WO2002050265A1 - Nouvelles proteines transmembranaires du type recepteur et adn correspondants - Google Patents

Nouvelles proteines transmembranaires du type recepteur et adn correspondants Download PDF

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Publication number
WO2002050265A1
WO2002050265A1 PCT/JP2001/011081 JP0111081W WO0250265A1 WO 2002050265 A1 WO2002050265 A1 WO 2002050265A1 JP 0111081 W JP0111081 W JP 0111081W WO 0250265 A1 WO0250265 A1 WO 0250265A1
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Prior art keywords
protein
salt
present
receptor protein
compound
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PCT/JP2001/011081
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English (en)
Japanese (ja)
Inventor
Kenichi Horikoshi
Yoshio Taniyama
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Takeda Chemical Industries, Ltd.
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Priority to AU2002222681A priority Critical patent/AU2002222681A1/en
Publication of WO2002050265A1 publication Critical patent/WO2002050265A1/fr

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • 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 a novel transmembrane receptor uniform protein derived from human leukocytes or a salt thereof and a DNA encoding the same.
  • Transmembrane receptor-like proteins are present on the surface of functional cells in living cells and organs, and play a physiologically important role as targets for molecules that regulate the functions of those cells and organs, such as bioactive substances. ing.
  • the receptor transmits a signal into the cell through binding to a physiologically active substance, and this signal causes various reactions such as suppression of activation and activation of the cell.
  • Yuichi Protein elucidating the functions of cells and organs in various living organisms, It will provide a very important tool for closely related drug development.
  • physiological functions are regulated under the regulation by many physiologically active substances.
  • Physiologically active substances are present in various parts of the body and regulate their physiological functions through their corresponding receptor proteins.
  • Clarifying the relationship between substances that regulate complex functions in living organisms and their specific receptor proteins is a very important tool for drug development. Also, In order to efficiently screen agonists and antagonists for receptor proteins and to develop pharmaceuticals, we will elucidate the functions of receptor protein genes expressed in vivo and express them in an appropriate expression system. It was necessary.
  • Transmembrane receptor protein is a new bioactive substance (ligand) that uses signal transduction as an index. It is useful for searching and searching for an agonist or an evening gonist for the reception. On the other hand, even if a physiological ligand is not found, the physiological effect of the receptor is analyzed from the inactivation experiment (knockout animal) of the receptor, and an agonist or an antagonist against the receptor is analyzed. It is also possible to make a second strike.
  • ligands for the receptor, agonists, or antagonists, etc. may be used for diseases associated with dysfunction of the receptor. It can be expected to be used as a prophylactic / therapeutic agent and diagnostic agent.
  • a decrease or increase in the function of the receptor in vivo due to a gene mutation of the transmembrane receptor protein often causes some disease.
  • not only administration of an antagonist or agonist to the receptor, but also introduction of the receptor gene into a living body (or a specific organ) or introduction of an antisense nucleic acid to the receptor gene It can also be applied to treatment.
  • the nucleotide sequence of the receptor is indispensable information for examining the presence or absence of a deletion or mutation in the gene, and the receptor gene is used to prevent or treat a disease associated with dysfunction of the receptor. And diagnostics. Disclosure of the invention
  • the present invention provides a novel transmembrane receptor uniform protein useful as described above. That is, a polynucleotide (DNA, R) containing a novel transmembrane receptor-like protein or a partial peptide thereof or a salt thereof, and a polynucleotide (DNA, RNA and a derivative thereof) encoding the protein or a partial peptide thereof. NA and derivatives thereof), a recombinant vector containing the polynucleotide, a transformant carrying the recombinant vector, a method for producing the protein or a salt thereof, the protein or a partial peptide or a partial peptide thereof.
  • Antibodies to salts, compounds that alter the expression level of the protein, methods for determining the ligand for the protein, methods for screening compounds that alter the binding of ligand to the protein (antagonists, agonists) or salts thereof The screening kit, the screening (Antagonists, agonists) or salts thereof that change the binding between the ligand and the protein obtained using the screening method or screening kit, and compounds that change the binding between the ligand and the protein ( (Antagonist, agonist) or a compound containing a compound capable of changing the expression level of the protein or a salt thereof.
  • the present inventors isolated a cDNA encoding a novel transmembrane receptor uniform protein derived from human peripheral blood leukocytes and analyzed its entire nucleotide sequence. Successfully analyzed. Then, when this base sequence was translated into an amino acid sequence, the first to fifth transmembrane regions were confirmed on a hydrophobicity plot, and the protein encoded by these cDNAs was a five-transmembrane receptor-like protein. Was confirmed. The present inventors have further studied based on these findings, and as a result, have completed the present invention.
  • transmembrane receptor-like protein or a salt thereof which comprises an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 3;
  • a ligand for the protein of (1) or a salt thereof obtainable by using the protein of (1) or the partial peptide of (3) or a salt thereof;
  • a compound capable of changing the binding property between the ligand obtainable by using the screening method according to (18) or the screening kit according to (19) and the protein or salt thereof according to (1) or a compound thereof. salt;
  • a compound or a compound thereof that alters the binding property between the ligand obtainable using the screening method according to (18) or the screening kit according to (19) and the protein or salt thereof according to (1).
  • a medicament comprising a salt;
  • a polynucleotide comprising a nucleotide sequence complementary to or substantially complementary to the polynucleotide of (5) or a part thereof;
  • a medicament comprising the compound according to (30) or a salt thereof.
  • an infectious disease a central disease, an endocrine disease, or a metabolic disease, which comprises administering to a mammal an effective amount of the compound according to (20), (29) or (30) or a salt thereof.
  • the protein is: (1) one or two or more in the amino acid sequence represented by SEQ ID NO: 1 (preferably about 1 to 30, more preferably about 1 to 10, and further preferably several (1) 2) amino acid sequence in which the amino acid sequence of SEQ ID NO: 1 or 2 or more (preferably 1 to 30) Amino acid sequence to which about 1 to 10 amino acids are added, more preferably about 1 to 10 amino acids, and still more preferably several (1 to 5) amino acids; or 3 or more amino acids in the amino acid sequence represented by SEQ ID NO: 1 (preferably Is an amino acid sequence in which about 1 to 30, more preferably about 1 to 10, and still more preferably several (1 to 5) amino acids have been substituted with other amino acids, or amino acids combining them
  • the protein or a salt thereof according to the above (1) which is a protein containing a sequence;
  • the protein is (1) one or two or more in the amino acid sequence represented by SEQ ID NO: 3 (preferably about 1 to 30, more preferably about 1 to 10, and more preferably several (1) Amino acid sequence in which the amino acid sequence of SEQ ID NO: 3 is 1 or 2 or more (preferably about 1 to 30, more preferably about 1 to 10, More preferably, an amino acid sequence to which several (1 to 5) amino acids have been added; 3 One or more (preferably about 1 to 30, more preferably 1 to 30) amino acids in the amino acid sequence represented by SEQ ID NO: 3 A protein containing an amino acid sequence in which about 1 to 10, more preferably several (1 to 5) amino acids have been substituted with another amino acid, or an amino acid sequence combining them; 1) described protein Other salts thereof;
  • a transmembrane receptor expressed in the cell membrane of the transformant by culturing the transformant according to (9) with a compound that activates the protein or salt thereof according to (1).
  • the transformant described in (9) above is cultured with the compound described in (1) above or a compound that activates the protein described in (1) or a salt thereof and a test compound, the cell membrane of the transformant is cultured. (1) measuring and comparing the cell stimulating activity mediated by the transmembrane receptor in contact with the transmembrane receptor expressed in A method for screening a compound or a salt thereof that alters the binding property to a protein or a salt thereof;
  • a compound or a salt thereof which can be obtained by using the screening kit according to any of (48) to (50), which changes the binding property between the ligand and the protein or salt thereof according to (1);
  • (52) Contains a compound or a salt thereof, which can be obtained by using the screening kit according to any one of (48) to (50), which changes the binding property between the ligand and the protein or salt thereof according to (1).
  • a medicament characterized in that: (53) The protein according to (1) or the above (1), wherein the antibody according to (11) is contacted with the protein according to (1) or the partial peptide according to (4) or a salt thereof. (4) Determination of the partial peptide or the salt thereof described in (4) (54) The antibody of (11) described above, a test solution and a labeled protein of (1) or a partial peptide of (4) or a labeled thereof.
  • test solution characterized by measuring the ratio of the labeled protein of the above (1) or the partial peptide of the above (4) or a salt thereof bound to the antibody in a competitive reaction with a salt.
  • FIG. 1 is a hydrophobicity plot of pTB 2184.
  • FIG. 2 is a hydrophobicity plot of pTB 2185.
  • FIG. 3 is a diagram showing the amino acid sequence of pTB 2184 in one-letter code.
  • FIG. 4 is a diagram showing the amino acid sequence of pTB 2185 in one-letter code.
  • the transmembrane receptor-like protein of the present invention (hereinafter sometimes abbreviated as the receptor protein of the present invention) has an amino acid sequence represented by SEQ ID NO: 1 or 3 (FIG. 3, This receptor protein has the same or substantially the same amino acid sequence as in Fig. 4).
  • the receptor protein of the present invention can be used, for example, in humans and other mammals (eg, guinea pigs, rats, mice, porpies, pigs, pigs, higgies, puppies, monkeys, etc.). All cells (eg spleen cells, nerve cells, glial cells, knee j3 cells, bone marrow cells, mesangial cells, Langerhans cells, epidermal cells, epithelial cells, endothelial 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, synovial cells, chondrocytes, bone cells , Osteoblasts, osteoclasts, mammary cells, hepatocytes or stromal cells, or precursors of these cells, stem cells or cancer cells), blood cells, or any tissue in which these cells are present
  • amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 3 include, for example, about 50% or more, preferably, the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 3.
  • Examples of the protein having an amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 3 of the present invention include, for example, the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 3
  • a protein having substantially the same amino acid sequence and having substantially the same activity as the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 3 is preferable.
  • substantially the same activity examples include a ligand binding activity and a signal transduction activity. Substantially the same indicates that their activities are the same in nature. Therefore, activities such as ligand binding activity and signal transduction activity are equivalent (eg, about 0.01 to 100 times, preferably about 0.5 to 20 times, and more preferably about 0.5 to 20 times. 2 times), but the degree of activity and protein Quantitative factors such as the molecular weight of the quality may be different.
  • the measurement of the activity such as the ligand binding activity and the signal information transduction action can be performed according to a known method.
  • the activity can be measured according to a ligand determination method ⁇ ⁇ ⁇ ⁇ screening method described later.
  • the receptor protein of the present invention includes: (1) one or two or more amino acids in the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 3 (preferably, about 1 to 30, more preferably 1 to 1); An amino acid sequence in which about 0, more preferably several (1 to 5) amino acids have been deleted; (2) 1 or 2 or more (preferably) in the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 3 Is an amino acid sequence having about 1 to 30 amino acids, more preferably about 1 to 10 amino acids, and still more preferably several (1 to 5) amino acids; 3 SEQ ID NO: 1 or SEQ ID NO: : 1 or 2 or more (preferably about 1 to 30, more preferably about 1 to 10, and more preferably several (1 to 5)) amino acids in the amino acid sequence represented by 3 An amino acid sequence substituted with another amino acid, or 4 Such as protein you containing the amino acid sequence which is a combination of these is also used.
  • the receptor protein of the present invention has an N-terminal (an amino terminal) at the left end and a C-terminal (a lipoxyl terminal) at the right end.
  • the C-terminal of the receptor protein of the present invention may be any of a hydroxyl group (one COOH), a carboxylate (—COO—), an amide (one C ⁇ NH 2 ) or an ester (one COOR). .
  • R in the ester e.g., methyl, Echiru, n- propyl, alkyl groups such as isopropyl, n- butyl, Shikurobe 'pentyl, C 3 _ 8 cycloalkyl group such as cyclohexyl, for example, phenyl, (6 _ 1 2 Ariru group such as ⁇ one naphthyl, for example, benzyl, full Eniru alkyl or ⁇ - naphthylmethyl etc. ⁇ - naphthyl such as phenethyl - C 7 one i 4, such as C _ 2 alkyl group!
  • a bivaloyloxymethyl group commonly used as an ester for oral use is used.
  • the receptor protein of the present invention has a lipoxyl group (or lipoxylate) other than the C-terminus
  • the carboxyl group is amidated or esterified.
  • Such proteins are also included in the receptor protein of the present invention.
  • the ester in this case, for example, the above-mentioned terminal ester and the like are used.
  • the receptor protein of the present invention is a protein as described above, with Amino group protecting groups Mechionin residues of N-terminal (e.g., formyl group, etc. 6 Ashiru groups such as any C 2 _ 6 Arukanoiru group of Asechiru) Protected, N-terminally cleaved in vivo, and daryumin group formed by pidal dimamine oxidation, Substituent on the side chain of amino acid in the molecule (eg, OH, SH, amino group, I imidazole group, indole group, etc.
  • Amino group protecting groups Mechionin residues of N-terminal (e.g., formyl group, etc. 6 Ashiru groups such as any C 2 _ 6 Arukanoiru group of Asechiru) Protected, N-terminally cleaved in vivo, and daryumin group formed by pidal dimamine oxidation, Substituent on the side chain of amino acid in
  • Guanijino group suitable protecting group (e.g., ho mill group, C 2 such Asechiru - those protected by such Ashiru group such as 6 Arukanoiru group), or a sugar It also includes complex proteins such as so-called glycoproteins with linked chains.
  • the receptor protein of the present invention include, for example, a receptor protein containing an amino acid sequence represented by SEQ ID NO: 1 or a receptor protein containing an amino acid sequence represented by SEQ ID NO: 3
  • the partial peptide of the receptor protein of the present invention (hereinafter sometimes abbreviated to partial peptide) in which the same protein is used include the above-mentioned partial peptides of the receptor protein of the present invention.
  • the receptor protein molecule of the present invention is a site that is exposed outside the cell membrane and has substantially the same activity (eg, ligand binding activity). Etc.) are used.
  • an extracellular region (hydrophilic site) in a hydrophobic plot analysis is used as a partial peptide of the receptor protein having the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 3.
  • a peptide containing a part analyzed to be Further, a peptide partially containing a hydrophobic (Hydrophobic) site can also be used.
  • a peptide containing individual domains may be used, but a peptide containing a plurality of domains at the same time may be used.
  • the number of amino acids of the partial peptide of the present invention is at least 20 or more, preferably 50 or more, more preferably 100 or more of the amino acid sequences constituting the receptor protein of the present invention.
  • a substantially identical amino acid sequence refers to an amino acid sequence of about 50% or more, preferably about 60% or more, more preferably about 70% or more, further preferably about 80% or more, and particularly preferably Represents an amino acid sequence having about 90% or more, most preferably about 95% or more homology.
  • the partial peptide of the present invention has the following features: 1) deletion of one or more (preferably about 1 to 10, more preferably several (1 to 5)) amino acids in the above amino acid sequence; (2) One or more (preferably about 1 to 20, more preferably about 1 to 10, and more preferably several (1 to 5)) amino acids are added to the above amino acid sequence. Or 3 one or more (preferably about 1 to 10, more preferably several, and more preferably about 1 to 5) amino acids in the above amino acid sequence are replaced with other amino acids It may be.
  • the C-terminus force Rupokishiru group (- COOH), the force Rupokishire one Bok (- COO -) - may be (CO NH 2) or an ester (-COOR), ⁇ bromide.
  • the partial peptide of the present invention has a lipoxyl group (or carboxylate) other than the C-terminus, the partial peptide of the present invention includes amidation or esterification of the lipoxyl group. .
  • the ester in this case, for example, the above-mentioned C-terminal ester and the like are used.
  • the partial peptide of the present invention has an N-terminal methionine residue in which the amino group of the methionine residue is protected by a protecting group, and the N-terminal side of which is cleaved in vivo.
  • the resulting G 1 n is pyroglutamine-oxidized, the one in which the substituent on the side chain of the amino acid in the molecule is protected by an appropriate protecting group, or the compound peptide such as a so-called glycopeptide to which a sugar chain is bound. included.
  • Examples of the salt of the receptor protein or a partial peptide thereof of the present invention include a physiologically acceptable salt with an acid or a base, and a physiologically acceptable acid addition salt is particularly preferable.
  • 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) , Succinic acid, tartaric acid, citric acid, malic acid, oxalic acid Acids, benzoic acid, methanesulfonic acid, benzenesulfonic acid) and the like are used.
  • inorganic acids eg, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid
  • organic acids eg, acetic acid, formic acid, propionic acid, fumaric acid, maleic acid
  • Succinic acid tartaric acid, citric acid, mal
  • the receptor protein of the present invention or a salt thereof can be produced from the aforementioned human or other mammalian cells or tissues by a known method for purifying a receptor protein, or can encode the receptor protein of the present invention described later. It can also be produced by culturing a transformant containing DNA ′. In addition, when the protein is produced from human or other mammalian tissues or cells, it can be produced according to the protein synthesis method described later or according to the method. The resulting extract can be purified and isolated by a combination of reverse chromatography, ion exchange chromatography, and other similar chromatographies.
  • a commercially available resin for protein synthesis can be usually used.
  • resins include, for example, chloromethyl resin, hydroxymethyl resin, benzhydrylamine resin, aminomethyl resin, 4-benzyloxybenzyl alcohol resin, 4-methylbenzhydrylamine resin, PAM Resin, 4-hydroxymethylmethylphenylacetamidomethyl resin, polyacrylamide resin, 4- (2 ', 4' dimethoxyphenylhydroxymethyl) phenoxy resin, 4- (2 ', 4, dimethoxyphenyl) Rumoc aminoethyl) phenoxy resin.
  • amino acids whose hamino group and side chain functional group are appropriately protected are condensed on the resin in accordance with the sequence of the target protein according to various known condensation methods.
  • the protein is cleaved from the resin, and at the same time, various protecting groups are removed.
  • an intramolecular disulfide bond formation reaction is carried out in a highly diluted solution to obtain a target protein or an amide thereof.
  • various activating reagents that can be used for protein synthesis can be used, and carbodiimides are particularly preferable.
  • carpimides examples include DCC, N, N'-diisopropyl carpimide, N-ethyl N '-(3-dimethylaminoprolyl) carpimide.
  • the protected amino acid may be added directly to the resin with a racemization inhibitor (eg, HOBt, HOOBt) or pre-protected as a symmetrical acid anhydride or HOBt ester or HOOBt ester.
  • the amino acid can be added to the resin after activation.
  • 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 protein condensation reaction.
  • acid amides such as N, N-dimethylformamide, N, N-dimethylacetamide and N-methylvinylidone
  • halogenated hydrocarbons such as methylene chloride and chloroform
  • alcohols such as trifluoroethanol.
  • 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; or an appropriate mixture thereof. Used.
  • the reaction temperature is appropriately selected from a range known to be usable for the protein 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, tertiary pentoxycarbonyl, isobornyloxycarbonyl, 4-methoxybenzyloxycarbonyl, C 1 Z, Br—Z, Damantyloxycarbonyl, trifluoroacetyl, phthaloyl, formyl, 2-ditrophenylsulfenyl, diphenylphosphinothioyl, Fmoc and the like are used.
  • the lipoxyl group can be, for example, alkyl esterified (for example, methyl, ethyl, propyl, butyl, tert-butyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, 2-adamantyl, etc.) Or cyclic alkyl esterification), aralkyl esterification (for example, benzyl ester, 412 trobenzyl ester, 4-methoxybenzyl ester, Benzyl ester, benzhydryl esterification), phenacyl esterification, benzyloxycarbonyl hydrazide, Yukari Sharybutoxycarbonyl hydrazide, trityl hydrazide and the like.
  • alkyl esterified for example, methyl, ethyl, propyl, butyl, tert-butyl, cyclopentyl, cyclohexy
  • the hydroxyl group of serine can be protected, for example, by esterification or etherification.
  • a group suitable for the esterification for example, a lower alkanol group such as an acetyl group, an aroyl group such as a benzoyl group, a group derived from carbonic acid such as a benzyloxycarbonyl group, an ethoxycarbonyl group and the like are used.
  • a group suitable for etherification include a benzyl group, a tetrahydroviranyl group, and a t-butyl group.
  • the protecting group of the phenolic hydroxyl group of tyrosine for example, B zl, C 1 2 - Bz l, as 2-nitrobenzyl, B r- Z, protecting group of the imidazole of histidine such as evening over tert-butyl is used, for example, Tos, 4-methoxy-2,3,6-trimethylbenzenesulfonyl, DNP, benzyloxymethyl, Bum, Boc, Trt, Fmoc and the like are used.
  • activated raw oxypoxyl groups include, for example, corresponding acid anhydrides, azides, and active esters [alcohols (eg, pen phenol, 2,4,5-trichloro phenol, 2,4 —Dinitrophenol, cyanomethyl alcohol, paranitrophenol, HONB, esters with N-hydroxysuccinimide, N-hydroxyphthalimide, HOB t)].
  • active esters eg, pen phenol, 2,4,5-trichloro phenol, 2,4 —Dinitrophenol, cyanomethyl alcohol, paranitrophenol, HONB, esters with N-hydroxysuccinimide, N-hydroxyphthalimide, HOB t
  • 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, methanesulfonic acid, or trifluoromethane.
  • a catalyst such as Pd-black or Pd-carbon, or hydrogen fluoride anhydride, methanesulfonic acid, or trifluoromethane.
  • the elimination reaction by the above-mentioned acid treatment is generally performed at a temperature of about 120 ° (: 4040 ° C.)
  • anisol for example, anisol
  • a force-thione scavenger such as phenol, thioanisole, meta-cresol, para-cresol, dimethyl sulfide, 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
  • the formyl group used as an indole protecting group of tributofan is 1,2-ethanedithiol, 1,4-1
  • 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 (protein) chain is added to the amino group side of the desired amino acid.
  • a protein in which only the protecting group for the N-terminal amino group of the peptide chain was removed and a protein in which only the protecting group for the C-terminal hepoxyl group were removed were prepared.
  • the condensation is carried out in a mixed solvent as described above. 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 protein. This crude protein is purified by using various known purification means, and the main fraction is freeze-dried to obtain an amide of the desired protein.
  • ester of a protein for example, after condensing the carboxy-terminal amino acid a-hydroxyl group with a desired alcohol to form an amino acid ester, the ester of the desired protein can be obtained in the same manner as the protein amide. You can get your body.
  • the partial peptide of the receptor protein of the present invention or a salt thereof can be produced according to a known peptide synthesis method, or by cleaving the receptor protein of the present invention with an appropriate peptide. can do.
  • 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, the present invention
  • the target peptide can be produced by condensing a partial peptide or an amino acid that can constitute the protein of the present invention with the remaining portion, and removing the protecting group when the product has a protecting group.
  • Known condensation methods and elimination of protecting groups include, for example, the methods described in the following 1 to 5.
  • the partial peptide of the present invention can be purified and isolated by a combination of ordinary purification methods, for example, solvent extraction, distillation, column chromatography, liquid chromatography, recrystallization, and the like.
  • the partial peptide obtained by the above method is a free form, it can be converted to an appropriate salt by a known method, and conversely, when it is obtained by a salt, it can be converted to a free form by a known method. Can be.
  • any polynucleotide may be used as long as it contains the nucleotide sequence (DNA or RNA, preferably DNA) encoding the receptor protein of the present invention.
  • the polynucleotide is RNA such as DNA or mRNA encoding the receptor protein of the present invention, and may be double-stranded or single-stranded. In the case of a double strand, it may be a double-stranded DNA, a double-stranded RNA or a hybrid of DNA: RNA. If single stranded, it may be the sense strand (ie, the coding strand) or the antisense strand (ie, the non-coding strand).
  • the DNA encoding the receptor protein of the present invention 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 vector used for the library may be any of bacteriophage, plasmid, cosmid, phagemid and the like.
  • the DNA can be directly amplified by Reverse Transcriptase Polymerase Chain Reaction (hereinafter abbreviated as RT-PCR method) using a preparation of tota1 RNA or mRNA fraction from the cells and tissues described above.
  • RT-PCR method Reverse Transcriptase Polymerase Chain Reaction
  • the DNA encoding the receptor protein of the present invention includes, for example, (1) a DNA containing the nucleotide sequence represented by SEQ ID NO: 2, and (2) a DNA containing the nucleotide sequence represented by SEQ ID NO: 2.
  • An activity substantially the same as that of a receptor uniform protein containing DNA that hybridizes with DNA under high stringent conditions and containing the amino acid sequence represented by SEQ ID NO: 3 eg, Ligand binding activity, may be of any if D N A that encodes a protein having a signal information transduction activity, etc.).
  • Examples of the DNA that hybridizes with the DNA containing the nucleotide sequence represented by SEQ ID NO: 2 or SEQ ID NO: 4 under high stringent conditions include, for example, the nucleotide sequence represented by SEQ ID NO: 2 or SEQ ID NO: 4 And a DNA containing a nucleotide sequence having a homology of about 70% or more, preferably about 80% or more, more preferably about 90% or more, and still more preferably about 95% or more.
  • Hybridization can be performed according to a known method or a method analogous thereto, for example, the method described in Molecular Cloning 2nd (J. Sambrook et al., Cold Spring Harbor Lab. Press, 1989). Can be. When using a commercially-available library, specify it in the attached instruction manual. It can be performed according to the method described above. More preferably, it can be performed under high stringent conditions.
  • 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 to 100 ° C.
  • the conditions at ⁇ 65 ° C are shown. In particular, the case where the sodium concentration is about 19 mM and the temperature is about 65 ° C is most preferable.
  • DNA encoding the receptor-like protein containing the amino acid sequence represented by SEQ ID NO: 1 a DNA containing the base sequence represented by SEQ ID NO: 2 or the like is used.
  • DNA encoding the receptor homologous protein containing the amino acid sequence represented by SEQ ID NO: 3 DNA containing the base sequence represented by SEQ ID NO: 4 and the like are used.
  • a part of the nucleotide sequence of the DNA encoding the receptor protein of the present invention or a polynucleotide containing a part of the nucleotide sequence complementary to the DNA is a partial peptide of the present invention described below.
  • the term is used to mean not only that the DNA is included but also that it contains RNA.
  • an antisense polynucleotide capable of inhibiting the replication or expression of the receptor protein of the present invention is cloned or determined and encodes the receptor protein of the present invention. It can be designed and synthesized based on DNA base sequence information.
  • a polynucleotide can hybridize with the RNA of the receptor protein gene of the present invention and can inhibit the synthesis or function of the RNA, or can inhibit the synthesis or function of the receptor gene of the present invention.
  • the expression of the receptor protein of the present invention can be regulated and controlled through the interaction with protein-related RNA.
  • Polynucleotides that are complementary to a selected sequence of the receptor protein-related RNA of the present invention, and polynucleotides that can specifically hybridize with the receptor-protein-associated RNA of the present invention, are in vivo and in vivo. It is useful for regulating and controlling the expression of the receptor protein protein of the present invention outside, and also for treating or diagnosing diseases.
  • the term "corresponding" means having homology or being complementary to a particular sequence of nucleotides, base sequences or nucleic acids, including genes.
  • nucleotide sequence or nucleic acid usually refers to the amino acid of the peptide (protein) in the direction derived from the sequence of the nucleotide (nucleic acid) or its complement.
  • end hairpin loop 5, end 6—base pair 'repeat of the receptor Yuichi protein gene of the present invention, 5, end untranslated region, polypeptide translation start codon, protein coding region, ORF translation stop codon,
  • the 3′-end untranslated region, the 3′-end palindrome region, and the 3′-end hairpin map may be selected as preferred regions of interest, but any region within the receptor protein gene of the present invention may be selected. sell.
  • Antisense polynucleotides are polydeoxynucleotides containing 2-deoxy D-report, polynucleotides containing D-report, and other types of N-glycosides of purine or pyrimidine bases.
  • Polynucleotides or other polymers having a non-nucleotide backbone eg, commercially available protein nucleic acids and synthetic sequence-specific nucleic acid polymers
  • polymers containing special bonds such as DNA or RNA (Including nucleotides having a configuration permitting the attachment of bases). They can be double-stranded DNA, single-stranded DNA, double-stranded RNA, single-stranded RNA, and even DNA: RNA hybrids, and can also be unmodified polynucleotides (or unmodified oligonucleotides).
  • Nucleotides as well as those with known modifications, eg, those with labels known in the art, capped, methylated, one or more natural nucleotides as analogs.
  • Substituted, modified with an intramolecular nucleotide for example, having an uncharged bond (eg, methylphosphonate, phosphotriester, phosphoramidate, olebamate, etc.), a charged bond or a sulfur-containing bond (Eg, phosphorothioate, phosphorodithioate, etc.), such as proteins (nucleases, nucleases, inhibitors) , Which has toxins, antibodies, signal peptides, poly-such as a single L one-lysine) or a sugar (e.g., side groups, such as monosaccharides, etc.), inter-force With chelating compounds (eg, acridine, psoralen, etc.), chelating compounds (eg, metals, radioactive metals,
  • nucleoside may include not only those containing purine and pyrimidine bases but also those having other modified heterocyclic bases. Such modifications may include methylated purines and pyrimidines, acylated purines and pyrimidines, or other heterocycles. Modified nucleotides and modified nucleotides may also be modified at the sugar moiety, e.g., where one or more hydroxyl groups have been replaced with a halogen, a force, an aliphatic group, or the like.
  • the antisense polynucleotide (nucleic acid) of the present invention is an RNA, a DNA or a modified nucleic acid (RNA, DNA).
  • modified nucleic acid include, but are not limited to, sulfur derivatives of nucleic acids, thiophosphate derivatives, and those resistant to degradation of polynucleoside amides and oligonucleoside amides.
  • the antisense nucleic acid of the present invention can be preferably designed according to the following policy. That is, to make the antisense nucleic acid more stable in the cell, to increase the cell permeability of the antisense nucleic acid, to have a greater affinity for the target sense strand, and to be more toxic if it is toxic. Make sense nucleic acid less toxic.
  • the antisense nucleic acids of the present invention may contain altered or modified sugars, bases, or bonds, may be provided in special forms such as ribosomes or microspheres, may be applied by gene therapy, It could be given in additional form.
  • the addition forms include polycations such as polylysine, which acts to neutralize the charge of the phosphate backbone, and interaction with cell membranes.
  • Hydrophobic substances such as lipids that increase or increase the uptake of nucleic acids (eg, phospholipids, cholesterol, etc.).
  • Preferred lipids for addition include cholesterol and its derivatives (eg, cholesteryl chromate formate, cholic acid, etc.).
  • nucleic acid can be attached to the 3 'end or 5' end of the nucleic acid, and can be attached via a base, sugar, or intramolecular nucleoside bond.
  • Other groups include cap groups specifically located at the 3 'or 5' end of nucleic acids that prevent degradation by nucleases such as exonucleases and RNases. .
  • Examples of such a capping group include, but are not limited to, hydroxyl-protecting groups known in the art, such as glycols such as polyethylene glycol and tetraethylene glycol.
  • the inhibitory activity of the antisense nucleic acid can be examined using the transformant of the present invention, the in vivo or in vitro gene expression system of the present invention, or the in vivo or in vitro translation system of the receptor protein of the present invention. .
  • the nucleic acid can be applied to cells by various known methods.
  • the DNA encoding the partial peptide of the present invention may be any DNA containing the above-described nucleotide sequence encoding the partial peptide of the present invention.
  • the DNA may be any of rally, the above-described cell / tissue-derived cDNA, the above-described cell / tissue-derived cDNA library, and synthetic DNA.
  • the vector used for the library may be any of pacteriophage, plasmid, cosmid, phagemid and the like. Alternatively, it can be directly amplified by reverse transcriptase polymerase chain reaction (hereinafter abbreviated as RT-PCR method) using a mRNA fraction prepared from the cells and tissues described above.
  • RT-PCR method reverse transcriptase polymerase chain reaction
  • examples of the DNA encoding the partial peptide of the present invention include: (1) a DNA having a partial nucleotide sequence of a DNA containing the nucleotide sequence represented by SEQ ID NO: 2 or SEQ ID NO: 4. Or (2) an amino acid represented by SEQ ID NO: 1 or SEQ ID NO: 3, which has DNA that hybridizes with DNA represented by SEQ ID NO: 2 or SEQ ID NO: 4 under high stringent conditions.
  • the sequence containing the sequence A DNA having a partial nucleotide sequence of a DNA encoding a protein having substantially the same activity (eg, ligand binding activity, signal signal transduction action, etc.) as the Sepuform protein is used.
  • Examples of the DNA that hybridizes with the DNA represented by SEQ ID NO: 2 or SEQ ID NO: 4 under high stringency conditions include, for example, about 70% or more, preferably, about 70% or more of the nucleotide sequence represented by SEQ ID NO: 2 or SEQ ID NO: 4.
  • DNA containing a base sequence having a homology of about 80% or more, more preferably about 90% or more, and still more preferably about 95% or more is used.
  • Cloning of the DNA completely encoding the receptor protein of the present invention or its partial peptide may be performed by cloning the base of the DNA encoding the peptide of the present invention. Amplified by PCR using a synthetic DNA primer having a partial base sequence of the sequence, or a DNA incorporated into an appropriate vector is a DNA encoding a partial or entire region of the receptor protein of the present invention. Selection can be carried out by hybridization with fragments or those labeled with synthetic DNA. The hybridization-screening method can be performed, for example, according to the method described in Molecular Cloning 2nd (J. Sambrook et al., Cold Spring Harbor Lab. Press, 1989). When a commercially available library is used, the procedure can be performed according to the method described in the attached instruction manual.
  • the DNA base sequence can be replaced by the ODA-LA PCR method using PCR or a known kit, for example, Mutan TM -supper Exress Km (Takara Shuzo), Mutan TM -K (Takara Shuzo), etc. Can be performed according to known methods such as the Gapped du 1 ex method and the Kunk e method, or a method analogous thereto.
  • the DNA encoding the cloned receptor protein may be used as it is depending on the purpose, or may be digested with restriction enzymes if desired. Or can be used with a linker added.
  • the DNA may have ATG as a translation initiation codon at its 5 'end, and may have TAA, TGA or TAG as a translation termination codon at its 3' end. These translation start codons and translation stop codons It can also be added using a DNA adapter.
  • the expression vector for the receptor protein of the present invention may be prepared, for example, by (a) cutting out a DNA fragment of interest from DNA (for example, cDNA) containing DNA encoding the receptor protein of the present invention; It can be prepared by ligating the fragment downstream of the promoter in an appropriate expression vector.
  • vectors examples include Escherichia coli-derived plasmids (eg, pCR4, pCR2.1, pBR322, pBR325, pUC12, pUC13), Bacillus subtilis-derived plasmids (eg, pUB110, pTP5, pC194), yeast-derived plasmids (Eg, pSH19, pSH15), bacteriophages such as ⁇ phage, animal viruses such as retrovirus, vaccinia virus, baculovirus, etc., ⁇ A1-11, pXTl, pRc / CMV, pRc / RSV, p cDNA I / Neo is used.
  • Escherichia coli-derived plasmids eg, pCR4, pCR2.1, pBR322, pBR325, pUC12, pUC13
  • Bacillus subtilis-derived plasmids eg, pUB110, p
  • the promoter used in the present invention may be any promoter as long as it is appropriate for the host used for gene expression.
  • SR promoter when animal cells are used as host, SR promoter, SV40 promoter, LTR promoter, CMV promoter, HSV-TK promoter, etc. are listed. Among them, CMV promoter, SR promoter, etc. It is preferable to use.
  • t rp promoter one When the host is Eshierihia genus bacterium, t rp promoter one, l ac flop port mode evening one, re cA promoter, AP L promoter, and l pp promoter of all, when the host is Bacillus, spol promoter , SP02 flop port motors, such as p en p promoter, if the host is a yeast, PH05 flop port motor, PGK promoter, GAP promoter Isseki one, etc. ADH promoter are preferred. When the host is an insect cell, polyhedrin promoter, P10 promoter, and the like are preferable.
  • the expression vector may contain, in addition to the above, an enhancer, a splicing signal, a poly-A addition signal, a selection marker, and an SV40 replication origin (hereinafter sometimes abbreviated as SV40 ori), if desired.
  • a selection marker for example, dihydrofolate reductase (hereinafter, dh fr And sometimes abbreviated) gene [methotrexate (MTX) resistance], ampicillin phosphorus resistant gene (hereinafter sometimes abbreviated as Amp r), neomycin resistant gene (hereinafter sometimes abbreviated as Ne o r And G418 resistance).
  • the target gene can be selected by using a thymidine-free medium.
  • a signal sequence suitable for the host is added to the N-terminal side of the receptor protein of the present invention.
  • the host is a bacterium belonging to the genus Escherichia, a PhoA • signal sequence, an OmpA • signal sequence, etc.
  • the host is a Bacillus genus, an amylase • signal sequence, a subtilisin • signal sequence, etc.
  • a transformant can be produced.
  • Hosts include, for example, Escherichia, Bacillus, yeast, insect cells,
  • Escherichia examples include Escherichia coli K12 ⁇ DH1 [Processings of the national academy] Natl. Acad. Sci. USA), 60, 160 (1968)], JMl 03 [Nuirec 'Acid's Risaichi, (Nucleic Acids Research), 9, 309 (1981)], JA221 [Janazore Journal of Molecular Biology], 120, 517 (1978)], HB 101 [Journal of Molecular Biology, Biology, 41, 459 (1969)], C 600 [Genetics, 39, 440 (1954)], DH5a CInoue, H., Nojima, H.
  • Bacillus bacteria include, for example, Bacillus subtilis MI114 [Gene, 24, 255 (1983)], 207-21 [Journal of Biochemistry, 95] , 87 (1 984)].
  • yeast examples include, for example, Saccharomyces cerevisiae AH22, AH22R—, A87-11 A, DKD—5D, 20B-12, and Schizosaccaromyces pombe (Scliizosacclmromyces pombe) NCYC1 913, NCYC 2036 , Pichia pastoris, etc. are used.
  • Insect cells include, for example, when the virus is AcNPV, a cell line derived from the larva of Spodoptera (Spodoptera frugiperda cell; S f cell), Tridioplusia ni ⁇ MGl cell derived from the midgut, High Five TM derived from the egg of Tridioplusia ni Cells, cells derived from Mamestra brassicae or cells derived from Estigmena acrea are used.
  • Sf cell include Sf9 cell (ATCC CRL1711), Sf21 cell (Vaughn, JL et al., In Vivo, 13, 213-217, (1977) ) Is used.
  • insects for example, silkworm larvae are used [Maeda et al., Nature, 315, 592 (1985)].
  • animal cells examples include monkey cells COS-7, Vero, Chinese Hamster cells CHO (hereinafter abbreviated as CHO cells), dh fr gene-deficient Chinese muscular cells, and CHO cells (hereinafter CHO (dhir) —) Cells), mouse L cells, mouse At T-20, mouse myeloma cells, rat GH3, human FL cells, etc. are used.
  • Transformation of insect cells or insects can be carried out according to the method described in, for example, Bio / Technology, 6, 47-55 (1988).
  • a liquid medium is suitable as the medium used for the culturing, and a carbon source necessary for the growth of the transformant is contained therein.
  • the carbon source include glucose, dextrin, soluble starch, and sucrose.
  • the nitrogen source include ammonium salts, nitrates, corn chip liquor, peptone, casein, meat extract, soybean meal, potato extract, and the like.
  • the inorganic or organic substance and the inorganic substance include 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 Experimen 'in', Molecular Genetics (Journal of Experiments in Mo 1 ecu lar Genetics), 431-433, Cold Spring Harbor Laboratory, New York 1972].
  • an agent such as 3-indolylacrylic acid can be added.
  • 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.
  • cultivation is usually performed at about 30 to 40 ° C for about 6 to 24 hours.
  • the culture medium used was 10% immobilized in Grace's Insect Medium (Grace, TCC, Nature, 195, 788 (1962)). Those to which additives such as serum 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.
  • examples of the medium include a MEM medium containing about 5 to 20% fetal bovine serum [Science, 122, 501 (1952)], a DMEM medium [Virology, 8, 396 (1959) 3, RPMI 1640 medium [Journal of the American Medical Association at ion, 199, 519 (1967) And 199 medium [Proceeding of the Society for the Biological Medicine, 73, 1 (1950)].
  • the pH is about 6-8.
  • Cultivation is usually carried out at about 30 ° (: ⁇ 40 ° C) for about 15 to 60 hours, and aeration and agitation are added as necessary.
  • the receptor protein of the present invention can be produced inside the cell, in the cell membrane, or outside the cell of the transformant.
  • the receptor protein of the present invention can be separated and purified from the culture by, for example, the following method.
  • the cells or cells are collected by a known method after culturing, suspended in an appropriate buffer, and subjected to ultrasound, lysozyme and Z or freezing. After the cells or cells are destroyed by thawing or the like, a method of obtaining a crude extract of the receptor protein by centrifugation or filtration is appropriately used. Evening as urea or hydrochloric guanidine in the buffers and protein denaturing agent may contain a surfactant such as tri Bokun X- 1 0 0 TM.
  • the receptor protein is secreted into the culture solution, after the culture is completed, the supernatant is separated from the cells or cells by a known method, and the supernatant is collected.
  • Purification of the receptor protein contained in the thus obtained culture supernatant or extract can be carried out by appropriately combining known separation and purification methods.
  • These known separation and purification methods mainly include methods using solubility such as salting out and solvent precipitation, dialysis, ultrafiltration, gel filtration, and SDS-polyacrylamide gel electrophoresis, mainly molecular weight.
  • Method using difference in charge such as ion exchange chromatography, method using specific affinity such as affinity mouth chromatography, reverse phase high-performance liquid mouth chromatography, etc.
  • a method utilizing a difference in hydrophobicity, a method utilizing an isoelectric point difference such as an isoelectric focusing method, and the like are used.
  • the receptor protein thus obtained When the receptor protein thus obtained is obtained in a free form, it can be converted into a salt by a known method or a method analogous thereto. It can be converted into a free form or another salt by an analogous method.
  • the receptor protein produced by the recombinant can be arbitrarily modified or the polypeptide can be partially removed by applying an appropriate protein modifying enzyme before or after purification.
  • Protein-modifying enzymes include, for example, trypsin, chymotrypsin, arginyl endopeptidase, protein Kinase, glycosidase and the like are used.
  • the activity of the receptor protein of the present invention or a salt thereof produced in this manner can be measured by a binding experiment with a labeled ligand and an enzyme immunoassay using a specific antibody. ⁇
  • An antibody against the receptor protein of the present invention or its partial peptide or a salt thereof may be a polyclonal antibody, as long as it can recognize the receptor protein of the present invention or its partial peptide or its salt. Any of monoclonal antibodies may be used.
  • An antibody against the receptor protein of the present invention or its partial peptide or a salt thereof may be obtained by using the receptor protein of the present invention as an antigen, It can be produced according to a known antibody or antiserum production method.
  • the receptor protein or the like of the present invention is administered to a mammal at a site capable of producing an antibody by administration, itself or together with a carrier or a diluent.
  • complete Freund's adjuvant / incomplete Freund's adjuvant may be administered in order to enhance the antibody-producing ability.
  • the administration is usually performed once every 2 to 6 weeks, for a total of about 2 to 10 times. Examples of mammals to be used include monkeys, rabbits, dogs, guinea pigs, mice, rats, sheep, goats, and mice and rats are preferably used.
  • a warm-blooded animal immunized with the antigen for example, a mouse 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.
  • a monoclonal antibody-producing hybridoma By fusing the antibody-producing cells contained in the above with myeloma cells, a monoclonal antibody-producing hybridoma can be prepared.
  • the antibody titer in the antiserum can be measured, for example, by reacting a labeled receptor protein or the like described below with the antiserum, and then measuring the activity of a labeling agent bound to the antibody.
  • the fusion operation is performed according to known methods, for example, the method of Koehler and Milstein [Nature, 256, 495 (1975)]. Can be implemented.
  • the fusion promoter include polyethylene glycol (PEG) and Sendai virus, and PEG is preferably used.
  • myeloma cells examples include NS-1, P3U1, SP2Z0 and the like, with P3U1 being preferred.
  • the preferred ratio between the number of antibody-producing cells (spleen cells) and the number of myeloma cells used is about 1: 1 to 20: 1, and the concentration of PEG (preferably PEG1000 to PEG6000) is about 10 to 80%.
  • PEG preferably PEG1000 to PEG6000
  • hybridoma culture on a solid phase eg, microplate
  • an antigen such as receptor protein is directly or adsorbed together with a carrier
  • the supernatant is added, and then an anti-immunoglobulin antibody (anti-mouse immunoglobulin antibody is used if the cell used for cell fusion is a mouse) or protein A labeled with a radioactive substance or an enzyme is added to the solid phase.
  • an anti-immunoglobulin antibody anti-mouse immunoglobulin antibody is used if the cell used for cell fusion is a mouse
  • protein A labeled with a radioactive substance or an enzyme is added to the solid phase.
  • a method for detecting bound monoclonal antibodies adding a hybridoma culture supernatant to a solid phase to which anti-immune glopurin antibody or protein A has been adsorbed, adding a receptor protein or the like labeled with a radioactive substance, an enzyme, etc.
  • a method for detecting the bound monoclonal antibody is exemplified.
  • the selection of the monoclonal antibody can be carried out according to a known method or a method analogous thereto. Usually, it can be carried out in a medium for animal cells to which HAT (hypoxanthine, aminopterin, thymidine) is added.
  • HAT hyperxanthine, aminopterin, thymidine
  • any medium can be used as long as it can grow a hybridoma.
  • RPMI 1640 medium containing 1 to 20%, preferably 10 to 20% fetal bovine serum, GIT medium containing 1 to 10% fetal bovine serum (Wako Pure Chemical Industries, Ltd.) or a hybridoma culture medium Serum medium (SFM-101, Nissui Pharmaceutical Co., Ltd.) or the like can be used.
  • the culture temperature is usually 20 to 40 ° (:, preferably, about 37 ° C.
  • the culture time is usually 5 days to 3 weeks, preferably 1 week to 2 weeks.
  • the culture is usually 5% carbon dioxide.
  • the antibody titer of the culture supernatant of the hybridoma 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 in the same manner as normal polyclonal antibodies. [Examples: salting out, alcohol precipitation, isoelectric focusing, electrophoresis, ion exchangers (ex. , DEAE), ultracentrifugation, gel filtration, antigen-binding solid phase, or specific antibody obtained by collecting only the antibody with an active adsorbent such as protein A or protein G, and dissociating the bond to obtain the antibody. Purification method].
  • the polyclonal antibody of the present invention can be produced according to a known method or a method analogous thereto. For example, a complex of an immunizing antigen (antigen such as the protein of the present invention) and a carrier protein is formed, and a mammal 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 receptor protein of the present invention and separating and purifying the antibody.
  • the type of carrier protein and the mixing ratio of the carrier and the hapten are determined by the efficiency of the antibody against the hapten immunized by cross-linking with the carrier. If possible, any material may be cross-linked at any ratio.For example, the weight ratio of peroxyserum albumin, pericyroglobulin, keyhole lysine, hemocyanin, etc. A method of pulling the hapten 1 at a rate of about 0.1 to 20, 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 or incomplete Freund's adjuvant may be administered in order to enhance the antibody-producing ability upon administration.
  • the administration can usually be performed once every about 2 to 6 weeks, for a total of about 3 to 10 times.
  • Polyclonal antibodies can be obtained from the blood, ascites, etc. of a mammal immunized by the above method. Preferably, it can be collected from blood.
  • 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 serum described above. Separation and purification of the polyclonal antibody can be performed according to the same immunoglobulin separation and purification method as the above-described separation and purification of the monoclonal antibody.
  • the receptor protein of the present invention or its salt, its partial peptide or its salt, and the DNA encoding the receptor protein of the present invention or its partial peptide are: (1) a ligand (agonist) for the receptor protein of the present invention; ), (2) a prophylactic and / or therapeutic agent for a disease associated with dysfunction of the receptor protein of the present invention, (3) a genetic diagnostic agent, (4) a receptor protein of the present invention or a portion thereof.
  • the use of the receptor binding assay system using the recombinant receptor protein expression system of the present invention alters the binding of a ligand to the receptor protein of the present invention, which is specific to humans and mammals.
  • Compounds eg, agonists, antagonists, etc.
  • Antagonists can be used as preventive and therapeutic agents for various diseases.
  • a receptor protein or a partial peptide of the present invention or a salt thereof hereinafter sometimes abbreviated as a receptor protein of the present invention, etc.
  • a DNA encoding a receptor protein of the present invention or a partial peptide thereof hereinafter, referred to as a peptide.
  • the use of an antibody against the receptor protein of the present invention or the like is specifically described below. .
  • the present invention provides a method for determining a ligand for the receptor protein of the present invention, which comprises contacting the receptor protein of the present invention or a salt thereof or the partial peptide of the present invention or a salt thereof with a test compound.
  • a test compound include, in addition to known ligands, for example, tissue extracts of humans or other mammals (eg, mice, rats, mice, rats, mice, etc.), cell culture supernatants, and the like. Used. For example, the tissue extract, cell culture supernatant, or the like is added to the receptor protein of the present invention, and fractionation is performed while measuring cell stimulating activity and the like, whereby a single ligand can be finally obtained.
  • the ligand determination method of the present invention uses the receptor protein of the present invention or a partial peptide thereof or a salt thereof, or constructs an expression system for a recombinant receptor protein, and By using the receptor binding assay system used, the receptor binding protein of the present invention can be bound to the cell stimulating activity (for example, production of intracellular cAMP, production of intracellular cGMP, This is a method for determining a compound (for example, a peptide, a protein, a non-peptidic compound, a synthetic compound, a fermentation product, etc.) having a phosphorylation promoting or suppressing activity, or a salt thereof.
  • a compound for example, a peptide, a protein, a non-peptidic compound, a synthetic compound, a fermentation product, etc.
  • the receptor protein of the present invention or a partial peptide thereof is brought into contact with a test compound, for example, the receptor It is characterized by measuring the binding amount of the test compound to the protein or the partial peptide, the cell stimulating activity, and the like.
  • the present invention provides
  • a method for determining a ligand for a receptor protein of the present invention which comprises measuring the amount of binding to a protein or a salt thereof;
  • ⁇ Receptor protein-mediated cell stimulating activity eg, intracellular cAMP production, intracellular cGMP production, intracellular protein
  • a method for determining a ligand for the receptor protein or a salt thereof of the present invention which comprises measuring the activity of promoting or suppressing the phosphorylation of the protein.
  • the receptor protein is Of the present invention characterized by measuring cell-mediated cell stimulating activity (eg, activity of promoting or suppressing intracellular cAMP production, intracellular cGMP production, intracellular protein phosphorylation, etc.).
  • cell-mediated cell stimulating activity eg, activity of promoting or suppressing intracellular cAMP production, intracellular cGMP production, intracellular protein phosphorylation, etc.
  • the receptor protein used in the ligand determination method may be any protein as long as it contains the above-described receptor protein of the present invention or the partial peptide of the present invention. Receptor proteins expressed in large amounts using are suitable.
  • the above-described expression method is used. It is preferable that the DNA encoding the receptor protein is expressed in mammalian cells or insect cells.
  • cDNA is usually used, but is not necessarily limited thereto.
  • a gene fragment or a synthetic DNA may be used.
  • the DNA fragment In order to introduce a DNA fragment encoding the receptor protein of the present invention into host animal cells and express them efficiently, the DNA fragment must be expressed in a baculovirus belonging to a baculovirus using an insect as a host.
  • Virus (nuclear polyhedrosis virus; NPV) polyhedrosis promoter SV40-derived promoter, rejuvenous virus promoter, metamouth thionine promoter, human human shock promoter, cytomegalovirus promoter It is preferable to incorporate it in the downstream such as SR Hipro Yuichi. Examination of the amount and quality of the expressed receptor can be performed by a known method. For example, the literature [Nambi, P. et al., The Journal of Biological Chemistry (J. Biol. Chem.), 267, 1955.55-19555, pp. 1 992 years].
  • the receptor protein of the present invention or a partial peptide thereof or a salt thereof may be a receptor protein or a partial peptide thereof or a salt thereof purified according to a known method. Or a cell containing the receptor protein or a cell membrane fraction thereof may be used.
  • the cells When cells containing the receptor protein of the present invention are used in the ligand determination method of the present invention, the cells may be immobilized with daltaraldehyde, formalin, or the like.
  • the immobilization method can be performed according to a known method.
  • Cells containing the receptor protein of the present invention include the receptor protein of the present invention. This refers to a host cell that has expressed a protein. Examples of the host cell include Escherichia coli, Bacillus subtilis, yeast, insect cells, animal cells, and the like.
  • the cell membrane fraction refers to a fraction containing a large amount of cell membrane obtained by a known method after cell disruption.
  • the cells can be crushed by crushing the cells with a P0 tter _E1 Vehjem homogenizer, crushing with a Warinda blender ⁇ polytron (Kinematica), crushing by ultrasonic waves, pressurizing with a French press, etc. Crushing by ejecting cells from thin nozzles.
  • fractionation by centrifugal force such as fractionation centrifugation and density gradient centrifugation is mainly used.
  • the cell lysate is centrifuged at a low speed (500 rpm to 300 rpm) for a short time (typically about 1 minute to 10 minutes), and the supernatant is further centrifuged at a high speed (150 rpm).
  • the suspension is usually centrifuged at pm to 300 rpm for 30 minutes to 2 hours, and the resulting precipitate is used as a membrane fraction.
  • the membrane fraction contains a large amount of expressed receptor protein and membrane components such as cell-derived phospholipids and membrane proteins.
  • the amount of the receptor protein of the cells or during the membrane fraction containing the receptions evening over protein is preferably from 1 0 3 to 1 0 8 molecules per cell, is 1 0 5-1 0 7 molecules Is preferred.
  • the receptor protein fraction is preferably a natural receptor protein fraction or a recombinant receptor protein fraction having an activity equivalent thereto.
  • equivalent activity refers to equivalent ligand binding activity, signal transduction activity and the like.
  • radioactive isotopes such as [ 3 H], [ 125 I], [ 14 C] and [ 35 S] are preferably used.
  • a cell or a cell containing the receptor protein of the present invention is used.
  • Prepare an overnight receptor preparation by suspending the membrane fraction of the cells in a buffer appropriate for the method of determination.
  • the buffer may be any buffer such as a phosphate buffer of pH 4 to 10 (preferably pH 6 to 8) or a buffer such as Tris-hydrochloric acid buffer, which does not inhibit the binding between the ligand and the receptor protein.
  • surfactants such as CHAPS, Tween-80 TM (Kao Ichi Atlas), digitonin, dexcholate, etc.
  • a protease inhibitor such as PMS F, leupeptin, E-64 (manufactured by Peptide Research Institute), pepstatin and the like may be added for the purpose of suppressing the degradation of the receptor or ligand by the protease.
  • a fixed amount (5000 cpm to 500,000 cpm) of a test compound labeled with [ 3 H], [ 125 I], [ 14 C], [ 35 S], etc., is added to the receptor solution of 0.0 lml to 1 Om1. Coexist. Prepare a reaction tube containing a large excess of unlabeled test compound to determine the amount of non-specific binding (NSB).
  • the reaction is carried out at about 0 ° (: to 50 ° (:, preferably at about 4 ° C to 37 ° C, for about 20 minutes to 24 hours, preferably for about 30 minutes to 3 hours.
  • the glass fiber filter paper After washing with an appropriate amount of the same buffer, the radioactivity remaining on the glass fiber filter paper is measured with a liquid scintillation counter or T-counter.
  • a test compound having a count (B-NSB) less than the binding amount (NSB) exceeding Ocpm can be selected as a ligand (agonist) for the receptor protein or its salt of the present invention.
  • the receptor protein-mediated cell stimulating activity (for example, intracellular cAMP production, intracellular cGMP Production, phosphorylation or the like of an intracellular protein, etc.) can be measured using a known method or a commercially available measurement kit. Specifically, first, cells containing the receptor protein of the present invention are cultured on a multiwell plate or the like. Before determining the ligand, replace the medium with a fresh medium or an appropriate buffer that is not toxic to cells, add the test compound, etc., incubate for a certain period of time, extract the cells, or collect the supernatant. , According to each method And quantify.
  • a substance that serves as an indicator of cell stimulating activity for example, the production of intracellular cAMP
  • an inhibitor for the degrading enzyme is added to perform the assay. You may.
  • activities such as cAMP production suppression can be detected as a production suppression effect on cells whose basic production has been increased by forskolin or the like.
  • the kit for determining a ligand that binds to the receptor protein of the present invention or a salt thereof includes the receptor protein of the present invention or a salt thereof, a partial peptide or a salt thereof of the present invention, a cell containing the receptor protein of the present invention, or It contains the membrane fraction of cells containing the receptor protein of the present invention.
  • kits for determining a ligand of the present invention include the following.
  • receptions evening CHO cells expressing an protein of the present invention passaged 5 X 10 5 cells / well in 12-well plates, and cultured for 2 days at 37 ° C, 5% C0 2 , 95% air .
  • Test compounds that are poorly soluble in water should be dissolved in dimethylformamide, DMSO, methanol, etc.
  • the same as the labeled compound is prepared at a concentration 100 to 1000 times higher.
  • Measurement method 1 After washing the receptor protein-expressing CHO cells of the present invention cultured in a 12-well tissue culture plate twice with 1 ml of the measurement buffer, add 490 l of the measurement buffer to each well. .
  • Examples of the ligand capable of binding to the receptor protein of the present invention or a salt thereof include substances specifically present in lung, leukocyte, testis, spleen and the like.
  • the receptor protein of the present invention encodes the receptor protein according to the action of the ligand.
  • the DNA can be used as a medicament such as an agent for preventing and / or treating a disease associated with dysfunction of the receptor protein of the present invention.
  • the receptor protein of the present invention is reduced in the living body (deficiency of the receptor protein protein)
  • (1) using the receptor protein of the present invention in the patient (B) by administering to the patient and expressing the DNA encoding the receptor protein of the present invention in the patient; or (b) After the DNA encoding the receptor protein of the present invention is introduced and expressed in a cell, the amount of the receptor protein in the patient's body is increased by transplanting the cell into the patient, and the like. The effect of can be fully exhibited. That is, DNA encoding the receptor protein of the present invention is safe and low toxic.
  • the present invention is useful as a prophylactic and / or therapeutic agent for diseases associated with dysfunction of the receptor protein of the present invention.
  • the receptor protein of the present invention is a G protein-coupled receptor protein, which is a la-adrenocept protein isomer 3 [British 'Journal of Pharmacol. (Br. J. Pharmacol.), 129 (8), 1569-15776 (2000)], a novel transmembrane receptor homologous protein having about 30% homology at the amino acid sequence level.
  • the D ⁇ encoding the receptor protein or the receptor protein of the present invention may be, for example, an infectious disease (eg, immune dysfunction, pneumonia, influenza, etc.), a central disease (eg, Alzheimer's disease, dementia, Food disorders), endocrine disorders (eg, hypertension, gonadal dysfunction, thyroid dysfunction, pituitary dysfunction, etc.), metabolic disorders (eg, diabetes, lipid metabolism, hyperlipidemia, etc.), cancer (eg, , Non-small cell lung cancer, ovarian cancer, prostate cancer, stomach cancer, bladder cancer, breast cancer, cervical cancer, colon cancer, rectal cancer, etc.), heart disease (eg, angina pectoris, myocardial infarction, etc.), respiratory system Diseases (eg, airway obstructive disease, infectious lung disease, etc.), Gastrointestinal diseases (eg, ulcer, polyposis, etc.), Immune system diseases (eg, systemic lupus erythematosus, rheumatism) Useful sexual disorders, etc.) of any prevention
  • the receptor protein of the present invention When used as the above-mentioned prophylactic / therapeutic agent, it can be formulated according to a conventional method.
  • DNA of the present invention when DN ⁇ encoding the receptor protein of the present invention (hereinafter sometimes abbreviated as DNA of the present invention) may be used as the above-mentioned prophylactic or therapeutic agent, the DNA of the present invention may be used alone or as a retrovirus. After insertion into a suitable vector such as a vector, an adenovirus vector, or an adenovirus associated virus vector, it can be carried out according to a conventional method.
  • the DNA of the present invention can be administered as it is or together with an adjuvant for promoting uptake, using a gene gun or a catheter such as Hydrate gel gel.
  • the DNA encoding the receptor protein of the present invention or (2) the DNA encoding the receptor protein may be used as a sugar-coated tablet, capsule, elixir, microcapsule, or the like, or water or Other drugs It can be used parenterally in the form of injections, such as sterile solutions with physiologically acceptable liquids or suspensions.
  • 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, etc. Swelling agents such as magnesium stearate, sweeteners such as sucrose, lactose or saccharine, and flavoring agents such as peppermint, cocoa oil or cherry.
  • the unit dosage form is a capsule, the above type of material can further contain a liquid carrier such as an oil or fat.
  • Sterile compositions for injection can be 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. it can.
  • aqueous liquid for injection include physiological saline, isotonic solution containing glucose and other adjuvants (eg, D-sorbitol, D-mannitol, sodium chloride, etc.) and the like. such as an alcohol (e.g., ethanol), polyalcohol (e.g., propylene glycol, polyethylene Dali call), a nonionic surfactant (eg, polysorbate one preparative 8 0 TM, HCO- 5 0), such as a combination You may.
  • the oily liquid for example, sesame oil, soybean oil and the like are used, and may be used in combination with solubilizers such as benzyl benzoate and benzyl alcohol.
  • prophylactic and therapeutic agents include, for example, buffers (for example, phosphate buffer and sodium acetate buffer), soothing agents (for example, benzalkonium chloride, procaine hydrochloride, etc.), stabilizers (for example, human serum Albumin, polyethylene glycol, etc.), preservatives (eg, benzyl alcohol, phenol, etc.), antioxidants and the like.
  • buffers for example, phosphate buffer and sodium acetate buffer
  • soothing agents for example, benzalkonium chloride, procaine hydrochloride, etc.
  • stabilizers for example, human serum Albumin, polyethylene glycol, etc.
  • preservatives eg, benzyl alcohol, phenol, etc.
  • antioxidants eg, benzyl alcohol, phenol, etc.
  • the prepared injection solution is usually filled in a suitable ampoule.
  • the preparations obtained in this way are safe and have low toxicity, such as, for example, humans and other mammals (eg, rats, mice, puppies
  • the dose of the receptor protein of the present invention varies depending on the administration subject, target organ, symptoms, administration method, and the like.
  • oral administration in general, for example, in an infectious disease patient (as 60 kg), About 0.1 to 100 mg, preferably about 1.0 to 5 Omg, more preferably about 1.0 to 2 Omg per day.
  • the single dose varies depending on the subject of administration, target organ, symptoms, administration method, etc.
  • it is usually used, for example, for infectious disease patients (60 kg)
  • the dose can be given in terms of 6 O kg.
  • the dosage of the DNA of the present invention may vary depending on the administration subject, target organ, symptoms, administration method, and the like. About 0.1 mg to 100 mg, preferably about 1.0 to 5 Omg, more preferably about 1.0 to 2 Omg.
  • the single dose varies depending on the administration target, target organ, symptoms, administration method, and the like.
  • it is usually, for example, an infectious disease patient (as 6 O kg)
  • it is convenient to administer about 0.01 to 3 Omg per day, preferably about 0.1 to 20 mg, more preferably about 0.1 to 1 Omg per day by intravenous injection. is there.
  • the dose can be administered in terms of 6 O kg.
  • the DNA of the present invention can be used as a probe to produce the receptor of the present invention in humans or other mammals (eg, rats, mice, rabbits, sheep, pigs, rabbits, cats, dogs, monkeys, etc.).
  • Abnormality (genetic abnormality) of DNA or mRNA encoding one protein or a partial peptide thereof can be detected, for example, damage, mutation or reduced expression of the DNA or mRNA.
  • the DNA or mRNA is useful as a diagnostic agent for a gene such as increase or overexpression.
  • the above-described genetic diagnosis using the DNA of the present invention can be carried out, for example, by the well-known Northern Eighth hybridization or PCR-SSCP method (Genomics, Vol. 5, pp. 874-879 (1989). ), Proceedings of the National Academy of Sciences of the United States of America, Vol. 86, Vol. 27, Procedings of the National Academy of Sciences of the United States of America. 6-2770 pages (1989)).
  • the DNA of the present invention can be used for screening a compound that changes the expression level of the receptor protein or its partial peptide of the present invention.
  • the present invention relates to, for example, (i) a non-human mammal's (1) blood, (2) a specific organ, (3) a tissue or cell isolated from an organ, or (U) a transformant of the present invention contained in a transformant or the like.
  • the measurement of the amount of mRNA of the receptor protein or its partial peptide of the present invention is specifically carried out as follows.
  • non-human mammals eg, mice, rats, rabbits, sheep, pigs, pigs, cats, dogs, monkeys, etc., more specifically, dementia rats, obese mice, arteriosclerosis ⁇ Drugs (eg, anti-dementia drugs, antihypertensive drugs, anti-cancer drugs, anti-obesity drugs, etc.) or physical stress (eg, flooding stress, electric shock, light / dark, low temperature, etc.)
  • blood or a specific organ eg, lung, testis, spleen, brain, lung, large intestine, etc.
  • tissue or cells isolated from the organ is obtained.
  • the mRNA of the receptor protein of the present invention or its partial peptide contained in the obtained cells can be obtained, for example, by extracting mRNA from cells or the like by a usual method, for example, For example, it can be quantified by using a technique such as TaqMan PCR, and can also be analyzed by performing Northern blot by a known means.
  • a transformant expressing the receptor protein of the present invention or a partial peptide thereof is prepared according to the above method, and the mRNA of the receptor protein of the present invention or the partial peptide thereof contained in the transformant is prepared. It can be quantified and analyzed in the same way.
  • Screening for a compound that changes the expression level of the receptor protein or its partial peptide of the present invention comprises:
  • a given time before drug or physical stress is applied to a normal or disease model non-human mammal (30 minutes to 24 hours before, preferably 30 minutes to 12 hours before, Preferably 1 hour to 6 hours before) or after a certain time (30 minutes to 3 days, preferably 1 hour to 2 days, more preferably 1 hour to 24 hours), or drug or physical
  • the test compound is administered at the same time as the target stress, and after a certain period of time after administration (30 minutes to 3 days, preferably 1 hour to 2 days, more preferably 1 hour to 24 hours), the cells
  • the amount of mRNA of the receptor protein of the present invention or its partial peptide contained in the present invention can be determined and analyzed,
  • the test compound is mixed in a medium and cultured for a certain period of time (1 day to 7 days, preferably 1 day to 3 days, more preferably 2 days to 3 days). A day after), the amount can be determined by quantifying and analyzing the amount of mRNA of the receptor protein of the present invention or its partial peptide contained in the transformant.
  • the compound or a salt thereof obtained by using the screening method of the present invention is a compound having an action of changing the expression level of the receptor protein or a partial peptide thereof of the present invention.
  • the cell stimulating activity through the receptor protein of the present invention for example, intracellular cAMP generation, intracellular cGMP) (Enhancement of the production, phosphorylation of intracellular proteins, etc.
  • (a) Expression of the receptor protein of the present invention or its partial peptide Is a compound that attenuates the cell stimulating activity by reducing
  • Such compounds include peptides, proteins, non-peptidic compounds, synthetic compounds
  • These compounds may be novel compounds or known compounds.
  • the compound that enhances the cell stimulating activity is useful as a safe and low toxic drug for enhancing the physiological activity of the receptor protein or the like of the present invention.
  • the compound that attenuates the cell stimulating activity is useful as a safe and low toxic drug for decreasing the physiological activity of the receptor protein of the present invention.
  • a compound or a salt thereof obtained by using the screening method of the present invention is used as a pharmaceutical composition, it can be carried out according to a conventional method.
  • tablets, capsules, elixirs, microcapsules, sterile solutions, suspensions, and night formulations can be prepared in the same manner as the above-mentioned drug containing the receptor protein of the present invention.
  • the preparations obtained in this way are safe and low toxic, for example, in humans and other mammals (eg, rats, mice, puppies, higgs, bush, puppies, cats, dogs, monkeys, etc.). Can be administered.
  • the dose of the compound or a salt thereof varies depending on the administration subject, target organ, symptoms, administration method, and the like.
  • oral administration in general, for example, in an infectious disease patient (60 kg), About 0.1- per day; L 00 mg, preferably about 1.0-5 Omg, more preferably about 1.0-2 Omg.
  • the single dose varies depending on the administration subject, target organ, symptoms, administration method, etc.
  • the dose can be administered in terms of 6 O kg.
  • a preventive and / or therapeutic agent for various diseases containing a compound that changes the expression level of the receptor protein or its partial peptide of the present invention
  • the receptor protein of the present invention is, as described above, Is considered to play some important role. Therefore, the compound that alters the expression level of the receptor protein or its partial peptide of the present invention can be used as a preventive and / or therapeutic agent for a disease associated with dysfunction of the receptor protein of the present invention. it can.
  • the compound when used as a prophylactic and / or therapeutic agent for a disease associated with dysfunction of the receptor protein of the present invention, it can be formulated according to a conventional method.
  • the compound can be used as a sugar-coated tablet, capsule, elixir, microcapsule or the like as needed, orally, or aseptic solution with water or another pharmaceutically acceptable liquid. It can be used parenterally or in the form of injections such as suspensions.
  • the compound is mixed with known physiologically acceptable carriers, flavoring agents, 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, etc. Swelling agents such as magnesium stearate, sweeteners such as sucrose, lactose or saccharin, and flavoring agents such as peppermint, cocoa oil or cherry.
  • the unit dosage form is a capsule, the above type of material can further contain a liquid carrier such as an oil or fat.
  • Sterile compositions for injection can be 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. it can.
  • aqueous liquid for injection include physiological saline, isotonic solution containing glucose and other adjuvants (eg, D-sorbitol, D-mannitol, sodium chloride, etc.) and the like.
  • an alcohol e.g., ethanol
  • polyalcohol e.g., propylene glycol, polyethylene Dali call
  • a nonionic surfactant eg, polysorbate 8 0 TM, HCO - 5 0
  • the oily liquid for example, sesame oil, soybean oil and the like are used, and may be used in combination with solubilizers such as benzyl benzoate and benzyl alcohol.
  • prophylactic and therapeutic agents include, for example, buffers (for example, phosphate buffer and sodium acetate buffer), soothing agents (for example, benzalkonium chloride, procaine hydrochloride, etc.), stabilizers (for example, human serum Albumin, polyethylene glycol, etc.), preservatives (eg, benzyl alcohol, phenol, etc.), antioxidants and the like.
  • buffers for example, phosphate buffer and sodium acetate buffer
  • soothing agents for example, benzalkonium chloride, procaine hydrochloride, etc.
  • stabilizers for example, human serum Albumin, polyethylene glycol, etc.
  • preservatives eg, benzyl alcohol, phenol, etc.
  • antioxidants eg, benzyl alcohol, phenol, etc.
  • the prepared injection solution is usually filled in a suitable ampoule.
  • the preparations obtained in this way are safe and low toxic, so they can be used, for example, in humans and other mammals (eg, rats, mice
  • the dose of the compound or a salt thereof varies depending on the administration subject, target organ, symptom, administration method, and the like. About 0.1 to 100 mg, preferably about 1.0 to 5 Omg, and more preferably about 1.0 to 2 Omg. In the case of parenteral administration, the single dose varies depending on the administration subject, target organ, symptoms, administration method, etc. ), It is convenient to administer about 0.01 to 3 Omg per day, preferably about 0.1 to 20 mg, more preferably about 0.1 to 1 Omg by intravenous injection. is there. For other animals, the dose can be administered in terms of 6 O kg.
  • the receptor protein and the like of the present invention have a binding property to a ligand, the ligand concentration in a living body can be quantified with high sensitivity.
  • the quantification method of the present invention can be used, for example, in combination with a competition method. That is, the ligand concentration in the subject can be measured by bringing the subject into contact with the receptor protein of the present invention. Specifically, for example, it can be used according to the method described in the following (1) or (2) or a method analogous thereto.
  • Hiroshi Irie Kelsha, published in 1949
  • a compound eg, agonist, antagonist, etc.
  • the ligand and the present invention can be used.
  • A) Cell stimulating activity via the receptor protein of the present invention for example, activity to promote or suppress intracellular CAMP production, intracellular cGMP production, intracellular protein phosphorylation, etc.
  • So-called agonist So-called agonist
  • the present invention relates to (i) the case where the receptor protein of the present invention or its partial peptide or a salt thereof is brought into contact with a ligand, and (ii) the receptor protein of the present invention or its partial peptide or a salt thereof.
  • a method for screening a salt is provided.
  • the screening method of the present invention is characterized in that, in the cases (i) and (ii), for example, the amount of a ligand bound to the receptor protein or the like, the cell stimulating activity, etc. are measured and compared. .
  • the present invention provides
  • the amount of binding to the ligand is measured and compared, and the binding between the ligand and the receptor protein of the present invention is changed.
  • a compound that activates the receptor protein or the like of the present invention eg, a ligand for the receptor protein or the like of the present invention
  • a cell containing the receptor protein or the like of the present invention e.g., a ligand for the receptor protein or the like of the present invention
  • Receptor-mediated cell stimulating activity eg, intracellular CAMP production, cell proliferation
  • C GMP production, activity of promoting or suppressing intracellular protein phosphorylation, etc. comparing the ligand with the receptor protein of the present invention, A method for screening the salt, and
  • a compound of the present invention in which a compound that activates the receptor protein or the like of the present invention (eg, a ligand for the receptor protein or the like of the present invention) is expressed on a cell membrane by culturing a transformant containing the DNA of the present invention.
  • a compound that activates the receptor protein or the like of the present invention eg, a ligand for the receptor protein or the like of the present invention
  • the present invention expresses on the cell membrane by culturing a transformant containing the DNA of the present invention with a compound that activates the receptor protein or the like of the present invention and a test compound when they are brought into contact with the DNA of the present invention.
  • Receptor-mediated cell stimulating activity for example, activity to promote or suppress intracellular cAMP production, intracellular cGMP production, intracellular protein phosphorylation, etc.
  • a method for screening a compound or a salt thereof that alters the binding property between the ligand and the receptor protein or the like of the present invention which is characterized by measuring and comparing
  • a transmembrane receptor A candidate compound is obtained using a cell, tissue or its cell membrane fraction containing Yuichi protein (primary screening), and then the candidate compound actually binds to human transmembrane receptor Yuichi protein and its ligand.
  • a test secondary screening to confirm whether or not to inhibit was required. If the cell, tissue or cell membrane fraction is used as it is, other receptor proteins are also mixed, and it has been difficult to actually screen an agonist or an antagonist for the target receptor protein.
  • the receptor protein of the present invention by using the receptor protein of the present invention, primary screening is not required, and a compound that inhibits binding between a ligand and the receptor protein can be efficiently screened. Furthermore, it is possible to easily evaluate whether the screened compound is an agonist or an engonist.
  • the receptor protein or the like of the present invention used in the screening method of the present invention may be any as long as it contains the above-mentioned receptor of the present invention.
  • Cell membrane fractions of mammalian organs containing one protein or the like are preferred.
  • human-derived organs are particularly difficult to obtain, it is suitable to use human-derived receptor protein expressed in large amounts using recombinants, etc., for screening.
  • the method described above is used to produce the receptor protein of the present invention and the like, but it is preferable to express the DNA of the present invention in mammalian cells and insect cells.
  • the DNA fragment encoding the protein portion of interest cDNA is used, but is not necessarily limited thereto.
  • a gene fragment or a synthetic DNA may be used.
  • the DNA fragment In order to introduce the DNA fragment encoding the receptor protein of the present invention into host animal cells and express them efficiently, the DNA fragment should be expressed in a nuclear polyhedrosis virus belonging to a baculovirus using an insect as a host.
  • a nuclear polyhedrosis virus belonging to a baculovirus using an insect as a host.
  • NPV nuclear polyhedrosis virus
  • polyhedrin promoter SV40-derived promoter overnight, retrovirus promoter, meta-mouth thionein promoter, human heat shock promoter overnight, cytomegalovirus promoter
  • It is preferably incorporated downstream of the SRQ; promoter.
  • the amount and quality of the expressed receptor can be examined by a known method. For example, it can be carried out according to the method described in the literature [Nambi, P. et al., The Journal of Biological Chemistry (J. Biol. Chem.), 267, 19555-19559, 1992]. it can.
  • the receptor protein or the like of the present invention may be a receptor protein or the like purified according to a known method, or may be a receptor protein or the like.
  • a cell containing a protein or the like may be used, or a membrane fraction of a cell containing the receptor protein or the like may be used.
  • the screening method of the present invention contains the receptor protein of the present invention or the like.
  • the cells may be fixed with dartartaldehyde, formalin, or the like.
  • the immobilization method can be performed according to a known method.
  • the cell containing the receptor protein or the like of the present invention refers to a host cell that expresses the receptor protein or the like, and the host cell is preferably Escherichia coli, Bacillus subtilis, yeast, insect cells, animal cells, or the like.
  • the cell membrane fraction refers to a cell membrane-rich fraction obtained by disrupting cells and then obtained by a known method.
  • Cell crushing methods include crushing cells with a Potter- ⁇ 1 vehjem homogenizer, crushing using a Waring blender ⁇ polytron (Kinematica), crushing by ultrasonic waves, French Examples include crushing by ejecting cells from a fine nozzle while applying pressure with a press or the like.
  • fractionation by centrifugal force such as fractionation centrifugation and density gradient centrifugation is mainly used.
  • the cell lysate is centrifuged at a low speed (500 rpm to 3000 rpm) for a short time (typically about 1 to 10 minutes), and the supernatant is further centrifuged at a higher speed (15000 rpm to 30000 rpm). After centrifugation for 30 minutes to 2 hours, the resulting precipitate is used as a membrane fraction.
  • the membrane fraction contains a large amount of expressed receptor proteins and membrane components such as cell-derived phospholipids and membrane proteins.
  • the amount of the receptor protein in the cell or membrane fraction containing the receptor protein or the like is preferably 10 3 to 10 8 molecules per cell, and more preferably 10 5 to 10 7 molecules per cell.
  • receptor protein fraction a natural receptor protein fraction or a recombinant receptor protein fraction having an activity equivalent thereto is desirable.
  • equivalent activity refers to equivalent ligand binding activity, signal transduction action, and the like.
  • labeled ligand a labeled ligand, a labeled ligand analog compound and the like are used.
  • ligands labeled with [ 3 H], [ 12S I], [ 14 C], [ 35 S] and the like are used.
  • a cell containing the receptor protein of the present invention or a membrane fraction of the cell is first screened.
  • Prepare the receptor protein preparation by suspending it in a suitable buffer.
  • Any buffer may be used as long as it does not inhibit the binding of the ligand to the receptor protein, such as a phosphate buffer having a pH of 4 to 10 (preferably pH 6 to 8) or a buffer such as Tris-HCl buffer.
  • CHAPS, Surfactants such as Tween-80 TM (Kaoichi Atlas), digitonin, dexcholate, etc. can also be added to the buffer.
  • protease inhibitors such as PMS F, leptin, E-644 (manufactured by Peptide Research Institute), and pepstatin can be added for the purpose of suppressing the degradation of receptors and ligands by proteases.
  • NBS non-specific binding
  • the reaction is carried out at about 0 ° C to 50 ° C, preferably about 4 ° C to 37 ° C, for about 20 minutes to 24 hours, preferably about 30 minutes to 3 hours.
  • the solution 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 using a liquid scintillation counter or a T-counter.
  • the specific binding amount (B—NSB) is For example, a test compound having 50% or less can be selected as a candidate substance having a competitive inhibitory ability.
  • cell stimulating activity via the receptor protein for example, cell Intracellular cAMP production, intracellular cGMP production, activity of promoting or inhibiting intracellular protein phosphorylation, etc.
  • cell stimulating activity via the receptor protein for example, cell Intracellular cAMP production, intracellular cGMP production, activity of promoting or inhibiting intracellular protein phosphorylation, etc.
  • cells containing the receptor protein or the like of the present invention are cultured on a multi-well plate or the like. Before performing screening, replace the medium with a fresh medium or an appropriate buffer that is not toxic to cells, add test compounds, etc., incubate for a certain period of time, and then extract cells or collect supernatant. Then, the generated product is quantified according to each method. If the production of a substance as an indicator of the cell stimulating activity is difficult to perform due to the presence of a degrading enzyme contained in the cell, an inhibitor for the degrading enzyme may be added to perform the assay. In addition, activities such as cAMP production suppression can be detected as a production suppression effect on cells whose basic production amount has been increased by forskolin or the like.
  • cells expressing an appropriate receptor protein are required.
  • a cell expressing the receptor protein of the present invention a cell line having the natural receptor protein of the present invention, a cell line expressing the above-mentioned recombinant receptor protein or the like is desirable.
  • test compounds for example, peptides, proteins, non-peptidic compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts, etc. are used. Or a known compound.
  • a screening kit for a compound or a salt thereof that alters the binding between a ligand and the receptor protein of the present invention or the like includes cells containing the receptor protein of the present invention, the receptor protein of the present invention, or the like, or And those containing a membrane fraction of cells containing the receptor protein of the present invention and the like.
  • Examples of the screening kit of the present invention include the following.
  • CHO cells expressing the receptor protein of the present invention were subcultured on a 12-well plate at 5 ⁇ 10 5 Z-wells, and cultured at 37 ° C., 5% CO 2 , and 95% air for 2 days.
  • the compound or a salt thereof obtained by using the screening method or the screening kit of the present invention is a compound having an action of changing the binding property between the ligand and the receptor protein of the present invention and the like.
  • a compound having a cell stimulating activity eg, an activity of promoting or suppressing intracellular cAMP production, intracellular cGMP production, intracellular protein phosphorylation, etc.
  • Agonist an activity of promoting or suppressing intracellular cAMP production, intracellular cGMP production, intracellular protein phosphorylation, etc.
  • the receptor protein of the present invention so-called “ Agonist), (mouth) a compound that does not have the cell stimulating activity (so-called angelic gonist),
  • Examples of the compound include peptides, proteins, non-peptidic compounds, synthetic compounds, and fermentation products. These compounds may be novel compounds, Known compounds may be used.
  • the agonist against the receptor protein or the like of the present invention has the same activity as the physiological activity of the ligand for the receptor protein or the like of the present invention, it is useful as a safe and low-toxic drug depending on the ligand activity.
  • the antagonist to the receptor protein or the like of the present invention can suppress the physiological activity of the ligand to the receptor protein or the like of the present invention, it is useful as a safe and low-toxic drug for suppressing the ligand activity.
  • the compound that enhances the binding force between the ligand and the receptor protein of the present invention is useful as a safe and low-toxic drug for enhancing the physiological activity of the ligand for the receptor protein of the present invention and the like.
  • the compound that decreases the binding force between the ligand and the receptor protein of the present invention is useful as a safe and low-toxic drug for reducing the physiological activity of the ligand for the receptor protein and the like of the present invention.
  • the compound or its salt obtained by using the screening method or the screening kit of the present invention is used as the above-mentioned pharmaceutical composition, it can be carried out according to a conventional method.
  • tablets, capsules, elixirs, microcapsules, sterile solutions, suspensions, and the like can be prepared in the same manner as in the above-described medicine containing the receptor protein of the present invention.
  • the preparations obtained in this way are safe and low toxic, for example, in humans and other mammals (eg, rats, puppies, sheep, pigs, puppies, cats, dogs, sal, etc.). Can be administered.
  • the dose of the compound or a salt thereof varies depending on the administration subject, target organ, symptoms, administration method, and the like.
  • oral administration in general, for example, in an infectious disease patient (as 60 kg), About 0.1 to 100 mg, preferably about 1.0 to 5 Omg, more preferably about 1.0 to 2 Omg per day.
  • parenteral administration the single dose varies depending on the administration subject, target organ, symptoms, administration method, etc.
  • About 0.01 to 30 mg, preferably about 0.1 to 20 mg, more preferably about 0.1 to 10 mg per day is administered by intravenous injection. Is good It is convenient.
  • the dose can be administered in terms of 60 kg.
  • a preventive and / or therapeutic agent for various diseases containing a compound (eg, agonist, angonist) that changes the binding property between the receptor protein and the ligand of the present invention.
  • a compound eg, agonist, angonist
  • the receptor protein of the present invention is considered to play some important role in vivo, for example, immune function, central function, circulatory function, digestive function, and cardiac function. Therefore, a compound (eg, agonist, angonist) that alters the binding property between the receptor protein of the present invention and the ligand and the ligand for the receptor protein of the present invention are dysfunctional of the receptor protein of the present invention. It can be used as a prophylactic and / or therapeutic agent for a disease associated with the disease.
  • a compound eg, agonist, angonist
  • the compound or ligand when used as an agent for preventing and / or treating a disease associated with dysfunction of the receptor protein of the present invention, it can be formulated according to a conventional method.
  • the compound or ligand can be sterilized with tablets or capsules, elixirs, microcapsules, etc., as required, or sugar-coated, or with water or other pharmaceutically acceptable liquids. It can be used parenterally in the form of injections, such as solutions or suspensions.
  • the compound is mixed with known physiologically acceptable carriers, flavoring agents, 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 this. 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, etc. Swelling agents such as magnesium stearate, sweeteners such as sucrose, lactose or saccharin, and flavoring agents such as peppermint, cocoa oil or cellulose.
  • the unit dosage form is a capsule, the above type of material can further contain a liquid carrier such as an oil or fat.
  • Sterile composition for injection Can be formulated according to the usual practice of dissolving or suspending an active substance in a vehicle such as water for injection, or a naturally occurring vegetable oil such as sesame oil or coconut oil.
  • a vehicle such as water for injection, or a naturally occurring vegetable oil such as sesame oil or coconut oil.
  • aqueous liquid for injection include physiological saline, isotonic solution containing glucose and other adjuvants (eg, D-sorbitol, D-mannitol, sodium chloride, etc.) and the like.
  • Agents for example, alcohols (eg, ethanol), polyalcohols (eg, propylene glycol, polyethylene daricol), nonionic surfactants (eg, Polysorbate 80 TM , HC-150) .
  • the oily liquid for example, sesame oil, soybean oil and the like are used, and may be used in combination with solubilizers such as benzyl benzoate and benzyl alcohol.
  • prophylactic and therapeutic agents examples include, for example, buffers (for example, phosphate buffer and sodium acetate buffer: solution), soothing agents (for example, benzalkonium chloride, procaine hydrochloride, etc.), stabilizers (for example, human Serum albumin, polyethylene glycol, etc.), preservatives (eg, benzyl alcohol, phenol, etc.), antioxidants and the like.
  • buffers for example, phosphate buffer and sodium acetate buffer: solution
  • soothing agents for example, benzalkonium chloride, procaine hydrochloride, etc.
  • stabilizers for example, human Serum albumin, polyethylene glycol, etc.
  • preservatives eg, benzyl alcohol, phenol, etc.
  • antioxidants antioxidants and the like.
  • the prepared injection solution is usually filled in a suitable ampoule.
  • the above-mentioned prophylactic / therapeutic agent can be used in combination with an appropriate drug, for example, as a DDS preparation specifically targeting an
  • the preparations obtained in this way are safe and have low toxicity, for example, in humans and other mammals (eg, rats, mice, puppies, sheep, puppies, puppies, cats, dogs, monkeys, etc.). Can be administered.
  • mammals eg, rats, mice, puppies, sheep, puppies, puppies, cats, dogs, monkeys, etc.
  • the dose of the compound or a salt thereof varies depending on the administration subject, target organ, symptom, administration method, and the like.
  • oral administration in general, for example, in an infectious disease patient (as 60 kg), one day is required.
  • the single dose varies depending on the administration subject, target organ, symptoms, administration method, etc.
  • the dose can be administered in terms of 60 kg.
  • the antibody of the present invention can specifically recognize the receptor protein of the present invention and the like, quantification of the protein of the present invention in a test solution, particularly quantification by a sandwich immunoassay, etc. Can be used for That is, the present invention provides, for example,
  • the present invention provides a method for quantifying the receptor protein of the present invention in a test solution.
  • one of the antibodies is an antibody that recognizes the N-terminal of the receptor protein of the present invention or the like, and the other antibody is an antibody that reacts with the C-terminal of the receptor protein or the like of the present invention. Is preferred.
  • the receptor protein of the present invention can be measured using a monoclonal antibody against the receptor protein of the present invention (hereinafter sometimes referred to as the monoclonal antibody of the present invention), and detection by tissue staining or the like can be performed. Can also.
  • the antibody molecule itself may be used, or the F (ab ') 2 , Fab', or Fab fraction of the antibody molecule may be used.
  • the assay method using an antibody against the receptor protein or the like of the present invention is not particularly limited, and may be an antibody or an antigen corresponding to the amount of antigen (eg, the amount of receptor protein) in the test solution.
  • Any method can be used to detect the amount of antibody-antigen complex by chemical or physical means and calculate this from a standard curve prepared using a standard solution containing a known amount of antigen. May be used.
  • nephrometry, a competitive method, an immunometric method and a sandwich method are preferably used, but in terms of sensitivity and specificity, It is particularly preferable to use the sandwich method described in (1).
  • a labeling agent used in a measuring 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, [1 2 5 I], [1 3 1 I], [3 H], and [1 4 C] used.
  • the above-mentioned enzyme a stable enzyme having a large specific activity is preferable. For example, / 3-galactosidase,] 3-darcosidase, alkaline phosphatase, peroxidase, and lignoic acid dehydrogenase are used.
  • the fluorescent substance for example, fluorescein, fluorescein isothiosinate and the like are used.
  • the luminescent substance for example, luminol, luminol derivative, luciferin, lucigenin and the like are used. Further, a biotin-avidin system can be used for binding the antibody or antigen to the labeling agent.
  • insolubilization of the antigen or antibody physical adsorption may be used, or a method using a chemical bond usually used for insolubilizing or immobilizing a protein or enzyme may be used.
  • the carrier for example, insoluble polysaccharides such as agarose, dextran, cell mouth, and the like, synthetic resins such as polystyrene, polyacrylamide, and silicon, and glass are used.
  • the test solution is reacted with the insolubilized monoclonal antibody of the present invention (primary reaction), and further reacted with the labeled monoclonal antibody of the present invention (secondary reaction).
  • primary reaction the insolubilized monoclonal antibody of the present invention
  • secondary reaction the labeled monoclonal antibody of the present invention
  • the primary reaction and the secondary reaction may be performed in the reverse order, may be performed simultaneously, or may be performed at staggered times.
  • the labeling agent and the method of insolubilization can be based on those described above.
  • the antibody used for the solid phase antibody or the labeling antibody does not necessarily need to be one type, and a mixture of two or more types of antibodies is used for the purpose of improving measurement sensitivity and the like. May be used.
  • the monoclonal antibody of the present invention used in the primary reaction and the secondary reaction is preferably an antibody having a different binding site such as a receptor protein.
  • the antibody used in the reaction and the secondary reaction is, for example, when the antibody used in the secondary reaction recognizes the C-terminal of the receptor protein, the antibody used in the primary reaction is preferably other than the C-terminal For example, an antibody that recognizes 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 is separated from (F) and the labeled antigen (B) bound to the antibody.
  • BZF separation The amount of labeling of either B or F is measured, and the amount of antigen in the test solution is quantified.
  • a soluble antibody is used as an antibody
  • BZF separation is carried out using polyethylene glycol
  • a liquid phase method using a second antibody against the above antibody or an immobilized antibody is used as the first antibody.
  • An immobilization method using a soluble antibody as the first antibody and using 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 label in either phase is measured to determine the amount of antigen in the test solution.
  • nephelometry the amount of insoluble sediment generated as a result of 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 precipitate is obtained, laser nephrometry utilizing laser scattering is preferably used.
  • the receptor protein of the present invention or a salt thereof can be quantified with high sensitivity by using the antibody of the present invention.
  • the receptor protein of the present invention or a salt thereof in a living body using the antibody of the present invention, it is possible to diagnose various diseases related to dysfunction of the receptor protein of the present invention. it can.
  • the antibody of the present invention can be used for specifically detecting the receptor protein 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 protein of the present invention, detection of the receptor protein of the present invention in each fraction at the time of purification, and receptor protein of the present invention in test cells It can be used for the analysis of the behavior of an object.
  • the antibody of the present invention can specifically recognize the receptor protein of the present invention or its partial peptide or a salt thereof, it changes the amount of the receptor protein of the present invention or its partial peptide in the cell membrane. It can be used for screening of compounds.
  • the present invention provides a method for screening a compound that changes the amount of the receptor protein of the present invention or its partial peptide in a cell membrane by confirming the protein on the cell membrane by quantifying the protein.
  • Transformants expressing the receptor protein of the present invention or a partial peptide thereof were sectioned, and then immunostaining was used to quantify the degree of staining of the receptor protein at the cell surface layer.
  • immunostaining was used to quantify the degree of staining of the receptor protein at the cell surface layer.
  • non-human mammals for example, mice, rats, rabbits, puppies, higgies, bushus, puppies, cats, dogs, monkeys, etc., more specifically, demented rats, obese mice, Drugs (eg, anti-dementia drugs, anti-hypertensive drugs, anti-cancer drugs, anti-obesity drugs, etc.) or physical stress (eg, flooding stress, electric shock, light / dark, low temperature, etc.)
  • Drugs eg, anti-dementia drugs, anti-hypertensive drugs, anti-cancer drugs, anti-obesity drugs, etc.
  • physical stress eg, flooding stress, electric shock, light / dark, low temperature, etc.
  • blood or a specific organ eg, lung, leukocyte, testis, spleen, etc.
  • tissue or cell isolated from the organ is obtained.
  • the obtained organs, tissues or cells are transferred to an appropriate buffer (for example, Tris-HCl buffer, phosphate buffer, Suspension, etc.)
  • a cell membrane fraction is obtained by techniques such as centrifugal separation, filtration, and column fractionation.
  • the cell membrane fraction refers to a fraction containing a large amount of cell membrane obtained by a known method after cell disruption.
  • Methods for crushing cells include crushing cells with a Potter-E lvehj em-type homogenizer, crushing with a single ring blender polytron (Kinema tica), crushing with ultrasonic waves, French press, etc. Crushing by ejecting cells from a thin nozzle while applying pressure.
  • fractionation by centrifugal force such as fractionation centrifugation and density gradient centrifugation is mainly used.
  • the cell lysate is centrifuged at low speed (500 rpm to 3000 rpm) for a short time (typically, about 1 minute to 10 minutes), and the supernatant is further centrifuged at a higher speed (15000 rpm to 30000 rpm). After centrifugation for 30 minutes to 2 hours, the resulting precipitate is used as a membrane fraction.
  • the membrane fraction is rich in expressed receptor protein and the like and membrane components such as cell-derived phospholipids and membrane proteins.
  • the receptor protein of the present invention or its partial peptide contained in the cell membrane fraction can be quantified by, for example, a sandwich immunoassay using the antibody of the present invention, Western blot analysis, or the like.
  • Such a sandwich immunoassay can be performed in the same manner as described above, and the Western plot can be performed by known means.
  • Screening for a compound that alters the amount of the receptor protein of the present invention or its partial peptide in the cell membrane is performed by:
  • test compound When the transformant is cultured according to a conventional method, the test compound is mixed in a medium and cultured for a certain period of time (1 day to 7 days, preferably 1 day to 3 days, more preferably 2 days to 3 days). After a day), it can be performed by quantifying the amount of the receptor protein of the present invention or its partial peptide in the cell membrane.
  • the confirmation of the receptor protein of the present invention or its partial peptide contained in the cell membrane fraction is specifically performed as follows.
  • non-human mammals eg, mice, rats, egrets, sheep, higgs, bush, horses, cats, dogs, monkeys, etc .; more specifically, dementia rats, obese mice, arteries, etc.
  • Drugs eg, anti-dementia drugs, antihypertensive drugs, anti-cancer drugs, anti-obesity drugs, etc.
  • physical stress eg, flooding stress, electric shock, light / dark, low temperature, etc.
  • blood or a specific organ eg, lung, leukocyte, testis, spleen, etc.
  • a tissue or cell isolated from the organ is obtained.
  • the obtained organ, tissue or cell is cut into a tissue section according to a conventional method, and immunostained using the antibody of the present invention.
  • the protein on the cell membrane By confirming the protein on the cell membrane by quantifying the degree of staining of the receptor protein on the cell surface, it is possible to quantitatively or qualitatively determine the receptor protein of the present invention or a portion thereof on the cell membrane. The amount of peptide can be confirmed.
  • the compound or a salt thereof obtained by using the screening method of the present invention is a compound having an action of changing the amount of the receptor protein or its partial peptide of the present invention in a cell membrane.
  • the cell stimulating activity mediated by the receptor protein of the present invention is a compound that reduces the cell stimulating activity by reducing the amount of the receptor protein of the present invention or its partial peptide in the cell membrane.
  • Examples of the compound include a peptide, a protein, a non-peptidic compound, a synthetic compound, and a fermentation product. These compounds may be a novel compound or a known compound.
  • the compound that enhances the cell stimulating activity is useful as a safe and low toxic drug for enhancing the physiological activity of the receptor protein or the like of the present invention.
  • the compound that reduces the cell stimulating activity is useful as a safe and low-toxic drug for reducing the physiological activity of the receptor protein of the present invention or the like.
  • a compound or a salt thereof obtained by using the screening method of the present invention is used as a pharmaceutical composition, it can be carried out according to a conventional method.
  • tablets, capsules, elixirs, microcapsules, sterile solutions, suspensions, and the like can be prepared in the same manner as the above-described pharmaceuticals containing the receptor protein of the present invention.
  • the preparations obtained in this way are safe and have low toxicity, for example, in humans and other mammals (eg, rats, mice, rabbits, sheep, pigs, pigs, cats, dogs, monkeys, etc.). Can be administered.
  • mammals eg, rats, mice, rabbits, sheep, pigs, pigs, cats, dogs, monkeys, etc.
  • the dose of the compound or a salt thereof varies depending on the administration subject, target organ, symptom, administration method, and the like.
  • oral administration in general, for example, in an infectious disease patient (as 60 kg), one day About 0.1 to 100 mg, preferably about 1.0 to 50 mg, more preferably about 1.0 to 2 Omg.
  • the single dose varies depending on the administration subject, target organ, symptoms, administration method, etc.
  • the amount converted per 60 kg can be administered.
  • the receptor protein of the present invention or its partial peptide in the cell membrane As described above, the receptor protein of the present invention is thought to play some important role in vivo, for example, in the lungs or immune function, as described above. Can be Therefore, the compound that alters the amount of the receptor protein of the present invention or its partial peptide in the cell membrane can be used as an agent for preventing and / or treating a disease associated with dysfunction of the receptor protein of the present invention.
  • the compound when used as an agent for preventing and / or treating a disease associated with dysfunction of the receptor protein of the present invention, it can be formulated according to a conventional method.
  • the compound can be used as a sugar-coated tablet, capsule, elixir, microcapsule or the like as needed, orally, or aseptic solution with water or another pharmaceutically acceptable liquid. It can be used parenterally or in the form of injections such as suspensions.
  • the compound is mixed with known physiologically acceptable carriers, flavoring agents, 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, etc. Swelling agents such as magnesium stearate, sweeteners such as sucrose, lactose or saccharin, and flavoring agents such as peppermint, cocoa oil or cellulose.
  • the unit dosage form is a capsule, the above type of material can further contain a liquid carrier such as an oil or fat.
  • Sterile compositions for injection can be 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. it can.
  • aqueous solution for injection for example, physiological saline, isotonic solution containing glucose and other adjuvants (eg, D-sorbitol, D-mannitol, sodium chloride, etc.) and the like are used.
  • Agent for example, alcohol (eg, It may be used in combination with ethanol), polyalcohol (eg, propylene glycol, polyethylene dalicol), nonionic surfactant (eg, polysorbate 80 TM , HCO-50).
  • oily liquid for example, sesame oil, soybean oil and the like are used, and may be used in combination with solubilizers such as benzyl benzoate and benzyl alcohol.
  • prophylactic and therapeutic agents include, for example, buffers (for example, phosphate buffer and sodium acetate buffer), soothing agents (for example, benzalkonium chloride, procaine hydrochloride, etc.), stabilizers (for example, human serum Albumin, polyethylene glycol, etc.), preservatives (eg, benzyl alcohol, phenol, etc.), antioxidants and the like.
  • buffers for example, phosphate buffer and sodium acetate buffer
  • soothing agents for example, benzalkonium chloride, procaine hydrochloride, etc.
  • stabilizers for example, human serum Albumin, polyethylene glycol, etc.
  • preservatives eg, benzyl alcohol, phenol, etc.
  • antioxidants eg, benzyl alcohol, phenol, etc.
  • the prepared injection solution is usually filled in a suitable ampoule.
  • the preparations obtained in this way are safe and have low toxicity, for example, in humans and other mammals (eg, rats, mice, rabbits,
  • the dose of the compound or a salt thereof varies depending on the administration subject, target organ, symptom, administration method, and the like.
  • oral administration in general, for example, in an infectious disease patient (as 60 kg), one day About 0.1 to 100 mg, preferably about 1.0 to 50 mg, more preferably about 1.0 to 2 Omg.
  • the single dose varies depending on the administration subject, target organ, symptoms, administration method, etc.
  • the amount converted per 60 kg can be administered.
  • the neutralizing activity of an antibody against the receptor protein of the present invention, its partial peptide, or a salt thereof against the receptor protein or the like means an activity of inactivating a signal transduction function involving the receptor protein. I do. Therefore, when the antibody has neutralizing activity, the receptor protein Signal transduction, for example, cell stimulating activity via the receptor protein (for example, activity to promote or suppress intracellular cAMP production, intracellular cGMP production, intracellular protein phosphorylation, etc.) Can be inactivated. Therefore, it can be used for prevention and / or treatment of diseases caused by overexpression of the receptor protein and the like.
  • transgenic animals expressing the receptor protein and the like of the present invention can be produced.
  • animals include non-human mammals (for example, rats, mice, egrets, sheep, pigeons, pigs, cats, cats, dogs, monkeys, etc.) (hereinafter sometimes abbreviated as animals). Particularly preferred are mice, egrets, and the like.
  • the DNA of the present invention When introducing the DNA of the present invention into a target animal, it is generally advantageous to use the DNA as a gene construct linked downstream of a promoter capable of expressing in animal cells.
  • a DNA construct of the present invention derived from an animal having a high homology to the DNA is linked to a gene construct linked downstream of various promoters capable of expressing the DNA in animal cells.
  • a DNA-introduced animal that highly produces the receptor protein of the present invention or the like can be produced by injecting the fertilized egg into a fertilized egg.
  • the promoter for example, a virus-derived promoter or a ubiquitous expression promoter such as metamouth thionein may be used, but a promoter of a gene specifically expressed in leukocytes is preferably used.
  • the presence of the receptor protein of the present invention in the germ cells of the produced animal after DNA introduction indicates that all the offspring of the produced animal have the receptor protein of the present invention in all of the germ cells and somatic cells Means that The progeny of this kind of animal that has inherited the gene has the receptor protein of the present invention in all of its germ cells and somatic cells.
  • the DNA-introduced animal of the present invention was confirmed to stably maintain the gene by mating.
  • breeding can be carried out in a normal breeding environment as the DNA-bearing animal.
  • homozygous animals having the transgene on both homologous chromosomes are obtained, and by crossing the male and female animals, all the offspring can obtain the DNA.
  • Breeding to have Since the animal into which the DNA of the present invention has been introduced expresses the receptor protein or the like of the present invention at a high level, it may be used as an animal for screening an agonist or an anthony gonist against the receptor protein of the present invention or the like. Useful.
  • the DNA-introduced animal of the present invention can also be used as a cell source for tissue culture.
  • tissue culture For example, by directly analyzing DNA or RNA in the tissue of the DNA-introduced mouse of the present invention, or by analyzing the tissue in which the receptor protein of the present invention expressed by a gene is present, the receptor of the present invention can be analyzed overnight. It can analyze protein, etc.
  • the cells of a tissue having the receptor protein of the present invention are cultured by standard tissue culture techniques, and these are used to study the function of cells from a generally difficult tissue such as brain or peripheral tissue. be able to.
  • a drug that enhances the function of various tissues can be selected.
  • the receptor protein of the present invention can be isolated and purified therefrom.
  • bases, amino acids, and the like are indicated by abbreviations based on the abbreviations by IU PAC-IUB Communication on Biochemical Nomenclature or conventional abbreviations in the field, and examples thereof are described below.
  • optical isomer of an amino acid the L-form is indicated unless otherwise specified.
  • dATP phosphate dTTP Deoxythymidine triphosphate
  • dGTP Deoxyguanosine triphosphate
  • dCTP Deoxycytidine triphosphate ATP Adenosine triphosphate
  • TC thiazolidine one 4 (R) also Ichiriki Rupokisamido group, this specification: referred substituents frequently used medium, the protecting groups and reagents the following symbols.
  • FIG 1 shows the amino acid sequence of the human-derived novel transmembrane receptor uniform protein PTB 2184 of the present invention.
  • FIG. 1 shows the nucleotide sequence of cDNA encoding the novel human transmembrane receptor-like protein pTB2184 of the present invention.
  • FIG. 1 shows the amino acid sequence of the novel human transmembrane receptor uniform protein pTB2185 of the present invention.
  • FIG. 1 shows the nucleotide sequence of cDNA encoding the novel human transmembrane receptor uniform protein pTB2185 of the present invention.
  • a human peripheral blood leukocyte cDNA (CLONTECH) was used as type II, and a PCR reaction was performed using two primers, primer 1 (SEQ ID NO: 5) and primer 2 (SEQ ID NO: 6).
  • the composition of the reaction solution in the reaction was as follows: cDNA 51 was used as type III, Pfu Turbo DNA Polymerase (ST RATAGENE) 1 / i 1 amount, Primer 1 (SEQ ID NO: 5) and Primer 1 (SEQ ID NO: 6) was added to each of 0.5 M, dNTPs was added to 200 M, and the buffer attached to the enzyme was added to 101, thereby obtaining the IOOI liquid volume.
  • the PCR reaction was performed at 96 for 1 minute, followed by 5 cycles of 96 ° C for 15 seconds, 72 ° C for 1 minute and 30 seconds, then 96 ° C for 15 seconds, 6530 seconds, and 72 ° C. The cycle of C ⁇ 1 minute was repeated 30 times, and finally, extension reaction at 72 ° C ⁇ 7 minutes was performed. Next, an adenine addition reaction was performed on the 3 ′ end of the PCR reaction product. First, the PCR reaction product of 912 base pairs was purified by agarose gel electrophoresis, and this was used as type ⁇ in the reaction.
  • the anti The composition of the reaction mixture in the reaction was 1/10 of the purified PCR reaction product described above, ExTaq DNA Polymerase (Takara Shuzo) 0.11, dNTPs 50 ⁇ , and buffer attached to the enzyme 5 1 In addition, the liquid volume was set at 301. The adenine addition reaction was performed at 72 ° C. for 15 minutes. The PCR reaction product to which the 3′-terminal adenine had been added was subcloned into a plasmid vector PCR 2.1 (In Vitrogen) according to the protocol of DNA Ligation Kit Ver. 2 (Takara Shuzo).
  • the novel transmembrane receptor uniform protein containing the amino acid sequence (SEQ ID NO: 3) encoded by the nucleotide sequence of the DNA represented by SEQ ID NO: 4 was named pTB2185.
  • the transformants were named Escherichia coli DH5 ⁇ 2184 and Escherichia coli DH5 «/ pTB2185, respectively.
  • the ninth Thr in the amino acid sequence of pTB2184 is replaced with Met, and the 140th Leu is replaced with Arg.
  • the C at the 26th position in the nucleotide sequence of the DNA encoding pTB2184 is substituted with T, and the T at the 419th position is substituted with G.
  • the transmembrane receptor uniform protein or its partial peptide or a salt thereof of the present invention are as follows: Ligand (ago 2) Acquisition of antibodies and antisera, 3) Construction of a recombinant receptor protein expression system, 4) Development of a receptor binding Atsushi system using the same expression system and screening of drug candidate compounds, 4) Structure Implementation of drug design based on comparison with chemically similar ligands / receptors, ⁇ ⁇ ⁇ ⁇ probes in genetic diagnosis ⁇ reagents for the preparation of PCR primers, 7production of transgenic animals or ⁇ gene therapy It can be used as a drug such as a drug.

Abstract

L'invention concerne de nouvelles protéines convenant, par exemple, au criblage d'un agoniste ou d'un antagoniste. D'une manière plus spécifique, l'invention concerne de nouvelles protéines transmembranaires du type récepteur contenant une séquence d'acide aminé identique ou sensiblement identique à la séquence d'acide aminé représentée par SEQ ID NO:1 ou SEQ ID NO:2 ; des sels desdites protéines ou des polynucléotides codant pour lesdites protéines. L'invention concerne en outre les utilisations médicales desdites protéines.
PCT/JP2001/011081 2000-12-19 2001-12-18 Nouvelles proteines transmembranaires du type recepteur et adn correspondants WO2002050265A1 (fr)

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WO2000022131A2 (fr) * 1998-10-13 2000-04-20 Arena Pharmaceuticals, Inc. Recepteurs non-endogenes de la proteine g humaine ayant une activite constitutive
WO2000078809A1 (fr) * 1999-06-21 2000-12-28 Smithkline Beecham Corporation Axor16, récepteur couplé à une protéine g

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