WO2003044182A1 - Cellules exprimant gob-5 et utilisation - Google Patents

Cellules exprimant gob-5 et utilisation Download PDF

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Publication number
WO2003044182A1
WO2003044182A1 PCT/JP2002/012051 JP0212051W WO03044182A1 WO 2003044182 A1 WO2003044182 A1 WO 2003044182A1 JP 0212051 W JP0212051 W JP 0212051W WO 03044182 A1 WO03044182 A1 WO 03044182A1
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protein
seq
amino acid
acid sequence
cells
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PCT/JP2002/012051
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English (en)
Japanese (ja)
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Atsushi Nakanishi
Shigeru Morita
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Takeda Chemical Industries, Ltd.
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Priority to AU2002366021A priority Critical patent/AU2002366021A1/en
Publication of WO2003044182A1 publication Critical patent/WO2003044182A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/502—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A61P11/06—Antiasthmatics
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics

Definitions

  • the present invention bronchial asthma, chronic obstructive pulmonary disease, on the usefulness mouse g ob- 5 expressing cells in the screening of an agent for the prophylaxis or treatment of diseases such as rhinitis.
  • Bronchial asthma is a chronic inflammatory disease of the respiratory tract that presents with narrowing of the airway and symptoms such as paroxysmal respiratory distress, wheezing, and coughing. Many cells are involved in its onset and progression, including airway epithelial cells, obesity cells, eosinophils, and T lymphocytes.
  • airway epithelial cells including airway epithelial cells, obesity cells, eosinophils, and T lymphocytes.
  • One of the most important features of bronchial asthma is that the airway is more responsive to stimuli (airway hyperresponsiveness)
  • This airway hyperresponsiveness is caused by inflammation of the airway, mainly the detachment of the airway epithelium by chemical mediators secreted from cells infiltrating the airway such as eosinophils, but also genetic and environmental factors are complicated. It is thought to have affected.
  • VCAM-1 and ICAM-1 Adhesion of VCAM-1 and ICAM-1 onto airway epithelial cells and capillary endothelial cells around the bronchi when triggering the inflammatory response of the airways due to external stimuli (allergens, air) or viral infections
  • the molecule is expressed [Journal of 'Alergy' and 'Tali-Cal Immunology (J. Allergy Clin. Immunol.), 96, 941 (1995)], and cytokines and chemoattractants are produced.
  • Bronchial asthma patients have enhanced function of Th2-type helper T cells, and Th2-type cytokins such as IL_3, IL-14, IL-5, IL-13, GM-CSF, eotaxin, Increased production of chemokines such as RAN TES.
  • Th2-type cytokins such as IL_3, IL-14, IL-5, IL-13, GM-CSF, eotaxin
  • Increased production of chemokines such as RAN TES.
  • IL-4 and IL-13 have an activity of inducing IgE production
  • IL-3 and IL-14 have an activity of inducing mast cell proliferation.
  • eosinophils are differentiated and proliferated by the action of IL_5, GM-CSF, etc., and infiltrate the respiratory tract by eotaxin and RANTE S [Allergy Asthma Proc.], 20 Vol.14, p.1 (1999)].
  • the epithelial cells that cover the trachea and bronchial mucous membrane are directly stimulated by the external submucosa. Not only does it have a barrier function that prevents it from being transmitted to the body, it also excretes secretions and foreign bodies, but it also controls the contraction of the trachea by secreting epithelial-derived smooth muscle relaxing factor.
  • Eosinophils that have infiltrated the airways of patients with bronchial asthma are activated and release intracellular granular proteins such as MBP (major basic protein) and ECP (eosinophil cation protein) by degranulation [ Compr. Ther., Vol. 20, pp. 65 1 (1 994)].
  • cytotoxic action of these granule proteins causes exfoliation and damage of epithelial cells.
  • Detachment of epithelial cells leads to exposure of sensory nerve terminals, increased epithelial permeability, and loss of epidermal-derived smooth muscle relaxant.
  • eosinophil-produced leukotriene C4 (LTC4) and platelet activating factor (PAF) increase the tone of bronchial smooth muscle. If these changes are repeated and chronic illness occurs, the bronchial wall may thicken, leading to airway hyperresponsiveness.
  • diseases caused by atopy include pollen derived from plants such as cedar, dust, 1
  • Allergens such as allergic rhinitis and hay fever are caused by various factors such as (2) allergens.
  • the specific manifestations of these atopic diseases include aqueous nasal discharge, nasal congestion, and sneezing.
  • the onset of these three features usually involves nasal itching within minutes of inhalation of the allergen, followed by a sneezing attack, as well as massive nasal discharge and nasal congestion.
  • Nasal congestion is persistent and lasts for several hours, and the nasal congestion may make nasal breathing difficult and, if severe, may lead to heavy head. Not only do these rhinitis symptoms cause significant discomfort to the patient over a long period of time, but the symptoms are causing a great disability to the patient's daily life.
  • Rhinitis also includes diseases caused by viral, bacterial, and fungal infections such as acute rhinitis, chronic rhinitis, and sinusitis, or allergic reactions.
  • the present invention provides a gob-5-expressing animal cell and a method for screening a therapeutic / prophylactic agent for diseases such as bronchial asthma, chronic obstructive pulmonary disease, and rhinitis using the same.
  • a therapeutic / prophylactic agent for diseases such as bronchial asthma, chronic obstructive pulmonary disease, and rhinitis using the same.
  • the introduction of the mouse-derived gob-5 expression plasmid pcDNA_gob-5 into CHO cells, BALB 3T3 cells or NCI-H292 cells> We succeeded in producing cells expressing mouse-derived gob-5 at high levels, and found that these cells were extremely useful for screening in bronchial asthma, chronic obstructive pulmonary disease, rhinitis, etc.
  • the present inventors have further studied based on these findings, and as a result, have completed the present invention.
  • gob5-CHO No. 51 (FERM BP-7805), gob5-NC capable of expressing a protein containing the amino acid sequence represented by SEQ ID NO: 1
  • a protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1, or a partial peptide thereof or a partial peptide thereof, wherein the cell described in (1) above is used.
  • gob 5 -CHO No. 51 (FERM BP-7805), gob 5 -NC I -H292 No. 3-7 (FERM BP) capable of expressing the protein containing the amino acid sequence represented by SEQ ID NO: 1 —7806) or gob 5— B ALB 3 T3 NO. 22 (FERM BP-7807), characterized by containing the cell represented by SEQ ID NO: 1, A screening kit for a compound or a salt thereof that inhibits the activity of a protein or a partial peptide thereof or a salt thereof containing substantially the same amino acid sequence,
  • the medicament according to the above (6) which is an agent for preventing or treating bronchial asthma or chronic obstructive pulmonary disease.
  • the medicament according to (6) which is a prophylactic or therapeutic agent for rhinitis.
  • a protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1 is a human protein.
  • Cells of warm-blooded animals eg, guinea pigs, rats, mice, -birds, egrets, stags, pits, hidges, magpies, monkeys, etc.
  • Bone marrow cells mesangial cells, Langerhans cells, epidermal cells, epithelial cells, goblet cells, endothelial cells, smooth muscle cells, fibroblasts, fiber cells, muscle cells, fat cells, immune cells (eg, macrophages, T cells, B cells, natural killer cells, mast cells, neutrophils, basophils, eosinophils, monocytes), megakaryocytes, synovial cells, chondrocytes, bone cells, osteoblasts Cells, osteoclasts, mammary cells,
  • the amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 1 is at least about 50%, preferably about 60% or more, preferably at least 60%, the amino acid sequence represented by SEQ ID NO: 1. About 70% or more, preferably about 80% or more, preferably about 90% or more, and preferably about 95% or more amino acid sequences having homology of about 95% or more. 12051
  • Examples of the protein having an amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 1 include, for example, a protein having an amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 1 described above. However, a protein having substantially the same activity as the protein having the amino acid sequence represented by SEQ ID NO: 1 is preferable.
  • Examples of substantially equivalent activities include, for example, chloride channel-like activities (eg, calcium-dependent chloride channel-like activities). Substantially the same means that those properties are the same (eg, physiologically or pharmacologically). Therefore, it is preferable that the chloride channel-like activities are equivalent (eg, about 0.1 to 100 times, preferably about 0.1 to 10 times, more preferably 0.5 to 2 times). However, the quantitative factors such as the degree of these activities and the molecular weight of the protein may be different.
  • the activity such as chloride channel-like activity can be measured according to a known method. For example, the method described in Genomics, Vol. 54, page 200 (1998) or according to it. It can be measured according to the method.
  • Examples of the protein having an amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 1 include, for example, an amino acid sequence represented by SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4. Contained proteins and the like.
  • Examples of the protein used in the present invention include: 1) one or more (preferably, 1 to 2) in the amino acid sequence represented by any one of SEQ ID NOs: 1 to 4; An amino acid sequence in which about 30 amino acids have been deleted, preferably about 1 to 10 amino acids, and more preferably about 1 to 5 amino acids; 2) SEQ ID NO: 1 to SEQ ID NO: 4 One or more (preferably about 1 to 30, preferably about 1 to 10, and more preferably a number (1 to 5)) amino acids in the amino acid sequence represented by the SEQ ID NO: 3) SEQ ID NO: 1 to SEQ ID NO: One or more amino acid sequences represented by any one of SEQ ID NOs: 4 (preferably about 1 to 30 amino acids, preferably 1 to 30 amino acids) Is about 1 to 10 amino acids, more preferably about 1 to 5 amino acids.
  • Acid sequence 4) SEQ ID NO: 1 to SEQ ID NO: 1 or 2 or more in the amino acid sequence shown in any of SEQ ID NO: 4 (preferred Or about 1 to 30 amino acids, preferably about 1 to 10 amino acids, and more preferably, an amino acid sequence in which several (1 to 5) amino acids have been substituted with other amino acids; or 5) So-called mucins, such as proteins containing combined amino acid sequences, are also included.
  • the position of the insertion, deletion or substitution is not particularly limited, but each of SEQ ID NO: 1 to SEQ ID NO: 4 In particular, positions other than the amino acid sequence common to the amino acid sequences represented by SEQ ID NO: 1 and SEQ ID NO: 2 are listed.
  • the left end is the N-terminus (amino terminus) and the right end is the C-terminus (carboxyl terminus) according to the convention of peptide labeling.
  • the protein used in the present invention including the protein containing the amino acid sequence represented by SEQ ID NO: 1, usually has a carboxy / le group (-COOH) or a carboxylate (one COO- ), But the C-terminal may be an amide (-CONH 2 ) or an ester (-COOR).
  • R in the ester e.g., methyl, Echiru, n- propyl, isopropyl, C, such as n- butyl, _ 6 alkyl groups, such as cyclopentyl,, C 3, such as cyclohexyl link port - 8 cycloalkyl group , for example, phenyl, - 1 2 Ariru group, e.g., benzyl, C 7 such as ⁇ - Nafuchiru ⁇ alkyl group such as a phenyl primary alkyl group or ⁇ - naphthylmethyl such phenethyl - - C 6, such as naphthyl I Ararukiru group And a piperoyloxymethyl group.
  • C such as n- butyl, _ 6 alkyl groups, such as cyclopentyl, C 3, such as cyclohexyl link port - 8 cycloalkyl group
  • the protein used in the present invention has a carboxyl group (or carbonyl group) other than the c-terminus
  • a protein in which the carboxyl group is amidated or esterified is also included in the protein used in the present invention.
  • the ester in this case, for example, the above-mentioned C-terminal ester and the like are used.
  • the amino group at the amino acid residue at the ⁇ -terminal (eg, methyoyun residue) is protected by a protecting group (eg, an acyl group such as a C alkanol such as a formyl group or an acetyl group).
  • a protecting group eg, an acyl group such as a C alkanol such as a formyl group or an acetyl group.
  • Protected, in vivo Pyroglutamine-oxidized N-terminal glutamine residue generated by cleavage, substituent on the side chain of amino acid in molecule eg, 1 OH, 1 SH, amino group, imidazole group, indole group, guar group) - Gino group, etc.
  • a suitable protecting group e.g., formyl group, those protected by Asechinore such ⁇ Bok 6 Ashiru groups such as C Bok 6 Arukanoinore group such group
  • a so-called glycoproteins sugar chain bound And other complex proteins e.g., formyl group, those protected by Asechinore such ⁇ Bok 6 Ashiru groups such as C Bok 6 Arukanoinore group such group
  • protein used in the present invention include, for example, a mouse-derived protein containing the amino acid sequence represented by SEQ ID NO: 1 (hereinafter sometimes abbreviated as gob-5), SEQ ID NO: 2 A human-derived protein containing the amino acid sequence represented by SEQ ID NO: 3; a human-derived protein containing the amino acid sequence represented by SEQ ID NO: 4 Proteins.
  • the partial peptide of the protein used in the present invention is the partial peptide of the protein used in the present invention described above, and preferably has the same properties as the protein used in the present invention described above. Any one may be used.
  • a peptide having an amino acid sequence of the 22nd to 914th amino acid sequence in the amino acid sequence represented a peptide having an amino acid sequence of the 30th to 93rd amino acid sequence in the amino acid sequence represented by SEQ ID NO: 3, SEQ ID NO:
  • the screening method or screening kit of the present invention described below includes a cell membrane binding site (eg, the 1st to 21st amino acid sequence in the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2).
  • a cell membrane binding site eg, the 1st to 21st amino acid sequence in the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2.
  • Peptides having a part of the 7th amino acid sequence are preferably used.
  • Peptides having 0 or more amino acid sequences are used.
  • one or more amino acids in the amino acid sequence are deleted. Or one or more (preferably, about 1 to 20; more preferably, about 1 to 10; more preferably, about 1 to 5) amino acids are added to the amino acid sequence.
  • one or more amino acids (preferably about 1 to 10, more preferably several, more preferably about 1 to 5) in the amino acid sequence May be substituted.
  • the partial peptide used in the present invention usually has a carboxyl group (one COOH) or a carboxylate (one COO) at the C-terminus.
  • the C-terminus has an amide (_) CO NH 2 ) or ester (-COOR).
  • the partial peptide used in the present invention has a carboxyl group (or carboxylate) in addition to the C-terminal, and the N-terminal amino acid residue (eg, , A methionine residue) whose amino group is protected with a protecting group, a glutamine residue formed by cleavage of the N-terminal side in vivo, and pyroglutamic acid residue, a substituent on the side chain of an amino acid in the molecule And those protected with an appropriate protecting group, or complex peptides such as so-called glycopeptides having a sugar chain bonded thereto.
  • the N-terminal amino acid residue eg, A methionine residue
  • the partial peptide used in the present invention can also be used as an antigen for producing an antibody.
  • Salts of proteins or partial peptides used in the present invention include salts with physiologically acceptable acids (eg, inorganic acids, organic acids) and bases (eg, alkali metal salts). And especially preferred are physiologically acceptable acid addition salts.
  • physiologically acceptable acids eg, inorganic acids, organic acids
  • bases eg, alkali metal salts
  • physiologically acceptable acid addition salts include, for example, salts with inorganic acids (eg, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid) or organic acids (eg, acetic acid, formic acid, propionic acid, fumaric acid, maleic acid) Succinic acid, tartaric acid, citric acid, malic acid, oxalic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid).
  • inorganic acids eg, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid
  • organic acids eg, acetic
  • the protein or its partial peptide or a salt thereof used in the present invention can be produced from human or warm-blooded animal cells or tissues by the known protein purification method described above. It can also be produced by culturing a transformant containing DNA. It can also be produced according to the peptide synthesis method described below.
  • the human or mammalian tissues or cells are homogenized, extracted with an acid, etc., and the resulting extract is subjected to reverse phase chromatography and ion exchange chromatography. Purification and isolation can be performed by combining chromatography such as the above.
  • a commercially available resin for protein synthesis can usually be used.
  • examples of such a tree include, for example, chloromethinole resin, hydrocyanine resin, benzhydrylamine resin, aminomethyl resin, 4-benzyloxybenzyl alcohol resin, 4-methylbenzhydrylamine resin, and PAM resin.
  • a protein or partial peptide 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 partial peptide or a compound thereof.
  • a sexi-dani reagent can be used, and in particular, carbodiimides are preferred.
  • the carbohydrates DCC, N, N'-diisopropylcarbodiimide, N-ethyl-N '-(3-dimethylaminoprolyl) carbodiimide and the like are used.
  • the protected amino acid is added directly to the resin together with a racemization inhibitor (eg, H ⁇ B t, HOOB t), or the corresponding acid anhydride or HOB t ester or HOOB t ester is added in advance.
  • the protected amino acid can be added to the resin after the activation.
  • the solvent used for activating the protected amino acid or for condensing with the resin can be appropriately selected from solvents known to be usable for the protein condensation reaction.
  • acid amides such as N, N-dimethylformamide, N, N-dimethylacetamide, N-methyl virolidone, halogenated hydrocarbons such as methylene chloride, methylformate and the like; Alcohols such as ethanol, sulfoxides such as dimethyl sulfoxide, ethers such as pyridine, dioxane, and tetrahydrofuran; -tolyls such as aceto-tolyl and propionitrile; and esters such as methyl acetate and ethyl acetate. And the like.
  • the reaction temperature is appropriately selected from a range that is known to be used for a protein bond formation reaction, and is usually appropriately selected from a range of about ⁇ 20 ° C. to 50 ° C.
  • the activated amino acid derivative is usually used in a 1.5 to 4-fold excess.
  • Examples of the protecting group for the starting amino group include Z, Boc, t-pentyloxycanoleponinole, isobornyloxycarbonyl, 4-methoxybenzyloxycarbonyl, C11Z, Br-Z Adamanchinoleoxycanoleponinole, trifluora cetyl, phthaloynole, honoleminole, 2-ethanol phenorelesnolefeninole, dipheninole phosphinoinoil, Fmoc and the like.
  • Carboxyl groups are, for example, alkyl esterified (eg, methyl, ethyl, Linear, branched or cyclic alkylesterification of propyl, butyl, t-butyl, cyclopentinole, cyclohexyl, cycloheptyl, cyclootatyl, 2-adamantyl, etc., aralkylesterification (for example, benzyl Esters, 4-Etrobenzinole Esthenol, 4-Methoxypenzinole Esthenol, 4-Methyl Benzinole Esters, Benzhydryl Esterification), Phenacil Esterification, Benzyloxyl-Polyhydrazide, T-toxy It can be protected by carbonyl hydrazide or trityl hydrazide.
  • alkyl esterified eg, methyl, ethyl, Linear, branched or cyclic alkylester
  • the hydroxyl group of serine can be protected, for example, by esterification or etherification.
  • a group suitable for this esterification for example, a group derived from carbonic acid such as a lower group such as an acetyl group and 6 ) an aroyl group such as an alkanol group and a benzoyl group, a benzyloxy carboel group, and an ethoxycarbonyl group are used.
  • Examples of a group suitable for ethereal dani include a benzyl group, a tetrahydridovinyl 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-- Torobenjiru, B r _ Z, such as t one-butyl is used.
  • Examples of the protecting group for imidazole of histidine include Tos, 4-methoxy-2,3,6-trimethylbenzenesulfonyl, DNP, benzyloxymethyl, Bum, Boc, Trt, and Fmoc. Used.
  • activated carboxyl groups of the raw materials include, for example, corresponding acid anhydrides, azides, and activated esters [alcohols (eg, pentachlorophenol, 2,4,5-trichlorophenol), 2,4-dinitro Phenol, cyanometinole alcohol, paranitrophenol, HONB, N-hydroxysuccinimide, N-hydroxyphthalimid, and esters with HOBt).
  • alcohols eg, pentachlorophenol, 2,4,5-trichlorophenol
  • 2,4-dinitro Phenol cyanometinole alcohol
  • paranitrophenol HONB
  • N-hydroxysuccinimide N-hydroxyphthalimid
  • esters with HOBt esters with HOBt
  • Methods for removing (eliminating) the protecting group include, for example, catalytic reduction in a hydrogen stream in the presence of a catalyst such as Pd-black or Pd-carbon, or hydrogen fluoride anhydride or methanesulfonic acid.
  • a catalyst such as Pd-black or Pd-carbon
  • bases such as sodium, piperidine, and piperazine, and reduction with sodium in liquid ammonia are also used.
  • the elimination reaction by the above acid treatment is generally carried out at a temperature of about 120 ° C. to 40 ° C.
  • anisol for example, anisol, phenol, thienoanisole, methcrezo monole
  • a force-thione scavenger such as Lacrezo 1 / re, dimethinoresnorole, 1,4-butanedithiole, 1,2-ethanedithiole or the like.
  • the 2,4-dinitrophenyl group used as the imidazole protecting group of histidine is removed by thiophenol treatment, and the formyl group used as the indole protecting group of tributofan is 1,2-ethanedithiol, 1,4_ In addition to deprotection by acid treatment in the presence of butanedithiol, etc., it is also removed by alkali treatment with dilute sodium hydroxide solution, dilute ammonia, etc.
  • the protection of the functional group which should not be involved in the reaction of the raw materials, the protecting group, the elimination of the protective group, the activation of the functional group involved in the reaction, and the like can be appropriately selected from known groups or known means.
  • an amide form of a protein or partial peptide for example, first, after amidation of the carboxyl group of the carboxy terminal amino acid is protected by amidation, a peptide (protein) chain is added to the amino side. After extending to the chain length, a protein or partial protein from which only the N-terminal ⁇ -amino group protecting group of the peptide chain has been removed and a partial peptide and only the C-terminal carboxyl group protecting group have been removed. A peptide is produced, and these proteins or peptides are condensed in a mixed solvent as described above. Details of the condensation reaction are the same as described above.
  • the crude protein or peptide can be purified using various known purification means, and the main fraction can be lyophilized to obtain an amide of the desired protein or peptide.
  • an ester of a protein or peptide for example, after condensing the ⁇ -hydroxyl group of the amino acid at the carboxy terminus with a desired alcohol to form an amino acid ester, Protein It is possible to obtain an ester of a quality or a peptide.
  • the partial peptide used in the present invention or a salt thereof can be produced according to a known peptide synthesis method or by cleaving the protein used in the present invention 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 partial peptide or amino acid that can constitute the partial peptide used in the present invention is condensed with the remaining portion, and when the product has a protecting group, the protecting group is eliminated to remove the target peptide. Pide can be manufactured. Examples of the known condensation method and elimination of the protecting group include the methods described in the following 1) to 5).
  • the partial peptide used in 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 or a method analogous thereto, and conversely, when the partial peptide is obtained by a salt, the known method or Can be converted into a free form or another salt by a method according to the above.
  • the polynucleotide (eg, DNA) encoding the protein used in the present invention may be any polynucleotide containing the above-described nucleotide sequence encoding the protein used in the present invention.
  • any of the genomic DNA, genomic DNA library, the above-mentioned cDNA derived from the cell's tissue, the above-mentioned cells, the cDNA library derived from Itotori, and synthetic DNA may be used.
  • Vectors used for library are bacteriophage, plasmid, And phagemid.
  • RT-PCR method reverse transcriptase polymerase chain reaction
  • DNA encoding the protein used in the present invention examples include, for example, DNA containing the nucleotide sequence represented by SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 or SEQ ID NO: 8, or SEQ ID NO: : 5, SEQ ID NO: 6, SEQ ID NO: 7 or SEQ ID NO: 8, containing a nucleotide sequence that hybridizes under high stringent conditions with the nucleotide sequence represented by SEQ ID NO: 8, SEQ ID NO: 1, SEQ ID NO: Any DNA may be used as long as it encodes a protein having substantially the same properties as the protein having the amino acid sequence represented by SEQ ID NO: 3 or SEQ ID NO: 4.
  • Examples of the DNA that can hybridize with the base sequence represented by SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8 under high stringent conditions include, for example, SEQ ID NO: 5, sequence About 50% or more, preferably about 60% or more, more preferably about 70% or more, more preferably about 80% with the nucleotide sequence represented by SEQ ID NO: 6, SEQ ID NO: 7 or SEQ ID NO: 8 As described above, DNA containing a nucleotide sequence having a homology of about 90% or more, most preferably about 95% or more is particularly preferably used.
  • Hybridization is performed according to a known method or a method analogous thereto, for example, the method described in Molecular Cloning 2nd (J. Sambrook et al., Cold Spring Harbor Lab. Press, 1989). be able to.
  • a commercially available library it can be performed according to the method described in the attached instruction manual. More preferably, it can be carried out under high stringency conditions.
  • High stringency conditions include, for example, a sodium concentration of about 19 to 40 mM, preferably about 19 to 20 mM, and a temperature of about 50 to 70 ° C, preferably about 60 to 100 ° C. The conditions at 65 ° C are shown. In particular, the sodium concentration is about 19 mM and the temperature is about 65. Is most preferred.
  • a protein containing the amino acid sequence represented by SEQ ID NO: 1 For example, DNA containing the nucleotide sequence represented by SEQ ID NO: 5 is used as the DNA encoding As the DNA encoding the protein containing the amino acid sequence represented by SEQ ID NO: 2, a DNA containing the base sequence represented by SEQ ID NO: 6 or the like is used. As the DNA encoding the protein containing the amino acid sequence represented by SEQ ID NO: 3, a DNA containing the base sequence represented by SEQ ID NO: 7 and the like are used. As the DNA encoding the protein containing the amino acid sequence represented by SEQ ID NO: 4, a DNA containing the base sequence represented by SEQ ID NO: 8 and the like are used.
  • any DNA may be used as long as it contains the base sequence encoding the partial peptide used in the present invention described above. Further, 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 lignitori, and synthetic DNA.
  • Examples of the DNA encoding the partial peptide used in the present invention include, for example, a DNA having a part of a DNA having a base sequence represented by SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 or SEQ ID NO: 8. Or a nucleotide sequence that hybridizes with the nucleotide sequence represented by SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 or SEQ ID NO: 8 under high stringent conditions, and substantially corresponds to the protein of the present invention. For example, a DNA containing a part of a DNA encoding a protein having a similar activity is used.
  • the DNA hybridizable to the base sequence represented by SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 or SEQ ID NO: 8 has the same significance as described above.
  • Hybridization methods and high stringency conditions are the same as described above.
  • the cloning means may be amplified by PCR using a synthetic DNA primer having a part of the nucleotide sequence encoding the protein of the present invention, or assembled into an appropriate vector.
  • the inserted DNA can be selected by hybridization with a DNA fragment encoding a part or the entire region of the protein of the present invention or a DNA fragment labeled with a synthetic DNA.
  • the method of hybridization can be performed, for example, according to the method described in Molecular Cloning 2nd (J. Sambrook et al., Cold Spring Harbor Lab. Press, 1989). When a commercially available library is used, it can be performed according to the method described in the attached instruction manual.
  • the nucleotide sequence of DNA can be replaced by ODA-LA PCR or Gapped using PCR or a known kit, for example, Mutan TM _super Express Km (Takara Shuzo), Mutan TM _K (Takara Shuzo) or the like. It can be performed according to a known method such as the duplex method or the Kunkel method, or a method analogous thereto.
  • the DNA encoding the cloned protein can be used as it is depending on the purpose, or can be used after digesting with a restriction enzyme or adding a linker, if desired.
  • the DNA may have ATG as a translation initiation codon at the 5 'end and TAA, TGA or TAG as a translation termination codon at the 3' end. These translation initiation codon and translation termination codon can also be added using an appropriate synthetic DNA adapter.
  • the expression vector for the protein of the present invention includes, for example, (a) cutting out a DNA fragment of interest from DNA encoding the protein of the present invention, and (b) ligating the DNA fragment downstream of a promoter in an appropriate expression vector. It can be manufactured by
  • Escherichia coli-derived plasmids eg, pBR322, pBR325, pUC12, pUC13
  • Bacillus subtilis-derived plasmids eg, pUB110, pTP5, pC194
  • yeast-derived vectors In addition to plasmids (eg, pSH1 9, pSH15), pacteriophages such as ⁇ phage, animal viruses such as retroviruses, vaccinia vires / res, paculoinores, etc., pAl-11, ⁇ 1, Rc / CMV, pRc / RSV, pcDNAI / Neo and the like are used.
  • the promoter used in the present invention may be any promoter as long as it is appropriate for the host used for gene expression. For example, house animal cells 1
  • SRa promoter When used mainly, SRa promoter, SV40 promoter, LTR motor, CMV promoter, HSV-TK promoter and the like can be mentioned. Of these, CMV (cytomegalovirus) promoter, SR promoter and the like are preferably used. .
  • CMV cytomegalovirus
  • the host is a bacterium belonging to the genus Escherichia, the trp promoter, lac promoter, recA promoter, P L promoter, lpp promoter, T7 promoter, etc .; if the host is a bacterium belonging to the genus Bacillus, the SPO1 promoter; When the host is yeast, such as SP02 promoter and penP promoter, PHO5 promoter, PGK promoter, GAP promoter, ADH promoter and the like are preferable. When the host is an insect cell, a polyhedrin promoter, a P10 promoter and the like are preferable.
  • the expression vector may further contain, if desired, an enhancer, a splicing signal, a polyA addition signal, a selection marker, an SV40 replication origin (hereinafter sometimes abbreviated as SV400 ri), and the like.
  • a selection marker include dihydrofolate reductase (hereinafter sometimes abbreviated as dhfr) gene [methotrexate (MTX) resistance], ampicillin phosphorus resistant gene (hereinafter sometimes abbreviated as Amp r), neomycin resistant gene (hereinafter sometimes abbreviated as Ne o r, G4 1 8 resistance).
  • dhfr gene is used as a selection marker using Chinese hamster cells deficient in the dhfr gene
  • the target gene can be selected using a thymidine-free medium.
  • a signal sequence suitable for the host is added to the N-terminal side of the protein of the present invention.
  • the host is a bacterium belonging to the genus Escherichia, a Ph0A signal sequence, an OmpA signal sequence, and the like. If the host is yeast, MFa signal sequence, SUC2 signal sequence, etc.If the host is an animal cell, insulin signal sequence, ainterferin signal sequence, antibody molecule, signal sequence Etc. can be used respectively.
  • a transformant can be produced.
  • Escherichia bacteria for example, Escherichia bacteria, Bacillus bacteria, yeast, insect cells, insects, animal cells, and the like are used.
  • Escherichia examples include, for example, Eschericia coli 12 and DH1 [Processing's of the National Academy ' (Proc. Natl. Acad. Sc i. USA) s 60, 160 (1968)], JM103 (Nucleic Acids Research, Vol. 9, 309 (1981)), JA 221 (Jananore) Journal of Molecular Biology), 120 vol., 517 (1978)), HB 101 (Journal of Op. Molecular Biology, 41, 459 (1969)), C600 [Genetics, 39 volumes, 440 (1954)] and the like are used.
  • Bacillus bacteria examples include, for example, Bacillus subtilis MI 114 [Gene, 24, 255 (1983)], 207-21 (Journal of Biochemistry), 95 , 87 (1984)].
  • yeast examples include, for example, Saccharomyces cerevisiae (Saccharomyces cerevisiae) AH22, AH22R-1, NA87-11A, DKD-5D, 20B-12, Schizosaccharomyces. Pichia pastoris KM71 or the like is used.
  • Insect cells include, for example, when the virus is Ac NPV, a cell line derived from the larva of Spodoptera (Spodoptera frugiperda cell; Sf cell), MGl cells derived from the midgut of Trichoplusia, and High Five TM derived from eggs of Trichoplusia ni Cells, cells derived from Mamestra brassicae or cells derived from Estigmena acrea are used. ⁇ If the virus is BmNP V, a silkworm-derived cell line (Bombyx mori N cell; BmNPV N cells) are used. Examples of the Sf cells include Sf9 cells (ATCC CRL1711) and Sf21 cells (Vaughn, JL et al., In Vivo, 13, 213-21 7 (1977)). Are used.
  • insects for example, silkworm larvae are used [Maeda et al., Nature, 315, 592 (1985)].
  • animal cells examples include monkey cell COS-7, Vero, Chinese Hamster cell CHO (hereinafter abbreviated as CHO cell), dh fr gene-deficient hamster cell CHO cell (hereinafter abbreviated as CHO (dh fr—) cell). ), Mouse L cells, mouse AtT-20, mouse myeloma cells, rat GH3, human FL cells, BALB 3T3 cells, NCI-H292 cells (ATCC No. CRL-1848), and the like.
  • CHO cell Chinese Hamster cell CHO
  • CHO (dh fr—) cell dh fr—) cell
  • Mouse L cells mouse AtT-20, mouse myeloma cells, rat GH3, human FL cells, BALB 3T3 cells, NCI-H292 cells (ATCC No. CRL-1848), and the like.
  • CHO cells, BALB 3T3 cells, and NCI-H292 cells are preferably used as hosts for the gob-5 expression vector.
  • Insect cells or insects can be transformed, for example, according to the method described in 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 (1995) (published by Shujunsha), Virology, Vol. 52, 456 (1973). Can be performed according to the method described in Thus, a transformant transformed with the expression vector containing the DNA encoding the protein can be obtained.
  • CHO cells transformed with the gob-5 expression vector eg, gob5-CHO No. 51; FERM BP-7805
  • NC transformed with the gob-5 expression vector I-H292 cells eg, gob 5 -NC I-H 292 No. 3-7; F ERM BP-7806
  • B ALB 3 T3 cells transformed with the gob-5 expression vector eg, gob 5—BALB 3 T 3 NO. 22; FERM BP-7807
  • a liquid medium is suitable as a medium used for the cultivation, and a carbon source necessary for the growth of the transformant is contained therein.
  • Nitrogen sources inorganic substances and others.
  • carbon sources include glucose, dextrin, soluble starch, and rice bran.
  • nitrogen sources include ammonium salts, nitrates, corn chip liquor, peptone, casein, meat extract, soybean meal, and potato extract.
  • the inorganic or organic substance and the inorganic substance include calcium chloride, sodium dihydrogen phosphate, magnesium chloride and the like.
  • yeast extract, vitamins, growth promoting factors and the like may be added.
  • the pH of the medium is preferably about 5-8.
  • a medium for culturing the genus Escherichia for example, an M9 medium containing glucose and casamino acids (Miller, Journal 'Op' Experimen, J., Journal of Experiments in Molecular Genetics), 431-433, Cold Spring Harbor Laboratory, New York (1972)].
  • a drug such as 3] 3-indolylacrylic acid can be added to make the promoter work efficiently.
  • the cultivation is usually carried out at about 15 to 43 ° C for about 3 to 24 hours, and if necessary, aeration and stirring may 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 may be added.
  • the culture medium may be, for example, Parkholder's minimal medium [Bostian, KL et al., Processings of the "National 'Academy' of Sciences”. Opc The USA (Proc. Natl. Acad. Sci. USA), 77 vol., 4505 (1980)] and an SD medium containing 0.5% casamino acid [Bitter, GA et al., Processings • Prob . Natl. Acad. Sci. USA, 81, 5330 (1984)].
  • the pH of the medium is adjusted to about 5-8. Culture is usually carried out at about 20 ° C to 35 ° C for about 24 to 72 hours, and aeration and / or agitation are added as necessary.
  • the culture medium When culturing an insect cell or a transformant in which the host is an insect, the culture medium is 10% inactivated in Grace's Insect Medium (Grace, ⁇ CC, Nature, 195, 788 (1962)). Those to which additives such as serum are appropriately added are used.
  • the pH of the medium is preferably adjusted to about 6.2 to 6.4. Culture is usually performed at about 27 ° C for about 3 to 5 days, and aeration and agitation are added as necessary.
  • 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, 501 (1952)], a DMEM medium [Virology, Vol. 8, 396 (1959)], RPMI 1640 medium [Journal of the American Medical Association], 199, 51 9 (1967)], 199 medium [Proceeding of the Society for the Biological Medicine, Medicine, 73 vol., 1 (1950)], etc. Used.
  • the pH is about 6-8. Cultivation is usually carried out at about 30 ° C to 40 ° C for about 15 to 60 hours, and aeration and stirring are added as necessary.
  • the protein of the present invention can be produced in the cells, in the cell membrane, or outside the cells of the transformant.
  • the following method Can be performed.
  • the cells or cells are collected by a known method, suspended in an appropriate buffer, and subjected to ultrasonic wave, lysozyme and / or freeze-thawing. After the cells or cells are destroyed by the method, a method of obtaining a crude extract of the protein by centrifugation or filtration is used as appropriate.
  • the buffer may contain a protein denaturant such as urea or guadin hydrochloride, or a surfactant such as Triton X-100 TM .
  • Purification of the protein contained in the culture supernatant or extract obtained in this manner can be performed by appropriately combining known separation and purification methods.
  • These known separation and purification methods include methods utilizing solubility such as salting out and solvent precipitation.
  • Methods that use specific affinity such as affinity chromatography, methods that use differences in hydrophobicity such as reversed-phase high-performance liquid chromatography, and methods that use differences in isoelectric points such as isoelectric focusing Etc. are used.
  • the protein obtained by force When the protein obtained by force is obtained in a free form, it can be converted to a salt by a known method or a method analogous thereto. It can be converted into a free form or another salt by an analogous method.
  • the protein produced by the recombinant can be arbitrarily modified or the polypeptide can be partially removed by applying an appropriate protein-modifying enzyme before or after purification.
  • an appropriate protein-modifying enzyme for example, trypsin, chymotrypsin, arginyl endopeptidase, protein kinase, glycosidase and the like are used.
  • the presence of the protein of the present invention produced by force can be measured by, for example, enzymatic immunoassay western blotting using a specific antibody.
  • the antibody against the protein used in the present invention may be any of a polyclonal antibody and a monoclonal antibody as long as it can recognize the protein used in the present invention.
  • An antibody against the protein used in the present invention can be produced according to a known antibody or antiserum production method using the protein of the present invention as an antigen. [Preparation of monoclonal antibody]
  • the protein used in the present invention is administered to a warm-blooded animal at a site where antibody production is possible by administration, or it is administered together with a carrier or a diluent.
  • Complete Freund's adjuvant or incomplete Freund's adjuvant may be administered to enhance the antibody-producing ability upon administration.
  • the administration is usually performed once every 2 to 6 weeks, for a total of about 2 to 10 times.
  • Examples of the warm-blooded animal to be used include monkeys, egrets, dogs, guinea pigs, mice, rats, sheep, goats, and chickens, and mice and rats are preferably used.
  • a warm-blooded animal immunized with an antigen for example, an individual with an antibody titer is selected from a mouse, and the spleen or lymph node is collected 2 to 5 days after the final immunization and contained in them.
  • an individual with an antibody titer is selected from a mouse, and the spleen or lymph node is collected 2 to 5 days after the final immunization and contained in them.
  • a monoclonal antibody-producing hybridoma can be prepared.
  • the antibody titer in the antiserum can be measured, for example, by reacting a labeled protein described below with the antiserum, and then measuring the activity of the labeling agent bound to the antibody.
  • the fusion operation can be performed according to a known method, for example, the method of Koehler and Milstein [Nature, 256, 495 (1975)].
  • the fusion promoter include polyethylene glycol (PEG) and Sendai virus, but PEG is preferably used.
  • myeloma cells examples include myeloma cells of warm-blooded animals such as NS-1, P3U1, SP20, and AP-1, but P3U1 is preferably used.
  • the preferred ratio between the number of antibody-producing cells (spleen cells) used and the number of myeloma cells used is about 1: 1 to 20: 1, and PEG (preferably PEG 1000 to PEG6000) Is added at a concentration of about 10 to 80%, and incubating at 20 to 40 ° C., preferably 30 to 37 ° C. for 1 to 10 minutes allows efficient cell fusion.
  • hybridoma culture supernatant is added to a solid phase (eg, a microplate) on which protein antigens are directly or adsorbed together with a carrier.
  • a solid phase eg, a microplate
  • an anti-immunoglobulin antibody labeled with a radioactive substance or enzyme (anti-mouse immunoglobulin antibody is used if the cells used for cell fusion are mouse) or protein A
  • a monoclonal antibody bound to the solid phase A monoclonal antibody bound to a solid phase is prepared by adding a hybridoma culture supernatant to a solid phase to which an anti-immunoglobulin antibody or protein A is adsorbed, adding a protein labeled with a radioactive substance, an enzyme, or the like. And a method for detecting antibody.
  • the selection of the monoclonal antibody can be performed according to a known method or a method analogous thereto. Usually, it can be performed in an animal cell culture medium supplemented with HAT (hypoxanthine, aminopterin, thymidine).
  • HAT hyperxanthine, aminopterin, thymidine
  • any medium can be used as long as it can grow a hybridoma.
  • a RPMI 1640 medium containing 1 to 20%, preferably 10 to 20% fetal bovine serum
  • a GIT medium containing 1 to 10% fetal bovine serum (Wako Pure Chemical Industries, Ltd.
  • serum-free medium for hybridoma culture SFM-101, Nissui Pharmaceutical Co., Ltd.).
  • the culture temperature is usually from 20 to 40 ° C, preferably about 37 ° C.
  • the culture time is generally 5 days to 3 weeks, preferably 1 week to 2 weeks.
  • the culture can be usually performed under 5% carbon dioxide gas.
  • the antibody titer of the hybridoma culture supernatant can be measured in the same manner as the measurement of the antibody titer in the antiserum described above.
  • Monoclonal antibodies can be separated and purified by known methods, for example, immunoglobulin separation and purification methods (eg, salting out method, alcohol precipitation method, isoelectric point precipitation method, electrophoresis method, ion exchanger (eg, DEAE)). Adsorption-desorption method, ultracentrifugation method, gel filtration method, antigen-binding solid phase or specific purification method in which the antibody is collected using an active adsorbent such as protein A or protein G and the bond is dissociated to obtain the antibody). Can be done. (Preparation of polyclonal antibody)
  • immunoglobulin separation and purification methods eg, salting out method, alcohol precipitation method, isoelectric point precipitation method, electrophoresis method, ion exchanger (eg, DEAE)
  • the polyclonal antibody against the protein of the present invention can be produced according to a known method or a method analogous thereto.
  • an immune antigen protein antigen
  • a complex thereof with a carrier protein is formed, and immunization is performed on a warm-blooded animal in the same manner as in the above-described method for producing a monoclonal antibody.
  • the antibody can be produced by collecting the substance containing the antibody against the protein and separating and purifying the antibody.
  • the type of carrier protein and the mixing ratio of the carrier and the hapten depend on the antibody against the hapten immunized by crosslinking the carrier. If efficient, any kind of cross-linking may be used at any ratio.For example, ⁇ serum albumin ⁇ ⁇ psiloglopurine, hemocyanin, etc. may be used at a weight ratio of about 0 to 1 for hapten. A method of coupling at a rate of 1 to 20, preferably about 1 to 5 is used. In addition, various condensing agents can be used for force coupling between the hapten and the carrier.
  • glutaraldehyde ⁇ carpoimide a maleimide active ester, an active ester reagent containing a thiol group or a dithioviridyl group, or the like is used.
  • the condensation product is administered to a warm-blooded animal itself or together with a carrier or diluent at a site where antibody production is possible.
  • Complete Freund's adjuvant / incomplete Freund's adjuvant may be administered in order to enhance the antibody-producing ability upon administration. The dose is usually given once every 2 to 6 weeks, about 3 to 10 times in total.
  • the polyclonal antibody can be collected from the blood, ascites, etc., preferably from the blood of a warm-blooded animal immunized by the above method.
  • the polyclonal antibody titer in the antiserum can be measured in the same manner as the measurement of the antibody titer in the antiserum 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-described separation and purification of the monoclonal antibody.
  • gob 5 CHO No. 51 (FERM BP—7805), gob 5-NC I-H292 No. 3— of the present invention, which can express the protein containing the amino acid sequence represented by SEQ ID NO: 1 7 (FERM BP— 7806) Describes a method for screening a drug candidate for a disease using the cell indicated by gob 5-BALB 3 T3 NO. 22 (FERM BP-7807).
  • gob-5 expressing cells referred to as gob-5 expressing cells.
  • the compound or a salt thereof that inhibits the activity or expression of the protein of the present invention may be used in lungs and airways such as bronchial asthma and chronic obstructive pulmonary disease. It can be used as a medicament such as an agent for preventing and treating lungs with inflammation of the chest and chest.
  • rhinitis eg, allergic rhinitis, hay fever, acute rhinitis, chronic rhinitis. It can be used as a prophylactic / therapeutic agent for rhinitis, hypertrophic rhinitis, atrophic rhinitis, dry rhinitis, vasomotor rhinitis, gangrenous rhinitis, sinusitis, etc.).
  • the mouse gob-5 expressing cell of the present invention is useful as a reagent for screening a compound or a salt thereof that inhibits the activity or expression of the protein used in the present invention.
  • a compound or a salt thereof that inhibits the activity (for example, chloride channel-like activity) of the protein of the present invention, which is characterized by using the mouse gob-5-expressing cell of the present invention (hereinafter, referred to as a salt thereof).
  • the screening method may be abbreviated as an inhibitor.
  • the chloride channel-like activities of the protein of the present invention in the cases (i) and (ii) are measured and compared.
  • ionomycin As a calcium activator, ionomycin, A23187 (calcimicin) and the like are used.
  • Test compounds include, for example, peptides, proteins, non-peptidic compounds, Examples thereof include a synthetic compound, a fermentation product, a cell extract, a plant extract, and an animal tissue extract. These compounds may be novel compounds or known compounds.
  • the mouse gob-5 expressing cells of the present invention are prepared by suspending them in a buffer suitable for screening.
  • the buffer may be any buffer which does not inhibit the chloride channel-like activity of the protein of the present invention, such as 11 to 4 to 10 (preferably 11 to 6 to 8) phosphate buffer and borate buffer. ! / It may be shifted.
  • the chloride channel-like activity of the protein of the present invention can be measured by a known method, for example, the method described in Genomics, Vol. 54, p. 200 (1998) or a method analogous thereto.
  • a compound or a salt thereof that inhibits the expression of the gene encoding the protein of the present invention may be used for bronchial asthma or chronic obstructive disease. It can be used as a medicament such as pulmonary disease, lung with airway inflammation, prevention of thoracic disease, and remedy.
  • compounds that inhibit the expression of the gene encoding the protein of the present invention or salts thereof also include rhinitis (eg, allergic rhinitis, It can be used as a prophylactic / therapeutic agent for hay fever, acute rhinitis, chronic rhinitis, hypertrophic rhinitis, atrophic rhinitis, dry rhinitis, vasomotor rhinitis, gangrene rhinitis, sinusitis, etc.).
  • rhinitis eg, allergic rhinitis, It can be used as a prophylactic / therapeutic agent for hay fever, acute rhinitis, chronic rhinitis, hypertrophic rhinitis, atrophic rhinitis, dry rhinitis, vasomotor rhinitis, gangrene rhinitis, sinusitis, etc.
  • the DNA of the present invention is useful as a reagent for screening a compound or a salt thereof (hereinafter, abbreviated as an inhibitor) that inhibits the expression of a gene encoding the protein of the present invention.
  • the screening methods include (iii) culturing the mouse gob-5 expressing cells of the present invention, and (iv) mouse gob-5 expression of the present invention in the presence of the test compound.
  • a method of screening for an inhibitor which is characterized by performing a comparison with the case where a current cell is cultured, may be mentioned.
  • the expression level of the gene (specifically, the amount of the protein of the present invention or the amount of mRNA encoding the protein) in (iii) and (iv) is measured and compared.
  • test compound those similar to the above can be mentioned.
  • the amount of the protein is measured by a known method, for example, using an antibody recognizing the protein of the present invention, and analyzing the protein present in a cell extract or the like according to a method such as Western analysis or ELISA method or a method analogous thereto.
  • the amount of mRNA that can be measured is determined by a known method, for example, using a nucleic acid containing SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8 or a part thereof as a probe. It can be measured according to Northern hybridization or a PCR method using the base sequence represented by SEQ ID NO: 5 or a part thereof as a primer or a method analogous thereto.
  • a test compound that inhibits the gene expression level in the case of the above (iv) by about 20% or more, preferably 30% or more, more preferably about 50% or more as compared with the case of the above (iii) can be selected as a compound that suppresses the expression of the gene encoding the protein of the present invention.
  • the screening kit of the present invention contains the mouse gob-5 expressing cell of the present invention.
  • the compound or a salt thereof obtained by using the screening method or the screening kit of the present invention may be a test compound described above, for example, a peptide, a protein, a non-peptide '("a raw compound, a synthetic compound, a fermentation product, a cell extract, A compound selected from plant extracts, animal tissue extracts, plasma, and the like, or a salt thereof, which is a compound or a salt thereof that inhibits the activity (eg, chloride channel-like activity) of the protein of the present invention. .
  • the compound or its salt that inhibits the activity of the protein of the present invention, or the compound or its salt that inhibits the expression of the gene encoding the protein of the present invention may be, for example, a lung, a lung associated with inflammation of the respiratory tract, a chest disease, or a respiratory tract.
  • Respiratory disease eg, chronic obstructive pulmonary disease (eg, chronic bronchitis, emphysema, diffuse panbronchiolitis, intrinsic asthma, etc.), bronchial asthma, cystic fibrosis, irritable pneumonia, pulmonary fibrosis, etc. It is useful as a medicament such as a prophylactic or therapeutic agent.
  • rhinitis eg, allergic rhinitis, hay fever, acute rhinitis, It is useful as a prophylactic and therapeutic agent for chronic rhinitis, hypertrophic rhinitis, atrophic rhinitis, dry pronasal rhinitis, vasomotor rhinitis, psoriasis rhinitis, sinusitis, etc.
  • rhinitis eg, allergic rhinitis, hay fever, acute rhinitis, It is useful as a prophylactic and therapeutic agent for chronic rhinitis, hypertrophic rhinitis, atrophic rhinitis, dry pronasal rhinitis, vasomotor rhinitis, psoriasis rhinitis, sinusitis, etc.
  • 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 prophylactic / therapeutic agent, it can be formulated according to a conventional method.
  • a conventional method for example, tablets, capsules, elixirs, micron capsules, sterile solutions, suspensions and the like can be used.
  • the preparations obtained in this way are safe and low toxic, and can be used, for example, in humans or warm-blooded animals (eg, mice, rats, puppies, sheep, stags, puppies, puppies, pumas, birds, cats, dogs, Can be administered orally or parenterally to monkeys, chimpanzees, etc.).
  • warm-blooded animals eg, mice, rats, puppies, sheep, stags, puppies, puppies, pumas, birds, cats, dogs, Can be administered orally or parenterally to monkeys, chimpanzees, etc.
  • the dose of the compound or a salt thereof varies depending on its action, target disease, subject to be administered, route of administration, and the like.
  • a compound or a compound thereof that inhibits the activity of the protein of the present invention for the purpose of treating bronchial asthma When a salt is orally administered, generally, in an adult (assuming a body weight of 60 kg), about 0.1 to 100 mg, preferably about 1.0 to 5 mg of the compound or a salt thereof per day is administered. 0 mg, more preferably about 1.0-20 mg.
  • the single dose of the compound or a salt thereof varies depending on the administration subject, target disease, and the like.
  • a compound or a compound that inhibits the activity of the protein of the present invention for the purpose of treating bronchial asthma When the salt is administered to an adult (with a body weight of 6 O kg) in the form of an injection, usually about 0.01 to 30 mg, preferably about 0 to 30 mg of the lig compound or its salt per day.
  • bases, amino acids and the like are indicated by abbreviations based on the abbreviations by IUPAC-IUB Commission on Biochemical Nomenclature or abbreviations used in the art. Examples thereof are described below.
  • amino acids may have optical isomers, L-form shall be indicated unless otherwise specified.
  • Tr p Tribute fan
  • SEQ ID NO: 8 This shows the nucleotide sequence of DNA encoding the human CLCA4 protein having the amino acid sequence represented by SEQ ID NO: 4.
  • Gob 5— CHO No. 51 obtained in Example 1 is an independent administrative agency of the Industrial Technology Research Institute of Chuo No. 6 (Zip code 305-8566), 1-1 1-1 Higashi, Tsukuba-shi, Ibaraki since January 19, 2001. Laboratory Deposited at the Patent Organism Depositary Center as FERM BP-7805.
  • Example 2 BALB 3 T 3 NO.22 obtained in Example 1 was obtained from January 19, 2001 at Tsukuba East 1-chome, Ibaraki Pref. 1 Central 1 (City No. 305 -8566) Deposited with the Patent Organism Depositary of the National Institute of Advanced Industrial Science and Technology as FE RM BP-7807.
  • Example 3 BALB 3 T 3 NO.22 obtained in Example 1 was obtained from January 19, 2001 at Tsukuba East 1-chome, Ibaraki Pref. 1 Central 1 (City No. 305 -8566) Deposited with the Patent Organism Depositary of the National Institute of Advanced Industrial Science and Technology as FE RM BP-7807.
  • Example 1 Example 1
  • lungs and lungs with inflammation of the respiratory tract can be used. Bronchitis, endogenous asthma, etc.), bronchial asthma, cystic fibrosis, irritable pneumonia, pulmonary fibrosis, etc.), rhinitis, etc. can do.

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  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Microbiology (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Food Science & Technology (AREA)
  • Zoology (AREA)
  • Biophysics (AREA)
  • Genetics & Genomics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Peptides Or Proteins (AREA)

Abstract

L'invention concerne des cellules exprimant gob-5. Plus spécifiquement, l'invention concerne une méthode de criblage d'un médicament prophylactique ou d'un remède pour les maladies respiratoires telles que l'asthme, la broncho-pneumopathie chronique obstructive ou la néphrite à l'aide de cellules exprimant gob-5 et d'autres éléments semblables.
PCT/JP2002/012051 2001-11-20 2002-11-19 Cellules exprimant gob-5 et utilisation WO2003044182A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU2002366021A AU2002366021A1 (en) 2001-11-20 2002-11-19 CELLS EXPRESSING gob-5 AND USE THEREOF

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP2001-354262 2001-11-20
JP2001354262 2001-11-20

Publications (1)

Publication Number Publication Date
WO2003044182A1 true WO2003044182A1 (fr) 2003-05-30

Family

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Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP2002/012051 WO2003044182A1 (fr) 2001-11-20 2002-11-19 Cellules exprimant gob-5 et utilisation

Country Status (2)

Country Link
AU (1) AU2002366021A1 (fr)
WO (1) WO2003044182A1 (fr)

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999044620A1 (fr) * 1998-03-03 1999-09-10 Magainin Pharmaceuticals, Inc. Facteurs associes a l'asthme servant de cibles pour le traitement d'allergies atopiques dont l'asthme et des troubles associes
WO2001038530A1 (fr) * 1999-11-24 2001-05-31 Takeda Chemical Industries, Ltd. Utilisation d'un gene associe a une maladie
WO2002014366A2 (fr) * 2000-08-16 2002-02-21 Universiteit Utrecht Genes impliques dans la reponse immunitaire observee dans l'asthme

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999044620A1 (fr) * 1998-03-03 1999-09-10 Magainin Pharmaceuticals, Inc. Facteurs associes a l'asthme servant de cibles pour le traitement d'allergies atopiques dont l'asthme et des troubles associes
WO2001038530A1 (fr) * 1999-11-24 2001-05-31 Takeda Chemical Industries, Ltd. Utilisation d'un gene associe a une maladie
WO2002014366A2 (fr) * 2000-08-16 2002-02-21 Universiteit Utrecht Genes impliques dans la reponse immunitaire observee dans l'asthme

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
GRUBER A.D. ET AL.: "Genomic cloning, molecular characterization and functional analysis of human CLCA1, the first human member of the family of Ca2+-activated C1 channel proteins", GENOMICS, vol. 54, no. 2, 1998, pages 200 - 214, XP002945247 *
KOMIYA T. ET AL.: "Cloning and identification of the gene gob-5, which is expressed in intestinal goblet cells in mice", BIOCHEM. BIOPHYS. RES. COMMUN., vol. 255, no. 2, 1999, pages 347 - 351, XP002936001 *
NAKANISHI A. ET AL.: "Role of gob-5 in mucus overproduction and airway hyperresponsiveness in asthma", PROC. NATL. ACAD. SCI. USA, vol. 98, no. 9, April 2001 (2001-04-01), pages 5175 - 5180, XP002227786 *

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