés de fixation de ladite protéine ou de son sel audit complexe glucidique ; un kit de criblage ; un composé ou son sel obtenu par criblage ; et un agent améliorant la résistance à l'insuline, un agent préventif ou un remède contre le diabète, etc., comprenant ce composé ou son sel.
PCT/JP2004/014802 2003-10-03 2004-09-30 Methode de criblage d'un agent ameliorant la resistance a l'insuline WO2005033700A1 (fr)

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Publication number Priority date Publication date Assignee Title
CN108196054A (zh) * 2017-07-27 2018-06-22 数字本草中医药检测有限公司 一种检测甘草酸的试纸条及其制备方法和应用
WO2020194317A1 (fr) * 2019-03-28 2020-10-01 Yeda Research And Development Co. Ltd. Méthode de traitement de troubles liés aux lipides

Citations (3)

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Publication number Priority date Publication date Assignee Title
JPH03170491A (ja) * 1989-11-14 1991-07-24 Fidia Spa 改良ガングリオシドおよびその官能基誘導体
JP2003503676A (ja) * 1999-06-01 2003-01-28 プロティオーム・サイエンシィズ・ピーエルシー インスリン抵抗性疾患に関する方法及び組成物
WO2003099331A1 (fr) * 2002-05-24 2003-12-04 Takeda Pharmaceutical Company Limited Agents renforçateurs de la résistance à l'insuline

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH03170491A (ja) * 1989-11-14 1991-07-24 Fidia Spa 改良ガングリオシドおよびその官能基誘導体
JP2003503676A (ja) * 1999-06-01 2003-01-28 プロティオーム・サイエンシィズ・ピーエルシー インスリン抵抗性疾患に関する方法及び組成物
WO2003099331A1 (fr) * 2002-05-24 2003-12-04 Takeda Pharmaceutical Company Limited Agents renforçateurs de la résistance à l'insuline

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108196054A (zh) * 2017-07-27 2018-06-22 数字本草中医药检测有限公司 一种检测甘草酸的试纸条及其制备方法和应用
WO2020194317A1 (fr) * 2019-03-28 2020-10-01 Yeda Research And Development Co. Ltd. Méthode de traitement de troubles liés aux lipides

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