WO2003088992A1 - Nouvelle methode de criblage - Google Patents

Nouvelle methode de criblage Download PDF

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Publication number
WO2003088992A1
WO2003088992A1 PCT/JP2003/005037 JP0305037W WO03088992A1 WO 2003088992 A1 WO2003088992 A1 WO 2003088992A1 JP 0305037 W JP0305037 W JP 0305037W WO 03088992 A1 WO03088992 A1 WO 03088992A1
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Prior art keywords
amino acid
acid sequence
protein
salt
seq
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PCT/JP2003/005037
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English (en)
Japanese (ja)
Inventor
Shuji Hinuma
Masaki Hosoya
Yugo Habata
Ryo Fujii
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Takeda Chemical Industries, Ltd.
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Priority to AU2003235310A priority Critical patent/AU2003235310A1/en
Publication of WO2003088992A1 publication Critical patent/WO2003088992A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy

Definitions

  • the present invention relates to the use of human-derived G protein-coupled receptor protein H7TBA62.
  • G proteins conjugated guanine nucleotide-binding proteins
  • G protein-coupled receptor proteins are present on the surface of various functional cells in living cells and organs, and serve as physiological targets for molecules that regulate the functions of those cells and organs, such as hormones, neurotransmitters and bioactive substances. Plays an important role.
  • the receptor transmits a signal into a cell through binding to a physiologically active substance, and this signal causes various reactions such as suppression of activation and activation of the cell.
  • physiological functions are regulated under the control of many hormones, hormone-like substances, neurotransmitters or bioactive substances.
  • physiologically active substances are present at various sites in the living body, and regulate their physiological functions through their corresponding receptor proteins.
  • receptor proteins In vivo There are many unknown hormones, neurotransmitters and other physiologically active substances, and the structure of their receptor proteins has not been reported so far. Furthermore, it is often unknown whether subtypes exist in known 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.
  • the functions of receptor protein genes expressed in vivo must be elucidated and expressed in an appropriate expression system. It was necessary.
  • G protein-coupled receptors with unknown functions and There are many so-called orphan receptors for which no ligand has been identified, and there is an urgent need to search for ligands and elucidate the functions of G protein-coupled receptors.
  • the G protein-coupled receptor is useful for searching for a new physiologically active substance (that is, a ligand) and for searching for an agonist or an antagonist for the receptor, using the signal transduction action as an index.
  • a physiological ligand that is, a ligand
  • These ligands, agonists or antagonists to these receptors can be expected to be used as preventive / therapeutic or diagnostic agents for diseases associated with dysfunction or hyperfunction of G protein-coupled receptors.
  • a decrease or increase in the function of the G protein-coupled receptor in a living body based on a gene mutation 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 corresponding to the receptor gene It can also be applied to treatment.
  • the nucleotide sequence of the receptor is essential information for examining the presence or absence of a deletion or mutation in the gene, and the gene of the receptor is used to prevent or treat a disease associated with dysfunction of the receptor. And diagnostics.
  • the present invention relates to the determination of a ligand for the orphan G protein-coupled receptor protein H7TBA62 of unknown function, and the use of the G protein-coupled receptor protein and its ligand. That is, the present invention provides a method for screening a compound (antagonist, agonist) or a salt thereof that alters the binding property between a ligand and the G protein-coupled receptor protein, a salt thereof, the screening kit, the screening method or the screening kit.
  • an object of the present invention to provide a drug containing a compound (antagonist, agonist) that changes the binding property to a receptor protein or a compound that changes the expression level of the G protein-coupled receptor protein or a salt thereof.
  • the present inventors have conducted intensive studies and found that one of the human H7TBA62 ligands is partially a-receptor-agonist to agonism of the drainage receptor agonist.
  • 5-HT1A / 5-HT1 It was found to be oxymetazoline known as a B receptor agonist.
  • the present inventors have further studied based on these findings, and as a result, have completed the present invention.
  • a vasoconstriction comprising a G protein-coupled receptor protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 2, a partial peptide thereof, or a salt thereof Agents, serotonin agonists, acetylcholine release enhancers, gastrointestinal motility enhancers, smooth muscle contractors, platelet aggregating agents, central nervous stimulants or inotropic agents,
  • a neural effect comprising a G protein-coupled receptor protein or a partial peptide thereof or a salt thereof containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 2
  • a G protein-coupled receptor protein or a partial peptide thereof or a salt thereof containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 2
  • Vasoconstrictor, serotonin agonist, acetylcholine release enhancer, gastrointestinal motility enhancer, smooth muscle contractor, platelet aggregating agent, central nervous system stimulant or inotropic agent containing polynucleotide
  • a polynucleotide encoding a G protein-coupled receptor protein or a partial peptide thereof containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 2 A denervating agent for neuroactive nasal depressive blood or mucosal hyperemia comprising a polynucleotide, or acute circulatory insufficiency or Prevention and treatment of hypotension,
  • a polynucleotide encoding a G protein-coupled receptor protein or a partial peptide thereof containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 2 A diagnostic agent for vasoconstriction or dilation, serotonin action, acetylcholine release, gastrointestinal motility, smooth muscle contraction, platelet aggregation, central nervous control or inotropic effect, comprising a polynucleotide,
  • Neuroactive nasal depressive blood containing the polynucleotide mucosal hyperemia, acute circulatory insufficiency, hypotension, peripheral circulatory disorders, hypertension, depression, obsessive-compulsive disorder, obsessive-compulsive disorder, panic disorder, anxiety disorder, Eating disorders, ischemic diseases, gastrointestinal symptoms associated with antineoplastic drug administration, acute myocardial infarction, acute ophthalmitis, chronic knee inflammation, adult respiratory distress syndrome, alcohol withdrawal syndrome, Alzheimer's disease, ankylosing spondylitis, arrhythmia , Cocaine withdrawal syndrome, dementia, diarrhea, gastritis, hepatitis, infectious diseases, obiate withdrawal syndrome, osteoarthritis, osteoporosis, osteophytosis, peptic ulcer, Perip
  • Vasodilator, vasoconstrictor, anti-serotonin, acetylcholine release inhibitor, gastrointestinal motility inhibitor, smooth muscle contraction inhibitor, platelet aggregation inhibitor or central nervous system inhibitor
  • a neural effect comprising an antibody against a G protein-coupled receptor protein or a partial peptide thereof or a salt thereof, having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 2
  • Nasal depressive blood mucosal hyperemia, acute circulatory insufficiency, hypotension, peripheral circulatory disorders, hypertension, depression, obsessive-compulsive disorder, obsessive-compulsive disorder, panic disorder, anxiety disorder, eating disorder, ischemic disorder, anti- Gastrointestinal symptoms associated with malignant drug administration, acute myocardial infarction, acute knee inflammation, chronic knee inflammation, adult respiratory distress syndrome group, alcohol withdrawal syndrome, Alzheimer's disease, ankylosing spondylitis, arrhythmia, co-in withdrawal syndrome, dementia , Diarrhea, gastritis, hepatitis, infectious disease, obiate withdrawal syndrome, osteoarthritis, osteoporosis, bone behcetosis, peptic ulcer, peripheral vascular disease,
  • G protein-coupled receptor protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 2, or a polynucleotide containing a polynucleotide encoding a partial peptide thereof Salts complementary to nucleotides
  • Peripheral circulatory disorder hypertension, depression, obsessive-compulsive disorder, obsessive-compulsive disorder, panic disorder, anxiety disorder, eating disorder, ischemic disease, anticancer drug administration containing base sequence or a part thereof
  • gastrointestinal symptoms acute myocardial infarction, acute tengitis, chronic inflammation, adult respiratory distress syndrome, alcohol withdrawal syndrome, Alzheimer's disease, ankylosing spondylitis, arrhythmia, cocaine withdrawal syndrome, dementia, diarrhea, gastritis, hepatitis , Infections, obiate withdrawal syndrome, osteoarthritis, osteoporosis, bone behcetosis, peptic ulcer, peripheral vascular disease, sequelae of
  • a G protein-coupled receptor protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 2, a partial peptide thereof or a salt thereof, and (2) oxymetazoline
  • a medicament comprising the compound of the above-mentioned [15] or a salt thereof,
  • a vasoconstrictor comprising a G protein-coupled receptor protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 2 or an agonist against a salt thereof, a serotonin agonist Agent, acetylcholine release promoter, gastrointestinal motility enhancer, smooth muscle constrictor, platelet aggregating agent, central nervous system stimulant or inotropic agent, [18] A neuroactive nasal depression comprising an agonist against a G protein-coupled receptor 1 protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 2, or a salt thereof Remover of blood or mucosal hyperemia, or preventive and therapeutic agent for circulatory insufficiency or hypotension,
  • a vasodilator comprising a G protein-coupled receptor protein having an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 2 or an antagonist to a salt thereof, vasoconstriction Inhibitors, anti-serotonin agents, acetylcholine release inhibitors, gastrointestinal motility inhibitors, smooth muscle contraction inhibitors, platelet aggregation inhibitors or central nervous system inhibitors,
  • Peripheral circulatory disorder, hypertension comprising an G protein-coupled receptor protein having an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 2 or an antagonist to a salt thereof Illness, depression, obsessive-compulsive disorder, obsessive-compulsive disorder, panic disorder, anxiety disorder, eating disorder, ischemic disease, gastrointestinal symptoms associated with administration of antineoplastic drugs, acute myocardial infarction, acute knee inflammation, chronic inflammation, Adult respiratory distress syndrome, alcohol withdrawal syndrome, Alzheimer's disease, ankylosing spondylitis, arrhythmia, cocaine withdrawal syndrome, dementia, diarrhea, gastritis, hepatitis, infectious disease, obiate withdrawal syndrome, osteoarthritis, osteoporosis, osteoporosis, Peptic ulcer, peripheral vascular disease, sequelae of myocardial infarction, reflux esophagitis, rheumatoid arthritis, schizophrenia, sepsis, sepsis Click, prophylactic
  • Intracellular c when a test compound is brought into contact with a cell containing a G protein-coupled receptor protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 2 c A method for screening an agonist against the receptor protein or a salt thereof, which comprises measuring an AMP production inhibitory activity;
  • a G protein-coupled receptor protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 2, a partial peptide thereof or a salt thereof, and (2) the receptor protein Or an agonist or antagonis to the receptor protein or a salt thereof, which comprises using a compound or a salt thereof that changes the binding property between the salt and oxymethazoline.
  • a G protein-coupled receptor protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 2, a partial peptide thereof or a salt thereof, and (2) the receptor protein or A screening kit for an agonist or an antagonist for the receptor protein or a salt thereof, which comprises a compound that changes the binding property of the salt to oxymethazoline or a salt thereof;
  • a medicament comprising an agonist or an antagonist to a G protein-coupled receptor protein or an salt thereof having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 2;
  • a polynucleotide containing a polynucleotide encoding a G protein-coupled receptor protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 2, or a polynucleotide encoding a partial peptide thereof The expression level of the G protein-coupled receptor protein is changed, blood vessel contraction or dilation, serotonin action, acetylcholine release, gastrointestinal motility, smooth muscle contraction, platelet aggregation or central nervous system.
  • a polynucleotide comprising a G protein-coupled receptor protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 2 or a polynucleotide comprising a polynucleotide encoding a partial peptide thereof Changes the expression level of the G protein-coupled receptor protein contained and modulates vasoconstriction or dilation, serotonin action, acetylcholine release, gastrointestinal motility, smooth muscle contraction, platelet aggregation or central nervous system
  • Vasoconstrictors, serotonin agonists, acetylcholine release promoters, gastrointestinal motility enhancers, smooth muscle contractors, platelet aggregating agents, central nervous stimulants or inotropic agents
  • a compound or a salt thereof that increases the expression level of a G protein-coupled receptor protein or a partial peptide thereof containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 2 An agent that removes neuroactive nasal depression or mucosal hyperemia, or prevents acute circulatory insufficiency or hypotension
  • a compound or a salt thereof, which decreases the expression level of a G protein-coupled receptor protein or a partial peptide thereof containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 2 A vasodilator, a vasoconstrictor, an anti-serotonin, an acetylcholine release inhibitor, a gastrointestinal motility inhibitor, a smooth muscle contraction inhibitor, a platelet aggregation inhibitor or a central nervous system inhibitor comprising
  • a compound or a salt thereof that reduces the expression level of a G protein-coupled receptor protein or a partial peptide thereof which has the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 2,
  • Contained peripheral circulatory disorders hypertension, depression, obsessive-compulsive disorder, obsessive-compulsive disorder, panic disorder, anxiety disorder, eating disorder, ischemic disease, gastrointestinal symptoms associated with antineoplastic drug administration, acute myocardial infarction , Acute inflammation, Chronic knee inflammation, Adult respiratory distress syndrome, Alcohol withdrawal syndrome, Alcheimer's disease, Ankylosing spondylitis, Arrhythmia, Cocaine withdrawal syndrome, Dementia, Diarrhea, Gastritis, Hepatitis, Infectious disease, Oviate withdrawal syndrome, Osteoarthritis , Osteoporosis, osteopethiosis, peptic ulcer, peripheral vascular disease, sequelae of myocardial infarction, reflux e
  • G protein-coupled receptor containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 2 containing oxymetazoline Signal transduction enhancer of puter protein,
  • a G protein-coupled receptor protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 2, a partial peptide thereof, or a salt thereof
  • a vascular harvest characterized by administering an effective amount of an agonist against a G protein-coupled receptor protein or a salt thereof containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 2
  • Contraction method serotonin activation method, acetylcholine release promotion method, gastrointestinal motility enhancement method, smooth muscle contraction method, platelet aggregation Method, central nervous system stimulant method, cardiotonic method, the method of removing the nerve-acting nasal depression blood or mucosal hyperemia or prevention and treatment method for acute circul
  • a G protein-coupled receptor protein or a partial peptide thereof, containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 2
  • An antibody against a salt (2) a G protein-coupled receptor protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 2 or a polynucleotide encoding a partial peptide thereof.
  • a polynucleotide comprising a nucleotide sequence complementary to the polynucleotide contained therein or a part thereof, or 3 a G protein comprising an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 2
  • G protein conjugate containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 2 for producing a remover or a preventive agent for treating acute circulatory failure or hypotension A receptor protein, a partial peptide thereof or a salt thereof, 2 a G protein-coupled receptor protein or a partial peptide thereof containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 2.
  • Antineoplastic drugs acute myocardial infarction, acute knee inflammation, chronic knee inflammation
  • Alcohol withdrawal syndrome Alzheimer's disease, ankylosing spondylitis, arrhythmia, cocaine withdrawal syndrome group, dementia, diarrhea, gastritis, hepatitis, infection, obiate withdrawal syndrome, osteoarthritis, osteoporosis, osteoporosis , Peptic ulcer, peripheral vascular disease, sequelae of myocardial infarction
  • H7TBA62 G protein-coupled receptor protein
  • H7TBA62-activating compound is contacted with H7TBA62-containing cells, and (ii) H7TBA62-activating compound and test compound contain H7TBA62.
  • a transformant obtained by transforming a compound that activates H7TBA62 with a recombinant vector containing DNA containing DNA encoding H7TBA62 by culturing the cell membrane of the transformant And H7TBA62 expressed in the cell membrane of the transformant were contacted with H7TBA62 expressed in H7TBA62.
  • a vasoconstrictor a serotonin agonist, characterized by using a G protein-coupled receptor protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 2 or a salt thereof.
  • Drugs Acetylcholine release enhancers, Gastrointestinal motility enhancers, Smooth muscle constrictors, Platelet aggregation agents, Central nervous system stimulants, Cardiotonic agents, Drugs for removing neuroactive nasal depressive blood or mucosal hyperemia, Acute circulatory failure or low
  • a vasoconstrictor, a serotonin agonist characterized by using a G protein-coupled receptor protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 2 or a salt thereof;
  • Drugs Acetylcholine release enhancers, Gastrointestinal motility enhancers, Smooth muscle constrictors, Platelet aggregating agents, Central nervous system stimulants, Cardiotonic agents, Drugs for removing neuroactive nasal depressive blood or mucosal hyperemia, or Acute circulatory failure or hypotension
  • Drugs Acetylcholine release enhancers, Gastrointestinal motility enhancers, Smooth muscle constrictors, Platelet aggregating agents, Central nervous system stimulants, Cardiotonic agents, Drugs for removing neuroactive nasal depressive blood or mucosal hyperemia, or Acute circulatory failure or hypotension
  • FIG. 1 shows the effect of oxymetazoline on the expression of a reporter (luciferase) gene in HEK293 cells transiently expressing H7TBA62.
  • the vertical axis shows luciferase activity (counts of seconds (cps)).
  • Controls show no forskolin addition and no oxymetazoline, forskolin shows no forskolin addition and no oxymethazoline addition, and forskolin + oxymethazoline shows forkolin and oxymethazoline addition.
  • the mouth shows cells into which only the vector has been introduced, and the figure shows cells into which H7TBA62 cDNA has been introduced.
  • FIG. 2 is a graph showing the activity of oxymetazoline for inhibiting the intracellular cAMP production of H7 TBA62-expressing HEK cells.
  • HEK shows the results for non-transfected HEK cells
  • HEK-H7 TBA62 shows the results for H7 TBA62-expressing HEK cells.
  • the vertical axis indicates the intracellular cAMP concentration (pmol).
  • Control No forskolin added ⁇ No oxymetazoline added, Forskolin added forskolin ⁇ No oxymetazoline added, Oxymetazoline: Forkolin added kamo ⁇ Oxymetazoline Indicates addition.
  • FIG. 3 shows the expression distribution of H7TBA62 mRNA in various human tissues. Copies / 25ngtotal RNA indicates the number of copies per 25ng of total RNA.
  • FIG. 4 shows the expression levels of H7TB A62 mRNA in prostate cancer cell lines and colon cancer cell lines.
  • CopiesZZSngTotal RNA indicates the number of copies per 25 ng of Total1 RNA.
  • DU145 and PC3 are the names of prostate cancer cell lines, respectively, and SW1417, WiDr, SW837 and SW1463 are the names of colon cancer cell lines, respectively.
  • the G protein-coupled receptor protein H7TBA62 used in the present invention is a receptor protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 2.
  • H7TBA62 is, for example, a human mammal (eg, a guinea pig, a rat, a mouse, a mouse, a heron, a pig, a sheep, a horse, a monkey, etc.), and any cell (eg, a spleen cell, a nerve cell, a glial cell, a knee).
  • a human mammal eg, a guinea pig, a rat, a mouse, a mouse, a heron, a pig, a sheep, a horse, a monkey, etc.
  • any cell eg, a spleen cell, a nerve cell, a glial cell, a knee.
  • Cells bone marrow cells, mesangial cells, Langerhans cells, epidermal cells, epithelial cells, endothelial cells, fibroblasts, fiber cells, muscle cells, adipocytes, immune cells (eg, macrophages, T cells, B cells, natural killer cells, Mast cells, neutrophils, basophils, eosinophils, monocytes), megakaryocytes, synoviocytes, chondrocytes, bone cells, osteoblasts, osteoclasts, breast cells, hepatocytes or stromal cells, Or progenitor cells of these cells, stem cells or cancer cells), blood cells, or any tissue in which these cells are present, such as the brain or various parts of the brain (Eg, sphere, nucleus, nucleus basalis, basal sphere, hippocampus, thalamus, hypothalamus, hypothalamus, cerebral cortex, medulla, cerebellum, occipital lobe, frontal lobe,
  • amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 2 is, for example, about 85% or more, preferably 90% or more, more preferably the amino acid sequence represented by SEQ ID NO: 2. Amino acid sequences having about 95% or more homology are exemplified.
  • Examples of the protein having an amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 2 of the present invention include, for example, a protein having an amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 2, A protein having substantially the same activity as the protein consisting of the amino acid sequence represented by SEQ ID NO: 2 is preferred.
  • Examples of substantially the same activity include a ligand binding activity and a signal information transmitting action. 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 (for example, about 0.01 to 100 times, preferably about 0.5 to 20 times, more preferably about 0.5 to 2 times). However, quantitative factors such as the degree of these activities and the molecular weight of the protein may be different.
  • the activity such as the ligand binding activity and the signal information transduction activity can be measured according to a method known per se.
  • the activity can be measured according to a screening method described later.
  • H7TBA62 a) one or two or more (preferably about 1 to 30 and more preferably about 1 to 10) in the amino acid sequence represented by SEQ ID NO: 2; More preferably, an amino acid sequence in which several (1 to 5) amino acids have been deleted; b) 1 or more (preferably 1 to 30) amino acids in the amino acid sequence represented by SEQ ID NO: 2 Degree, more preferably about 1 to 10, more preferably several (1 to 5) amino acids; c) 1 or 2 in the amino acid sequence represented by SEQ ID NO: 2 An amino acid sequence in which at least (preferably about 1 to 30 amino acids, more preferably about 1 to 10 amino acids, and still more preferably several (1 to 5) amino acids) have been substituted with other amino acids, or d ) A protein containing an amino acid sequence obtained by combining them is also used.
  • H 7 TBA 62 is represented on the left end in accordance with the convention of peptide labeling.
  • the N terminal (amino terminal) and the right terminal are the C terminal (carboxyl terminal).
  • R in the ester e.g., methyl, Echiru, n- propyl Honoré
  • C physician 6 alkyl group such as isopropyl or n _ heptyl, for example, cyclo pentyl, C 3 _ 8 cycloalkyl group such as cyclohexyl, for example, Fuweniru, alpha-C 6, such as naphthyl - 12 Ariru group, e.g., benzyl, phenyl one CI- 2 alkyl or c such phenethyl - such as naphthylmethyl ct- naphthyl
  • H7TBA62 has a carboxyl group (or carboxylate) at a position other than the C-terminus
  • those in which the carboxyl group is amidated or esterified are also included in H7TBA62.
  • the ester in this case, for example, the above-mentioned C-terminal ester and the like are used.
  • the H7TBA62 in proteins mentioned above, Amino group protecting groups Mechio two emissions residues of N-terminal (e.g., formyl group, etc. C ⁇ e Ashiru group such as C 2 _ 6 Al force Noiru group such Asechiru ), The N-terminal side is cleaved in vivo, the resulting daltamyl group is pyroglutamic acid, the substituent on the side chain of the amino acid in the molecule (for example, 1 OH, --SH, amino group, imidazole group, indole group, Guanijino group, etc.) a suitable protecting group (e.g., shall formyl group, and is protected by ⁇ such C i _ 6 Ashiru group such as C 2 _ 6 Arukanoiru groups such as cetyl) Or a complex protein such as a so-called glycoprotein to which a sugar chain is bound.
  • a suitable protecting group e.g., shall formyl group, and is
  • H7TBA62 As a specific example of H7TBA62, for example, H7TBA62 derived from a human consisting of the amino acid sequence represented by SEQ ID NO: 2 and the like are used. Human H7TB A62 is a known protein described in EP 885960-A2.
  • H7TBA62 partial peptide (hereinafter sometimes abbreviated as partial peptide) May be any peptide having the partial amino acid sequence of H7TBA62 described above.
  • partial peptide an exposed site which has substantially the same receptor binding activity as H7 TBA62 may be used.
  • the partial peptide of H7TBA62 having the amino acid sequence represented by SEQ ID NO: 2 was analyzed as an extracellular region (hydrophilic site) in a hydrophobicity plot analysis.
  • Peptide containing a moiety 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.
  • the c- substantially identical amino acid sequence which is preferably a peptide or the like refers to an amino acid sequence having about 85% or more, preferably about 90% or more, more preferably about 95% or more homology with these amino acid sequences. .
  • the partial peptide of the present invention has one or more (preferably about 1 to 10, more preferably several (1 to 5)) amino acids in the above amino acid sequence, Or one or more (preferably about 1 to 20, more preferably about 1 to 10, and still more preferably several (1 to 5)) amino acids are added to the amino acid sequence, or However, even if one or more or more (preferably about 1 to 10, more preferably several, and more preferably about 1 to 5) amino acids in the amino acid sequence are substituted with other amino acids, Good.
  • the partial peptide of the present invention may have a carboxyl group (one COOH), a carboxylate (one COO-), an amide (one CONH 2 ) or an ester (one COOR) at the C-terminus.
  • the carboxyl group is Those that have been midified or esterified are also included in the partial peptides of the present invention.
  • 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, as in the case of H7TBA62 described above, one in which the amino group of the N-terminal methionine residue is protected by a protecting group, and one in which the N-terminal is cleaved in vivo.
  • Pyroglutamine-oxidized glutamic acid residues those in which the substituent on the side chain of the amino acid in the molecule is protected with an appropriate protecting group, or those in which a sugar chain is bound, such as a so-called glycopeptide.
  • Peptides are also included.
  • Examples of the salt of H 7 TB A62 or a partial peptide thereof include a physiologically acceptable salt with an acid or a base, and a physiologically acceptable acid addition salt is particularly preferable.
  • Examples of such salts include salts with inorganic acids (eg, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid), and organic acids (eg, acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, Salts with conodic acid, tartaric acid, citric acid, malic acid, oxalic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid) 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, Salts with conodic acid, tartaric acid, citric acid, malic acid,
  • H7TBA62 or a salt thereof can be produced from the above-described human mammalian cells or tissues by a method for purifying a receptor protein known per se, or a DNA encoding H7TBA62 described later. Can also be produced by culturing a transformant containing Also, the protein can be produced by the protein synthesis method described later or according to it.
  • the human mammal tissues or cells are homogenized, extracted with an acid or the like, and the extract is subjected to reversed-phase mouth chromatography, ion exchange chromatography. Purification and isolation can be achieved by a combination of chromatography methods.
  • a commercially available resin for protein synthesis can be usually used.
  • a commercially available resin for protein synthesis can be usually used.
  • examples of such luster include, for example, chloromethyl resin, hydroxymethyl resin, benzhydrylamine resin, aminomethyl resin, 4-benzyloxybenzyl alcohol resin, 4-methylbenzhydrylamine resin, PAM resin, Droxymethylmethylphenylacetamide methyl resin, polyacrylamide resin, 4- ( 2 ', 4'-dimethoxyphenyl-hydroxymethyl) phenoxy resin, 4_ (2,, 4'-dimethoxyphenyl-Fmocaminoethyl) phenoxy resin, and the like.
  • an amino acid having an ⁇ -amino group and a side chain functional group appropriately protected is condensed on the resin according to the sequence of the target protein according to various condensation methods known per se.
  • 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 the target protein or its amide.
  • carbodiimides are particularly preferable.
  • carbodimids DCC, N ,, '-diisopropylcarbodiimid, ⁇ ethyl_ ⁇ '-(3-dimethylaminoprolyl) carbodiimide and the like are used.
  • Activation by these means involves the ability to add the protected amino acid directly to the resin together with the racemization inhibitor additives (eg, HOBt, HOOBt), or the activity of the protected amino acid as a symmetric anhydride or HOBt ester or HOOBt ester. It can be added after the chemical conversion.
  • the solvent used for the activation of the protected amino acid and the condensation 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, N-methylpyrrolidone, halogenated hydrocarbons such as dimethylene chloride and chloroform, trifluoromethane Alcohols such as ethanol, sulfoxides such as dimethyl sulfoxide, ethers such as pyridine, dioxane, and tetrahydrofuran; nitriles such as acetonitrile and propionitrile; esters such as methyl acetate and ethyl acetate; A mixture of the above is used.
  • the reaction temperature is appropriately selected from the range known to be usable for the protein bond formation reaction, and is usually selected from the range of about 120 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 pentyloxycarbonyl, isobornyloxycarbonyl, 4-methoxybenzyloxycarbonyl, C1-Z, Br-Z, Adamantyloxycarbonyl, trifluoroacetyl, phthaloyl, formyl, 2-nitrophenyl-nolesulfenyl, diphenylphosphinothioyl, Fmoc and the like are used.
  • the carboxyl group is, for example, alkyl esterified (for example, methyl, ethyl, propynole, butynole, tertiary butynole, cyclopentyl, cyclohexynole, cycloheptyl, cyclooctyl, 2-adamantyl, etc.
  • alkyl esterified for example, methyl, ethyl, propynole, butynole, tertiary butynole, cyclopentyl, cyclohexynole, cycloheptyl, cyclooctyl, 2-adamantyl, etc.
  • cyclic alkyl esterification aralkyl esterification (for example, benzyl estenole, 4-nitrobenzolene estenole, 4-methylethoxybenzinole estezole, 4-benzobenzyl ester, benzhydryl) Esterification), phenacyl estenolide, benzinoleoxy carboni ⁇ hydrazide, tert-butoxyca ⁇ / bonyl hydrazide, tritinorehydrazide, etc.
  • the hydroxyl group of serine can be protected, for example, by esterification or etherification.
  • a lower alkanol group such as an acetyl group, an arylo 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 tetrahydrobiranyl group, and a t-butyl group.
  • the protecting group of the phenolic hydroxyl group of tyrosine for example, B z 1, C 1 2 - B zl, 2- nitrobenzyl, B r _ Z, such as tertiary butyl Ru is used.
  • Examples of the protective group for histidine imidazole include Tos, 4-methoxy-1,2,3,6-trimethylbenzenesulfonyl, DNP, benzyloxymethyl, Bum, Boc, Trt, and Fmoc. .
  • activated carboxyl groups of the raw materials include, for example, corresponding acid anhydrides, azides, active esters [alcohols (eg, pentachlorophenol, 2,4,5-trichlorophenol, 2 , 4—dinitrophenol, cyanome Esters with Tinoleanoreco ⁇ /, para-trophenol, HONB, 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, methanesnolefonic acid, Acid treatment with trifluoromethanesulfonic acid, trifluoroacetic acid or a mixture thereof, base treatment with diisopropylethylamine, triethylamine, piperidine, piperazine, etc., and reduction with sodium in liquid ammonia Also used.
  • the elimination reaction by the above-mentioned acid treatment is generally carried out at a temperature of about 120 to 40 ° C.
  • a cation scavenger such as dimethi / resnolefide, 1,4-butanedithiol, and 1,2-ethanedithiol.
  • the 2,4-dinitrophenyl group used as an imidazole protecting group of histidine is removed by thiophenol treatment, and the formyl group used as an indole protecting group of tributofan is 1,2-ethanedithiol, 1,4 above.
  • alkali treatment with dilute sodium hydroxide solution or dilute ammonia.
  • the protection of the functional group which should not be involved in the reaction of the raw materials, the protection 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 having a desired chain length is added to the amino group side.
  • a protein in which only the protecting group for the amino group at the ⁇ -terminus of the peptide chain was removed and a protein in which only the protecting group for the carboxyl group at the C-terminus were removed were prepared. Condensation in a mixed solvent. Details of the condensation reaction are the same as described above. After purifying the protected protein obtained by the condensation, all the protecting groups are removed by the above method, and the desired crude protein is removed. You can get protein. This crude protein is purified using various known purification means, and the main fraction is freeze-dried to obtain an amide of the desired protein.
  • the ester of the desired protein can be obtained in the same manner as the amide of the protein. You can get the body.
  • the partial peptide of ⁇ 7 ⁇ 62 or a salt thereof can be produced according to a peptide synthesis method known per se, or by cleaving ⁇ 7 ⁇ 62 with an appropriate peptidase.
  • a peptide synthesis method for example, any of a solid phase synthesis method and a liquid phase synthesis method may be used. That is, the desired peptide can be produced by condensing a partial peptide or amino acid capable of constituting 7-62 with the remaining portion and, when the product has a protecting group, removing the protecting group. Examples of known condensation methods and elimination of protecting groups include the methods described in the following a) to e).
  • the partial peptide of the present invention can be purified and isolated by a combination of ordinary purification methods such as solvent extraction, distillation, column chromatography, liquid chromatography, and recrystallization.
  • the partial peptide obtained by the above method is a free form, it can be converted to an appropriate salt by a known method.
  • it is obtained by a salt it is converted to a free form by a known method. be able to.
  • the polynucleotide encoding H7TB A62 includes the above-mentioned H7TB
  • the polynucleotide is DNA encoding H7TBA62, RNA such as mRNA, and may be double-stranded or single-stranded. In the case of double-stranded, it may be double-stranded DNA, double-stranded RNA or DNA: RNA hybrid. In the case of a single strand, it may be a sense strand (ie, a coding strand) or an antisense strand (ie, a non-coding strand).
  • the mRN of H7TBA62 can be obtained by the method described in the well-known extramural empirical medicine “New PCR and its application” 15 (7), 1997 or a method analogous thereto. A can be quantified.
  • the DNA encoding H7TBA62 may be any of genomic DNA, a genomic DNA library, the above-described cDNA derived from cells and tissues, the above-described cDNA library derived from cells and tissues, and synthetic DNA.
  • the vector used for the library may be any of pacteriophage, plasmid, cosmid, and phagemid. Alternatively, it can also be directly amplified by Reverse Transcriptase Polymerase Chain Reaction (hereinafter abbreviated as RT-PCR method) using a preparation of total RNA or mRNA fraction from the above-mentioned cell's tissue.
  • the DNA encoding H7TBA62 includes, for example, a DNA containing the nucleotide sequence represented by SEQ ID NO: 1 or a DNA that hybridizes under high stringent conditions to the nucleotide sequence represented by SEQ ID NO: 1
  • Examples of the DNA capable of hybridizing with the base sequence represented by SEQ ID NO: 1 include, for example, about 85% or more, preferably about 90% or more, more preferably about 95% or more homology with the base sequence represented by SEQ ID NO: 1.
  • DNA containing a base sequence having a property is used.
  • Hybridization can be performed by a method known per se or a method analogous thereto, for example, Molecular Cloning 2nd Edition (J. Sarabrook et al., Cold Spring Harbor Lab. Press, 1989). When a commercially available library is used, it can be performed according to the method described in the attached instruction manual. 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 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 having the base sequence represented by SEQ ID NO: 1 is used.
  • a part of the nucleotide sequence of DNA encoding H7TBA62 or a polynucleotide containing a part of the nucleotide sequence complementary to the DNA is defined as the following partial peptide of the present invention. It is used to mean not only DNA but also RNA.
  • an antisense polynucleotide capable of inhibiting the replication or expression of the H7TBA62 gene has been cloned or determined.
  • the base of DNA encoding H7TBA62 has been determined.
  • Such a polynucleotide (nucleic acid) can hybridize with RNA of the H7TBA62 gene, and has the ability to inhibit the synthesis or function of the RNA or the H7TBA62 gene through interaction with H7TBA62-related RNA.
  • corresponding means having homology or being complementary to a specific sequence of nucleotides, base sequences or nucleic acids including genes.
  • the “correspondence” between a nucleotide, base sequence or nucleic acid and a peptide (protein) is a nucleotide (Nucleic acid) usually refers to the amino acids of a peptide (protein) in a directive derived from its sequence or its complement.
  • H7TBA62 gene 5 end hairpin loop, 5 'end 6—base pair' repeat, 5 'end untranslated region, polypeptide translation start codon, protein coding region, ORF translation start codon, 3' end untranslated region, 3
  • the terminal palindrome region and the three-terminal hairpin loop may be selected as preferred regions of interest, but any region within the H7TBA62 gene may be selected as the region of interest.
  • Antisense polynucleotides are 2-deoxy-D-ribose-containing polydeoxyribonucleotides, D-ribose-containing polyribonucleotides, N-daricosides of purine or pyrimidine bases, and others.
  • polymers having a non-nucleotide backbone eg, commercially available protein nucleic acids and synthetic sequence-specific nucleic acid polymers
  • polymers containing special bonds provided that the polymer is (Including nucleotides having a configuration that permits base pairing and base attachment as found in DNA and RNA).
  • They can be double-stranded DNA, single-stranded DNA, double-stranded RNA, single-stranded RNA, and even DNA: RNA hybrids, and can be unmodified polynucleotides (or unmodified oligonucleotides), or even known.
  • Modified e.g., labeled in the art, capped, methylated, substituted for one or more natural nucleotides with an analog, molecule Internally modified, such as those having an uncharged bond (eg, methylphosphonate, phosphotriester, phosphoramidate, olebamate, etc.), a charged or sulfur-containing bond (eg, phosphorothioate) Acetate, phospholipid dithioate, etc., for example, proteins (nucleases, nucleases, inhibitors, toxins) Has side-chain groups such as amino acids, antibodies, signal peptides, poly-L-lysine, etc.
  • 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 and a force, a lunar aliphatic group, or an ether, amine, etc. It may be converted to a functional group.
  • the antisense 'polynucleotide (nucleic acid) of the present invention is RNA, DNA, or a modified nucleic acid (RNA, DNA).
  • modified nucleic acids include, but are not limited to, sulfur derivatives and thiophosphate derivatives of nucleic acids, and those that are resistant to degradation of polynucleoside amides / 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 make the antisense nucleic acid more cell-permeable, to have a greater affinity for the target sense strand, and to be more toxic if it is toxic. Minimize the toxicity of sense nucleic acids.
  • the antisense nucleic acids of the present invention may contain altered or modified sugars, bases, or bonds, are provided in special forms such as ribosomes and microspheres, are applied by gene therapy, Can be given in a written form.
  • polycations such as polylysine, which acts to neutralize the charge of the phosphate skeleton, and cell membranes
  • Hydrophobic substances such as lipids (eg, phospholipids, cholesterol, etc.) that enhance the interaction or increase the uptake of nucleic acids are included.
  • Preferred lipids for addition include cholesterol and its derivatives (eg, cholesteryl chromate formate, cholic acid, etc.).
  • nucleic acid may be attached to the 3 'end of the nucleic acid at the 5' end, and may be attached via a base, sugar, or intramolecular nucleoside linkage.
  • Other groups include cap groups specifically arranged at the 3 'end or 5' end of nucleic acids for preventing degradation by nucleases such as exonuclease and RNase.
  • capping groups include, but are not limited to, hydroxyl protecting groups known in the art, including 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 G protein-coupled receptor protein. it can.
  • the nucleic acid can be applied to cells by various methods known per se.
  • 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.
  • it 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 batteriophage, plasmid, cosmid, phagemid and the like.
  • it can also be directly amplified by Reverse Transcriptase Polymerase Chain Reaction (hereinafter abbreviated as RT-PCR method) using an mRNA fraction prepared from the cells and tissues described above.
  • RT-PCR method Reverse Transcriptase Polymerase Chain Reaction
  • the DNA encoding the partial peptide of the present invention includes, for example, (1) DNA having a partial base sequence of DNA having the base sequence represented by SEQ ID NO: 1, or (2) SEQ ID NO: It has a nucleotide sequence that hybridizes under stringent conditions with the nucleotide sequence represented by 1 and has substantially the same activity as H7TBA62 consisting of the amino acid sequence represented by SEQ ID NO: 2 (eg, Riga DNA having a partial base sequence of DNA encoding a receptor protein having a signal binding activity, a signal transduction action, etc.) is used.
  • SEQ ID NO: 1 eg, Riga DNA having a partial base sequence of DNA encoding a receptor protein having a signal binding activity, a signal transduction action, etc.
  • Examples of the DNA capable of hybridizing with the nucleotide sequence represented by SEQ ID NO: 1 include, for example, about 85% or more, preferably about 90% or more, and more preferably about 95% or more homology with the nucleotide sequence represented by SEQ ID NO: 1.
  • DNA containing a base sequence having a property is used.
  • H7TBA62 Cloning of DNA that completely encodes H7TBA62 or its partial peptide (hereinafter sometimes abbreviated as H7TBA62 in some cases) is performed by PCR using a synthetic DNA primer having a partial base sequence of H7TBA62.
  • the DNA can be selected by amplification or hybridization with a DNA fragment coding for a part or the entire region of H7TBA62 or labeled using synthetic DNA.
  • the hybridization method can be carried out according to, for example, the method described in Molecular Cloning 2nd Edtion (J. Sambrook et al., Old Spring Harbor Lab. Press, 1989).
  • it can be performed according to the method described in the attached instruction manual.
  • Conversion of the nucleotide sequence of DNA is performed using PCR or a known kit, such as Mutan TM -super Express Km (Takara Shuzo Co., Ltd.) or Mutan TM -K (Takara Shuzo Co., Ltd.).
  • the method can be carried out according to a method known per se, such as the LAP CR method, the Gapped dup 1 ex method, the Kun ke 1 method, or a method analogous thereto.
  • the cloned DNA encoding H7TBA62 can be used as it is or, if desired, after digestion with a restriction enzyme or adding a linker.
  • the DNA may have ATG as a translation initiation codon at its 5 'end and TAA, TGA or TAG as a translation stop codon at its 3' end. These translation initiation codon and translation termination codon can be added using an appropriate synthetic DNA adapter.
  • the expression vector of H7TBA62 is, for example, A target DNA fragment is cut out from the DNA to be prepared, and (mouth) the DNA fragment can be produced by ligating the DNA fragment downstream of a promoter in an appropriate expression vector.
  • Plasmids derived from Escherichia coli eg, pBR322, pBR325, pUC12, pUC13
  • Bacillus subtilis-derived plasmids eg, pUB110, pTP5, pC194
  • yeast In addition to origin plasmids (eg, pSH19, pSH15), bacteriophages such as phage, retroviruses, vaccinia virus, animals such as baculoinores, etc., pAl—11, pXT1, p Rc / CMV, pRc / RSV, pcDNAIZNeo and the like are used.
  • the promoter used in the present invention may be any promoter as long as it is appropriate for the host used for gene expression.
  • SRa promoter when an animal cell is used as a host, SRa promoter, SV40 promoter, LTR promoter, CMV promoter, HSV-TK promoter and the like can be mentioned.
  • the CMV promoter SR ⁇ promoter and the like.
  • the host is Eshierihia genus bacterium, trp promoter, 1 ac promoter, re cA promoter, LP L promoter, lpp promoter one coater is, when the host is Bacillus, SPO 1 promoter, SP 02 promoter, When the host is yeast, such as the pen P promoter, the PH 05 promoter, PGK promoter, GAP promoter, ADH promoter and the like are preferred.
  • a polyhedrin promoter, a P10 promoter and the like are preferable.
  • the expression vector may further include, if desired, a enhancer, a splicing signal, a poly (A) addition signal, a selection marker, an SV40 replication origin (hereinafter sometimes abbreviated as SV40 ori), and the like.
  • a selection marker for example, dihydrofolate reductase (hereinafter sometimes abbreviated as dhfr) gene [Mesotorekise Ichito (MTX) resistance], phosphorus resistant gene (hereinafter sometimes abbreviated as Amp r), Neomycium Down resistance gene (hereinafter sometimes abbreviated as N eo r, G4 1 8 resistance).
  • dhfr dihydrofolate reductase
  • MTX phosphorus resistant gene
  • Amp r phosphorus resistant gene
  • N eo r Neomycium Down resistance gene
  • a signal sequence suitable for the host is added to the N-terminal side of the receptor protein of the present invention. If the host is a bacterium belonging to the genus Escherichia, a Ph0A. Signal sequence, an OmpA. Signal sequence, etc., if the host is a bacterium belonging to the genus Bacillus, an ⁇ -amylase signal sequence, a subtilisin signal sequence, etc. If the host is yeast, MFa 'signal sequence, SUC2 signal sequence, etc. If the host is an animal cell, insulin signal sequence, ⁇ -interferon signal sequence, antibody molecule, signal sequence Etc. can be used respectively.
  • a transformant can be produced using the thus constructed vector containing DNA encoding 7TB A62.
  • Examples of the host include Escherichia, Bacillus, yeast, insect cells, insects, animal cells, and the like.
  • bacterium belonging to the genus Escherichia include, for example, Escherichia coli K12, DH1 [Procedures, Op., The National, Academy of Sciences, Obs. p roc. Natl. Acad. Sci . USA), 60 Certificates, 1 60 (1 96 8)], JM1 0 3 [Nukeriekku 'Ashizzu • research (Nucleic Acids research), 9 Certificates, 30 9 (1 98 1)] , JA 221 [Journal of Molecular Biology, 120, 5 17 (1978)], ⁇ 101 [Journal of Molecular Biology] , 41, 459 (1969)], C600 [Genetics, 39, 440 (1954)], etc. are used.
  • Bacillus bacteria examples include, for example, Bacillus subtilis MI114 (Gene, 24, 255 (1983)), 207—21 (Journal of Biochemistry ( Journal of Biochemistry), 95, 87 (1 984)].
  • yeast examples include, for example, Saccharomyces cerevisiae AH 22, AH 22 R ⁇ , NA 87—11 A, DKD—5D, 20 B—12, Schizosaccharomyces porabe NC YC 191 3, NCYC 2036, Pichia pastoris, etc. are used.
  • Insect cells include, for example, when the virus is Ac NPV, a cell line derived from the larva of night moth (Spodoptera frugiperda cell; S f cell), MG 1 cell derived from the midgut of Trichoplusia, and High derived from the egg of Trichoplusia ni Five TM 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)) and the like. Is used.
  • insects for example, silkworm larvae are used [Maeda et al., Nature, Vol. 315, 592 (1985)].
  • animal cells examples include monkey cell COS-7, Vero, Chinese hamster cell CHO (hereinafter abbreviated as CHO cell), dhfr gene-deficient Chinese hamster cell CHO (hereinafter abbreviated as CHO (dhfr-) cell).
  • CHO cell Chinese hamster cell CHO
  • dhfr- dhfr gene-deficient Chinese hamster cell CHO
  • Mouse L-cells mouse AtT-20, mouse myeloma cells, rat GH3, human FL cells, human HEK 293 cells, and the like.
  • Transformation of Bacillus spp. Can be performed, for example, according to the method described in Molecular & General Genetics, Vol. 168, 111 (1979).
  • Insect cells or insects can be transformed according to the method described in, for example, Bio / Technology, 6, 47-55 (1988).
  • Transformation of animal cells is described, for example, in Cell Engineering Separate Volume 8, New Cell Engineering Experimental Protocol. 263-267 (1 995) (published by Shujunsha), Virology, Vol. 52, 456 (1973). Can be performed according to the method described in In this way, a transformant transformed with the expression vector containing DNA encoding H7TBA62 is obtained.
  • a liquid medium is suitable as a medium used for culturing, and a carbon source necessary for the growth of the transformant is contained therein.
  • carbon sources include glucose, dextrin, soluble starch, sucrose, etc.
  • nitrogen sources include ammonia salt, nitrate, corn chip liquor, peptone, casein, meat extract, soybean meal, potato extract
  • the inorganic or organic substance such as a liquid and the inorganic substance include calcium chloride, sodium dihydrogen phosphate, and magnesium chloride.
  • yeast extract, vitamins, growth promoting factors and the like may be added.
  • the pH of the medium is preferably about 5-8.
  • Examples of a medium for culturing Escherichia bacteria include, for example, M9 medium containing glucose and casamino acids [Miller, Journal “Ob” “Etasperimen” In ”Molecular Neatix ( Journal of Experiments in Molecular Genetics), 431-433, Cold Spring Harbor Laboratory, New York 1972]. If necessary, an agent such as 3i3-indolylacrylic acid can be added to make the promoter work efficiently if necessary.
  • culture is usually performed at about 15 to 43 ° C for about 3 to 24 hours. The process can be continued and, if necessary, aeration or stirring can be added.
  • the cultivation is usually performed at about 30 to 40 ° C for about 6 to 24 hours, and if necessary, aeration and stirring can be applied.
  • the medium When culturing a transformant in which the host is yeast, for example, the medium may be Burkholder's minimal medium [Bostian, K. Shira, Processing's of the National Academy of Cultures. Saienshiizu, O blanking tHE yOU SA (p roc. Natl. Acad. Sci. USA), 77 Certificates, 4505 (1980)] or 0. SD medium containing 5% casamino acid [Bitter, GA et al., professional sheathing Nat'l Acad. Sci. USA, 81, 5330 (1984)].
  • the pH of the medium is preferably adjusted to about 5-8.
  • the cultivation is usually performed at about 20 to 35 ° C for about 24 to 72 hours, and aeration and agitation are added as necessary.
  • the medium used is 10% serum inactivated in Grace's Insect Medium (Grace, TCC, Nature, 195, 788 (1962)).
  • a material to which an additive such as is appropriately added is 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.
  • the medium When culturing a transformant in which the host is an animal cell, the medium may be, for example, a MEM medium containing about 5 to 20% fetal bovine serum [Science, 122 vol., 501 (1952)], a DMEM medium [Virology, 8 volumes, 396 (1959)], RPMI 1640 medium [Journal of the American Medical Association, The Journal of the American Medical Association] 199, 519 (1967) 199 medium [Proceding of the Society for the Biological Medicine] (Proceeding of the Society for the Biological Medicine), 73, 1 (1950)].
  • the pH is about 6-8. Cultivation is usually performed at about 30 to 40 ° C for about 15 to 60 hours, and aeration and stirring are added as necessary.
  • H7TBA62 can be produced in the cells, in the cell membrane, or outside the cells of the transformant.
  • H 7 TB A62 can be separated and purified from the above culture by, for example, the following method.
  • H7TBA62 When extracting H7TBA62 from cultured cells or cells, after culturing, cells or cells are collected by a known method, suspended in an appropriate buffer, and sonicated, lysozyme and / or freeze-thaw, etc. Alternatively, a method of obtaining a crude extract of H7TBA62 by centrifugation or filtration after disrupting cells is used as appropriate.
  • the buffer may contain a protein denaturant such as urea or guanidine hydrochloride, or a surfactant such as Triton X-100 TM.
  • the H7TBA62 contained in the thus obtained culture supernatant or extract can be purified by appropriately combining known separation and purification methods.
  • These known separation and purification methods mainly include methods utilizing solubility such as salting out and solvent precipitation, dialysis, ultrafiltration, gel filtration, and SDS-polyacrylamide gel electrophoresis, and the like.
  • Method using difference in charge such as ion exchange chromatography, method using specific novelty such as affinity mouth chromatography, hydrophobicity such as reversed-phase high-performance liquid chromatography, etc.
  • a method utilizing the difference between isoelectric points, such as an isoelectric focusing method, and the like, are used.
  • H 7 TBA62 thus obtained is obtained in a free form, it can be converted to a salt by a method known per se or a method analogous thereto, and conversely, when it is obtained as a salt, a method known per se Alternatively, it can be converted to a free form or another salt by a method analogous thereto.
  • H7TBA62 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.
  • an appropriate protein modifying enzyme for example, trypsin, chymotrypsin, anoregininole endopeptidase, protein kinase, glycosidase and the like are used.
  • the activity of the resulting H7TBA62 is determined by binding experiments with labeled ligands. And Enzymymnoassay using a specific antibody.
  • the antibody to H7TBA62 may be any of a polyclonal antibody and a monoclonal antibody as long as it can recognize H7TBA62.
  • An antibody against H7TBA62 can be produced by using H7TBA62 as an antigen according to a method for producing an antibody or antiserum known per se.
  • H7TBA62 is administered to a mammal at a site capable of producing an antibody by administration itself or together with a carrier or diluent.
  • Complete Freund's adjuvant or incomplete Freund's adjuvant may be administered to enhance antibody production during administration.
  • the administration is usually performed once every 2 to 6 weeks, for a total of about 2 to 10 times.
  • Examples of the mammal to be used include monkeys, egrets, dogs, guinea pigs, mice, rats, sheep, goats, and mice and rats are preferably used.
  • a warm-blooded animal immunized with the antigen for example, an individual with an antibody titer is selected from a mouse, and the spleen or lymph node is collected 2 to 5 days after the final immunization.
  • an individual with an antibody titer is selected from a mouse, and the spleen or lymph node is collected 2 to 5 days after the final immunization.
  • a monoclonal antibody-producing hybridoma can be prepared.
  • the antibody titer in the antiserum can be measured, for example, by reacting the labeled receptor protein described below with the antiserum, and then measuring the activity of the labeling agent bound to the antibody.
  • the fusion operation can be carried out according to a known method, for example, the method of Köhler and Milstein [Nature, Vol. 256, p. 495 (1975)].
  • the fusion promoter include polyethylene glycol (PEG) and Sendai virus, but 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 PE G (preferably PEG 1000 to PEG 6000) Force Sl Added at a concentration of about 0 to 80%, and incubated at about 20 to 40 ° C, preferably about 30 to 37 ° C for about 1 to 10 minutes for efficiency. Cell fusion can be performed well.
  • a hybridoma culture supernatant is added to a solid phase (eg, a microplate) on which the antigen of the receptor protein is adsorbed directly or together with a carrier.
  • a solid phase eg, a microplate
  • an anti-immunoglobulin antibody labeled with a radioactive substance or an enzyme is used if the cells used for cell fusion are mice or protein A, and the monoclonal antibody bound to the solid phase is added.
  • Detection method A hybridoma culture supernatant is added to a solid phase to which anti-immunoglobulin antibody or protein A is adsorbed, and a receptor protein labeled with a radioactive substance or an enzyme is added. Detection method and the like can be mentioned.
  • the selection of the monoclonal antibody can be carried out according to a method known per se 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.
  • RPMI1640 medium containing 1-20%, preferably 10-20% fetal bovine serum
  • GIT medium containing 1-10% fetal bovine serum (Wako Pure Chemical Industries, Ltd.) or for hybridoma culture
  • a serum-free medium SFM-101, Nissui Pharmaceutical Co., Ltd.
  • the cultivation temperature is usually 20 to 40 ° (:, preferably, about 37 ° C.
  • the cultivation time is usually 5 days to 3 weeks, preferably, 1 week to 2 weeks.
  • 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, such as immunoglobulin separation and purification [eg, salting out, alcohol precipitation, isoelectric precipitation, electrophoresis, ion exchangers (eg, DEAE) adsorption / desorption method, ultracentrifugation method, gel filtration method, antigen-bound solid phase or protein A or protein G, etc.
  • immunoglobulin separation and purification eg, salting out, alcohol precipitation, isoelectric precipitation, electrophoresis, ion exchangers (eg, DEAE) adsorption / desorption method, ultracentrifugation method, gel filtration method, antigen-bound solid phase or protein A or protein G, etc.
  • Specific purification method in which an antibody alone is collected with the active adsorbent and the bond is dissociated to obtain the antibody].
  • the polyclonal antibody of the present invention can be produced by a method known per se or a method analogous thereto. For example, a complex of an immunizing antigen (H7TBA62 antigen) 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. It can be produced by collecting body contents and separating and purifying the antibody.
  • an immunizing antigen H7TBA62 antigen
  • carrier protein a mammal is immunized in the same manner as in the above-described method for producing a monoclonal antibody. It can be produced by collecting body contents 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 fact that the antibody is efficiently used for the hapten immunized by cross-linking the carrier. If possible, any material may be cross-linked at any ratio.For example, ⁇ serum anolebumin, ⁇ cysiglobulin, keyhorne limpet 'hemocyanin, etc., in a weight ratio of about 0 to hapten 1 per hapten : A method of pulling at a rate of! -20, preferably about 1-5 is used. In addition, various condensing agents can be used for force coupling between the hapten and the carrier.
  • an active ester reagent containing a daltalaldehyde carbodiimide, a maleimide active ester, a thiol group, or a dithioviridyl group is used.
  • the condensation product is administered to a warm-blooded animal at a site where antibody production is possible or together with a carrier or diluent.
  • Complete Freund's adjuvant ⁇ Incomplete Freund's adjuvant may be administered in order to enhance antibody production 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.
  • the polyclonal antibody can be collected from blood, ascites, or the like, preferably from blood, of the mammal immunized by the above method.
  • the measurement of the polyclonal antibody titer in the antiserum can be performed in the same manner as the measurement of the antibody titer in the serum described above. Separation and purification of the polyclonal antibody can be performed according to the same method for separation and purification of immunoglobulin as in the above-mentioned separation and purification of the monoclonal antibody.
  • One of the ligands of H7TBA62 is known as partial ⁇ -adrenergic receptor agonist ⁇ ⁇ mouth ⁇ ⁇ 5- ⁇ ⁇ ⁇ 1 ⁇ / 5- ⁇ 1 ⁇ receptor agonist, This is the oxymetazoline used.
  • DNA encoding ⁇ 7 ⁇ 62 (hereinafter sometimes abbreviated as D D of the present invention), an antibody to ⁇ 7 ⁇ 62 (hereinafter sometimes abbreviated as the antibody of the present invention), an antisense DNA to the DNA of the present invention (
  • the antisense DNA of the present invention has the following uses.
  • H7TBA62 is thought to be involved in vasoconstriction, serotonergic action, acetylcholine release, gastrointestinal motility, smooth muscle contraction, platelet aggregation, central nervous control or cardiotonic action.
  • DNA encoding a ) H7TBA62 or b) H7TBA62 can be converted into a vasoconstrictor, a serotonin agonist, an acetylcholine release promoter, a gastrointestinal motility enhancer, a smooth muscle contractor, a platelet aggregating agent, a central nerve. It can be used as a stimulant or a cardiotonic, and can be used as a medicament such as a preventive and / or therapeutic agent for diseases associated with these dysfunctions of H7TBA62.
  • H7TBA62 deficiency when there is a patient in whom the physiological action of the ligand cannot be expected due to a decrease in H7TBA62 in the living body (H7TBA62 deficiency), a) H7TBA62 is administered to the patient to replace the amount of the receptor B) (ii) administering the DNA encoding H7TBA62 to the patient and expressing it, or (mouth) inserting and expressing the DNA encoding H7TBA62 in the target cells, By transplanting the cells into the patient, the amount of the receptor in the patient's body can be increased, and the effect of the ligand can be sufficiently exerted.
  • H7TBA62-encoding DNA is a safe and low-toxic receptor dysfunction, particularly vasoconstriction, serotonin action, acetylcholine release, gastrointestinal motility, smooth muscle contraction, platelet aggregation, central nervous control or cardiotonic effect, etc. Failure It is useful as a prophylactic and / or therapeutic agent for diseases related to
  • H7TBA62 or the DNA of the present invention can be used, for example, as an agent for removing neuroactive nasal depressive blood or mucosal hyperemia, or as an agent for preventing or treating diseases such as acute circulatory insufficiency or hypotension. it can.
  • H7TBA62 or the DNA of the present invention may be used, for example, for cancer (eg, prostate cancer, colorectal cancer, etc.), infectious disease, pain, anorexia nervosa, bulimia, asthma, Parkinson's disease, acute heart failure, hypotension, hypertension, urinary retention , Osteoporosis, angina, myocardial infarction, ulcer, allergy, benign prostatic hypertrophy, mental and neurological disorders (anxiety, schizophrenia, depression, delirium, dementia, severe mental retardation and motor abnormalities, Huntington's chorea, It can be used as a prophylactic and therapeutic agent for diseases such as Jill de la llelet syndrome), ophthalmic drug, and topical vasoconstrictor for nasal drops.
  • cancer eg, prostate cancer, colorectal cancer, etc.
  • infectious disease e.g., pain, anorexia nervosa, bulimia, asthma, Parkinson's disease, acute heart failure, hypotension, hypertension, urinary
  • H7TBA62 When H7TBA62 is used as the above drug, it can be formulated according to conventional means.
  • the DNA of the present invention when used as the above medicine, the DNA of the present invention is used alone or after being inserted into an appropriate vector such as a retrovirus vector, an adenovirus vector, an adenovirus associated virus vector, and the like. Can be implemented in accordance with The DNA of the present invention can be administered as it is or together with an auxiliary agent for promoting uptake by a gene gun or a catheter such as a hydrogel catheter.
  • the DNA of the present invention may be orally administered as tablets, forcepsels, elixirs, microforces, etc., which are sugar-coated as required, or water or It can be used parenterally in the form of a sterile solution with other pharmaceutically acceptable solutions, or in the form of injections such as suspensions.
  • additives that can be mixed with tablets, capsules, etc.
  • Binders such as latin, corn starch, tragacanth, gum arabic, excipients such as crystalline cellulose, swelling agents such as corn starch, gelatin, alginic acid, lubricants such as magnesium stearate, sucrose, lactose or Sweetening agents such as saccharin, flavoring agents such as peppermint, cocoa oil or cherry are used.
  • the unit dosage form is a capsule
  • the above-mentioned material of the type may further contain a liquid carrier such as oil and fat.
  • Sterile compositions for injection can be formulated according to standard pharmaceutical practice, such as dissolving or suspending the active substance in a vehicle such as water for injection, or naturally occurring vegetable oils such as sesame oil or coconut oil.
  • a vehicle such as water for injection, or naturally occurring vegetable oils such as sesame oil or coconut oil.
  • a aqueous solution for injection for example, physiological saline, isotonic solution containing glucose and other auxiliary agents (for example, D-sorbitol, D-mannitol, sodium chloride, etc.) and the like are used.
  • an alcohol e.g., ethanol
  • polyalcohol e.g., propylene grayed recall, polyethylene glycol
  • nonionic surfactant eg, poly Sol Pies 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 benzy
  • the above-mentioned medicines include, for example, buffering agents (for example, phosphate buffer, sodium acetate buffer), soothing agents (for example, benzalkonium chloride, proactive hydrochloride, etc.), stabilizers (for example, , Human serum albumin, polyethylene glycol, etc.), preservatives (eg, benzyl alcohol, phenol, etc.), antioxidants and the like.
  • buffering agents for example, phosphate buffer, sodium acetate buffer
  • soothing agents for example, benzalkonium chloride, proactive 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, so they can be used, for example, in human mammals (eg rats,
  • the dose of ⁇ 7 ⁇ ⁇ ⁇ 62 varies depending on the subject of administration, target organ, symptoms, administration method, etc.
  • patients with acute circulatory insufficiency body weight 60 kg
  • the single dose depends on the subject, target organ, symptoms, administration method, etc.
  • the dose can be administered in terms of weight per 6 O kg.
  • the dosage of the DNA of the present invention varies depending on the administration subject, target organ, symptoms, administration method, and the like, in the case of oral administration, in general, for example, in an acute circulatory failure patient (with a body weight of 60 kg), About 0.1 to per day: I 0 Omg, preferably about 1.0-5 Omg, more preferably about 1.0-2 Omg.
  • the single dose varies depending on the administration target, target organ, symptoms, administration method, etc.
  • it is usually used, for example, in patients with acute circulatory failure (body weight 6).
  • the DNA and antisense DNA of the present invention can be used as a probe to produce H7TBA62 or H7TBA62 in humans or mammals (eg, rat, mouse, rabbit, sheep, sheep, pigeon, pig, cat, dog, monkey, etc.). Since abnormalities (gene abnormalities) in the DNA or mRNA encoding the partial peptide can be detected, for example, damage, mutation or reduced expression of the DNA or mRNA, increase in the DNA or mRNA, or It is useful as a gene diagnostic agent for overexpression.
  • the above-mentioned genetic diagnosis using the DNA or the antisense DNA of the present invention can be carried out, for example, by the known Northern hybridization or PCR-SSCP method (Genomics, Vol. 5, 874- 879 (1 989), Proceedings of the National Academy of Sciences or the United States of America, 86 Vol., Pp. 2766-2770 (1 989 )).
  • H7TBA62 For example, if a decrease in H7TBA62 expression is detected by Northern hybridization, dysfunction of H7TBA62, in particular, vasoconstriction, serotonin action, acetylcholine release, gastrointestinal motility, smooth muscle contraction, platelet aggregation, central location It can be diagnosed that the patient is likely to be suffering from a disease associated with insufficiency such as neuromodulation or inotropic activity or is likely to suffer in the future.
  • insufficiency such as neuromodulation or inotropic activity
  • H7TBA62 when overexpression of H7TBA62 is detected by Northern hybridization, for example, diseases caused by overexpression of H7TBA62, particularly vasoconstriction, serotonin action, acetylcholine release, gastrointestinal motility, smooth muscle contraction, platelets It can be diagnosed that the patient is likely to be suffering from a disease associated with abnormal hyperactivity such as aggregation, central nervous regulation or inotropic effect, or is likely to suffer in the future.
  • H7TBA62 dysfunction diseases associated with H7TBA62 dysfunction include, for example, neuroactive nasal depression or mucosal hyperemia, or acute circulatory failure or hypotension.
  • H7TBA62 Diseases caused by overexpression of H7TBA62 include, for example, peripheral circulatory disorders, hypertension, depression, obsessive-compulsive disorder, obsessive-compulsive disorder, panic disorder, anxiety disorder, eating disorder, ischemic disease, anticancer drug administration Gastrointestinal symptoms associated with, acute myocardial infarction, acute knee inflammation, chronic inflammation, adult respiratory distress syndrome, alcohol withdrawal syndrome, Alzheimer's disease, stiff spondylitis, arrhythmia, cocaine withdrawal syndrome, dementia, diarrhea, gastritis, hepatitis, Infectious disease, Obiate withdrawal syndrome, osteoarthritis, osteoporosis, osteoporotic disease, peptic ulcer, peripheral vasculopathy, sequelae of myocardial infarction, reflux esophagitis, rheumatoid arthritis, schizophrenia, sepsis, septic shock, Examples include ulcerative colitis or unstable angina.
  • the DNA and antisense DNA of the present invention include, for example, cancer (eg, prostate cancer, colorectal cancer, etc.), infectious disease, pain, anorexia nervosa, bulimia, asthma, Parkinson's disease, acute heart failure, hypotension, Hypertension, urinary retention, osteoporosis, angina, myocardial infarction, ulcer, allergy, benign prostatic hypertrophy, mental and neurological disorders (anxiety, schizophrenia, depression, delirium, dementia, severe mental retardation and motor abnormalities, Huntington's chorea, It can also be used as a diagnostic agent for diseases such as Jill de la Rellet syndrome.
  • cancer eg, prostate cancer, colorectal cancer, etc.
  • infectious disease e.g., pain, anorexia nervosa, bulimia, asthma, Parkinson's disease, acute heart failure, hypotension, Hypertension, urinary retention, osteoporosis, angina, myocardial infarction, ulcer, allergy, benign prostatic hypertrophy,
  • the DNA of the present invention can be used for screening a compound that changes the expression level of H7TBA62.
  • the present invention relates to, for example, H7TBA62 contained in (i) a) blood of a non-human mammal, b) a specific organ, c) a tissue or cell isolated from an organ, or (ii) a transformant.
  • the present invention provides a method for screening a compound that changes the expression level of H7TBA62 by measuring the amount of mRNA.
  • the measurement of the mRNA amount of H7TBA62 is specifically performed as follows.
  • non-human mammals for example, mice, rats, egrets, sheep, pigs, 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, flooded stress, electric shock, After a certain period of time, blood or specific organs (eg, brain, liver, kidney, etc.), or tissues or cells isolated from the organs are obtained.
  • the mRNA of H7TBA62 contained in the obtained cells can be quantified by, for example, extracting mRNA from cells or the like by an ordinary method and using, for example, a technique such as TaqMan PCR. Analysis can also be performed by performing Northern blotting by known means.
  • a transformant expressing H7TBA62 is prepared according to the method described above, and the mRNA of H7TBA62 contained in the transformant can be quantified and analyzed in the same manner.
  • test compound When culturing the transformant according to a conventional method, the test compound is mixed in the medium, and after culturing 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 later), it can be performed by quantifying and analyzing the amount of H7TBA62 mRNA contained in the transformant.
  • the compound obtained by using the screening method of the present invention or a salt thereof is a compound having an action of changing the expression level of H7TBA62, and specifically, (a) the expression level of H7TBA62 A compound that enhances H7TBA62-mediated cell stimulating activity by increasing the amount of H7TBA62, and (a) a compound that attenuates the cell-stimulating activity by decreasing the expression level of H7TBA62.
  • Cell stimulating activities include, for example, arachidonic acid release, acetylcholine release, intracellular Ca 2+ release, intracellular cAMP production, intracellular cGMP production, inositol phosphate production, cell membrane potential fluctuation, intracellular protein phosphorylation And the activity of promoting or suppressing the activation of C-fOS, the decrease of pH, and the like. Among them, the activity of suppressing the production of intracellular cAMP is preferred.
  • Examples of the compound include a peptide, a protein, a non-peptidic compound, a synthetic compound, and a fermentation product. These compounds may be novel compounds or known compounds.
  • H7TBA62 Compounds that alter the expression level of H7TBA62 obtained by the above screening method include dysfunction of H7TBA62, particularly vasoconstriction, serotonin action, acetylcholine release, gastrointestinal motility, and smooth muscle contraction. It can be used as a prophylactic and / or therapeutic agent for diseases related to abnormalities such as platelet aggregation, central nervous regulation or cardiotonic effect, ophthalmic drug, and local vasoconstrictor for nasal drops.
  • diseases related to abnormalities such as platelet aggregation, central nervous regulation or cardiotonic effect, ophthalmic drug, and local vasoconstrictor for nasal drops.
  • compounds that increase the expression level of H7TBA62 and enhance cell stimulating activity are useful as safe and low-toxic preventive and therapeutic agents for diseases related to H7TBA62 dysfunction. It is. Compounds that decrease the expression level of H7TBA62 and attenuate the cell stimulating activity are useful as safe and low-toxic preventive / therapeutic agents for diseases caused by excessive expression of H7TBA62.
  • H7TBA62 dysfunction diseases associated with H7TBA62 dysfunction include, for example, neuroactive nasal depression or mucosal hyperemia, or acute circulatory failure or hypotension.
  • H7TBA62 Diseases caused by overexpression of H7TBA62 include, for example, peripheral circulatory disorders, hypertension, depression, obsessive-compulsive disorder, obsessive-compulsive disorder, panic disorder, anxiety disorder, eating disorder, ischemic disease, Gastrointestinal symptoms associated with malignant drug administration, acute myocardial infarction, acute tengitis, chronic knee inflammation, adult respiratory distress syndrome, alcohol withdrawal syndrome, Alzheimer's disease, ankylosing spondylitis, arrhythmia, cocaine withdrawal syndrome, dementia, diarrhea, Gastritis, hepatitis, infectious disease, obiate withdrawal syndrome, osteoarthritis, osteoporosis, bone pechet disease, peptic ulcer, peripheral vasculopathy, sequelae of myocardial infarction, reflux esophagitis, rheumatoid arthritis, schizophrenia, sepsis, sepsis These include shock, ulcerative colitis or unstable angina.
  • compounds that alter the expression level of H7TBA62 obtained by the above screening method include, for example, cancer (eg, prostate cancer, colorectal cancer, etc.), infectious disease, pain, anorexia nervosa, bulimia, asthma, Parkinson Disease, acute heart failure, hypotension, hypertension, urinary retention, osteoporosis, angina, myocardial infarction, ulcer, allergy, benign prostatic hypertrophy, mental and neurological disorders (anxiety, schizophrenia, depression, delirium, dementia, severe It can also be used as a prophylactic / therapeutic agent for diseases such as mental retardation and dyskinesia of symptoms, Huntington's chorea, and Jill 'de la Allette syndrome.
  • cancer eg, prostate cancer, colorectal cancer, etc.
  • infectious disease e.g., pain, anorexia nervosa, bulimia, asthma, Parkinson Disease, acute heart failure, hypotension, hypertension, urinary retention, osteoporosis, angina, myocardial in
  • 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 formulated according to a conventional method.
  • the compound can be used as a tablet, capsule, elixir, microcapsule, or the like, if necessary, orally sterilized with water or other pharmaceutically acceptable liquid. It can be used parenterally in the form of injections, such as solutions or suspensions.
  • the compound is generally used together with known physiologically acceptable carriers, flavoring agents, excipients, vehicles, preservatives, stabilizers, binders, and the like. It can be manufactured by admixing it in the unit dosage form required for the approved formulation. The amount of the active ingredient in these preparations is such that an appropriate dose in the specified range can be obtained.
  • excipients that can be incorporated into tablets, forceps, etc.
  • binders such as gelatin, corn starch, tragacanth, gum arabic, excipients such as crystalline cenorellose, corn starch, gelatin, alginic acid, etc.
  • a bulking agent such as sucrose, a lubricant such as magnesium stearate, a sweetening agent such as sucrose, lactose or saccharin, and a flavoring agent such as peppermint, cocoa oil or cellulose are used.
  • the unit dosage form is a capsule, the above-mentioned material of the type may further contain a liquid carrier such as oil and fat.
  • Sterile compositions for injection can be formulated according to standard pharmaceutical practice, such as dissolving or suspending the active substance in a vehicle such as water for injection, or naturally occurring vegetable oils 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.).
  • alcohols e.g., ethanol
  • polyalcohol e.g., propylene grayed recall, polyethylene glycol
  • nonionic surfactant eg, Porisoru base one preparative 8 0 TM, HCO- 5 0
  • the oily liquid for example, sesame oil, soybean oil and the like are used, and they may be used in combination with solubilizers such as benzyl benzoate and benzyl alcohol.
  • the above-mentioned medicines include, for example, buffering agents (for example, phosphate buffer, sodium acetate buffer), soothing agents (for example, benzalkonium chloride, pro-proin hydrochloride, etc.), stabilizers (for example, Serum albumin, polyethylene glycol, etc.), preservatives (eg, benzyl alcohol, phenol, etc.), antioxidants, etc.
  • buffering agents for example, phosphate buffer, sodium acetate buffer
  • soothing agents for example, benzalkonium chloride, pro-proin hydrochloride, etc.
  • stabilizers for example, 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, so they can be used, for example, in human mammals (e
  • the dose of the compound or its salt depends on the administration target, target organ, symptoms, administration method, etc.
  • oral administration for example, in patients with acute circulatory insufficiency (with a body weight of 60 kg), about 0:! To 100 mg per day, preferably about 1.0 5050 mg, more preferably about 1.0-20 mg.
  • the single dose varies depending on the administration target, target organ, symptoms, administration method, etc.
  • It is convenient to administer about 0.01 to 30 mg, preferably about 0.1 to 2 Omg, more preferably about 0.1 to 10 mg per day by intravenous injection. is there.
  • the dose can be administered in terms of weight per 60 kg.
  • the antibody of the present invention can specifically recognize H7TBA62, it can be used for quantification of the receptor in a test solution, particularly for quantification by sandwich immunoassay.
  • one antibody is an antibody that recognizes the N-terminal of H7TBA62 and the other antibody is an antibody that reacts with the C-terminal of the receptor. .
  • the receptor can be quantified using a monoclonal antibody against H7TBA62, and detection can also be carried out by using a dye, such as staining with a silkworm.
  • a dye such as staining with a silkworm.
  • the antibody molecule itself may be used, or the F (ab ') 2 , Fab', or Fab fraction of the antibody molecule may be used.
  • the method for quantifying H7TBA62 using the antibody of the present invention is particularly limited. Instead, the amount of antibody, antigen, or antibody-antigen complex corresponding to the amount of antigen (eg, H7TBA62) in the test solution is detected by chemical or physical means, and this is Any measurement method can be used as long as it is a measurement method calculated from a standard curve prepared using a standard solution containing an antigen. For example, it is particularly preferable to use a sandwich method described later in terms of sensitivity and specificity in which nephrometry, a competitive method, an immunometric method and a sandwich method are suitably used.
  • a labeling agent used in a measurement method using a labeling substance for example, a radioisotope, an enzyme, a fluorescent substance, a luminescent substance and the like are used.
  • the radioisotope for example, [1 2 5 I], [1 3 1 I], [3 H], and [1 4 C] used.
  • the enzyme is preferably a stable enzyme having a large specific activity.
  • j3-galactosidase,] 3-darcosidase, alkaline phosphatase, peroxidase, malate dehydrogenase and the like are used.
  • the fluorescent substance for example, fluorescamine, fluorescein isothiosinate and the like are used.
  • the luminescent substance for example, luminol, luminol derivative, luciferin, lucigenin and the like are used.
  • a biotin-avidin system can be used for binding the antibody or antigen to the labeling agent.
  • the carrier include insoluble polysaccharides such as agarose, dextran, and cellulose; synthetic resins such as polystyrene, polyacrylamide, and silicon; and glass.
  • a test solution is reacted with the insolubilized monoclonal antibody of the present invention (primary reaction), and further reacted with another labeled monoclonal antibody of the present invention (secondary reaction).
  • primary reaction By measuring the activity of the labeling agent, the amount of H 7 TBA 62 in the test solution can be determined.
  • 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 in accordance with those described above.
  • the antibody used for the solid phase antibody or the labeling antibody does not necessarily need to be one kind, and the measurement sensitivity is not limited. A mixture of two or more types of antibodies may be used for the purpose of improving the antibody.
  • the monoclonal antibody of the present invention used for the primary reaction and the secondary reaction is preferably an antibody having a different site to which H7TBA62 binds. That is, the antibody used in the primary reaction and the secondary reaction is preferably used, for example, when the antibody used in the secondary reaction recognizes the C-terminal of H7TBA62, the antibody used in the primary reaction is preferably used. For example, an antibody that recognizes other than the C-terminal, for example, the N-terminal, is used.
  • the monoclonal antibody of the present invention can be used in a measurement system other than the sandwich method, for example, a competition method, an immunometric method, or a nephrometry.
  • the antigen in the test solution and the labeled antigen are allowed to react competitively with the antibody, and then the unreacted labeled antigen (F) and the labeled antigen (B) bound to the antibody are separated.
  • BZF separation The labeling amount of either B or F is measured, and the amount of antigen in the test solution is quantified.
  • a soluble antibody is used as the antibody
  • BZF separation is carried out using polyethylene glycol
  • a solid phase antibody is used as the first antibody
  • One antibody is a soluble one
  • a solid-phase method using a solid-phased antibody as the second antibody is used.
  • the antigen in the test solution and the solid-phased antigen are subjected to a competitive reaction with a certain amount of the labeled antibody, and then separated from the solid phase and the liquid phase.
  • the excess amount of the labeled antibody is allowed to react, then the immobilized antigen is added, and the unreacted labeled antibody is bound to the solid phase, and then the solid phase and the liquid phase are separated.
  • the amount of label in either phase is measured to quantify the amount of antigen in the test solution.
  • the amount of insoluble sediment produced 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 sediment is obtained, laser nephrometry utilizing laser scattering is preferably used.
  • H7TBA62 can be quantified with high sensitivity by using the antibody of the present invention.
  • a decrease in the concentration of H7TBA62 is detected by quantifying the concentration of H7TBA62 using the antibody of the present invention, for example, dysfunction of H7TBA62, particularly central and peripheral nerve function It can be diagnosed that the person is likely to have the disease associated with the abnormality or is likely to have the disease in the future. If an increase in the concentration of H7TBA62 is detected, for example, the GP
  • H7TBA62 dysfunction diseases associated with H7TBA62 dysfunction include, for example, neuroactive nasal depression or mucosal hyperemia, or acute circulatory failure or hypotension.
  • H7TBA62 Diseases caused by overexpression of H7TBA62 include, for example, peripheral circulatory disorder, hypertension, depression, obsessive-compulsive disorder, obsessive-compulsive disorder, panic disorder, anxiety disorder, eating disorder, ischemic disease, anticancer drug administration Digestive symptoms associated with acute myocardial infarction, acute Knee inflammation, chronic inflammation, adult respiratory distress syndrome, alcohol withdrawal syndrome, Alzheimer's disease, ankylosing spondylitis, arrhythmia, cocaine withdrawal syndrome, dementia, diarrhea, gastritis, hepatitis, infection, obiate withdrawal syndrome, osteoarthritis, bone Osteoporosis, osteopeetosis, peptic ulcer, peripheral vasculopathy, sequelae of myocardial infarction, reflux esophagitis, rheumatoid arthritis, schizophrenia, sepsis, septic shock, ulcerative colitis or unstable angina Can be
  • the antibody of the present invention may be used, for example, for cancer (eg, prostate cancer, colorectal cancer, etc.), infectious disease, pain, anorexia nervosa, bulimia, asthma, Parkinson's disease, acute heart failure, hypotension, hypertension, urinary retention, Osteoporosis, angina, myocardial infarction, ulcer, allergy, benign prostatic gland hypertrophy, psychiatric / neurological disorders (anxiety, schizophrenia, depression, delirium, dementia, severe mental retardation and motor abnormalities, Huntington's chorea It can also be used as a diagnostic agent for diseases such as the Jill de la 'lett syndrome group).
  • cancer eg, prostate cancer, colorectal cancer, etc.
  • infectious disease e.g., pain, anorexia nervosa, bulimia, asthma, Parkinson's disease, acute heart failure, hypotension, hypertension, urinary retention, Osteoporosis, angina, myocardial infarction, ulcer
  • H7TBA62 Binding of oxymetazoline to H7TBA62 inhibits the production of intracellular cAMP. Therefore, H7TBA62 is useful as a reagent for searching for or determining agonists other than oxymetazoline (including natural ligands) for H7TBA62 using the activity of inhibiting the production of intracellular cAMP as an index.
  • the present invention is characterized in that the activity of inhibiting the intracellular cAMP production via H7TBA62 when the test compound is brought into contact with cells containing H7TBA62 is measured.
  • test compounds known ligands (for example, angiotensin, bombesin, cannabinoid, cholecystokinin, glutamine, serotonin, melatonin, neuropeptide Y, opioid, purine, vasopressin, oxytocin, PACAP (e.g., , PACAP 27, PACAP 38), secretin, glucagon, calcitonin, adrenomedullin, somatostatin, GHRH, CRF, ACTH, GRP, PTH, VIP (Vasoactive Intestinal and Rerated Polypeptide), somatostatin , Dopamine, Motilin, amylin, bradykinin, CGRP (calcitonin gene relayed peptide), leukotriene, pancreastatin, prostaglandin, thromboxane, adenosine, adrenaline, chemokine-perfamily (eg, IL-8, GRO a, GRO
  • Tissue extract, cell culture supernatant, and low-molecular-weight synthetic compounds are added to H7TBA62, and fractionated while measuring cell stimulating activity and the like, to finally obtain a single ligand.
  • H7 TBA62 By using H7 TBA62 or constructing a recombinant H7TBA62 expression system and using a receptor binding assay using this expression system, it has ligand-like activity against H7TBA62
  • Compounds that change the binding property between oxymetazoline and H7TBA62 eg, peptides, proteins, non-peptidic compounds, synthetic compounds, fermentation products, etc.
  • Such compounds include (a) a compound having a cell stimulating activity via H7TBA62 (so-called agonist against H7TBA62), and (mouth) a compound having no cell-stimulating activity (so-called H7TBA62).
  • Antagonist Antagonist
  • a compound that enhances the binding strength between oxymetazoline and H7TBA62 or (2) a compound that decreases the binding strength between oxymetazoline and H7TBA62.
  • the present invention is characterized in that a comparison is made between (i) the case where H7TBA62 is brought into contact with oxymetazoline and (ii) the case where H7TBA62 is brought into contact with oxymetazoline and a test compound.
  • a method for screening a compound or a salt thereof that alters the binding property between oxymetazoline and H7TBA62 is provided.
  • the screening method of the present invention is characterized in that, in the cases (i) and (ii), for example, the amount of oxymetazoline bound to H7TBA62, the cell stimulating activity, and the like are measured and compared.
  • Cell stimulating activities include, for example, arachidonic acid release, acetylcholine release, intracellular Ca 2+ release, intracellular cAMP generation, intracellular cGMP generation, inositol phosphate production, cell membrane potential fluctuation, intracellular protein phosphorylation And c-fos activation, pH-lowering, and other activities that promote or suppress the activity, among which intracellular c-AMP production-suppressing activity is preferred.
  • the present invention provides
  • a compound that activates H7TBA62 for example, oxymetazoline
  • a compound that activates H7TBA62 and a test compound are contacted with cells that contain H7TBA62, A method for screening a compound or a salt thereof that changes the binding property between a ligand and the GPCR, wherein the cell stimulating activity mediated by H7TBA62 is measured and compared, and
  • H7TBA62 eg. oxymetazoline
  • a compound that activates H7TBA62 eg, oxymetazoline
  • H7TBA62 eg, oxymetazoline
  • a cell stimulating activity via the receptor was measured when the compound and the test compound were brought into contact with H7TBA62 expressed on the cell membrane by culturing a transformant containing the DNA of the present invention.
  • a method for screening a compound or a salt thereof, which alters the binding property between a ligand and H7TBA62 e.g, oxymetazoline
  • a compound that changes the binding property between oxymetazoline and H7TBA62 or a salt thereof can be used instead of oxymetazoline.
  • the compound that changes the binding property between oxymetazoline and H7TBA62 or a salt thereof can be obtained, for example, by performing the below-described squaring method of the present invention using oxymetazoline as a ligand.
  • oxymetazoline and H7TBA62 The term "oxymetazoline” is used to refer to a compound or a salt thereof that alters the binding property.
  • any H7 TBA62 may be used as long as it contains the above-mentioned H7 TBA62.
  • Cell membrane fractions are preferred.
  • human-derived H7TBA62 expressed in large amounts using recombinants is suitable for screening.
  • the above-mentioned method is used for producing H7TBA62, but it is preferably carried out by expressing the DNA of the present invention in mammalian cells or insect cells.
  • the complementary DNA is used as the DNA fragment encoding the protein portion of interest, 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 H7TBA62 into host animal cells and to express them efficiently, the DNA fragment must be expressed in a nuclear polyhedrosis disease belonging to Nokukuro nores using insects as a host.
  • NP V .nuclear polyhedrosis virus
  • SV40-derived promoter SV40-derived promoter
  • Letrowinores promoter meta-oral thionine promoter
  • human shock promoter cytomegalovirus promoter
  • SRa promoter SRa promoter
  • the amount and quality of the expressed receptor can be examined by a method known per se. 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 protein containing ⁇ 62 may be ⁇ 62 purified according to a method known per se, or a cell containing the receptor may be used. Alternatively, a membrane fraction of cells containing the receptor may be used.
  • the cell when a cell containing ⁇ 62 is used, the cell may be fixed with dartal aldehyde, formalin, or the like.
  • the immobilization method can be performed according to a method known per se.
  • the H7TBA62-containing cell refers to a host cell that has expressed the receptor, and the host cell is preferably Escherichia coli, Bacillus subtilis, yeast, insect cells, animal cells, and the like.
  • the cell membrane fraction refers to a cell membrane-rich fraction obtained by disrupting cells and then obtained by a method known per se.
  • Cells can be crushed by crushing the cells with a Potter-Elvehjem homogenizer, crushing using a Warlinda Blender Polytron (manufactured by Kinematica), crushing by ultrasonic waves, pressing the cells while pressing with a French press, etc. This includes crushing caused by jetting from a narrow nozzle.
  • fractionation by centrifugal force such as fractionation centrifugation or density gradient centrifugation is mainly used.
  • the cell lysate is centrifuged at a low speed (500-3000 rpm) for a short time (typically about 1-10 minutes), and the supernatant is further centrifuged at a higher speed (15000-30000 rpm) for 30 minutes to 2 hours.
  • the resulting precipitate is used as the membrane fraction.
  • the membrane fraction is rich in the expressed H7TBA62 and membrane components such as cell-derived phospholipids and membrane proteins.
  • the amount of H7TBA62 in the cells or membrane fraction containing H7TBA62 is preferably 10 3 to 10 8 molecules per cell, and more preferably 10 5 to 10 7 molecules per cell. .
  • the H7TBA62 fraction is preferably a natural H7TBA62 fraction or a recombinant GPCR fraction having an activity equivalent thereto.
  • “equivalent activity” means equivalent ligand binding activity, signal transduction action, and the like.
  • labeled oxymetazoline labeled oxymetazoline or a salt thereof, or a labeled oxymetazoline analog is used.
  • oxymetazoline labeled with [ 3 H], [ 125 I], [ 14 C], [ 35 S], etc. which is used.
  • the GPCR sample is prepared by suspending in a buffer.
  • the buffer may be any buffer that does not inhibit the binding between oxymetazoline and H7 TBA62, such as a phosphate buffer of pH 4 to 10 (preferably pH 6 to 8) and a buffer of Tris-monohydrochloride.
  • CHAP S, Twe e ⁇ - 8 ⁇ ⁇ M - can (Kao Atlas Co.), digitonin, also be added to the server Ffa a surfactant such as Dokishikoreto.
  • a protease inhibitor such as PMS F, leptin, E-64 (manufactured by Peptide Research Laboratories), or pepstatin can be added for the purpose of suppressing the degradation of receptor ligand by a protease.
  • the reaction is carried out at about 0-50 ° C, preferably about 4-37 ° C, for about 20 minutes to 24 hours, preferably for about 30 minutes to 3 hours.
  • the reaction solution is filtered through a glass fiber filter, 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 ⁇ -counter.
  • the count ( ⁇ .-NSB) obtained by subtracting the non-specific binding amount (NSB) from the count ( ⁇ ⁇ ⁇ 0 ) when there is no antagonist is 100%
  • the specific binding amount (B—NSB) 1 A test compound which is 50% or less can be selected as a scavenger having competitive inhibitory ability.
  • a known method for measuring the cell stimulating activity through H7TBA62 can be used.
  • it can be measured using a commercially available measurement kit.
  • cells containing H7TBA62 are cultured in a multiwell plate or the like. Before performing screening, use fresh medium Alternatively, replace with a suitable buffer that is not toxic to the cells, add the test compound, etc., incubate for a certain period of time, extract the cells or collect the supernatant, and quantitate the resulting product according to each method I do. If the production of a substance (for example, arachidonic acid) as an indicator of cell stimulating activity is difficult due to a degrading enzyme contained in cells, an inhibitor for the degrading enzyme is added to perform the assay. Well ,. In addition, 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.
  • a substance for example, arachidonic acid
  • cells expressing the appropriate H7TBA62 are required.
  • a cell expressing H7TBA62 a cell line having natural H7TBA62, a cell line expressing the above-mentioned recombinant H7TBA62, and the like are desirable.
  • test compounds for example, peptides, proteins, non-peptidic compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts, etc. are used, and these compounds are novel compounds. Or a known compound.
  • test compound a compound designed to bind to the ligand binding boxet based on the atomic coordinates of the active site of H7TBA62 and the position of the ligand binding boxet is preferably used.
  • the atomic coordinates of the active site of H7TBA62 and the position of the ligand binding pocket can be measured using a known method or a method analogous thereto.
  • Whether the compound that alters the binding property between oxymetazoline and H7TBA62 is an agonist or an antagonist can be confirmed using the above-described agonist screening method for H7TBA62.
  • Screening kits for compounds or salts thereof that alter the binding between oxymetazoline and H7TBA62 include those containing H7TBA62, cells containing H7TBA62, or membrane fractions of cells containing H7TBA62 It is.
  • screening kit of the present invention examples include the following. 1. Screening reagent
  • CHO cells expressing H7TBA62 were subcultured on a 12-well plate at 5 ⁇ 10 5 wells, and cultured at 37 ° C., 5% CO 2 , 95% air for 2 days.
  • the compound obtained by using the screening method or the screening kit of the present invention or a salt thereof is a compound having an action of changing the binding property between a ligand and H7TBA62.
  • H7TBA62 A compound having a cell stimulating activity via a cell (so-called agonist against H7TBA62), a compound having no cell stimulating activity via a cell (a so-called antagonist against H7TBA62), (c) enhancing the binding force between a ligand and H7TBA62 Or (2) a compound that decreases the binding force between the ligand and H7TBA62.
  • 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 agonist against H7TBA62 has the same physiological activity as oxymetazoline, which has a ligand-like activity on H7TBA62, and is therefore useful as a safe and low-toxic drug according to the physiological activity of the ligand of H7TBA62.
  • an antagonist to H7TBA62 can suppress the biological activity of a ligand to H7TBA62, it is useful as a safe and low-toxic drug for suppressing the biological activity of H7TBA62 ligand.
  • a compound that enhances the binding force between oxymetazoline and H7TBA62 can enhance the biological activity of the ligand for H7TBA62, so it can be used as a safe and low-toxic drug depending on the biological activity of the H7TBA62 ligand. Useful.
  • Compounds that decrease the binding strength between oxymetazoline and H7TBA62 can reduce the bioactivity of the ligand for H7TBA62, It is useful as a safe and low toxic drug for suppressing the biological activity of TBA62 ligand.
  • a compound or a salt thereof which is obtained by using the screening method or the screening kit of the present invention, which enhances the binding force between agonist or oxymethazoline for H7TBA62 and H7TBA62, It can be used as a vasoconstrictor, serotonergic agent, acetylcholine release promoter, gastrointestinal motility enhancer, smooth muscle contractor, platelet aggregating agent, central nervous system stimulant or inotropic agent.
  • those compounds or salts thereof are used, for example, as agents for removing neuroactive nasal depression or mucosal congestion, or for preventing or treating acute circulatory insufficiency or hypotension, ophthalmic drugs, and topical nasal drops.
  • Useful as a vasoconstrictor Useful as a vasoconstrictor.
  • the compound or a salt thereof which is obtained by using the screening method or the screening kit of the present invention, which decreases the binding force between antagonist or oxymethazoline and H7TBA62 to H7TBA62, or a salt thereof is used for vasodilation.
  • Agent vasoconstriction inhibitor, anti-serotonin agent, acetylcholine release inhibitor, gastrointestinal motility inhibitor, smooth muscle contraction inhibitor, platelet aggregation inhibitor or central nervous system inhibitor.
  • those compounds or salts thereof include, for example, peripheral circulatory disorder, hypertension, depression, obsessive-compulsive disorder, obsessive-compulsive disorder, panic disorder, anxiety disorder, eating disorder, ischemic disease, antineoplastic tumor Gastrointestinal symptoms associated with drug administration, acute myocardial infarction, acute knee inflammation, chronic knee inflammation, adult respiratory distress syndrome, alcohol withdrawal syndrome, Alhaima's disease, ankylosing spondylitis, arrhythmia, cocaine withdrawal syndrome, dementia, diarrhea, gastritis , Hepatitis, infectious disease, opiate withdrawal syndrome, osteoarthritis, osteoporosis, bone Pechet disease, peptic ulcer, peripheral vasculopathy, sequelae of myocardial infarction, reflux esophagitis, rheumatoid arthritis, schizophrenia, sepsis It is useful as a preventive and therapeutic agent for septic shock, ulcerative colitis or unstable angina.
  • compounds or salts thereof obtained using the screening method or screening kit of the present invention include, for example, cancer (eg, prostate cancer, colorectal cancer, etc.), infectious disease, pain, anorexia nervosa, bulimia, asthma, Parkinson Disease, acute heart failure, hypotension, hypertension, urinary retention, osteoporosis, angina, myocardial infarction, ulcer, allergy, benign prostate hypertrophy, mental and neurological disorders (anxiety, schizophrenia, depression, delirium, dementia) , Severe It is useful as a prophylactic and therapeutic agent for diseases such as mental retardation and motor abnormalities, Huntington's chorea, and Jill de 'La' Lettette syndrome.
  • cancer eg, prostate cancer, colorectal cancer, etc.
  • infectious disease e.g., pain, anorexia nervosa, bulimia, asthma, Parkinson Disease, acute heart failure, hypotension, hypertension, urinary retention, osteoporosis, angina, myocardial infarction,
  • a compound or a salt thereof 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 formulated according to a conventional method.
  • the compound can be used as a tablet, capsule, elixir, microcapsule, or the like, if necessary, orally sterilized with water or other pharmaceutically acceptable liquid. 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 doing. The amount of the active ingredient in these preparations is adjusted so that an appropriate dose in the specified range can be obtained.
  • Additives that can be mixed into tablets, capsules, etc . include binders such as gelatin, corn starch, tragacanth, gum arabic, excipients such as crystalline cellulose, corn starch, gelatin, alginic acid Swelling agents such as sucrose, lubricating agents such as magnesium stearate, sweetening agents such as sucrose, lactose or saccharin, and flavoring agents such as peppermint, cocoa oil or cellulose.
  • the unit dosage form is a capsule
  • the above-mentioned material of the type may further contain a liquid carrier such as oil and fat.
  • Sterile compositions for injection can be formulated according to standard pharmaceutical practice, such as dissolving or suspending the active substance in a vehicle such as water for injection, or naturally occurring vegetable oils such as sesame oil or coconut oil.
  • a vehicle such as water for injection, or naturally occurring vegetable oils 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 salt, etc.) and the like. aid such as an alcohol (e.g., ethanol), polyalcohol (e.g., propylene grayed recall, polyethylene glycol), nonionic surfactant (eg, Porisonore Pies 8 0 TM, HCO - 5 0 ) such as in combination with I'm sorry.
  • the oily liquid for example, sesame oil, soybean oil, and the like are used. It may be used in combination with alcohol, etc.
  • the above-mentioned medicines include, for example, buffering agents (for example, phosphate buffer, sodium acetate buffer), soothing agents (for example, benzalkonium chloride, pro-proin hydrochloride, etc.), stabilizers (for example, Serum albumin, polyethylene glycol, etc.), preservatives (eg, benzyl alcohol, phenol, etc.), antioxidants and the like.
  • buffering agents for example, phosphate buffer, sodium acetate buffer
  • soothing agents for example, benzalkonium chloride, pro-proin hydrochloride, etc.
  • stabilizers for example, 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, so they can be used, for example, in human mammals (
  • 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 for example, in an acute circulatory failure patient (with a body weight of 60 kg), About 0.1 to 100 mg per day, preferably about 1.0 to 50 mg, more preferably about 1.0 to 20 mg.
  • parenteral administration the single dose varies depending on the administration target, target organ, symptoms, administration method, and the like.
  • the dose can be administered in terms of the weight per 60 kg.
  • H7TBA62 it is possible to confirm whether or not various drugs exert a pharmacological effect via H7TBA62.
  • a vasoconstrictor (i) a vasoconstrictor, (ii) a serotonin agonist, (iii) acetylcholine release promoter, (iv) a gastrointestinal motility enhancer, (V ) Smooth muscle contractors, (vi) platelet aggregating agents, (vii) central nervous system stimulants, (viii) cardiotonic agents, (ix) neuroactive nasal depressive blood or mucosal hyperemia, (X) acute circulatory failure or Prevention and treatment of hypotension, (xi) ophthalmic drug, (xii) local vasoconstrictor for nasal drops, (xiii) vasodilator, (xiv) vasoconstrictor, (XV) Rotonin drugs, ( XV i) acetylcholine release inhibitor, (Xvii) gastrointestinal motility inhibitor, (xviii) smooth muscle contraction inhibitor, (Xix) platelet aggregation inhibitor, (XX) central nervous system inhibitor or (xxi) Perip
  • Serotonin agonists Serotonin agonists, (iii) acetylcholine release enhancer, (iv) gastrointestinal motility enhancer, (V) smooth muscle constrictor, (vi) platelet aggregating agent, (vii) central nervous system stimulant, (viii) inotropic Drugs, (ix) drugs for removing neuroactive nasal depressive blood or mucosal hyperemia, (X) drugs for preventing and treating acute circulatory insufficiency or hypotension, (xi) ophthalmic drugs or (xii) local vasoconstriction for nasal drops A method for confirming that the drug is an agonist against the receptor protein or a salt thereof,
  • H7TBA62 vasodilator, (ii) vasoconstrictor, (iii) anti-serotonin, (iv) acetylcholine release inhibitor
  • drugs include, for example, cancer (eg, prostate cancer, colon cancer, etc.), infectious disease, pain, anorexia nervosa, bulimia, asthma, Parkinson's disease, acute heart failure, hypotension, high blood pressure, urinary retention, osteoporosis , Angina, myocardial infarction, ulcer, allergy, benign prostatic hyperplasia, mental and neurological disorders (anxiety, schizophrenia, depression, delirium, dementia, severe mental retardation and motor abnormalities, Huntington's chorea, Jill ⁇ Prevention of diseases such as La Marette's syndrome ⁇ Remedies can also be used.
  • cancer eg, prostate cancer, colon cancer, etc.
  • infectious disease e.g., pain, anorexia nervosa
  • bulimia asthma
  • Parkinson's disease acute heart failure
  • hypotension high blood pressure
  • urinary retention osteoporosis
  • Angina myocardial infarction
  • ulcer allergy
  • benign prostatic hyperplasia mental and neurological disorders
  • This confirmation method can be carried out by using the above drug in place of the test compound in the above-described method for screening a compound that alters the binding 14 between oxymetazoline and H7TBA62.
  • the kit for a confirmation method of the present invention is a kit for screening a compound that changes the binding property between the ligand and H7TBA62, which contains the above drug in place of the test compound.
  • the antibody of the present invention can specifically recognize H7TBA62, it can be used for screening a compound that changes the amount of H7TBA62 in a cell membrane.
  • Sections of a) blood of a non-human mammal b) a specific organ, c) tissue or cells isolated from the organ, and then immunohistochemically stain the cells or cells on the cell surface.
  • a method for screening a compound that changes the amount of H7TBA62 in a cell membrane by confirming the protein on the cell membrane by quantifying the degree of staining of the receptor protein.
  • H7TBA62-expressing transformants and the like were sectioned, and the degree of staining of the receptor protein on the cell surface was quantified by immunostaining to confirm the protein on the cell membrane.
  • non-human mammals eg, mice, rats, egrets, sheep, pigs, mice, cats, dogs, monkeys, etc .; more specifically, dementia rats, obese mice, arteries
  • 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 specific organs eg, the liver, kidney, etc.
  • tissues or cells isolated from the organs are obtained.
  • the obtained organ, tissue or cell is suspended in, for example, an appropriate buffer (for example, Tris-HCl buffer, phosphate buffer, or Hessian buffer) to destroy the organ, tissue or cell.
  • an appropriate buffer for example, Tris-HCl buffer, phosphate buffer, or Hessian buffer
  • a cell membrane fraction is obtained by using a surfactant (for example, Triton X100 TM , Tween 20 TM, etc.), and further using a technique such as centrifugation, filtration, or column fractionation.
  • the cell membrane fraction refers to a fraction abundant in cell membrane obtained by disrupting cells and then obtained by a method known per se.
  • Cell crushing methods include a method of crushing cells with a Potter-Elvehiem homogenizer and a pelleting blender. Crushing with a retron (Kinematica), ultrasonic crushing,
  • fractionation by centrifugal force such as fractionation centrifugation or density gradient centrifugation is mainly used.
  • the cell lysate is centrifuged at a low speed (500-3000 rpm) for a short time (usually about 1-10 minutes), and the supernatant is further spun at a high speed (15000-30000 rpm) for 30 minutes-2 hours. Centrifuge and use the resulting precipitate as the membrane fraction.
  • the membrane fraction is rich in expressed H7TBA62 and membrane components such as cell-derived phospholipids and membrane proteins.
  • H7TBA62 contained in the cell membrane fraction can be quantified by, for example, a sandwich immunoassay using the antibody of the present invention, an eastern blot analysis, or the like.
  • Such a sandwich immunoassay can be carried out in the same manner as described above, and the estant blot can be carried out by means known per se.
  • a transformant expressing H7TBA62 is prepared according to the method described above, and H7TBA62 contained in the cell membrane fraction can be quantified.
  • Screening of a compound that alters the amount of H7 TBA62 in the cell membrane can be performed by: (i) A certain period of time (30 minutes to 24 hours before drug or physical stress is applied to a normal or disease model non-human mammal) Preferably 30 minutes to 12 hours before, more preferably 1 hour to 6 hours before, or after a certain time (30 minutes to 3 days after, preferably 1 hour to 2 days after, more preferably 1 hour after Or a test compound is administered simultaneously with the drug or physical 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 2 days) After 24 hours) can be performed by quantifying the amount of H7TBA62 in the cell membrane,
  • test compound When the transformant is cultured according to a conventional method, the test compound is mixed in a medium, and after culturing 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 later), it can be performed by quantifying the amount of H7TBA62 in the cell membrane.
  • the confirmation of H7TBA62 contained in the cell membrane fraction is specifically performed as follows. Do.
  • non-human mammals e.g., mouse, rat, egret, sheep, pig, pig, cat, dog, monkey, etc., more specifically, dementia rat, obese mouse, artery 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)
  • 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
  • blood or specific organs eg, brain, liver, kidney, etc.
  • tissues or cells isolated from the organs are 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 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 H7TBA62 in the cell membrane. Specifically, (a) increasing the amount of H7TBA62 in the cell membrane A compound that enhances the cell stimulating activity via the G protein-coupled receptor, and (a) a compound that decreases the cell stimulating activity by decreasing the amount of H7TBA62 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.
  • a compound that enhances the cell stimulating activity by increasing the amount of H7TBA62 in the cell membrane is useful as a safe and low-toxic prophylactic / therapeutic agent for diseases associated with H7TBA62 dysfunction.
  • a compound that attenuates the cell stimulating activity by reducing the amount of H7TBA62 in the cell membrane is useful as a safe and low-toxic prophylactic / therapeutic agent for diseases caused by overexpression of H7TBA62.
  • compounds or salts thereof that increase the amount of H 7 TBA 62 include vasoconstrictors, serotonin agonists, acetylcholine release enhancers, gastrointestinal motility enhancers, smooth muscle contractors, platelet aggregating agents Can be used as a central nervous system stimulant or cardiotonic.
  • these compounds or salts thereof are used, for example, as agents for removing neuroactive nasal depressive blood or mucosal hyperemia, or for preventing or treating acute circulatory insufficiency or hypotension, ophthalmic drugs, and topical nasal drops.
  • Useful as a vasoconstrictor Useful as a vasoconstrictor.
  • Compounds or salts thereof that reduce the amount of H7TBA62 in the cell membrane include vasodilators, vasoconstrictors, anti-serotonins, acetylcholine release inhibitors, gastrointestinal motility inhibitors, smooth muscle contraction inhibitors, It can be used as a platelet aggregation inhibitor or central nervous system depressant.
  • those compounds or salts thereof include, for example, peripheral circulatory disorder, hypertension, depression, obsessive-compulsive disorder, obsessive-compulsive disorder, panic disorder, anxiety disorder, eating disorder, ischemic disease, anti-malignant disease Gastrointestinal symptoms associated with tumor drug administration, acute myocardial infarction, acute knee inflammation, chronic knee inflammation, adult respiratory distress syndrome, alcohol withdrawal syndrome, Alzheimer's disease, ankylosing spondylitis, arrhythmia, cocaine withdrawal syndrome, dementia, diarrhea, Gastritis, hepatitis, infectious disease, oviate withdrawal syndrome, osteoarthritis, osteoporosis, osteophytosis, peptic ulcer, peripheral vascular disease, sequelae of myocardial infarction, reflux esophagitis, rheumatoid arthritis, schizophrenia, sepsis, It is useful as a prophylactic and therapeutic agent for septic shock, ulcerative colitis or unstable angina.
  • compounds or salts thereof that alter the amount of H 7 TBA 62 in cell membranes include, for example, cancer (eg, prostate cancer, colorectal cancer, etc.), infectious diseases, pain, anorexia nervosa, bulimia, asthma, Parkinson's disease , Acute heart failure, hypotension, hypertension, urinary retention, osteoporosis, angina, myocardial infarction, ulcer, allergy, benign prostatic hypertrophy, mental and neurological disorders (anxiety, schizophrenia, depression, delirium, It is useful as a preventive / therapeutic agent for diseases such as dementia, severe symptoms of mental retardation and motor abnormalities, Huntington's chorea, and Jill de la Allette syndrome.
  • cancer eg, prostate cancer, colorectal cancer, etc.
  • infectious diseases e.g., pain, anorexia nervosa, bulimia, asthma, Parkinson's disease , Acute heart failure, hypotension, hypertension, urinary retention, osteoporosis, angina, myo
  • 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 formulated according to a conventional method.
  • the compound can be administered orally as tablets, capsenoles, elixirs, microcapsules, etc., optionally coated with sugar, water or water or the like. It can be used parenterally in the form of injections, such as sterile solutions with other pharmaceutically acceptable 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 doing. The amount of the active ingredient in these preparations is such that an appropriate dose in the specified range can be obtained.
  • Such leavening agents, lubricants such as magnesium stearate, sweeteners such as sucrose, lactose or saccharin, and flavoring agents such as peppermint, cocoa oil or cherry are used.
  • the unit dosage form is a capsule, the above-mentioned material of the type may further contain a liquid carrier such as oil and fat.
  • Sterile compositions for injection can be formulated according to standard pharmaceutical practice, such as dissolving or suspending the active substance in a vehicle such as water for injection, or naturally occurring vegetable oils such as sesame oil or coconut oil. .
  • aqueous liquid for injection examples include physiological saline, isotonic solution containing glucose and other adjuvants (eg, D-sorbitol, D-mannitol, sodium salt, etc.) and the like. aid such as an alcohol (e.g., ethanol), polyalcohol (e.g., propylene grayed recall, polyethylene glycol), nonionic surfactant (eg, Porisoru Pies 8 0 TM, HCO - 5 0 ) such as in combination with Is also good.
  • 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.
  • the above preventive and therapeutic agents include, for example, buffers (for example, phosphate buffer, sodium acetate buffer), soothing agents (for example, benzalkonium chloride, prochloride hydrochloride, etc.), stabilizers (for example, It may be combined with human serum albumin, polyethylene glycol, etc.), preservatives (eg, benzyl alcohol, phenol, etc.), antioxidants and the like.
  • buffers for example, phosphate buffer, sodium acetate buffer
  • soothing agents for example, benzalkonium chloride, prochloride hydrochloride, etc.
  • stabilizers for example, It may be combined with 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 into a suitable ampoule.
  • the preparations obtained in this way are safe and low toxic, so they can be used, for example
  • 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 for example, in an acute circulatory failure patient (with a body weight of 60 kg), About 0.1 to 100 mg per day, preferably about 1.0 to 50 mg, more preferably about 1.0 to 2 Omg.
  • parenteral administration the single dose varies depending on the administration target, target organ, symptoms, administration method, etc.
  • it is usually used, for example, in patients with acute circulatory failure (body weight 6 O kg ), It is convenient to administer about 0.01 to 30 mg, preferably about 0.1 to 2 Omg, more preferably about 0.1 to 10 mg per day by intravenous injection.
  • the dose can be administered in terms of the weight per 60 kg.
  • the neutralizing activity of an antibody against H7TBA62 means an activity of inactivating a signal transmission function involving the receptor. Therefore, when the antibody has a neutralizing activity, signal transduction involving the receptor, for example, cell stimulating activity via the receptor (eg, arachidonic acid release, acetylcholine release, intracellular Ca 2+ release, cell CAMP production, intracellular cGMP production, inositol phosphate production, cell membrane potential fluctuations, intracellular protein phosphorylation, activation of c-fos, activity to promote or suppress pH reduction, etc.) Can be activated.
  • cell stimulating activity via the receptor eg, arachidonic acid release, acetylcholine release, intracellular Ca 2+ release, cell CAMP production, intracellular cGMP production, inositol phosphate production, cell membrane potential fluctuations, intracellular protein phosphorylation, activation of c-fos, activity to promote or suppress pH reduction, etc.
  • an antibody against H7TBA62 may be a disease caused by overexpression of the receptor (eg, peripheral circulatory disorder, hypertension, depression, obsessive-compulsive disorder, obsessive-compulsive disorder, panic disorder, anxiety) Disorders, eating disorders, ischemic diseases, gastrointestinal symptoms associated with the administration of antineoplastic drugs, acute myocardial infarction, acute knee inflammation, chronic otitis, adult respiratory distress syndrome, alcohol withdrawal syndrome, Alzheimer's disease, ankylosing spondylitis , Arrhythmia, cocaine withdrawal syndrome, dementia, diarrhea, gastritis, hepatitis, infectious disease, obiate withdrawal syndrome, osteoarthritis, osteoporosis, osteophytosis, peptic ulcer, peripheral Vasculopathy, sequelae of myocardial infarction, reflux esophagitis, rheumatoid arthritis, schizophrenia, sepsis, septic shock, ulcerative colitis or unstable angina)
  • the receptor eg, peripheral circulatory
  • antibodies to H7TBA62 include, for example, cancer (eg, prostate cancer, colon cancer, etc.), infectious diseases, pain, anorexia nervosa, bulimia, asthma, Parkinson's disease, acute heart failure, hypotension, hypertension , Urinary retention, osteoporosis, angina, myocardial infarction, ulcer, allergic benign, benign prostatic hyperplasia, mental and neurological disorders (anxiety, schizophrenia, depression, delirium, dementia, severe mental retardation and motor abnormalities) , Huntington's chorea, Jill 'de' la-lett syndrome) can also be used as a preventive and therapeutic agent for such diseases. (10) A medicine comprising the antisense DNA of the present invention
  • the antisense DNA of the present invention may be used for diseases caused by overexpression of H7TBA62 (eg, peripheral circulatory disorder, hypertension, depression, obsessive-compulsive disorder, obsessive-compulsive disorder, panic disorder, anxiety disorder, eating) Disorders, ischemic diseases, antidepressant symptoms associated with antineoplastic drug administration, acute myocardial infarction, acute knee inflammation, chronic knee inflammation, adult respiratory distress syndrome, alcohol withdrawal syndrome, Alzheimer's disease, ankylosing spondylitis, arrhythmia , Cocaine withdrawal syndrome, dementia, diarrhea, gastritis, hepatitis, infectious disease, obiate withdrawal syndrome, osteoarthritis, osteoporosis, osteopethyosis, peptic ulcer, peripheral vascular disease, sequelae of myocardial infarction, reflux esophagitis , Rheumatoid arthritis, schizophrenia, sepsis, septic shock, diseases such as ulcerative colitis or unstable angina).
  • the antisense DNA of the present invention can be used, for example, for cancer (eg, prostate cancer, colorectal cancer, etc.), infectious disease, pain, anorexia nervosa, bulimia, asthma, Parkinson's disease, acute heart failure, hypotension, hypertension, Urinary retention, osteoporosis, angina, myocardial infarction, ulcer, allergy, benign prostatic hypertrophy, mental and neurological disorders (anxiety, schizophrenia, depression, delirium, dementia, severe mental retardation and motor abnormalities, Huntington It can also be used as a prophylactic / therapeutic agent for diseases such as chorea and Jill 'de la llelet syndrome.
  • cancer eg, prostate cancer, colorectal cancer, etc.
  • infectious disease e.g., pain, anorexia nervosa, bulimia, asthma, Parkinson's disease, acute heart failure, hypotension, hypertension, Urinary retention, osteoporosis, angina, myocardial infarction, ulcer,
  • the antisense DNA when used, the antisense DNA is used alone or inserted into an appropriate vector such as a retrovirus vector, an adenovirus vector, an adenovirus associated virus vector, and the like. It can be implemented according to the means.
  • the antisense DNA can be administered as it is or in the form of a formulation together with a physiologically acceptable carrier such as an adjuvant for promoting uptake, and administered using a gene gun or a catheter such as a hydrogel catheter.
  • the antisense DNA can also be used as a diagnostic oligonucleotide probe for examining the presence and expression of the DNA of the present invention in tissues and cells.
  • the present invention relates to a non-DNA having the exogenous DNA of the present invention (hereinafter abbreviated as the exogenous DNA of the present invention) or its mutant DNA (sometimes abbreviated as the exogenous mutant DNA of the present invention).
  • the exogenous DNA of the present invention or its mutant DNA (sometimes abbreviated as the exogenous mutant DNA of the present invention).
  • the exogenous DNA of the present invention or its mutant DNA (sometimes abbreviated as the exogenous mutant DNA of the present invention).
  • a human mammal Provide a human mammal.
  • Non-human mammals having the exogenous DNA of the present invention or the mutant DNA thereof include unfertilized eggs, fertilized eggs, germ cells including spermatozoa and their progenitor cells, and the like.
  • the calcium phosphate method, the electric pulse method, and the ribofection method are used. It can be produced by transferring the target DNA by a method such as a coagulation method, a coagulation method, a microinjection method, a particle gun method, or a DEAE-dextran method.
  • the exogenous DNA of the present invention can be transferred to somatic cells, organs of living organisms, tissue cells, and the like, and used for cell culture, tissue culture, and the like.
  • the DNA-transferred animal of the present invention can also be produced by fusing the cells with the above-mentioned germ cells by a cell fusion method known per se.
  • non-human mammals for example, porcupines, pigs, sheep, sheep, goats, magpies, dogs, cats, guinea pigs, hamsters, mice, rats and the like are used.
  • mice for example, pure strains such as C57BLZ6 strain, DBA2 strain, etc.
  • a hybrid line a Be CSFi line, a BDFi line, a BSDS Fi line, 88-no ( : line, ICR line, etc.) or a rat (for example, Wistar, SD, etc.) are preferable.
  • Examples of the “mammal” in the recombinant vector that can be expressed in mammals include human and the like in addition to the above-mentioned non-human mammals.
  • the exogenous DNA of the present invention refers to the DNA of the present invention once isolated and extracted from the mammal, not the DNA of the present invention originally possessed by non-human mammals.
  • mutant DNA of the present invention those having a mutation (for example, mutation) in the base sequence of the original DNA of the present invention, specifically, base addition, deletion, substitution with another base, etc. DNA that has been used is used, and also includes abnormal DNA.
  • the abnormal DNA means a DNA that expresses abnormal H7TBA62, and for example, DNA that expresses a receptor that suppresses the function of normal H7TBA62 is used.
  • the exogenous DNA of the present invention may be derived from a mammal of the same species or a different species as the animal of interest.
  • a promoter capable of being expressed in animal cells e.g, egrets, dogs, cats, guinea pigs, hamsters, rats, mice, etc.
  • the human DNA of the present invention when transferred, it can be derived from various mammals (eg, egrets, dogs, cats, guinea pigs, hamsters, rats, mice, etc.) having the DNA of the present invention having high homology thereto.
  • Microinjection of a DNA construct eg, a vector, etc. to which the human DNA of the present invention is bound downstream of various promoters capable of expressing the same DNA into a fertilized egg of a target mammal, for example, a mouse fertilized egg. It is possible to create a DNA transgenic mammal that highly expresses the DNA of the present invention.
  • H7TBA62 expression vectors include E. coli-derived plasmid and Bacillus subtilis.
  • plasmid derived from yeast plasmid derived from yeast, bacteriophage such as phage, retrovirus such as Moroni leukemia virus, and animal virus such as vaccinia virus or baculovirus may be used.
  • a plasmid derived from E. coli, a plasmid derived from Bacillus subtilis, or a plasmid derived from yeast are preferably used.
  • promoters that regulate the above-mentioned DNA expression include: (1) a promoter of DNA derived from a virus (eg, simian virus, cytomegalovirus, Moroni leukemia virus, J. virus, breast cancer virus, poliovirus, etc.); (2) Promoters derived from various mammals (such as humans, egrets, dogs, cats, cats, hamsters, rats, mice, etc.), such as albumin, insulin II, peropkin II, elastase, erythropoietin, Endothelin, muscle creatine kinase, glial fibrillary acidic protein, daltathione S-transferase, platelet-derived growth factor] 3, keratin K 1, 1:10 and 1 ⁇ : 14, collagen types I and II , Cyclic AMP-dependent protein kinase] 3 I-subunit, dystrophin, liquor Lithate-resistant al-force phosphatase, atrial natriuretic factor,
  • cytomegalovirus promoter capable of high expression in the whole body, human Topepuchido chain elongation factor 1 alpha promoter one (E F- 1 ⁇ ), such as human and two Wa tri] 3 ⁇ cutin promoter one is It is.
  • the above vector is a messenger R of interest in DN It preferably has a sequence that terminates the transcription of NA (generally referred to as terminator 1).
  • terminator 1 a sequence of each DNA derived from a virus and various mammals can be used.
  • SV40 terminator is used.
  • the splicing signal of each DNA, the enhancer region, a part of the intron of eukaryotic DNA, etc. are placed 5 'upstream of the promoter region, between the promoter region and the translation region, in order to further express the target exogenous DNA.
  • it can be connected to the 3 'downstream of the translation area depending on the purpose.
  • the normal H7TBA62 translation region is derived from human or various mammals (eg, egrets, dogs, cats, guinea pigs, hamsters, rats, mice, etc.), monkeys, kidneys, thyroid cells, Raw material from fibroblast-derived DNA and all or part of genomic DNA from various commercially available genomic DNA libraries, or complementary DNA prepared by known methods from liver, kidney, thyroid cells, and fibroblast-derived RNA Can be obtained as
  • a translation region in which a normal H7TBA62 translation region obtained from the above cells or tissues is mutated by a point mutation induction method can be prepared from the exogenous abnormal DNA.
  • the translation region can be prepared as a DNA construct that can be expressed in a transposed animal by a conventional DNA engineering technique in which it is ligated downstream of the aforementioned promoter and, if desired, upstream of the transcription termination site.
  • Transfer of the exogenous DNA of the present invention at the fertilized egg cell stage is ensured to be present in all germ cells and somatic cells of the target mammal.
  • the presence of the exogenous DNA of the present invention in the germinal cells of the produced animal after DNA transfer means that the progeny of the produced animal has the exogenous DNA of the present invention in all of its germ cells and somatic cells. Means to keep.
  • the progeny of this type of animal that inherits the exogenous DNA of the present invention has the exogenous DNA of the present invention in all of its germinal and somatic cells.
  • the non-human mammal to which the exogenous normal DNA of the present invention has been transferred should be confirmed to stably maintain the exogenous DNA by mating, and be subcultured as an animal having the DNA in a normal breeding environment. Can be done.
  • the transfer of the exogenous DNA of the present invention at the fertilized egg cell stage is ensured to be present in excess in all germinal and somatic cells of the target mammal.
  • Excessive presence of the exogenous DNA of the present invention in the germinal cells of the produced animal after DNA transfer indicates that all the offspring of the produced animal have the exogenous DNA of the present invention in all of their germ cells and somatic cells. Means that.
  • the offspring of such animals that have inherited the exogenous DNA of the present invention have an excess of the exogenous DNA of the present invention in all of their germinal and somatic cells.
  • the non-human mammal having the normal DNA of the present invention expresses the normal DNA of the present invention at a high level, and eventually promotes the function of H7TBA62 by promoting the function of the endogenous normal DNA. It may develop and can be used as a disease model animal. For example, using the normal DNA transgenic animal of the present invention to elucidate the pathological mechanism of H7TBA62 hyperactivity and H7TBA62-related diseases, and to study methods for treating these diseases. Is possible.
  • the mammal into which the exogenous normal DNA of the present invention has been transferred has an increased symptom of free H7TBA62, it can be used for a screening test for a therapeutic agent for a disease associated with H7TBA62. .
  • a non-human mammal having the exogenous abnormal DNA of the present invention was confirmed to stably retain the exogenous DNA by mating, and was subcultured as an animal having the DNA in a normal breeding environment. You can do it. Further, the desired foreign DNA can be incorporated into the above-mentioned plasmid and used as a raw material.
  • a DNA construct with a promoter can be prepared by ordinary DNA engineering techniques. Transfer of the abnormal DNA of the present invention at the fertilized egg cell stage is ensured to be present in all germ cells and somatic cells of the target mammal.
  • the presence of the abnormal DNA of the present invention in the germ cells of the animal produced after the transfer of the DNA means that the offspring of the animal produced have the abnormal DNA of the present invention in all of the germ cells and somatic cells. .
  • the offspring of this type of animal that inherited the exogenous DNA of the present invention All germ cells and somatic cells have the abnormal DNA of the present invention.
  • the non-human mammal having the abnormal DNA of the present invention expresses the abnormal DNA of the present invention at a high level, and eventually inhibits the function of endogenous normal DNA, thereby ultimately inactivating the inactive form of H7TBA62. It can be used as a disease model animal. For example, using the abnormal DNA transgenic animal of the present invention, it is possible to elucidate the pathological mechanism of H7TBA62 function-inactive refractory disease and to examine a method for treating this disease.
  • the abnormal DNA highly expressing animal of the present invention revealed that the abnormal GPCR of the present invention inhibits the function of normal GPCR (dominant negative action) in H7TBA62 function-inactive refractory disease. Model.
  • the mammal to which the foreign abnormal DNA of the present invention has metastasized has an increased symptom of free H7TB A62, it can be used for a therapeutic drug screening test for H7TBA62 function-inactive refractory disease.
  • each organ is removed from the DNA-transferred animal of the present invention, and after minced, it is possible to obtain free DNA-transferred cells, culture them, or systematize the cultured cells using a protease such as trypsin. It is. Furthermore, it is possible to identify H7TBA62-producing cells, investigate their relationship to apoptosis, differentiation or proliferation, or investigate their signal transduction mechanisms, and investigate their abnormalities.This is an effective study to elucidate H7TBA62 and its effects. Material.
  • the DNA transgenic animal of the present invention uses the DNA transgenic animal of the present invention, to develop a therapeutic agent for diseases associated with H7TBA62, including inactive refractory H7TBA62, using the above-described test methods and quantitative methods, etc. It is possible to provide an effective and rapid screening method for the therapeutic agent for the disease. Further, using the DNA-transferred animal of the present invention or the exogenous DNA expression vector of the present invention, it is possible to examine and develop a DNA therapy for H7TBA62-related diseases.
  • the present invention provides a non-human mammal embryonic stem cell in which the DNA of the present invention is inactivated, and a non-human mammal deficient in expression of the DNA of the present invention.
  • a non-human mammal deficient in expression of the DNA of the present invention wherein the DNA is inactivated
  • a reporter gene eg, a / 3-galactosidase gene derived from Escherichia coli
  • the reporter gene of the present invention is inactivated.
  • the non-human mammal according to item (6) which can be expressed under the control of a promoter for DNA,
  • a non-human mammalian embryonic stem cell in which the DNA of the present invention has been inactivated is a DNA that is capable of suppressing DNA expression by artificially mutating the DNA of the present invention possessed by the non-human mammal.
  • the DNA substantially does not have the ability to express H7TBA62 by substantially losing the activity of H7TBA62 encoded by the DNA (hereinafter referred to as the knockout DNA of the present invention).
  • the knockout DNA of the present invention refers to embryonic stem cells of non-human mammals (hereinafter abbreviated as ES cells).
  • non-human mammal the same one as described above is used.
  • the method for artificially mutating the DNA of the present invention can be performed, for example, by deleting a part or all of the DNA sequence and inserting or substituting another DNA sequence by a genetic engineering technique.
  • the knockout DNA of the present invention can be produced by, for example, shifting the reading frame of a codon or disrupting the function of a promoter or exon by these mutations.
  • Non-human mammalian embryonic stem cells in which the DNA of the present invention is inactivated include:
  • the DNA of the present invention possessed by the target non-human mammal is isolated, and its exon portion is a drug resistance gene represented by the neomycin H4 gene, hygromycin resistance gene, or 1acZ ( ⁇ -galactosidase).
  • Gene or cat (chloramphenicol acetyltransferase gene) to insert a reporter gene, etc., to disrupt exon function, or to terminate gene transcription in the intron between exons.
  • a DNA strand having a DNA sequence constructed as described above (hereinafter abbreviated as targeting vector 1) is introduced into the chromosome of the animal by, for example, a homologous recombination method, and the obtained ES cell is placed on the DNA of the present invention or Southern hybridization analysis using the neighboring DNA sequence as a probe or the DNA sequence on the targeting vector and the DNA sequence of the neighboring region other than the DNA of the present invention used for the production of a targeting vector Can be obtained by analyzing the knockout ES cells of the present invention by analyzing the PCR method using the primers as primers.
  • a DNA sequence for example, a polyA addition signal
  • the original ES cells for inactivating the DNA of the present invention by the homologous recombination method or the like for example, those already established may be used as described above, and the method of the known Evans and Kaufma may be used. It may be newly established according to. For example, in the case of mouse ES cells, currently, 129 ES cells are generally used, but since the immunological background is not clear, an alternative pure immunogenic genetic background is used. For example, for the purpose of obtaining ES vesicles in which C57B LZ6 and C57B / Z6 mice were obtained, the number of eggs collected by C57B / LZ6 mice was reduced by crossing with 0882.
  • BDFi mice have the advantage of high number of eggs collected and robust eggs, and also have C57BLZ6 mice as their background. It can be used advantageously because it is possible to replace its genetic background with C57BL / 6 mice by backcrossing with / 6 mice.
  • blastocysts 3.5 days after fertilization are generally used. Early embryos can be obtained.
  • Either male or female ES cells may be used, but male ES cells are generally more convenient for producing breeding line chimeras. It is also desirable to discriminate between males and females as soon as possible in order to reduce the complexity of culturing.
  • An example of a method for determining the sex of ES cells is a method of amplifying and detecting a gene in the sex-determining region on the Y chromosome by PCR.
  • this method conventionally, about 10 6 cells to the karyotyping In contrast, the number of ES cells in one colony (approximately 50) is sufficient, so the primary selection of ES cells in the early stage of culture can be performed by gender discrimination. By enabling the selection of cells, labor in the initial stage of culture can be significantly reduced.
  • the secondary selection can be performed, for example, by confirming the number of chromosomes by the G-banding method.
  • the number of chromosomes in the obtained ES cells is desirably 100% of the normal number.
  • Embryonic stem cell lines obtained in this way usually have very good proliferative properties, but must be carefully subcultured because they tend to lose their ontogenetic potential.
  • a suitable feeder cell such as STO fibroblasts
  • a carbon dioxide incubator preferably 5% carbon dioxide, 95% air or 5%
  • LIF 1-1 000 OU / ml
  • trypsin ZEDTA solution usually 0.001 to 0.5% trypsin Z
  • 0.1 to 5 mM EDTA preferably, about 0.1% trypsin / ImM EDTA
  • Such subculture is usually performed every 1 to 3 days. At this time, it is desirable to observe the cells, and if morphologically abnormal cells are found, discard the cultured cells.
  • ES cells are differentiated into various types of cells, such as parietal, visceral, and cardiac muscle, by monolayer culture up to high density or suspension culture until cell clumps are formed under appropriate conditions.
  • a DNA-deficient cell of the present invention obtained by differentiating the ES cell of the present invention is H7TB in vitro. Useful in A62 or H7TB A62 cell biology studies.
  • the non-human mammal deficient in DNA expression of the present invention can be distinguished from a normal animal by measuring the mRNA level of the animal using a known method and indirectly comparing the expression level.
  • non-human mammal those similar to the aforementioned can be used.
  • the non-human mammal deficient in expression of the DNA of the present invention can be obtained, for example, by introducing the targeting vector prepared as described above into a mouse embryonic stem cell or a mouse egg cell, whereby the DNA of the target targeting vector of the present invention becomes inactive.
  • the DNA of the present invention can be knocked out by homologous recombination of the converted DNA sequence with the DNA of the present invention on the chromosome of mouse embryonic stem cells or mouse egg cells by gene homologous recombination. .
  • the cells in which the DNA of the present invention has been knocked out include a DNA sequence on a Southern hybridization analysis or a targeting vector using the DNA sequence on or near the DNA of the present invention as a probe, and a mouse used for the targeting vector. It can be determined by analysis by PCR using the DNA sequence of the neighboring region other than the DNA of the present invention as a primer.
  • a non-human mammalian embryonic stem cell is used, a cell line in which the DNA of the present invention has been inactivated by gene homologous recombination is cloned, and the cell is used at an appropriate time, for example, at the 8-cell stage.
  • the chimeric embryo is injected into a non-human mammal embryo or blastocyst, and the produced chimeric embryo is transplanted into the uterus of the pseudo-pregnant non-human mammal.
  • the produced animal is a chimeric animal composed of both a normal cell having the DNA locus of the present invention and an artificially mutated cell having the DNA locus of the present invention.
  • all the tissues are more artificial than the population obtained by crossing such a chimeric individual with a normal individual. It can be obtained by selecting individuals composed of cells having the DNA locus of the present invention in which a mutation has been added to, for example, by judging coat color or the like.
  • the individual obtained in this manner is usually an H7TBA62 heterozygous expression-deficient individual, and mated with an H7TBA62 hetero-expression-defective individual to obtain an H7TBA62 homo-expression-deficient individual from their offspring. be able to.
  • a transgenic non-human mammal having a targeting vector introduced into a chromosome can be obtained by injecting a DNA solution into the nucleus of an egg by a microinjection method. Compared to non-human mammals, they can be obtained by selecting those having a mutation in the DNA locus of the present invention by homologous recombination of the gene.
  • the acquisition and maintenance of the germ line may be performed according to a conventional method. That is, by crossing male and female animals having the inactivated DNA, a homozygous animal having the inactivated DNA on both homologous chromosomes can be obtained. The obtained homozygous animal can be efficiently obtained by rearing the mother animal in such a manner that one normal individual and a plurality of homozygous animals are obtained. By mating male and female heterozygous animals, homozygous and heterozygous animals having the inactivated DNA are bred and subcultured.
  • the non-human mammal embryonic stem cells in which the DNA of the present invention has been inactivated are extremely useful for producing the non-human mammal deficient in expression of the DNA of the present invention.
  • the non-human mammal deficient in DNA expression of the present invention lacks various biological activities that can be induced by H7TBA62, it can serve as a model for a disease caused by inactivation of the biological activities of H7TBA62. However, it is useful for investigating the causes of these diseases and examining treatment methods.
  • the non-human mammal deficient in DNA expression of the present invention can be used for screening for a compound having a therapeutic / preventive effect against diseases caused by DNA deficiency or damage of the present invention.
  • the present invention is characterized in that a test compound is administered to a non-human mammal deficient in expressing the DNA of the present invention, and changes in the animal are observed and measured.
  • Compounds that have therapeutic and preventive effects on Provides a method of screening for its salts.
  • Examples of the non-human mammal deficient in expression of DNA of the present invention used in the screening method include the same as described above.
  • Test compounds include, for example, peptides, proteins, non-peptidic compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts, and plasma.These compounds are novel compounds. Or a known compound.
  • a non-human mammal deficient in expression of the DNA of the present invention is treated with a test compound and compared with a non-treated control animal, and changes in organs, tissues, disease symptoms, and the like of the animal are indexed.
  • the therapeutic and prophylactic effects of the test compound can be tested.
  • test compound for example, oral administration, intravenous injection, or the like is used, and it can be appropriately selected according to the symptoms of the test animal, properties of the test compound, and the like.
  • the dose of the test compound can be appropriately selected according to the administration method, the properties of the test compound, and the like.
  • the blood glucose level of the test animal decreases by about 10% or more, preferably about 30% or more, more preferably about 50% or more.
  • the compound can be selected as a compound having a therapeutic / preventive effect on the above-mentioned diseases.
  • the compound obtained by the screening method is a compound selected from the test compounds described above, and is a disease caused by H7TBA62 deficiency or damage (eg, vasoconstriction, serotonin action, acetylcholine release, Safety against disorders caused by dysfunctions such as gastrointestinal motility, smooth muscle contraction, platelet aggregation, central nervous control or inotropic effects (eg, neuroactive nasal depression, mucosal hyperemia, acute circulatory failure, hypotension) It can be used as a low-toxicity treatment / prophylactic agent, ophthalmic drug, topical vasoconstrictor for nasal drops, etc. Further, a compound derived from the compound obtained by the above-mentioned screening can also be used.
  • H7TBA62 deficiency or damage eg, vasoconstriction, serotonin action, acetylcholine release, Safety against disorders caused by dysfunctions such as gastrointestinal motility, smooth muscle contraction, platelet aggregation, central nervous control or inotropic
  • compounds obtained using the screening method include, for example, cancer (eg, prostate cancer, colorectal cancer, etc.), infectious disease, pain, anorexia nervosa, bulimia, asthma, Parkinson's disease, acute heart failure, hypotension, Hypertension, urinary retention, osteoporosis, angina, myocardial infarction, Diseases such as ulcers, allergies, benign prostatic hyperplasia, psychiatric disorders (anxiety, schizophrenia, depression, delirium, dementia, severe symptomatic mental retardation and motor abnormalities, Huntington's chorea, Jill de la Rollett syndrome) It can also be used as a prophylactic and therapeutic agent.
  • cancer eg, prostate cancer, colorectal cancer, etc.
  • infectious disease e.g., pain, anorexia nervosa, bulimia, asthma, Parkinson's disease, acute heart failure, hypotension, Hypertension, urinary retention, osteoporosis, angina, myocardial infarction
  • Diseases such
  • the compound obtained by the screening method may form a salt.
  • the salt of the compound include physiologically acceptable acids (eg, inorganic acids, organic acids, etc.) and bases (eg, alkali metals). And the like, and particularly preferred are physiologically acceptable acid addition salts.
  • physiologically acceptable acids eg, inorganic acids, organic acids, etc.
  • bases eg, alkali metals
  • physiologically acceptable acid addition salts include salts with inorganic acids (eg, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid, etc.) and organic acids (eg, acetic acid, formic acid, propionic acid, fumaric acid, maleic acid) Succinic acid, tartaric acid, citric acid, malic acid, oxalic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid, etc.).
  • inorganic acids eg, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric
  • a drug containing the compound or a salt thereof obtained by the screening method can be produced in the same manner as the above-mentioned drug containing a compound that changes the binding property between H7TBA62 and a ligand.
  • the preparations obtained in this way are safe and low toxic, for example, in humans or mammals (eg, rats, mice, guinea pigs, egrets, sheep, pigs, horses, cats, cats, animals). And monkeys and monkeys).
  • the dose of the compound or a salt thereof varies depending on the target disease, the administration subject, the administration route, and the like.
  • the compound when the compound is orally administered, generally, for example, patients with acute circulatory insufficiency (body weight 6 O kg ) In the range of about 0.1 to 100 mg, preferably about 1.0 to 50 mg, more preferably about 1.0 to 20 mg per day.
  • the single dose varies depending on the administration target, target organ, symptoms, administration method, etc.
  • the dose can be administered in terms of weight per 60 kg.
  • the present invention provides a test compound administered to a non-human mammal deficient in expression of the DNA of the present invention, and detects the expression of a reporter gene.
  • the non-human mammal deficient in expressing DNA of the present invention may be a non-human mammal deficient in expressing DNA of the present invention, wherein the DNA of the present invention is inactivated by introducing a reporter gene.
  • a reporter gene that can be expressed under the control of a promoter for the DNA of the present invention is used.
  • test compound examples include the same compounds as described above.
  • the same one as described above is used, and a galactosidase gene (1 acZ), a soluble alkaline phosphatase gene or a luciferase gene is suitable.
  • H7TBA62 when a part of the DNA region encoding H7TBA62 is replaced with a j3-galactosidase gene (1 ac Z) derived from Escherichia coli, a tissue that originally expresses H7TBA62 is used instead of H7TBA62.
  • Galactosidase is expressed.
  • a reagent that is a substrate for 3-galactosidase such as 5-bromo-4-chloro-3-indolyl / 3-galatatopyranoside (X-gal)
  • X-gal 5-bromo-4-chloro-3-indolyl / 3-galatatopyranoside
  • H7TBA62-deficient mice or tissue sections are fixed with dartal aldehyde, washed with phosphate buffered saline (PBS), and stained with X-ga1 at room temperature or around 37 ° C. After reacting for about 30 minutes to 1 hour, wash the tissue specimen with ImM EDTAZPBS solution to stop the] -galactosidase reaction and observe coloration. I'll do it. Further, mRNA encoding 1 ac Z may be detected according to a conventional method.
  • the compound or a salt thereof obtained by using the above-mentioned screening method is a compound selected from the above-mentioned test compounds, and is a compound that promotes or inhibits the promoter activity of DNA of the present invention.
  • the compound obtained by the screening method may form a salt.
  • the salt of the compound include physiologically acceptable acids (eg, inorganic acids) and bases (eg, organic acids). And the like, and particularly preferably a physiologically acceptable acid addition salt.
  • physiologically acceptable acids eg, inorganic acids
  • bases eg, organic acids
  • a physiologically acceptable acid addition salt examples include salts with inorganic acids (eg, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid, etc.) and organic acids (eg, acetic acid, formic acid, propionic acid, fumaric acid, maleic acid) , Conodic acid, tartaric acid, citric acid, malic acid, oxalic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid, etc.).
  • inorganic acids eg, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid, etc.
  • organic acids eg,
  • the compound of the present invention that promotes the promoter activity against DNA or a salt thereof can promote the expression of H7TBA62 and promote the function of the receptor. It is useful as a drug such as a preventive and therapeutic drug for illness, ophthalmic drug, and local vasoconstrictor for nasal drops.
  • the compound or its salt of the present invention that inhibits the promoter activity against DNA can inhibit the expression of H7TBA62 and inhibit the function of the receptor. It is useful as a drug for prevention and treatment of diseases that cause illness.
  • H7TBA62 dysfunction diseases associated with H7TBA62 dysfunction include, for example, neuroactive nasal depression, mucosal hyperemia, acute circulatory failure, hypotension and the like.
  • H7TBA62 Diseases caused by overexpression of H7TBA62 include, for example, peripheral circulatory disorders, hypertension, depression, obsessive-compulsive disorder, obsessive-compulsive disorder, panic disorder, anxiety disorder, eating disorder, ischemic disease, and anti-malignant disease.
  • Gastrointestinal symptoms associated with oncologic administration acute myocardial infarction, acute inflammation, chronic inflammation, adult respiratory distress syndrome, alcohol withdrawal syndrome, Alzheimer's disease, ankylosing spondylitis, arrhythmia, cocaine withdrawal syndrome, dementia, diarrhea, gastritis , Hepatitis, infectious disease, obiate withdrawal syndrome, osteoarthritis, osteoporosis, osteophytosis, Examples include peptic ulcer, peripheral vasculopathy, sequelae of myocardial infarction, reflux esophagitis, rheumatoid arthritis, schizophrenia, sepsis, septic shock, ulcerative colitis or unstable angina.
  • the compound of the present invention that promotes or inhibits the promoter activity for the DNA or a salt thereof includes, for example, cancer (eg, prostate cancer, colorectal cancer, etc.), infectious disease, pain, anorexia nervosa, bulimia, asthma, Parkinson's disease, acute heart failure, hypotension, hypertension, urinary retention, osteoporosis, angina, myocardial infarction, ulcer, allergy, benign prostatic hypertrophy, mental and neurological disorders (anxiety, schizophrenia, depression, delirium, dementia, It can also be used as a prophylactic / therapeutic agent for diseases such as severe symptomatic mental retardation and motor abnormalities, Huntington's chorea, and Jill de la Allette syndrome.
  • cancer eg, prostate cancer, colorectal cancer, etc.
  • infectious disease e.g., pain, anorexia nervosa, bulimia, asthma, Parkinson's disease, acute heart failure, hypotension, hypertension, urinary retention, osteoporosis, an
  • a compound derived from the compound obtained by the above-mentioned stalling can be used in the same manner.
  • a medicament containing the compound or a salt thereof obtained by the screening method can be produced in the same manner as the above-mentioned medicament containing a compound that alters the binding property between H7TBA62 or a salt thereof and a ligand.
  • the preparations obtained in this way are safe and low toxic, for example, in humans or mammals (eg, rats, mice, guinea pigs, egrets, sheep, pigs, horses, cats, cats, animals). And monkeys and monkeys).
  • the dosage of the compound or a salt thereof varies depending on the target disease, the administration subject, the administration route, and the like.
  • a compound that promotes or inhibits the promoter activity of DNA of the present invention is orally administered,
  • about 0.1 to 10 Omg per day preferably about 1.0 to 50 mg, more preferably about 1.0 to 20 mg per day.
  • the single dose varies depending on the administration target, target organ, symptoms, administration method, etc. 6 O kg)
  • 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. It is convenient.
  • the dose can be administered in terms of weight per 60 kg.
  • the non-human mammal deficient in expression of the DNA of the present invention is extremely useful for screening a compound or a salt thereof that promotes or inhibits the activity of the promoter for the DNA of the present invention.
  • the present invention can greatly contribute to investigating the cause of various diseases caused by insufficient expression of DNA and preventing and developing therapeutic agents.
  • genes encoding various proteins can be ligated downstream thereof and injected into egg cells of an animal to produce a so-called transgenic animal (transgenic animal). For example, it would be possible to specifically synthesize its receptor and examine its action in living organisms.
  • a low-molecular-weight molecule that specifically promotes or suppresses the production ability of H7TBA62 itself in the body It can be used as a compound search system.
  • DNA Deoxyribonucleic acid
  • RNA ribonucleic acid
  • mRNA messenger ribonucleic acid
  • dCTP Deoxycytidine triphosphate ATP Adenosine triphosphate EDTA Entetraacetic acid SD S Sodium dodecyl sulfate G 1 y Glycine
  • Trt Trityl
  • HOB t 1—Hydroxybenztriazole
  • sequence numbers in the sequence listing in the present specification indicate the following sequences.
  • the nucleotide sequence of cDNA encoding human-derived ⁇ 7 ⁇ 62 is shown.
  • SEQ ID NO: 4 shows the nucleotide sequence of a primer used in the PCR reaction in Example 3.
  • Example 3 shows the nucleotide sequence of a primer used in the PCR reaction in Example 3.
  • Example 4 shows the nucleotide sequence of a probe used in the PCR reaction in Example 3.
  • Expression plasmid for animal cells pAKKO— 111 H (Biochem. Biophys. Acta, Hinuma, S. eta 1.1129, p. 251—p. 259-1, p.
  • the expression vector plasmid pAK_H7TBA62 which was prepared by inserting H7TBA62 cDNA into a plasmid vector (identical to 1.1H) by a known method, and pAKKO—without inserting a specific gene. H was transfected into HEK293 cells by the following method.
  • Escherichia coli JM109 was transformed with these vectors, the resulting colonies were isolated and cultured, and then plasmid was prepared using QIAGEN Plasmid Max Kit (Qiagen).
  • a plasmid of pCRE-Luc (C1tech) in which a luciferase gene was linked as a reporter downstream of the cAMP response element (CRE) was prepared in the same manner.
  • HEK293 cells were seeded on a 96-we11 black plate (Betaton Dickinson) coated with collagen I at 100,000 cells / 611 and a culture volume of 100 ⁇ l, and cultured at room temperature.
  • the medium for culturing in a plate was prepared by adding only 10% of fetal serum (FBS, Gibco BRL) to DMEM (Dulbeccos modified Eagles medium, Gibco BRL). Using. Each plasmid is pAK-H7TBA62 and Porter plasmid pCRE-Luc was added at a ratio of 9: 1 to 240 ⁇ of Opti-MEM-I (Gibco BRL). This was mixed with an equal amount of 240 ⁇ l of ⁇ pti-MEM-I to which 101 ribofectamine 2000 (Gibco BRL) was added, and mixed as described in the manual attached to Lipofectamine 2000. To form a complex of ribosome and plasmid. These were added to the culture solution of HEK293 cells, each of which contained 25 / ilZwe11, and cultured at 37 ° C under 5% CO 2 conditions.
  • FBS fetal serum
  • DMEM Dulbeccos modified Eagles medium
  • H7TBA62 stable expression HEK cells and HEK-H7TBA62 were prepared by a method known per se.
  • HEK—H7 TBA62 or HEK cells not transfected with the receptor gene were coated with collagen I at a concentration of 100,000 ce 11 s / we 11 96-we 11 black plate (vector) (Deckinson) and cultured at 37 ° C., 5% CO 2 for 1 mm and used for Atsusei.
  • the sample buffer was HBSS in 0.1% BSA and 0.2 mM I BMX (Sigma). Was used.
  • AB IPRISM 7700 Sequ en c eDe t e c t or (Applied Biosystems) was used.
  • the primer used for quantification of the expression level [5'-TCGGCACTGGACTTTCACT-3 '(SEQ ID NO: 3),
  • [5'-TGCAAGATGGTTCTGACGGCCA-3 '(SEQ ID NO: 5)] is based on the nucleotide sequence of human H7TBA62 (SEQ ID NO: 1), and is dedicated to Primer Ex Exclusive software Designed using press (Applied Biosystems).
  • the cDNA used as type I was synthesized by reverse transcription of 1 ⁇ g of total RNA derived from various human tissues using random primers. The reverse transcription reaction was performed using Super Scriptl (GI BCO BRL) as a reverse transcriptase according to the attached protocol.
  • the reaction solution of AB I PR I SM 7700 S equenc e Detector was TaqMan Universal PCR Master Mix (Applied Biosystems) 12.51, each primer was 0.9 ⁇ , and the probe was 0.25 ⁇ and the cD ⁇ solution were mixed together and adjusted to 25 ⁇ 1 with distilled water.
  • the reaction in the AB I PR I SM 7700 Sequence Dector is 2 minutes at 50 ° C, 10 minutes at 95 ° C, and 40 cycles of 95 ° C * 15 seconds, 60 ° C for 1 minute. It was repeated.
  • Figure 3 shows the distribution of mRNA expression in various human tissues.
  • the prostate cancer cell line D High expression of mRNA was detected in U45, PC3, and colon cancer cell lines such as SW1417, WiDr, SW837, and SW1463 ( Figure 4).
  • DNA encoding H7TBA62, its partial peptide or a salt thereof, or H7TBA62 or its partial peptide is a vasoconstrictor, a serotonin agonist, an acetylcholine release promoter, a gastrointestinal motility enhancer, a smooth muscle contractor, a platelet aggregation. It is useful as a bulk drug, central nervous system stimulant or cardiotonic agent, for example, for the removal of neuroactive nasal depression and mucosal hyperemia, or for the prevention and treatment of acute circulatory insufficiency and hypotension.
  • H7TBA62 its partial peptide or its salt and oxymetazoline having ligand-like activity, it is possible to efficiently screen a compound that changes the binding property between the ligand and H7TBA62, its partial peptide or its salt. it can.

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Abstract

Selon l'invention, l'utilisation d'une protéine récepteur couplé aux protéines G comprenant une séquence d'acide aminé identique ou sensiblement identique à la séquence d'acide aminé représentée par SEQ ID NO:2 ou son sel, conjointement avec de l'oxymétazoline, permet d'isoler par criblage un agoniste ou un antagoniste de cette protéine récepteur.
PCT/JP2003/005037 2002-04-22 2003-04-21 Nouvelle methode de criblage WO2003088992A1 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2011229529A (ja) * 2004-06-21 2011-11-17 Takeda Chem Ind Ltd 糖尿病および肥満調節におけるｇｐｒ１００受容体の使用

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0885960A2 (fr) * 1997-06-18 1998-12-23 Smithkline Beecham Corporation Récepteur couplé à la protéine g (H7TBA62)
WO1999028300A1 (fr) * 1997-12-04 1999-06-10 Allergan Sales, Inc. Derives imidazoles substitues ayant une activite de type agoniste vis a vis des recepteurs adrenergiques alpha 2b ou 2b/2c

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0885960A2 (fr) * 1997-06-18 1998-12-23 Smithkline Beecham Corporation Récepteur couplé à la protéine g (H7TBA62)
WO1999028300A1 (fr) * 1997-12-04 1999-06-10 Allergan Sales, Inc. Derives imidazoles substitues ayant une activite de type agoniste vis a vis des recepteurs adrenergiques alpha 2b ou 2b/2c

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LIMON-BOULEZ ISABELLE ET AL.: "Partial agonist clonidine mediates alpha2-AR subtypes specific regulation of cAMP accumulation in adenylyl cyclase II transfected DDT1-MF2 cells", MOLECULAR PHARMACOLOGY, vol. 59, no. 2, February 2001 (2001-02-01), pages 331 - 338, XP002971526 *
OGIMOTO GOICHI ET AL.: "G protein-coupled receptors regulate NA+, K+-ATPase activity and endocytosis by modulating the recruitment of adaptor protein 2 and clathrin", PROC. NATL. ACAD. SCI. USA, vol. 97, no. 7, 28 March 2000 (2000-03-28), pages 3242 - 3247, XP002971527 *
UMLAND SHELBY P. ET AL.: "Receptor reverse analysis of the human alpha2c-adrenoceptor using (35S)GTPgammaS and cAMP functional assays", EUROPEAN JOURNAL OF PHARMACOLOGY, vol. 441, no. 3, 12 January 2001 (2001-01-12), pages 211 - 221, XP002971525 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2011229529A (ja) * 2004-06-21 2011-11-17 Takeda Chem Ind Ltd 糖尿病および肥満調節におけるｇｐｒ１００受容体の使用

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