WO2003062426A1

WO2003062426A1 - Novel protein, its dna and use thereof

Info

Publication number: WO2003062426A1
Application number: PCT/JP2003/000408
Authority: WO
Inventors: Atsushi Nakanishi; Shigeru Morita
Original assignee: Takeda Chemical Industries, Ltd.
Priority date: 2002-01-21
Filing date: 2003-01-20
Publication date: 2003-07-31

Abstract

A protein and a polynucleotide which are useful as diagnostic markers for respiratory diseases, nephritis, digestive diseases, etc. Inhibitors obtained by screening with the use of the above protein or polynucleotide or a neutralizing antibody inhibiting the activity of the protein are usable as, for example, preventives/remedies for the above-described diseases.

Description

Description New protein, its DNA and its application Technical field

The present invention relates to a novel protein, DNA encoding the protein, a method for screening a compound that inhibits the activity of the protein, a compound obtained by the screening method, and the like. More specifically, the present invention relates to a novel protein useful as a prophylactic / therapeutic agent or diagnostic agent for respiratory diseases, digestive diseases, and the like. Background art

Chronic obstructive pulmonary disease (chronic bronchitis, emphysema) is considered to become the central disease of respiratory illness with the aging of the smoking generation and prolonged life expectancy.

Bronchial asthma is a chronic inflammatory disease of the respiratory tract, with airway narrowing and paroxysmal dyspnea, wheezing, and coughing. Many cells are involved in its development and progression, including airway epithelial cells, mast 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 hyperreactivity is caused by inflammation of the airway, mainly by detachment of the airway epithelium by chemical mediators secreted from cells infiltrating the airway such as eosinophils, but genetic factors and environmental factors also have complex effects Is believed to be. When an inflammation reaction in the airways is triggered by external stimuli (allergens, exhausts) or viral infection, adhesion molecules such as VCAM-K ICAM-1 are found on airway epithelial cells and capillary endothelial cells around the bronchi. (J. Allergy Cl in. I Thigh unol., 96, 941 (1995)), and the site force and chemical migrants Produced. Patients with bronchial asthma have enhanced function of Th2-type helper T cells, and Th2-type cytokines such as IL-3, IL-4, IL_5, IL-13, and GM-CSF. Chemokine production increases. IL-4 and IL-13 induce IgE production, and IL-3 and IL-4 increase mast cells. There is a breeding induction effect. In addition, eosinophils are differentiated and proliferated by the action of IL-5, GM-CSF, etc., and infiltrate the respiratory tract by eotaxin and RANTES (Allergy Asthma Proc.), 20, 141 (1999)].

The DNA and protein of human-derived CLCA (Cl-channel, Ca ²⁺ -activated) 4 belong to the power channel-dependent chloride channel protein family, although they belong to FEBS Let ters, Vol. 455, p. 295. (1999)], No association with chronic obstructive pulmonary disease (C0PD; Chronic Obstructive Pulmonary disease) or bronchial asthma is known.

'On the other hand, the nucleotide sequence of the human CLCA1 gene has been reported in Genomics, Vol. 54, p. 200 (1998), Biochemical and Biophysical Research Communications, Biochem Biophys Res Commun, Vol. This gene has been reported to be associated with bronchial asthma and chronic obstructive pulmonary disease (W001 / 38530). The one that shows homology to the base sequence of the human CLCA1 gene is the base sequence of the mouse gob-5 gene (Biochemical and Biophysical Research Communication (Biochem.

Biophys. Res. Commun.), 255, 347 (1999)], and the base sequence of porcine CLCA1 gene (Physiol. Genomics, 3, 101 (2000)).

There is a need for the development of prophylactic and therapeutic agents and diagnostics for chronic obstructive pulmonary disease and bronchial asthma with few side effects. Disclosure of the invention

The present inventors have conducted intensive studies to solve the above-mentioned problems, and as a result, have found a novel gene belonging to a calcium-dependent chloride channel family, and have completed the present invention.

That is, the present invention

(1) a protein containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 17 or a salt thereof,

(2) a protein consisting of the amino acid sequence represented by SEQ ID NO: 1 or a protein thereof; salt,

(3) a protein comprising an amino acid sequence represented by SEQ ID NO: 17 or a salt thereof;

(4) a protein or a salt thereof having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 19,

(5) a protein containing the amino acid sequence represented by SEQ ID NO: 19 or a salt thereof,

(6) The partial peptide of the protein according to the above (1) or a salt thereof,

(7) a partial peptide of the protein according to (4) or a salt thereof,

(8) a polynucleotide containing a polynucleotide encoding the protein or a partial peptide thereof according to (1),

(9) a polynucleotide containing a polynucleotide encoding the protein or a partial peptide thereof according to the above (4),

(10) the polynucleotide according to the above (8) or (9), which is a DNA; (11) a polynucleotide having a base sequence represented by SEQ ID NO: 2 or SEQ ID NO: 18,

(12) the polynucleotide according to (10), which comprises the nucleotide sequence represented by SEQ ID NO: 20;

(13) a recombinant vector containing the polynucleotide according to (8) or (9) above,

(14) a transformant transformed with the recombinant vector according to (13),

(15) culturing the transformant according to (14) above, producing and accumulating the protein or partial peptide thereof or a salt thereof according to (1) or (4), and collecting the resulting protein; A method for producing the protein or partial peptide thereof or a salt thereof according to the above (1) or (4),

(16) a medicament comprising the protein of (1) or (4) or a partial peptide thereof or a salt thereof,

(17) an antibody against the protein according to (1) or (4) or a partial peptide thereof or a salt thereof, (18) a medicament comprising the antibody according to (17),

(19) a diagnostic agent comprising the antibody according to the above (17),

(20) an antisense polynucleotide comprising a nucleotide sequence complementary or substantially complementary to the nucleotide sequence of the polynucleotide according to (8) or (9) or a part thereof,

(21) comprising the antisense polynucleotide according to (20) above.

(22) The protein according to (1) or (4) or a partial peptide thereof or a salt thereof, wherein the protein according to (1) or (4) or a partial peptide thereof or a salt thereof is used. A method for screening for a compound that inhibits the activity of a salt of

(23) The protein according to (1) or (4) or a partial peptide or a partial peptide thereof, which contains the protein according to the above (1) or (4) or a partial peptide thereof or a salt thereof. Kits for screening compounds or salts thereof that inhibit the activity of those salts,

(24) The activity of the protein of (1) or (4) or a partial peptide thereof or a salt thereof, which can be obtained using the screening method of (22) or the screening kit of (23). Inhibiting compounds or salts thereof,

(25) a medicament comprising the compound according to (24) or a salt thereof,

(26) a method for screening a compound or a salt thereof that inhibits the expression of the protein gene according to (1) or (4), characterized by using the polynucleotide according to (8) or (9);

(27) A kit for screening a compound or a salt thereof that inhibits the expression of the protein gene according to (1) or (4), which comprises the polynucleotide according to (8) or (9). ,

(28) A compound or a salt thereof that inhibits the expression of the protein gene according to (1) or (4), which can be obtained using the screening method according to (26) or the screening kit according to (27). (29) a medicament comprising the compound according to (28) or a salt thereof,

(30) A method for screening a compound or a salt thereof that inhibits the expression of a protein according to (1) or (4), which comprises using the antibody according to (17).

(31) A kit for screening a compound or a salt thereof, which inhibits the expression of the protein according to (1) or (4), which comprises the antibody according to (17).

(32) A compound or a salt thereof that inhibits the expression of the protein according to (1) or (4), which can be obtained using the screening method according to (30) or the screening kit according to (31).

(33) A medicine comprising the compound or salt thereof according to (32) above, (34) The above-mentioned (16), (18), which is an agent for preventing or treating respiratory diseases, rhinitis or digestive diseases. (21), (25), (29) or the drug according to (33), (35) the above-mentioned (34) which is an agent for preventing or treating chronic obstructive pulmonary disease, bronchial asthma, inflammatory bowel disease or reflux esophagitis. ) Described medicament,

(36) The diagnostic agent according to (19) above, which is a diagnostic agent for respiratory disease, rhinitis or gastrointestinal disease.

(37) The diagnostic agent according to the above (36), which is a diagnostic agent for chronic obstructive pulmonary disease, bronchial asthma, inflammatory bowel disease or reflux esophagitis.

(38) Chronic obstructive pulmonary disease, bronchial asthma, inflammatory bowel characterized by administering to a mammal an effective amount of the compound according to (24), (28) or (32) or a salt thereof. How to prevent or treat disease or reflux esophagitis,

(39) The compound or salt thereof according to the above (24), (28) or (32) for producing a prophylactic or therapeutic agent for chronic obstructive pulmonary disease, bronchial asthma, inflammatory bowel disease or reflux esophagitis. Provide use and so on.

Moreover, ,

(40) the protein is (a) the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 17; (b) one or two amino acids in the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 17; Or more (preferably about 1 to 30, preferably 1 to 10 An amino acid sequence in which about 1 amino acid has been deleted, more preferably a number (1 to 5) amino acids, and (c) 1 or 2 or more amino acids in the amino acid sequence represented by SEQ ID NO: 1 or 17 (Preferably about 1 to 30, preferably about 1 to 10, and more preferably a number (1 to 5) of amino acid sequences; (SEQ ID NO: 1 or SEQ ID NO: 1) One or more (preferably about 1 to 30, preferably about 1 to 10, and more preferably a number of (1 to 5)) amino acids were inserted into the amino acid sequence represented by 7 An amino acid sequence, (e) one or two or more amino acids in the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 17 (preferably, about 1 to 30, preferably about 1 to 10) An amino acid sequence in which the number (1 to 5) of amino acids is more preferably replaced by another amino acid Or (f) thereof (b) ~ protein in the above (1) containing the amino acid sequence that is a combination of (e) wherein the protein,

(41) a polynucleotide that hybridizes with the polynucleotide according to (8) or (9) above under high stringent conditions,

(42) A diagnostic agent or the like containing the polynucleotide according to the above (8) or (9) is also provided. BEST MODE FOR CARRYING OUT THE INVENTION

A protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1 or 17 of the present invention, and the same or substantially the same as the amino acid sequence represented by SEQ ID NO: 19 Proteins containing substantially the same amino acid sequence (hereinafter sometimes referred to as the protein of the present invention) are human warm non-human animals (eg, guinea pigs, rats, mice, chickens, egrets, bush, Cells (eg, hepatocytes, spleen cells, neurons, glial cells, kidney B cells, bone marrow cells, mesangial cells, Langerhans cells, epidermal cells, epithelial cells, goblet cells, etc.) Endothelial cells, smooth muscle cells, fibroblasts, fibrocytes, muscle cells, adipocytes, immune cells (eg, macrophages, Τ cells, Β cells, natura Killer cells, mast cells, neutrophils, basophils, eosinophils, monocytes), megakaryocytes, synovial cells, chondrocytes, osteocytes, osteoblasts, osteoclasts, mammary gland cells, hepatocyte Vesicles or stromal cells, or their precursors, stem cells, or cancer cells) or any tissue in which these cells are present, such as the brain, various parts of the brain (eg, olfactory bulb, amygdala, basal sphere, hippocampus) , Thalamus, hypothalamus, cerebral cortex, medulla, cerebellum), spinal cord, pituitary, stomach, kidney, kidney, liver, gonad, thyroid, gall bladder, bone marrow, adrenal gland, skin, muscle, lung, digestive tract (eg, Large intestine, small intestine), blood vessels, heart, thymus, spleen, submandibular gland, peripheral blood, prostate, testicle, ovary, placenta, uterus, bone, joint, skeletal muscle, etc. It may be.

The amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 1 is about 70% or more, preferably about 80% or more, preferably about 90% or more as the amino acid sequence represented by SEQ ID NO: 1. % Or more, more preferably about 95% or more, and even more preferably about 97% or more.

'As a protein having an amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 1, for example, a protein having an amino acid sequence substantially the same as the amino acid sequence represented by the aforementioned SEQ ID NO: 1 And a protein having substantially the same activity as the protein having the amino acid sequence represented by SEQ ID NO: 1.

The amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 17 includes about 70% or more, preferably about 80% or more, preferably Amino acid sequences having about 90% or more, more preferably about 95% or more, more preferably about 97% or more homology, and the like. Examples of the protein having an amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 17 include, for example, an amino acid sequence substantially the same as the amino acid sequence represented by the aforementioned SEQ ID NO: 17 And a protein having substantially the same activity as the protein having the amino acid sequence represented by SEQ ID NO: 17 is preferable.

The amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 19 includes about 70% or more, preferably about 80% or more, preferably Amino acid sequences having a homology of about 90% or more, more preferably about 95% or more, and even more preferably about 97% or more. Examples of the protein having an amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 19 include, for example, an amino acid sequence substantially the same as the amino acid sequence represented by the aforementioned SEQ ID NO: 19 And a protein having substantially the same activity as the protein having the amino acid sequence represented by SEQ ID NO: 19.

Examples of substantially the same activity include chloride channel activity (eg, calcium-dependent chloride channel activity and the like). Substantially homogenous indicates that the properties are homologous in nature (eg, physiologically or pharmacologically). Therefore, it is preferable that the chloride channel activities are equivalent (eg, about 0.01 to: L 0 times, preferably about 0,0 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 activity can be measured according to a known method. For example, the activity can be measured according to the method described in Genomics, Vol. 54, p. 200 (1998) or a method analogous thereto. it can.

Examples of the protein of the present invention include (1) (a) one or more (preferably about 1 to 30 and preferably 1 to 30) amino acids in the amino acid sequence represented by SEQ ID NO: 1. About 10 amino acids, more preferably an amino acid sequence in which the number of amino acids (1 to 5) has been deleted, and (b) one or two or more amino acids (preferably About 30 amino acids, preferably about 1-10 amino acids, and more preferably about (1-5) amino acids; (c) the amino acid sequence represented by SEQ ID NO: 1 An amino acid sequence into which one or more (preferably about 1 to 30, preferably about 1 to 10, and more preferably a number (1 to 5)) amino acids have been inserted; ) SEQ ID NO: 1 or 2 or more in the amino acid sequence represented by 1 (preferably about 1 to 30 About 1 to 10 amino acids, more preferably about 1 to 5 amino acids are substituted with another amino acid, or (e) a protein containing an amino acid sequence combining them. So-called mutein,

(2) (a) one or two or more amino acids in the amino acid sequence represented by SEQ ID NO: 17 (preferably Preferably, the amino acid sequence has about 1 to 30 amino acids, preferably about 1 to 10 amino acids, more preferably about 1 to 5 amino acids, and (b) SEQ ID NO: 17. An amino acid sequence in which one or more (preferably about 1 to 30, preferably about 1 to 10, and more preferably about 1 to 5) amino acids have been added to the amino acid sequence to be (C) one or more amino acid sequences represented by SEQ ID NO: 17 (preferably about 1 to 30, preferably about 1 to 10, more preferably about 1 to 5 ) Amino acid sequence; (d) one or more (preferably about 1 to 30; preferably 1 to 1) amino acids in the amino acid sequence represented by SEQ ID NO: 17 An amino acid sequence in which about 0, more preferably a number (1 to 5) amino acids have been substituted with another amino acid, or (e So-called mucins, such as proteins containing an amino acid sequence,

(3) (a) 1 or 2 or more in the amino acid sequence represented by SEQ ID NO: 19 (preferably, about 1 to 30, preferably about 1 to 10, more preferably (B) amino acid sequence represented by SEQ ID NO: 1 or 2 or more (preferably 1 to 30 amino acids, preferably 1 to 5 amino acids); An amino acid sequence having about 1 to 10 amino acids, more preferably (1 to 5) amino acids, and (c) one or two or more amino acids (preferably, an amino acid sequence represented by SEQ ID NO: 19) An amino acid sequence having about 1 to 30 amino acids, preferably about 1 to 10 amino acids, and more preferably a number (1 to 5) of amino acids inserted therein; (d) SEQ ID NO: 19 1 or 2 or more in the amino acid sequence (preferably about 1 to 30, preferably about 1 to 10, more preferably 1 to 5) (E) an amino acid sequence obtained by substituting an amino acid with another amino acid, or (e) so-called mutein such as protein containing an amino acid sequence obtained by combining them.

In the present specification, the protein has a N-terminus at the left end (amino terminus) and a C-terminus at the right end (capillary end) according to the convention of peptide labeling. The protein of the present invention, including the protein containing the amino acid sequence represented by SEQ ID NO: 1, has a C-terminus having a lipoxyl group (-C00H), a carboxylate (-C00-), and an amide (-C0NH). ₂ ) or ester (-C00R). Here, as R in the ester, e.g., methyl, Echiru, n- propyl Le, isopropyl, CM alkyl group, such as n- butyl, for example, C _3. ₈ cycloalkyl groups cyclopentyl Le, cyclohexane, etc. cyclohexyl, for example, phenyl, alpha-Na C ₆ such Fuchiru - ₁₂ § Li Ichiru group, e.g., benzyl, phenyl, such as phenethyl - C, _ ₂ alkyl or flight one naphthylmethyl etc. a- Nafuchiru C _M such alkyl Le group C ₇ _ ₁₄ Ararukiru group, such as pin bar opening Iruokishimechiru group is used. When the protein of the present invention has a lipoxyl group (or carboxylate) other than the C-terminus, the protein of the present invention includes a lipoxyl group amidated or esterified. As the ester in this case, for example, the above-mentioned C-terminal ester and the like are used.

Furthermore, in the protein of the present invention, the amino group at the N-terminal amino acid residue (eg, methionine residue) is protected by a protecting group (eg, C Hi acetyl group such as alkanol such as formyl group and acetyl group). N-terminal glutamine residue generated by cleavage in vivo, which has been oxidized with lipamine, Substituents on the side chains of amino acids in the molecule (eg, -0H, -SH, amino groups , imidazo Ichiru group, I Ndoru group is protected with Guanijino group, etc.) a suitable protecting group (e.g., formyl group, C WINCH ₆ Ashiru groups such as C Bok ₆ Al force Noiru group such as § Se methyl group) And complex proteins such as so-called glycoproteins to which sugar chains are bound.

Specific examples of the protein of the present invention include, for example, a protein containing an amino acid sequence represented by SEQ ID NO: 1, a protein containing an amino acid sequence represented by SEQ ID NO: 17, SEQ ID NO: 19 And a protein containing the amino acid sequence represented by

The partial peptide of the protein of the present invention is the partial peptide of the protein of the present invention described above, and preferably any peptide having the same properties as the protein of the present invention described above. For example, among the constituent amino acid sequences of the protein of the present invention, 20 or more, preferably 50 or more, more preferably 70 or more, more preferably 100 or more, and most preferably 200 or more Peptides having the above amino acid sequences are used. Further, in the partial peptide of the present invention, one or more (preferably about 1 to 10, more preferably about 1 to 5) amino acids in the amino acid sequence are deleted, or 1 or 2 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; or One or two or more (preferably about 1 to 20, more preferably about 1 to 10, and more preferably a number (1 to 5)) amino acids are inserted into the amino acid sequence, or Even if one or two 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 another amino acid, Good.

In the partial peptide of the present invention, the C-terminus force Rupokishiru group (-C00H), Karupoki Shireto (-C00-), can be any of the amide (_C0NH ₂₎ or ester (-C00R).

Further, the partial peptide of the present invention includes, as in the case of the protein of the present invention described above, a peptide having a carbonyl group (or carboxylate) other than the C-terminal, an N-terminal amino acid residue (eg, methionine). The amino group of the system is protected with a protecting group, the N-terminal is cleaved in vivo, the glutamine residue is oxidized by pyridalamine, and the substituent on the side chain of the amino acid in the molecule is Also included are those protected with an appropriate protecting group, and complex peptides such as so-called glycopeptides to which a sugar chain is bound.

The partial peptide of the present invention can also be used as an antigen for producing an antibody. As the salt of the protein or partial peptide of the present invention, salts with physiologically acceptable acids (eg, inorganic acids, organic acids) and bases (eg, alkali metal salts) are used. Acceptable acid addition salts are preferred. Such salts include, for example, salts with inorganic acids (eg, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid) or organic acids (eg, acetic acid, formic acid, propionic acid, fumaric acid, maleic acid) Succinic acid, tartaric acid, citric acid, malic acid, oxalic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid).

The protein of the present invention or a partial peptide thereof or a salt thereof can be prepared by the above-mentioned human The protein can be produced from a warm-blooded animal cell or tissue by a known method for purifying a protein, or can be produced by culturing a transformant containing a DNA encoding the protein. It can also be produced according to the peptide synthesis method described below.

When producing from tissues or cells of a human or non-human mammal, the tissues or cells of a human or non-human mammal are homogenized, extracted with an acid or the like, and the extracted liquid is subjected to reverse phase chromatography and ion exchange. Purification and isolation can be performed by combining chromatography such as chromatography.

For the synthesis of the protein or partial peptide of the present invention, or a salt thereof, or an amide thereof, a commercially available resin for protein synthesis can be generally used. Examples of such resins include chloromethyl resin, hydroxymethyl resin, benzylhydrylamine resin, aminomethyl resin, 4-benzyloxybenzyl alcohol resin, 4-methylbenzhydrylamine resin, and PAM resin. 4-Hydroxymethylmethylphenylacetamidomethyl resin, polyacrylamide resin, 4- (2 ', 4, -dimethoxyphenyl-hydroxymethyl) phenoxy resin, 4- (2,4, dimethoxyphenyl) Lu Fmoc aminoethyl) phenoxy resin. Using such a resin, an amino acid having an amino group and a side chain functional group appropriately protected is condensed on the resin in accordance with the sequence of the target protein according to various known condensation methods. At the end of the reaction, the protein or partial peptide is cleaved from the resin, and at the same time, various protecting groups are removed.In addition, an intramolecular disulfide bond formation reaction is carried out in a highly diluted solution to obtain the target protein or partial peptide or an amide thereof. get.

Regarding the condensation of the protected amino acids described above, various activating reagents that can be used for protein synthesis can be used, and carbodiimides are particularly preferable. As the carbodiimides, DCC, N, N'-diisopropylcarbodiimide, N-ethyl-N, 1- (3-dimethylaminoprolyl) carbodiimide and the like are used. Activation by these involves adding the protected amino acid directly to the resin along with a racemization inhibitor additive (eg, HOBt, H〇〇Bt), or by adding a symmetrical anhydride or HOBt ester or H〇OB. Pre-protected amino as t-ester The acid can be added to the resin after activation.

The solvent used for the condensation of the protected amino acid with the activated resin can be appropriately selected from solvents known to be usable for the protein condensation reaction. For example, acid amides such as N, N-dimethylformamide, N, N-dimethylacetoamide, N-methylpyrrolidone, halogenated hydrocarbons such as methylene chloride, methylform, and trifluoromethyl Alcohols such as ethanol, sulfoxides such as dimethyl sulfoxide, ethers such as pyridine, dioxane, and tetrahydrofuran; nitriles such as acetonitrile and propionitrile; esters such as methyl acetate and ethyl acetate; or an appropriate mixture thereof. Etc. are 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 ° C to 50 ° C. The activated amino acid derivative is usually used in a 1.5 to 4-fold excess. Test Bok results with two Nhidorin reaction, condensation is also sufficient Repeat _c reactions which can perform sufficient condensation by repeating the condensation reaction without removal of the protecting group to be insufficient When condensation cannot be obtained, acetylation of unreacted amino acid using acetic anhydride or acetylimidazole can prevent the subsequent reaction from being affected.

Examples of the protecting group for the amino group of the starting material include Z, Boc, t-pentyloxycarbonyl, isoboriloxycarbonyl, 4-methoxybenzyloxycarbonyl, C1-1Z, Br—Z, and adamantylo. Xycarponyl, trifluoroacetyl, phthaloyl, formyl, 2-nitrophenylsulfenyl, diphenylphosphinothioyl, Fmoc and the like are used.

The lipoxyl group can be, for example, alkyl esterified (eg, methyl, ethyl, propyl, butyl, t-butyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, 2-adamantyl, etc.) Alkyl esterification), aralkyl esterification (e.g., benzyl ester, 412-methyl benzyl ester, 4-methoxybenzyl ester, 4-methyl benzyl ester, benzhydryl esterification), phenacyl esterification, benzene Ziroxycarponyl hydrazide, t-butoxycarbonyl hydrazide, It can be protected by trityl hydrazide or the like.

The hydroxyl group of serine can be protected, for example, by esterification or etherification. Suitable groups for this esterification include, for example, lower ((^ -6) alkanoyl groups such as acetyl group, aroyl groups such as benzoyl group, and groups derived from carbonic acid such as benzyloxycarbonyl and ethoxycarbonyl groups. Examples of a group suitable for etherification include a benzyl group, a tetrahydropyranyl group, and a t-butyl group.

The protecting group of the phenolic hydroxyl group of tyrosine, for _{example, Bz 1, C 1 2 -} B zl, 2- nitrobenzyl, B r- Z, such as t one-butyl is used.

Examples of the protecting group for histidine imidazole include Tos, 4-methoxy-1,2,3,6-trimethylbenzenesulfonyl, DNP, benzyloxymethyl, Bum, Boc, Trt, and Fmoc.

Examples of the activated form of the raw oxypoxy group include, for example, the corresponding acid anhydride, azide, and active ester [alcohol (for example, pen phenol, 2,4,5-trichloro phenol, 2, 4-dinitrophenol, cyanomethyl alcohol, paranitrophenol, HONB, N-hydroxysuccinimide, N-hydroxyphthalimide, and esters with HOB t)]. As the activated amino group of the raw material, for example, a corresponding phosphoramide is used.

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, methanesulfonic acid, trifluoromethane, or the like. Acid treatment with sulfonic acid, trifluoroacetic acid or a mixture thereof, base treatment with diisopropylethylamine, triethylamine, piperidine, piperazine, etc., and reduction with sodium in liquid ammonia, etc. Used. The elimination reaction by the above-mentioned acid treatment is generally carried out at a temperature of about 20 ° C to 40 ° C. In the acid treatment, for example, anisol, phenol, thioanisole, methacrylol, paracresol, dimethylsulfide, It is effective to add a cation scavenger such as 1,4-butanedithiol or 1,2-ethanedithiol. Also, histidine imida The 2,4-dinitrophenyl group used as the zole protecting group is removed by thiophenol treatment, and the formyl group used as the indole protecting group for tributofan is removed from the above 1,1,1-ethanedithiol and 1,4-butanedithiol. In addition to deprotection by acid treatment in the presence, it is also removed by alkali treatment with dilute sodium hydroxide solution, dilute ammonia, or the like.

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.

As another method for obtaining an amide form of a protein or partial peptide, for example, first, amidation and protection of the α-hydroxyl group of the amino acid at the carboxy terminal, and then a peptide (protein) chain having a desired chain length on the amino group side Then, a protein or partial peptide from which only the protecting group of the α-amino group at the の -terminal of the peptide chain is removed and a protein or partial peptide from which only the protecting group of the C-terminal lipoxyl group is removed Then, these proteins or peptides are condensed in a mixed solvent as described above. Details of the condensation reaction are the same as described above. After purifying the protected protein or peptide obtained by the condensation, all the protecting groups are removed by the above method, and a desired crude protein or peptide can be obtained. This crude protein or peptide is purified using various known purification means, and the main fraction is freeze-dried to obtain an amide of the desired protein or peptide.

In order to obtain an ester of a protein or peptide, for example, after condensing the α-lipoxyl group of the terminal amino acid with a desired alcohol to form an amino acid ester, in the same manner as the amide of a protein or peptide, It is possible to obtain an ester of the desired protein or peptide.

The partial peptide of the present invention or a salt thereof can be produced according to a known peptide synthesis method or by cleaving the protein of the present invention with an appropriate beptidase. As a method for synthesizing a peptide, for example, any of a solid phase synthesis method and a liquid phase synthesis method may be used. That is, the remaining peptide is condensed with the partial peptide or amino acid that can constitute the partial peptide of the present invention, and the product is protected. When the compound has a group, the target peptide can be produced by removing the protecting group. Known condensation methods and elimination of protecting groups include, for example, the methods described in the following (a) to (e).

(a) M. Bodanszky and M.A.Ondetti, Peptide Synthesis, Interscience Publishers, New York (1966)

(b) Schroeder and Luebke, The Peptide, Academic Press, New York (1965)

(c) Nobuo Izumiya et al., Fundamentals and experiments of peptide synthesis, Maruzen Co., Ltd. (1975)

(d) Haruaki Yajima and Shunpei Sakakibara, Laboratory of Biochemistry 1, Protein Chemistry IV, 205, (1977)

(e) Supervision of Haruaki Yajima, Development of Continuing Pharmaceuticals, Vol. 14, Peptide Synthesis, Hirokawa Shoten Also, after the reaction, conventional purification methods, such as solvent extraction, distillation, column chromatography, liquid chromatography, and liquid chromatography 'recrystallization' The partial peptide of the present invention can be purified and isolated by combining these methods. When the partial peptide obtained by the above method is an isomer, it can be converted into 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 encoding the protein of the present invention may be any polynucleotide containing a nucleotide sequence (DNA or RNA, preferably DNA) encoding the protein of the present invention described above. Is also good. The polynucleotide is RNA such as DNA or mRNA encoding the protein of the present invention, 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 (that is, a coding strand) or an antisense strand (that is, a non-coding strand).

Using the polynucleotide encoding the protein of the present invention, for example, the method described in the known experimental medicine special edition “New PCR and its Application” 15 (7), 1997, or a method analogous thereto, or mRNA can be quantified. The DNA encoding the protein of the present invention includes the aforementioned protein of the present invention. It may be any as long as it contains a nucleotide sequence encoding a protein. Also, genomic DNA, genomic DNA library, cDNA derived from the above-mentioned cells and tissues, and cells and tissues derived from the above-mentioned cells Any of a cDNA library or synthetic DNA may be used.

The vector used for the library may be any of bacteriophage, plasmid, cosmid, phagemid and the like. Alternatively, it can be directly amplified by Reverse Transcriptase Polymerase Chain Reaction (hereinafter abbreviated as RT-PCR method) using a preparation of the entire RNA or mRNA fraction from the above-mentioned cell'tissue.

Examples of the DNA encoding the protein of the present invention include: (1) a DNA containing the nucleotide sequence represented by SEQ ID NO: 2 or a highly stringent DNA with the nucleotide sequence represented by SEQ ID NO: 2 A DNA encoding a protein having a nucleotide sequence that hybridizes under the conditions and having substantially the same properties as a protein having the amino acid sequence represented by SEQ ID NO: 1, (2) SEQ ID NO: 18 A DNA containing the nucleotide sequence represented by SEQ ID NO: 17, or a nucleotide sequence hybridizing under high stringent conditions with the nucleotide sequence represented by SEQ ID NO: 18; A DNA encoding a protein having substantially the same properties as the contained protein, (3) a DNA containing the nucleotide sequence represented by SEQ ID NO: 20, or a nucleotide sequence represented by SEQ ID NO: 20; DNA containing a nucleotide sequence that hybridizes under high stringent conditions and encoding a protein having substantially the same properties as a protein containing the amino acid sequence represented by SEQ ID NO: 19 Any one may be used. Examples of DNA that can be hybridized with the nucleotide sequence represented by SEQ ID NO: 2 under high stringency conditions include, for example, about 70% or more, preferably about 80% or more of the nucleotide sequence represented by SEQ ID NO: 2. For example, DNA containing a base sequence having a homology of about 90% or more, more preferably about 95% or more, and more preferably about 97% or more is used.

Examples of the DNA that can hybridize with the base sequence represented by SEQ ID NO: 18 under high stringent conditions include, for example, the base sequence represented by SEQ ID NO: 18 DNA containing a base sequence having about 70% or more, preferably about 80% or more, preferably about 90% or more, more preferably about 95% or more, more preferably about 97% or more with the sequence. Is used.

Examples of the DNA that can hybridize with the nucleotide sequence represented by SEQ ID NO: 20 under high stringency conditions include, for example, about 70% or more, preferably about 80% or more of the nucleotide sequence represented by SEQ ID NO: 20 DNA containing a base sequence having homology of preferably about 90% or more, more preferably about 95% or more, and more preferably about 97% or more is used.

Hybridization is performed according to a known method or a method analogous thereto, for example, a method described in Molecular Cloning 2nd (J. Sambrook et al., Cold Spring Harbor Lab. Press, 1989). be able to. 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 stringency conditions.

The high stringent conditions include, for example, a sodium concentration of about 19 to 40 mM, preferably about 19 to 2 OmM, and a temperature of about 50 to 70 ° (:, preferably about 60 to 65 ° C.). In particular, a sodium concentration of about 19 mM and a temperature of about 65 ° C. are most preferable.

More specifically, the DNA encoding the protein containing the amino acid sequence represented by SEQ ID NO: 1 is a DNA containing the base sequence represented by SEQ ID NO: 2, represented by SEQ ID NO: 17 The DNA encoding the protein containing the amino acid sequence represented by SEQ ID NO: 18 includes the DNA containing the base sequence represented by SEQ ID NO: 18, and the DNA encoding the protein containing the amino acid sequence represented by SEQ ID NO: 19 For example, a DNA containing the base sequence represented by SEQ ID NO: 20 is used. As the DNA encoding the partial peptide of the present invention, any DNA may be used as long as it contains the above-described nucleotide sequence encoding the partial peptide of the present invention. Further, it may be any of genomic DNA, genomic DNA library, cDNA derived from the above-mentioned cells and tissues, cDNA library derived from the above-described cells and tissues, and synthetic DNA. Examples of the DNA encoding the partial peptide of the present invention include a DNA having a part of a DNA having a base sequence represented by SEQ ID NO: 2, SEQ ID NO: 18 or SEQ ID NO: 20; SEQ ID NO: 2, SEQ ID NO: 18 or SEQ ID NO: 20 contains a nucleotide sequence that hybridizes under high stringent conditions with the nucleotide sequence represented by SEQ ID NO: 20; SEQ ID NO: 1, SEQ ID NO: 17 or A DNA containing a part of a DNA encoding a protein having substantially the same activity as the protein containing the amino acid sequence represented by SEQ ID NO: 19 is used.

The DNA hybridizable with the nucleotide sequence represented by SEQ ID NO: 2, SEQ ID NO: 18 or SEQ ID NO: 20 has the same significance as described above.

The same hybridization method and high stringency conditions as described above are used.

Means for cloning DNA that completely encodes the protein or partial peptide of the present invention (hereinafter, in the description of the cloning and expression of DNAs encoding them, these may be simply referred to as the protein of the present invention). Amplification by PCR using a synthetic DNA primer having a part of the nucleotide sequence encoding the protein of the present invention, or DNA integration into an appropriate vector Alternatively, selection can be performed by hybridization with a DNA fragment encoding the entire region or a DNA fragment labeled with a synthetic DNA. Hybridization can be carried out, 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 DNA base sequence can be converted by PCR or a known kit such as Mutan ^™ _super Express Km (Takara Shuzo Co., Ltd.) or Mutan ™ -K (Takara Shuzo Co., Ltd.) using the 0DA-LA PCR method. It can be performed according to a known method such as the gapped duplex method or the Kunkel method, or a method analogous thereto.

The DNA encoding the cloned protein may be used as is, or may be digested with restriction enzymes or added with a linker, if desired. Can be. The DNΑ may have ATG as a translation initiation codon at its 5 ′ end, and may have TAA, TGA or TAG as a translation termination codon at its 3 ′ end. These translation initiation codon and translation termination codon can also be added using an appropriate synthetic DNA adapter.

The expression vector of the protein of the present invention may be prepared, for example, by (a) cutting out a DNA fragment of interest from DNA encoding the protein of the present invention, and (mouth) converting the DNA fragment into a promoter in an appropriate expression vector. It can be manufactured by connecting downstream of

Examples of the vector include a plasmid derived from Escherichia coli (eg, pBR322, pBR325, pUC12, pUC13), a plasmid derived from Bacillus subtilis (eg, pUB110, TP5, pC194), a plasmid derived from yeast ( For example, pSH19, pSH15), bacteriophages such as phage λ, animal viruses such as retrovirus, vaccinia virus, baculovirus, etc., ρΑ1-11, ρΧTl, pRc / CMV, pRc / RSV, pc DN AI ZNeo is used.

The promoter of the present invention may be any promoter as long as it is suitable for the host used for gene expression. For example, when an animal cell is used as a host, SRo! Promoter, SV40 promoter, LTR promoter, CMV promoter, HSV-TK Promoter and the like can be mentioned.

Of these, it is preferable to use the CMV (cytomegalovirus) promoter, the SRa promoter, etc. When the host is Eshierihia genus, when trp promoter one, lac promoter, rec A promoter, XP _L pro motor-, l pp promoter, T 7 promoter, the host is Bacillus, SP_〇 1 When the host is yeast, such as a promoter, SP02 promoter, penP promoter, etc., PH05 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.

In addition to the above, the expression vector may include an enhancer, A signal containing a signal, a poly-A addition signal, a selection marker, an SV40 replication origin (hereinafter sometimes abbreviated as SV400ri) and the like can be used. Selection methods include, for example, dihydrofolate reductase (hereinafter sometimes abbreviated as dhfr) gene (methotrexate (MTX) resistance), ampicillin resistance gene (hereinafter sometimes abbreviated as Amp ¹ ) And neomycin resistance gene (hereinafter sometimes abbreviated as Ne ο ¹ · G418 resistance), etc. Particularly, when the dh fr gene is used as a selection marker using Chinese hamster cells lacking the dh fr gene. Alternatively, the target gene can be selected by using a medium containing no thymidine.

If necessary, a signal sequence suitable for the host is added to the N-terminal side of the protein of the present invention. When the host is a genus Escherichia, the PhoA * signal sequence and OmpA signal sequence are used.When the host is a Bacillus genus, the human amylase signal sequence and subtilisin signal sequence are used. If the host is an animal cell, an insulin signal sequence, an α-interferon signal sequence, an antibody molecule, a signal sequence, etc. Available for each. A transformant can be produced using the vector containing D ベクター which encodes the protein of the present invention thus constructed.

As the host, for example, Escherichia bacteria, Bacillus bacteria, yeast, insect cells, insects, animal cells, and the like are used.

Specific examples of the genus Escherichia include, for example, Escherichia coli

(Escherichia coli) K12 · DH 1 [Proc. Natl. Acad. Sci. USA], 60 Vol. 160 (1968)], JM103 [Nucleic Acids Research ', Vol. 9, 309 (1981)], JA221 [Journal of Molecular Biology]

(Journal of Molecular Biology), 120 volumes, 517 (1978)], ΗΒ101 [Journal of Molecular Biology, 41 volumes, 459 (1969)], C 600 [Genetics, 39 volumes] , 440 (195 4)] is used.

Examples of Bacillus bacteria include, for example, Bacillus subtilis MI 114 [Gene, 24, 255 (1983)], 207-21 [Journal of Biochemistry, 95] , 87 (1984)].

Examples of yeast include, for example, Saccharomyces cerevisiae AH22, AH22R-, NA87-11A, DKD-5D, 20B-12 Shizosaccharomyces' bomb (Schizosaccharomyces pombe) NC YC 1913, NCYC 2036 , Pichia pastoris K M71 and the like are used.

As insect cells, for example, when the virus is Ac NPV, a cell line derived from a larva of night moth (Spodoptera frugiperda cell; Sf cell), MGl cell derived from the midgut of Trichoplusia ni, High derived from egg of Trichoplusia ni Five ™ cells, cells derived from Mamestra brassicae or cells derived from Estigmena acrea are used. When the virus is BmNPV, a silkworm-derived cell line (Bombyx mori N cell; BmN cell) or the like is used. Examples of the Sf cells include Sf9 cells (ATCC CRL1711), Sf21 cells (Vaughn, J.L., et al., In Vipo).

Vivo), 13, 213-217, (1977)).

As insects, for example, silkworm larvae are used [Maeda et al., Nature, 315, 592 (1985)].

Examples of animal cells include monkey cell COS-7, Vero, Chinese Hams Yuichi cell CHO '(hereinafter abbreviated as CHO cell), dh fr gene-deficient Chinese eight-muster cell CHO (hereinafter CHO (dh fr- ) Cells), mouse L cells, mouse AtT-20, mouse myeloma cells, rat GH3, and human FL cells.

For example, to transform a microorganism belonging to the genus Escherichia, for example, Proc. Natl. Acad. Sci. USA, Proc. Natl. Acad. Sci. USA , 69, 2110 (1972) and Gene, 17, 107 (1982). Monkey

Transformation of Bacillus can be performed, for example, according to the method described in Molecular & General Genetics, Vol. 168, 11 (1979).

To transform yeast, for example, the Method in Enzymo

(Methods in Enzymology), Vol. 194, 182-187 (1991), Proc. Natl. Acad., "The Processing of the National Academy of the National Academy of Sciences" Sci. USA), Vol. 75, 1929 (1 978).

To transform insect cells or insects, e.g., bio / technology

(Bio / Technology), 6, 47-55 (1988).

To transform animal cells, for example, see Cell Engineering Separate Volume 8 New Cell Engineering Experiment Protocol. 263-267 (1995) (published by Shujunsha), Virology, 52, 456 (1973). The method 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.

When culturing a transformant whose host is a bacterium belonging to the genus Escherichia or Bacillus, 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. , Nitrogen sources, inorganic substances and others. Examples of carbon sources include glucose, dextrin, soluble starch, and sucrose.Examples of nitrogen sources include ammonium salts, nitrates, corncheap, lica-peptone, casein, meat extract, soybean meal, and potato extract. Examples of the inorganic or organic substance such as a liquid and the inorganic substance include calcium chloride, sodium dihydrogen phosphate, and magnesium chloride. In addition, yeast extract, vitamins, growth promoting factors and the like may be added. The pH of the medium is preferably about 5 to 8.

As a medium for cultivating a bacterium belonging to the genus Escherichia, for example, an M9 medium containing glucose and casamino acid [Miller, Journal of ed. Preferred is "in 'molecular" Genetics (Journal of Experiments in Molecular Genetics), 431-433, Cold Spring Harbor Laboratory, New York 1972). If necessary, a drug such as 3) 3-indolylacrylic acid can be added to make the promoter work efficiently if necessary.

When the host is a bacterium belonging to the genus Escherichia, culturing is usually performed at about 15 to 43 ° C for about 3 to 24 hours, and if necessary, aeration and stirring may be added.

When the host is a bacterium belonging to the genus Bacillus, 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.

When culturing a transformant whose host is yeast, for example, Burkholder's minimal medium [Bostian, KL et al., Processings-of-the-National Academy of Cultures] Proc. Natl. Acad. Sci. USA, 77, 4505 (1980)] and an SD medium containing 0.5% casamino acid (Bitter, GA et al., Proc. Zing's of the National Academy of Sciences, of the USA (Proc. Natl. Acad. Sci. USA), 81, 5330 (198 4)]. The pH is preferably adjusted to about 5 to 8. Culture is usually performed at about 20 to 35 ° C for about 24 to 72 hours, and aeration and stirring are added as necessary.

When culturing a transformant in which the host is an insect cell or an insect, the medium used is Grace's Insect Medium (Grace, T.C.,

(Nature), 195, 788 (1962)), an immobilized additive such as 10% pepsin serum or the like is used as needed. Preferably, the pH of the medium is 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.

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)], DMEM medium [Virology, 8, 39

6 (1959)], RPM I 1640 medium [Journal of the America. 'The Medical' Association (The Journal of the American Medical Association) 199, May 19 (1967)], 199 Medium [Proceding-The 'The Society for the Biological Medicine'

(Proceeding of the Society for the Biological Medicine), Vol. 73, 1 (1950)]. Preferably, the pH is about 6-8. Culture is usually about 30-40. Perform at C for about 15-60 hours, adding aeration and agitation as needed.

As described above, the protein of the present invention can be produced in cells, cell membranes or extracellular cells of the transformant.

The protein of the present invention can be separated and purified from the culture by, for example, the following method.

When extracting the protein of the present invention from cultured cells or cells, after culturing, the cells or cells are collected by a known method, suspended in an appropriate buffer, and then subjected to ultrasound, lysozyme and Z or freezing. After the cells or cells are disrupted by thawing or the like, a method of obtaining a crude protein extract by centrifugation or filtration is appropriately used. The buffer may contain a protein denaturant such as urea or guanidine hydrochloride, or a surfactant such as Triton X-100 ^™ . If the protein is secreted into the culture, after the culture is completed, the supernatant is separated from the cells or cells by a known method, and the supernatant is collected.

The protein contained in the culture supernatant or the extract obtained in this manner can be purified 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, dialysis, ultrafiltration, gel filtration, and SDS-polyacrylamide gel electrophoresis. Methods using molecular weight differences, methods using charge differences such as ion-exchange chromatography, methods using specific affinity such as affinity chromatography, reverse-phase high-performance liquid chromatography, etc. A method utilizing a difference in hydrophobicity, a method utilizing a difference in isoelectric point such as isoelectric focusing, and the like are used.

If the protein thus obtained is obtained in a free form, The salt can be converted into a salt by a method or a method analogous thereto, and when the salt is obtained, it can be converted into a free form or another salt by a known method or a method analogous thereto.

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. As the protein modifying enzyme, for example, trypsin, chymotrypsin, arginyl endopeptidase, proteinase, glycosidase and the like are used.

The presence of the protein of the present invention thus produced can be measured by enzyme immunoassay using a specific antibody, Western plotting, or the like.

An antibody against the protein or partial peptide of the present invention or a salt thereof may be any of a polyclonal antibody and a monoclonal mononal antibody as long as it can recognize the protein or partial peptide of the present invention or a salt thereof.

An antibody against the protein or partial peptide of the present invention or a salt thereof (hereinafter, these may be simply abbreviated as the protein of the present invention in the description of the antibody) uses the protein of the present invention as an antigen, and is a known antibody. It can be produced according to a method for producing an antibody or antiserum.

[Preparation of monoclonal antibody]

(a) Preparation of monoclonal antibody-producing cells

The protein of the present invention is administered to a warm-blooded animal at a site capable of producing an antibody upon administration, itself or together with a carrier or diluent. Complete Freund's adjuvant or incomplete Freund's adjuvant may be administered in order to enhance antibody production 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 used include monkeys, rabbits, rabbits, dogs, guinea pigs, mice, rats, sheep, goats, and chickens, and mice and rats are preferably used.

When producing monoclonal antibody-producing cells, select a warm-blooded animal immunized with the antigen, for example, an individual with an antibody titer from a mouse, and spleen 2 to 5 days after the final immunization. A monoclonal antibody-producing hybridoma can be prepared by collecting fl cells or lymph nodes and fusing the antibody-producing cells contained therein with myeloma cells of the same or different species. 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 a labeling agent bound to the antibody. The fusion operation can be carried out according to known methods, for example, the method of Köhler and Milstein (Nature, 256, 495 (1975)). Examples of the fusion promoter include polyethylene glycol (PEG) and Sendai virus, and PEG is preferably used.

Examples of myeloma cells include myeloma cells of warm-blooded animals such as NS-1, P3U1, SP 2/0, and AP-1. P3U1 is preferably used. <Antibody-producing cells used ( The preferred ratio of the number of spleen cells to the number of myeloma cells is about 1 ::! To 20: 1, and PEG (preferably PEG 1000 to PEG6 000) is added at a concentration of about 10 to 80%. Incubation at 40, preferably 30 to 37 ° C for 1 to 10 minutes allows efficient cell fusion.

Various methods can be used to screen monoclonal antibody-producing hybridomas. For example, a hybridoma culture supernatant is added to a solid phase (eg, microplate) on which a protein antigen is adsorbed directly or together with a carrier, and then 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 mice) or protein A is added to the monoclonal antibody bound to the solid phase. Detection method, Anti-immunoglobulin antibody or protein A is adsorbed on a solid phase to which a hybridoma culture supernatant is added, and a protein labeled with a radioactive substance or an enzyme is added, and a monoclonal antibody bound to the solid phase is detected. And the like.

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 a medium for animal cells supplemented with HAT (hypoxanthine, aminopterin, thymidine). As a selection and breeding medium, any medium can be used as long as it can grow a hybridoma. example For example, RPMI 1640 medium containing 1 to 20%, preferably 10 to 20% fetal bovine serum, and GIT medium containing 1 to 10% fetal bovine serum (Wako Pure Chemical Industries, Ltd.) Alternatively, a serum-free culture medium for hybridoma culture (SFM-101, Nissui Pharmaceutical Co., Ltd.) can be used. The cultivation temperature is usually from 20 to 40 ° (:, preferably, about 37 ° C. The culturing time is usually from 5 days to 3 weeks, preferably from 1 week to 2 weeks. 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.

(b) Purification of monoclonal antibody

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 only the antibody is collected using an active adsorbent such as protein A or protein G and the bond is dissociated to obtain the antibody) It can be carried out.

(Preparation of polyclonal antibody)

The polyclonal body of the present invention can be manufactured according to a known method or a method analogous thereto. For example, a immunizing antigen (protein antigen) itself or a complex thereof with a carrier protein is formed, and a warm-blooded animal is immunized in the same manner as in the above-described method for producing a monoclonal antibody. It can be produced by collecting an antibody-containing substance and separating and purifying the antibody.

Regarding the complex of immunizing antigen and carrier-1 protein used to immunize warm-blooded animals, the type of carrier protein and the mixing ratio between carrier-1 and hapten are determined by the antibody against hapten immunized by cross-linking with carrier. As long as it can be efficiently carried out, any kind may be cross-linked at any ratio. For example, serum serum albumin, psiloglopurine, hemocyanin, etc. are used in a weight ratio of about 0.1 to 1 for hapten. A method of pulling at a rate of about 20 to 20 and preferably about 1 to 5 is used.

Also, use various condensing agents for the force coupling between the hapten and the carrier. Glutaraldehyde, carbodiimide, maleimide active ester, an active ester reagent containing a thiol group or a dithioviridyl group, etc. are 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 or incomplete Freund's adjuvant may be administered to increase antibody production ability during administration.Administration is usually performed once every about 2 to 6 weeks, for a total of about 3 to 10 times. . The polyclonal antibody can be collected from the blood, ascites, etc., preferably from the blood of a warm-blooded animal immunized by the above method.

The measurement of the polyclonal antibody titer in the antiserum can be performed in the same manner as the measurement of the antibody titer in the antiserum described above. Separation and purification of polyclonal antibodies

—It can be performed according to the same immunoglobulin separation and purification method as that of the null antibody.

Complementary to the nucleotide sequence of a DNA encoding the protein or partial peptide of the present invention (hereinafter, these DNAs may be abbreviated as the DNA of the present invention in the description of the antisense polynucleotide), or The antisense polynucleotide having a substantially complementary base sequence or a part thereof includes a base sequence complementary to or substantially complementary to the base sequence of the DNA of the present invention or a part thereof, Any antisense polynucleotide may be used as long as it has an action of suppressing the expression of the DNA, but antisense DNA is preferable.

The nucleotide sequence substantially complementary to the DNA of the present invention is, for example, the entire nucleotide sequence or a part of the nucleotide sequence complementary to the DNA of the present invention (that is, the complementary strand of the DNA of the present invention). A base sequence having about 70% or more, preferably about 80% or more, more preferably about 90% or more, and most preferably about 95% or more homology with the base sequence is exemplified. In particular, of the entire nucleotide sequence of the complementary strand of the DNA of the present invention, the complementary sequence of the nucleotide sequence of the portion encoding the N-terminal portion of the protein of the present invention (for example, the nucleotide sequence near the start codon) is approximately 70%. Antisense polynucleotides having a homology of at least about 80%, preferably at least about 80%, more preferably at least about 90%, and most preferably at least about 95% are suitable. Specifically, a nucleotide sequence complementary to or substantially complementary to the nucleotide sequence of DNA having the nucleotide sequence represented by SEQ ID NO: 2, SEQ ID NO: 18 or SEQ ID NO: 20, or a part thereof An antisense polynucleotide having, preferably, a nucleotide sequence complementary to the nucleotide sequence of DNA having the nucleotide sequence represented by SEQ ID NO: 2, SEQ ID NO: 18 or SEQ ID NO: 20, or a part thereof Antisense polynucleotide (more preferably, a nucleotide sequence complementary to the nucleotide sequence of DNA having the nucleotide sequence represented by SEQ ID NO: 2, SEQ ID NO: 18 or SEQ ID NO: 20, or a portion thereof. Antisense polynucleotide) and the like.

The number of antisense polynucleotides is usually about 10 to 40, preferably 15 to

It is composed of about 30 bases.

To prevent degradation by hydrolases such as nucleases, the phosphate residues (phosphates) of each nucleotide constituting the antisense DNA should be chemically modified, for example, phosphorothioate, methylphosphonate, and phosphorodithionate. It may be substituted with a phosphoric acid residue. These antisense polynucleotides can be produced using a known DNA synthesizer or the like.

According to the present invention, an antisense polynucleotide (nucleic acid) corresponding to the protein gene of the present invention, which can inhibit the replication or expression of the gene, has been cloned or encoded. It can be designed and synthesized based on DNA base sequence information. Such antisense polynucleotides can hybridize with the RNA of the protein gene of the present invention, inhibit the synthesis or function of the RNA, or interact with the protein-related RNA of the present invention. Thus, the expression of the protein gene of the present invention can be regulated and controlled. Polynucleotides that are complementary to a selected sequence of the protein-related RNA of the present invention, and that can specifically hybridize with the protein-related RNA of the present invention, are those of the present invention in vivo and in vitro. It is useful for regulating and controlling the expression of protein genes, and is also useful for treating or diagnosing diseases and the like. The term "corresponding" refers to a nucleotide, base sequence or specific sequence of a nucleic acid, including or homologous to a gene, Means that The "correspondence" between a nucleotide, nucleotide sequence or nucleic acid and a protein usually refers to the amino acids of the protein (as specified by the instructions) derived from the nucleotide (nucleic acid) sequence or its complement. 5 'end hairpin loop of protein gene, 5' end 6—base pair repeat, 5 'end untranslated region, polypeptide translation start codon, protein coding region, ORF translation stop codon, 3' end untranslated region, A 3'-end palindrome region or a 3'-end hairpin loop may be selected as a preferred region of interest, but any region within a protein gene may be selected as a target.

Regarding the relationship between the target nucleic acid and a polynucleotide complementary to at least a part of the target region, if the target nucleic acid can hybridize with the target region, the target nucleic acid is included in the polynucleotide of the target region. On the other hand, it can be said that it is “antisense”. Antisense polynucleotides are polynucleotides containing 2-dexoxy-D-reports, polynucleotides containing D-ribose, or other types of polynucleotides that are N-glycosides of purine or pyrimidine bases. Other polymers with non-nucleotide backbones (eg, commercially available protein nucleic acids and synthetic sequence-specific nucleic acid polymers) or other polymers containing special bonds (provided that the polymer is not contained in DNA or RNA) Pairing of bases as found (contains nucleotides having a configuration that allows base attachment)). They may be double-stranded DNA, single-stranded DNA, double-stranded RNA, single-stranded RNA, DNA: RNA hybrid, and may further comprise unmodified polynucleotides (or unmodified oligonucleotides). ), Those with known modifications, e.g., those with labels known in the art, capped, methylated, one or more natural nucleotides replaced with analogs Or modified with an intramolecular nucleotide, for example, having an uncharged bond (eg, methylphosphonate, phosphotriester, phosphoramidate, olebamate, etc.), a charged bond or a sulfur-containing bond (eg, Those with rotioate, phosphorodithioate, etc., such as proteins (eg, nucleases, nuclease inhibitors, toxins, antibodies) Signal peptides, poly-one L one lysine, etc.) or a sugar (e.g., Monosakka Compounds having side-chain groups such as amides, etc., compounds having an interactive compound (eg, acridine, psoralen, etc.), chelating compounds (eg, metals, radioactive metals, boron, oxidizable metals) ), Those containing an alkylating agent, and those having a modified bond (for example, a nucleic acid of the Hyanoma type 1). Here, "nucleoside", "nucleotide" and "nucleic acid" 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 by halogens, aliphatic groups, etc., or ethers, amines, etc. It may have been converted to a functional group.

The antisense polynucleotide of the present invention is an RNA, a DNA or a modified nucleic acid (RNA, DNA). Specific examples of the modified nucleic acid include a sulfur derivative of a nucleic acid, a thiophosphate derivative, and a polynucleoside amide / polynucleoside amide that is resistant to degradation of oligonucleoside amide. The antisense polynucleotide of the present invention can be designed, for example, as follows. In other words, it makes the antisense polynucleotide more stable in the cell, enhances the cell permeability of the antisense polynucleotide, increases the affinity for the target sense strand, and In some cases, the toxicity of the antisense polynucleotide is reduced. Many such modifications have been reported, for example, in Pharm Tech Japan, vol. 8, p. 247 or p. 395, 1992, Antisense Research and Appli cations, CRC Press, 1993.

The antisense polynucleotides of the present invention may contain altered or modified sugars, bases, or bonds, are provided in special forms such as ribosomes, microspheres, are applied by gene therapy, It can be provided in an added form. Such additional forms include polycations such as polylysine, which acts to neutralize the charge on the phosphate backbone, and lipids, which enhance the interaction with cell membranes and increase the uptake of nucleic acids ( For example, Hydrophobic substances such as phospholipid and cholesterol). Preferred lipids for addition include cholesterol and its derivatives (eg, cholesteryl chloroformate, cholic acid, etc.). These can be attached to the 3 'or 5' end of the nucleic acid and can be attached via bases, sugars, intramolecular nucleoside bonds. Other groups include capping groups specifically located at the 3, 5 'or 5' end of nucleic acids to prevent degradation by nucleases such as exonucleases and RNases . Such 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 polynucleotide 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 protein of the present invention. . Hereinafter, the protein or partial peptide of the present invention or a salt thereof (hereinafter, sometimes abbreviated as the protein of the present invention), the DNA encoding the protein or partial peptide of the present invention (hereinafter, referred to as the DNA of the present invention) Abbreviated), an antibody against the protein or partial peptide of the present invention or a salt thereof (hereinafter may be abbreviated as the antibody of the present invention), and an antisense polynucleotide of the DNA of the present invention (hereinafter referred to as the present invention). May be abbreviated as an antisense polynucleotide).

The protein of the present invention can be used as a disease marker because its expression is increased in a tissue-specific manner in chronic obstructive pulmonary disease and rhinitis. Therefore, the protein of the present invention may be used, for example, for respiratory diseases such as pulmonary thoracic disease accompanied by inflammation of the lungs and airways [eg, chronic obstructive pulmonary disease (chronic bronchitis, emphysema), diffuse panbronchiolitis, Bronchial asthma, cystic fibrosis, irritable pneumonia, etc.), allergic conjunctivitis, rhinitis

(Eg, allergic rhinitis, hay fever, acute rhinitis, chronic rhinitis, hypertrophic rhinitis, atrophic rhinitis, dry pronasitis, vasomotor rhinitis, gangrene rhinitis, sinusitis, etc.), gastrointestinal diseases (eg, Irritable bowel syndrome, inflammatory bowel disease, ulcerative colitis, Crohn's disease, reflux It is useful as a marker for early diagnosis of esophagitis, etc.), judgment of symptom severity, and prediction of disease progression.

Antisense polynucleotide of a gene encoding the protein of the present invention (protein gene of the present invention), a compound or a salt thereof that inhibits the activity of the protein of the present invention, a compound that inhibits the expression of the gene of the protein of the present invention, or Pharmaceuticals containing a salt thereof or an antibody against the protein of the present invention include, for example, respiratory diseases such as lungs, lungs accompanied by inflammation of the respiratory tract, and chest diseases [eg, chronic obstructive pulmonary disease (chronic bronchitis, emphysema) , Diffuse panbronchiolitis, bronchial asthma, cystic fibrosis, irritable pneumonia, etc.), allergic conjunctivitis, rhinitis (eg, allergic rhinitis, pollinosis, acute rhinitis, chronic rhinitis, hypertrophic rhinitis, atrophic rhinitis , Dry rhinitis, vasomotor rhinitis, gangrene rhinitis, sinusitis, etc., gastrointestinal disorders (eg, irritable bowel syndrome, inflammatory bowel disease) 'Ulcerative colitis, Crohn's disease, etc. reflux esophagitis) prevention of such - can be used as a therapeutic agent. Preferred are preventive and therapeutic agents for respiratory diseases, gastrointestinal diseases and the like.

[1] Screening of drug candidate compounds for disease

The protein of the present invention and the gene of the present invention have an increased tissue-specific expression in chronic obstructive pulmonary disease and rhinitis, and have a mucus production promoting action and an alveolar wall destruction promoting action in the lung, airway or nasal mucosa. The compound or its salt that inhibits the activity of the protein of the present invention and the compound or its salt that inhibits the expression of the protein gene of the present invention can be used, for example, for respiratory diseases such as lung-thoracic diseases accompanied by inflammation of the lungs and airways. Respiratory tract disease [eg, chronic obstructive pulmonary disease (chronic bronchitis, emphysema), diffuse panbronchiolitis, bronchial asthma, cystic fibrosis, irritable pneumonia, etc.], allergic conjunctivitis, rhinitis (eg, allergy) Rhinitis, hay fever, acute rhinitis, chronic rhinitis, hypertrophic rhinitis, atrophic rhinitis, dry pronasitis, vasomotor rhinitis, gangrene rhinitis, sinusitis, etc.), Prevention of gastrointestinal diseases (eg, irritable bowel syndrome, inflammatory bowel disease, ulcerative colitis, Crohn's disease, reflux esophagitis, etc.) · Therapeutic agents, preferably prevention of respiratory diseases, gastrointestinal diseases, etc. · It can be used safely as a low-toxic drug such as a therapeutic agent. Therefore, the protein of the present invention inhibits the activity of the protein of the present invention. The polynucleotide encoding the protein is useful as a reagent for screening a compound or a salt thereof, and the polynucleotide encoding the protein is useful as a reagent for screening a compound or a salt thereof that inhibits expression of the protein gene of the present invention. is there. That is, the present invention provides (1) a compound that inhibits the activity of the protein of the present invention (eg, chloride channel activity and the like) characterized by using the protein of the present invention, or a salt thereof (hereinafter, abbreviated as an inhibitor). (2) a compound that inhibits expression of the protein gene of the present invention, which comprises using a polynucleotide encoding the protein of the present invention, or a salt thereof.

(Hereinafter sometimes abbreviated as an inhibitor), (3) a compound or a salt thereof that inhibits the expression of the protein of the present invention characterized by using the antibody of the present invention (hereinafter referred to as an inhibitor) (May be abbreviated as).

Specific examples of the screening method include: (i) the case where cells having the ability to produce the protein of the present invention are activated with a calcium activator; and (ii) the case where cells having the ability to produce the protein of the present invention are tested. A method for screening for an inhibitor, which is characterized by comparing with a case where a mixture of compounds is activated with a calcium activator, is mentioned.

In the above screening method, for example, the chloride channel activity of the protein of the present invention in the cases (0 and (ii)) is measured and compared.

The calcium activator may be added to cells having the ability to produce the protein of the present invention after being mixed with the test compound, or the calcium activator may be added to cells having the ability to produce the protein of the present invention. After that, the test compound may be added.

The cells having the ability to produce the protein of the present invention and the test compound may be mixed before or after the cells having the ability to produce the protein of the present invention are activated with a calcium activator. A mixture of a test compound and a calcium activator may be added to cells having the ability to produce the protein of the present invention. As a calcium activator, ionomycin, A2187 (calcimycin) and the like are used. The chloride channel activity of the protein of the present invention can be determined according to a known method, for example, the method described in Genomics, vol. 54, p. 200 (1998) or a method analogous thereto.

Test compounds include, for example, peptides, proteins, non-peptidic compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, and animal tissue extracts, and these compounds are novel compounds. Or a known compound.

In order to carry out the above screening method, cells having the ability to produce the protein of the present invention are prepared by suspending them in a buffer suitable for screening. The buffer may be any one of phosphate buffer and borate buffer having a pH of about 4 to 10 (preferably, a pH of about 6 to 8).

Specific examples of the screening method include (iii) culturing cells capable of producing the protein of the present invention and (iv) culturing a mixture of cells capable of producing the protein of the present invention and a test compound. Then, the expression level of the protein gene of the present invention (specifically, the amount of the protein of the present invention or the amount of mRNA encoding the protein) is measured and compared.

The amount of the protein of the present invention can be measured by a known method, for example, using an antibody recognizing the protein of the present invention to detect the protein present in a cell extract or the like. It can be measured in accordance with the corresponding method.

The expression level of the protein gene of the present invention can be determined by a known method, for example, Northern blotting, reverse transcription-polymerase chain reaction (RT-PCR), real-time PCR analysis system (manufactured by ABI, TaqMan polymerase chain).

r eac Uon) or a method analogous thereto.

Test compounds include, for example, peptides, proteins, non-peptidic compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, and animal tissue extracts, and these compounds are novel compounds. Or a known compound. To carry out the above screening method, cells having the ability to produce the protein of the present invention are prepared by suspending them in a buffer suitable for screening. The buffer may be a buffer that does not inhibit the chloride channel activity of the protein of the present invention, such as a phosphate buffer having a pH of about 4 to 10 (preferably, a pH of about 6 to 8) or a borate buffer. Either may be used.

Specific examples of the screening method include (ν) ′ when a cell capable of producing the protein of the present invention is cultured and (vi) when a mixture of a cell capable of producing the protein of the present invention and a test compound is cultured. Make a comparison with the case. In the above screening method, the expression level of the protein of the present invention (specifically, the amount of the protein of the present invention) in the cases (V) and (vi) is measured using the antibody of the present invention. (Eg, detection of expression, quantification of expression level, etc.) and compare.

Test compounds include, for example, peptides, tanks, non-peptidic compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts, etc., and these compounds are new compounds. Or a known compound.

As a cell having the ability to produce the protein of the present invention, for example, a host (transformant) transformed with a vector containing a DNA encoding the protein of the present invention described above is used. As the host, for example, animal cells such as CHO cells are preferably used. For the screening, for example, a transformant in which the protein of the present invention is expressed on a cell membrane by culturing by the above-mentioned method is preferably used.

For example, a test compound that inhibits chloride channel activity in the case of the above (ii) by about 20% or more, preferably 30% or more, and more preferably about 50% or more compared to the case of the above (i) It can be selected as a compound that inhibits the activity of the protein of the present invention or a salt thereof. For example, the expression level of the protein gene of the present invention in the case (iv) is about 20% or more, preferably 30% or more, more preferably about 50%, as compared with the case (iii). The test compound that inhibits the above can be selected as a compound that inhibits the expression of the protein gene of the present invention or a salt thereof.

For example, the expression level of the protein of the present invention in the case of the above (vi) is

A test compound that inhibits about 20% or more, preferably 30% or more, more preferably about 50% or more of that in the case of (V) as a compound that inhibits expression of the protein of the present invention or a salt thereof. You can choose.

The screening kit of the present invention includes a protein or a partial peptide of the present invention or a salt thereof, a polypeptide encoding a protein or a partial peptide of the present invention, an antibody of the present invention, or a protein or partial base of the present invention. It contains cells that have the ability to produce peptides.

Compounds or salts thereof obtained by using the screening method or the screening kit of the present invention include the test compounds described above, for example, peptides, proteins, non-peptidic compounds, synthetic compounds, fermentation products, cell extracts, and plant extracts. Liquid, animal tissue extract, plasma, or a compound thereof, or a salt thereof, wherein the activity of the protein of the present invention (eg, chloride channel activity, etc.), the expression of the protein gene of the present invention, or A compound or a salt thereof that inhibits expression of the protein of the invention.

As the salt of the compound, those similar to the aforementioned salts of the protein of the present invention are used. '

Compounds or salts thereof obtained using the screening method or screening kit of the present invention include, for example, respiratory diseases such as lungs, lungs with inflammation of the airways, and chest diseases [eg, chronic obstructive pulmonary diseases (chronic bronchial Inflammation, emphysema), diffuse panbronchiolitis, bronchial asthma, cystic fibrosis, irritable pneumonia, etc.), allergic conjunctivitis, rhinitis (eg, allergic rhinitis, pollinosis, acute rhinitis, chronic rhinitis, hypertrophic) Rhinitis, atrophic rhinitis, dry rhinitis, vasomotor rhinitis, gangrene rhinitis, sinusitis, etc., gastrointestinal diseases (eg, irritable bowel syndrome, inflammatory bowel disease, ulcerative colitis, Crohn's disease, Prophylactic and therapeutic agents for reflux esophagitis, etc., preferably respiratory disease, digestion It is useful as a medicine for the prevention and treatment of tract diseases and the like.

The compound or a salt thereof obtained by using the screening method or the screening kit of the present invention can be safely administered by itself or as a suitable drug. The medicament used for the above-mentioned administration contains the above-mentioned compound or a salt thereof and a pharmacologically acceptable carrier, diluent or vehicle, and is provided as a pharmaceutical composition suitable for oral or parenteral administration. Is done.

For example, compositions for oral administration include solid or liquid dosage forms, specifically tablets (including sugar-coated tablets and film-coated tablets), pills, granules, powders, and capsules (including soft capsules). Syrups, emulsions, suspensions and the like. Such a composition is produced by a known method and contains a carrier, diluent or excipient commonly used in the pharmaceutical field. For example, lactose, starch, sucrose, magnesium stearate and the like are used as carriers and excipients for tablets.

As compositions for parenteral administration, for example, injections, suppositories, etc. are used. Injections are in the form of intravenous injections, subcutaneous injections, intradermal injections, intramuscular injections, infusions, etc. Is included. Such injections are prepared according to a known method, for example, by dissolving, suspending or emulsifying the above antibody or a salt thereof in a sterile aqueous or oily liquid usually used for injections. As an aqueous liquid for injection, for example, physiological saline, isotonic solution containing glucose and other adjuvants, and the like, suitable solubilizing agents, for example, alcohol (eg, ethanol), polyalcohol (eg, Propylene glycol, polyethylene glycol), a nonionic surfactant [eg, polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated catalyst)], and the like. As the oily liquid, for example, sesame oil, soybean oil and the like are used, and benzyl benzoate, benzyl alcohol and the like may be used in combination as a solubilizing agent. The prepared injection is usually filled into a suitable ampoule. Suppositories used for rectal administration are prepared by mixing the antibody or a salt thereof with a conventional suppository base.

The above-mentioned oral or parenteral pharmaceutical composition is conveniently prepared in a unit dosage form so as to be compatible with the dosage of the active ingredient. Such dosage unit dosage form Examples include tablets, pills, capsules, injections (ampoules), suppositories, etc., usually 5 to 500 mg per dosage unit, especially 5 to 100 mg for injections, and other dosage forms. Preferably, it contains 10-250 mg of the above antibody.

For example, compositions for nasal administration include inhalants, nasal drops, aerosols and the like.

When the preparation obtained by a conventional method is used as an inhaler, it is converted into a powder inhaler, a suspension for inhalation, a solution for inhalation or a capsule inhaler using a known method, and an appropriate inhaler for use. , And powder inhalants are particularly preferably used.

The average particle size of the powder inhalant is not particularly limited, but is preferably about 0.1 to 20 im, and particularly preferably about 1 to 5 m.

-The particle size of the powder inhaler is not particularly limited, but the amount of particles having a size of about 25 m or more is preferably about 5% or less, particularly preferably about 1% or less.

When using an inhalant, a commercially available inhaler may be used as a device for application. For example, ventolin / rotacaps (VENTOLIN)

R0TACAPS; Daraxo Co., Ltd., Spin Heller (registered trademark, Fujisawa Pharmaceutical Co., Ltd.), INTAL SPINCAPS (Fisons Co., Ltd.), Atovento and Verotech's ATROVENT AND BER0TEC

INHALETTEN; BERINGER INGELHEIM, FORADIL (F0RADIL; Ciba), BENTODISKS (Daraxo), publisher (registered trademark, Teijin Ltd.), BRICANYL TURBUHALER ; Astra) and MI AT INSUFFLATOR. As a route of administration, it is usually preferable to inhale directly into the nose and mouth using an inhaler.

The preparations obtained in this way are safe and low toxic and can be used, for example, in humans or in warm-blooded animals (eg, mice, rats, puppies, higgs, bushus, puppies, puppies, birds, cats, dogs). , Monkeys, chimpanzees, etc.) orally or parenterally. The dose of the compound or its salt that inhibits the activity of the protein of the present invention may vary depending on its action, target disease, subject to be administered, administration route and the like.For example, for the purpose of treating chronic obstructive pulmonary disease, When the compound or a salt thereof is administered orally, in general, for an adult (assuming a body weight of 60 kg), about 0.1 to 100 mg, preferably about 1.0 to 50 mg, of the compound or its salt per day is used. More preferably, about 1.0 to 20 mg is administered. When administered parenterally, the single dose of the above compound or a salt thereof varies depending on the administration subject, target disease, etc., for example, the activity of the protein of the present invention for the purpose of treating chronic obstructive pulmonary disease. When a compound or a salt thereof is administered to an adult (with a body weight of 60 kg) usually in the form of an injection, the compound or a salt thereof is about 0.01 to 30 mg, preferably about 0.1 to 20 mg per day. Degree, more preferably about

It is convenient to administer about 0.1 to 10 mg by intravenous injection. In the case of other animals, the dose can be administered in terms of weight per 60 kg.

[2] Quantification of the protein of the present invention, its partial peptide or its salt

An antibody against the protein of the present invention (hereinafter sometimes abbreviated as the antibody of the present invention) can specifically recognize the protein of the present invention, and therefore, the quantification of the protein of the present invention in a test solution. In particular, it can be used for quantification by sandwich immunoassay.

That is, the present invention

(i) Competitively reacting the antibody of the present invention with a test solution and the labeled protein of the present invention, and measuring the ratio of the labeled protein of the present invention bound to the antibody. A method for quantifying the protein of the present invention in a test solution, which comprises:

(ii) After reacting the test solution with the antibody of the present invention insolubilized on the carrier and another labeled antibody of the present invention simultaneously or continuously, measuring the activity of the labeling agent on the insolubilized carrier A method for quantifying the protein of the present invention in a test solution is provided. '' In the quantification method (ii) above, it is desirable that one antibody is an antibody that recognizes the N-terminal of the protein of the present invention and the other antibody is an antibody that reacts with the C-terminal of the protein of the present invention. . In addition, the protein of the present invention can be quantified using a monoclonal antibody against the protein of the present invention (hereinafter sometimes referred to as the monoclonal antibody of the present invention), and can also be detected by tissue staining or the like. . For these purposes, the antibody molecule itself may be used, or the FCab ') ₂ , Fab \ 5 or Fab fraction of the antibody molecule may be used.

The method for quantifying the protein of the present invention using the antibody of the present invention is not particularly limited, and may be an antibody, an antigen, or an antibody-antigen complex corresponding to the amount of antigen (eg, the amount of protein) in the test solution. Any measurement method may be used as long as the amount is detected by chemical or physical means and the amount is measured from a standard curve prepared using a standard solution containing a known amount of antigen. For example, nephelometry, a competition method, an immunometric method and a sandwich method are preferably used, but it is particularly preferable to use a sandwich method described later in terms of sensitivity and specificity.

Examples of the labeling agent used in the assay method using the labeling substance, for example, a radioactive isotope (e.g., ^[125 1], ^[131 1], [¾], etc. ^[14 c]), fluorescent substances [e.g., Shianin 5 Fluorescent dyes (eg, Cy2, Cy3, Cy5, Cy5.5, Cy7 (manufactured by Amersham Biosciences), etc.), fluorescamine, fluorescein sotiocinet, etc.), enzymes (eg, β-galactosidase, β-darcosidase, alpholipophorase, peroxidase, malate dehydrogenase, etc., luminescent substances (eg, luminol, luminol derivatives, reciferin, lucigenin, etc.), piotin, lanthanide, etc. Can be Furthermore, a biotin-avidin system can be used for binding the antibody or antigen to the labeling agent.

For the insolubilization of an antigen or an antibody, physical adsorption may be used, or a method using a chemical bond usually used for insolubilizing and immobilizing a protein or an enzyme may be used. Examples of the carrier include insoluble polysaccharides such as agarose, dextran, and cellulose; synthetic resins such as polystyrene, polyacrylamide, and silicon; and glass.

In the sandwich method, the test solution is reacted with the insolubilized monoclonal antibody of the present invention (primary reaction), and further reacted with another labeled monoclonal antibody of the present invention (secondary reaction). Measuring the activity of the above labeling agent Thus, the mass of the protein of the present invention 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 the same as those described above. Also, in the immunoassay by the sandwich method, the antibody used for the solid phase antibody or the labeling antibody is not necessarily required to be one type, and two or more types of antibodies are used for the purpose of improving measurement sensitivity and the like. Mixtures may be used.

In the method for measuring the protein of the present invention by the sandwich method of the present invention, the monoclonal antibody of the present invention used in the primary reaction and the secondary reaction differs in the binding site of the protein of the present invention. Antibodies are preferably used. That is, when the antibody used in the primary reaction and the secondary reaction is, for example, the antibody used in the secondary reaction recognizes the C-terminal of the protein of the present invention, the antibody used in the primary reaction is preferably An antibody that recognizes other than the C-terminal, for example, the N-terminal, is used.

The monoclonal sample of the present invention can be used in a measurement system other than the sandwich method, for example, a competitive method, an immunometric method, or a nephrometry.

In the competition method, after the antigen in the test wave and the labeled antigen are allowed to react competitively with the antibody, the unreacted labeled antigen (F) is separated from the labeled antigen (B) bound to the antibody ( B / F separation), measure the amount of labeling for either B or F, and quantify the amount of antigen in the test solution. In this reaction method, a soluble antibody is used as an antibody, B / F separation is performed using polyethylene glycol, a liquid phase method using a second antibody against the antibody, a solid phase antibody is used as the first antibody, or An immobilization method using a soluble antibody as the first antibody and an immobilized antibody as the second antibody is used.

In the immunometric method, the antigen in the test wave and the immobilized antigen are subjected to a competitive reaction with a certain amount of the labeled antibody, and then the solid phase and the liquid phase are separated. The original and an excess amount of the labeled antibody are allowed to react, and then the immobilized antigen is added to bind the unreacted labeled antibody to the solid phase, and then the solid phase and the liquid phase are separated. Next, the amount of label in either phase is measured to quantify the amount of antigen in the test solution.

In nephelometry, the amount of insoluble sediment generated as a result of an antigen-antibody reaction in a gel or in a solution is measured. The amount of antigen in the test solution is small, Even when only a sediment is obtained, a laser nephrometry utilizing scattering by a laser is preferably used.

In applying these individual immunoassays to the quantification method of the present invention, no special conditions, procedures, and the like need to be set. The protein measurement system of the present invention may be constructed by adding ordinary technical considerations of those skilled in the art to ordinary conditions and operation methods in each method. For details of these general technical means, reference can be made to reviews and documents.

For example, edited by Hiro Irie, "Radio Imuno Atsushi" (Kodansha, published in Showa 49) Edited by Hiro Irie "Radio Imno Atssey" (Kodansha, published in Showa 54), edited by Eiji Ishikawa et al. "The Enzyme Immunoassay Method" (2nd edition) (Ed.Ishikawa et al., Ed. Ishikawa et al.), 2nd Edition (Ed.) (3rd Edition) (Medical Publishing, published in 1962), "Methods in ENZYMOLOGYJ Vol. 70 (Immunochemical Techniques (Part A)), Ibid. Vol. 73 (Immunochemical Techniques (Part B)), Ibid. Vol. 74 (Immunochemical Techniques (Part C)), ibid.Vol. 84 (Immunochemical Techniques (Part C)

Vol. 92 (Immunochemical Techniques (Part E: Monoclonal Ant ibodies and General Immunoassay Methods)), ibid.Vol. 121 (Immunochemical Techniques (Part I: Hybridoma Technology and

Monoclonal Ant ibodies)) (above, published by Academic Press).

As described above, the protein of the present invention can be quantified with high sensitivity by using the antibody of the present invention.

Furthermore, when an increase in the concentration of the protein of the present invention is detected by quantifying the concentration of the protein of the present invention using the antibody of the present invention, for example, lung / thoracic disease accompanied by inflammation of the lung airways Respiratory diseases (eg, chronic obstructive pulmonary disease (chronic bronchitis, emphysema), diffuse panbronchiolitis, bronchial asthma, cystic fibrosis, irritable pneumonia, etc.), allergic conjunctivitis, rhinitis (eg, Allergic rhinitis, hay fever, acute rhinitis, chronic rhinitis, hypertrophic rhinitis, atrophic rhinitis, dry pronasitis, vasomotor rhinitis, gangrene rhinitis, sinusitis, etc., gastrointestinal diseases (eg, irritable bowel) Syndrome, flame (Eg, inflammatory bowel disease, ulcerative colitis, Crohn's disease, reflux esophagitis, etc.) or are likely to be affected in the future.

Further, the antibody of the present invention can be used for detecting the protein of the present invention present in a subject such as a body fluid or a tissue. In addition, preparation of an antibody column used for purifying the protein of the present invention, detection of the protein of the present invention in each fraction at the time of purification, analysis of the behavior of the protein of the present invention in test cells, etc. Can be used for

[3] Gene diagnostics

The DNA of the present invention can be used, for example, as a probe to produce human or warm-blooded animals (eg, rats, mice, guinea pigs, egrets, birds, higgies, pigs, pigs, dogs, cats, cats). (DNA, monkey, chimpanzee, etc.) can detect an abnormality (gene abnormality) in DNA or mRNA that encodes the protein of the present invention or a partial peptide thereof. Mutations or decreased expression or increases in the DNA or mRNA are useful as diagnostic agents for gene expression such as overexpression.

The above-described genetic diagnosis using the DNA of the present invention includes, for example, the well-known Northern hybridization and PCR-SSCP method (Genomics, Vol. 5, pp. 874-879 (1989), Proc. Proceedings of the National Academy

National Academy of Sciences of the United States of America), Vol. 86, pp. 2766-2770 (1989)).

For example, when overexpression is detected by Northern hybridization or when a DNA mutation is detected by PCR-SSCP method, for example, lung and chest diseases with inflammation of the lungs and airways Respiratory diseases such as chronic obstructive pulmonary disease (chronic bronchitis, emphysema), diffuse panbronchiolitis, bronchial asthma, cystic fibrosis, irritable pneumonia, etc., allergic conjunctivitis, rhinitis (eg , Allergic rhinitis, hay fever, acute rhinitis, chronic rhinitis, hypertrophic rhinitis, atrophic rhinitis, dry pronasitis, vasomotor rhinitis, gangrene rhinitis, sinusitis, etc., gastrointestinal diseases (eg, hypersensitivity) Bowel syndrome, It can be diagnosed as being highly likely to be a disease such as inflammatory bowel disease, ulcerative colitis, Crohn's disease, or reflux esophagitis.

[4] A drug containing an antisense polynucleotide

The antisense polynucleotide of the present invention, which can complementarily bind to the DNA of the present invention and suppresses the expression of the DNA, has low toxicity and has the activity of the protein of the present invention or the DNA of the present invention in vivo. · Functions (eg, chloride channel activity, etc.) can be suppressed, such as lungs · lungs with inflammation of the airways · respiratory diseases such as chest diseases [eg, chronic obstructive pulmonary disease '(chronic bronchitis, Pulmonary emphysema), diffuse panbronchiolitis, bronchial asthma, cystic fibrosis, irritable pneumonia, etc.), alegiitis conjunctivitis, rhinitis (eg, allergic rhinitis, hay fever, acute rhinitis, chronic rhinitis, hypertrophic) Rhinitis, atrophic rhinitis, dry rhinitis, vasomotor rhinitis, gangrene rhinitis, sinusitis, etc., gastrointestinal diseases (eg, irritable bowel syndrome, inflammatory bowel disease, ulcerative colitis, Crohn's disease) Reflux esophagitis, etc) agent for the prophylaxis or treatment of such can preferably be used as a prophylactic or therapeutic agent for respiratory diseases, gastrointestinal disorders.

When the antisense polynucleotide is used as the prophylactic or therapeutic agent, it can be formulated and administered according to a known method.

For example, when the antisense polynucleotide is used, the antisense polynucleotide may be used alone or in a suitable vector such as a retrovirus vector, an adenovirus vector, an adenovirus associated virus vector, or the like.

And then orally or parenterally administered to humans or non-human mammals (eg, rats, puppies, higgins, pigs, puppies, cats, dogs, monkeys, etc.) according to standard procedures be able to. The antisense polynucleotide can be administered as it is, or can be formulated with a physiologically acceptable carrier such as an adjuvant to promote uptake, and administered with a gene gun or a catheter such as a hide mouth gel catheter. Alternatively, they can be aerosolized and administered topically into the trachea as an inhalant.

The dosage of the antisense polynucleotide may vary depending on the target disease, the target of administration, the route of administration, and the like. For example, the present invention relates to the treatment of chronic obstructive pulmonary disease. When the antisense polynucleotide is locally administered into the trachea as an inhalant, generally, in an adult (body weight 60 kg), about 0.1 to 100 mg of the antisense polynucleotide is administered per day.

Further, the antisense polynucleotide can also be used as a diagnostic oligonucleotide probe for examining the presence or the expression status of the DNA of the present invention in tissues or cells.

The present invention further provides

(a) a double-stranded RNA containing a part of the RNA encoding the protein of the present invention,

(b) a medicament comprising the double-chain R NA,

(c) a lipozyme containing a part of RNA encoding the protein of the present invention; and (d) a medicament containing the lipozyme.

These double-stranded RNAs (RNAi; RNA interference method), lipozymes, and the like can suppress the expression of the polynucleotide (eg, DNA) of the present invention in the same manner as the above-mentioned antisense polynucleotides. Can inhibit the activity or function (eg, chloride channel activity, etc.) of the peptide of the present invention or the polynucleotide (eg, DNA) of the present invention in, for example, lungs with lung and airway inflammation, Chest disease (eg, chronic obstructive pulmonary disease (chronic bronchitis, emphysema), diffuse panbronchiolitis, bronchial asthma, cystic fibrosis, irritable pneumonia, etc.), inflammatory bowel disease, allergic conjunctivitis, rhinitis (Eg, allergic rhinitis, hay fever, acute rhinitis, chronic rhinitis, hypertrophic rhinitis, atrophic rhinitis, dry pronasitis, vasomotor rhinitis, gangrenous rhinitis, sinusitis, etc.) It can be used as anti-therapeutic agent.

The double-stranded RNA can be designed and manufactured based on the sequence of the polynucleotide of the present invention according to a known method (eg, Nature, 411, 494, 2001). The lipozyme can be designed and produced based on the sequence of the polynucleotide of the present invention according to a known method (eg, TRENDS in Molecular Medicine, Vol. 7, pp. 221, 2001). For example, it can be produced by linking a known lipozyme to a part of RNA encoding the peptide of the present invention. As a part of the RNA encoding the peptide of the present invention, a portion (RNA fragment) close to the cleavage site on the RNA of the present invention, which can be cleaved by a known lipozyme, can be mentioned. When the above-mentioned double-stranded RNA or lipozyme is used as the above-mentioned prophylactic / therapeutic agent, it can be formulated and administered in the same manner as for the antisense polynucleotide. [5] A drug containing the antibody of the present invention

Antibodies of the present invention having an activity of neutralizing the activity of the protein of the present invention include, for example, respiratory diseases such as lung and chest diseases accompanied by inflammation of the lungs and airways [eg, chronic obstructive pulmonary disease (chronic bronchitis, Emphysema), diffuse panbronchiolitis, bronchial asthma, cystic fibrosis, irritable pneumonia, etc.), allergic conjunctivitis, rhinitis (eg, allergic rhinitis, hay fever, acute rhinitis, chronic rhinitis, hypertrophic rhinitis, Atrophic rhinitis, dry rhinitis, vasomotor rhinitis, gangrene rhinitis, sinusitis, etc., gastrointestinal diseases (eg, irritable bowel syndrome, inflammatory bowel disease, ulcerative colitis, Crohn's disease, reflux disease) It can be used as a medicine for diseases such as esophagitis.

The prophylactic / therapeutic agent for the above-mentioned diseases containing the antibody of the present invention has low toxicity, and can be used directly as a liquid or as a pharmaceutical composition of an appropriate dosage form in human or non-human mammals (eg, rat, egret, hidge) Or parenteral administration to mice, dogs, cats, dogs, monkeys, etc.). The dosage varies depending on the administration subject, target disease, symptoms, administration route and the like.For example, when used for the treatment of chronic obstructive pulmonary disease in adults, the antibody of the present invention is used as a single dose. Usually, about 0.01 to 20 mg / kg body weight, preferably about 0.1 to 10 mg / kg body weight, more preferably about 0.1 to 5 rag / kg body weight, about 1 to 5 times a day, preferably about 1 to 5 rag / kg body weight. It is convenient to administer by intravenous injection about once to three times a day. In the case of other parenteral administration and oral administration, an equivalent dose can be administered. If the symptoms are particularly severe, the dose may be increased accordingly.

The antibodies of the present invention can be administered by themselves or as a suitable pharmaceutical composition. The pharmaceutical composition used for the administration contains the antibody or a salt thereof and a pharmacologically acceptable carrier, diluent or excipient. Such compositions are provided in dosage forms suitable for oral or parenteral administration.

That is, for example, as a composition for oral administration, a solid or liquid agent Examples include tablets (including sugar-coated tablets and film-coated tablets), pills, granules, powders, capsules (including soft capsules), syrups, emulsions, and suspensions. Such a composition is produced by a known method and contains a carrier, diluent or excipient commonly used in the pharmaceutical field. For example, lactose, starch, sucrose, magnesium stearate and the like are used as carriers and excipients for tablets.

As compositions for parenteral administration, for example, injections, suppositories, etc. are used. Injections are in the form of intravenous injections, subcutaneous injections, intradermal injections, intramuscular injections, infusions, etc. Is included. Such injections are prepared according to a known method, for example, by dissolving, suspending or emulsifying the above antibody or a salt thereof in a sterile aqueous or oily liquid usually used for injections. As an aqueous liquid for injection, for example, physiological saline, isotonic solution containing glucose and other adjuvants, and the like, suitable solubilizing agents, for example, alcohol (eg, ethanol), polyalcohol (eg, It may be used in combination with propylene glycol, polyethylene glycol), a nonionic surfactant [eg, polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated catalyst)], and the like. As the oily liquid, for example, sesame oil, soybean oil, and the like are used, and benzyl benzoate, benzyl alcohol, and the like may be used in combination as a solubilizing agent. The prepared injection is usually filled in a suitable ampoule. A suppository for rectal administration is prepared by mixing the above antibody or a salt thereof with a usual suppository base.

The above-mentioned oral or parenteral pharmaceutical composition is conveniently prepared in a unit dosage form so as to be compatible with the dosage of the active ingredient. Examples of such dosage unit forms include tablets, pills, capsules, injections (ampoules), and suppositories, and usually 5 to 500 mg per dosage unit form, and especially 5 to 500 mg for injections.

It is preferred that 100 mg and other dosage forms contain 10 to 250 mg of the above antibody.

Each of the above-mentioned compositions may contain other active ingredients as long as the composition does not cause an undesirable interaction with the above-mentioned antibody. [6] A drug containing the protein or DNA of the present invention

The protein (preferably a partial peptide) of the present invention may be used, for example, for respiratory diseases such as lungs, lungs with respiratory tract inflammation, and chest diseases [eg, chronic obstructive pulmonary disease (chronic bronchitis, emphysema), diffuse pan- Bronchiolitis, bronchial asthma, cystic fibrosis, irritable pneumonia, etc.), allergic conjunctivitis, rhinitis (eg, allergic rhinitis, pollinosis, acute rhinitis, chronic rhinitis, hypertrophic rhinitis, atrophic rhinitis, dryness Pronasalitis, vasomotor rhinitis, gangrenous rhinitis, sinusitis, etc., gastrointestinal disorders (eg, irritable bowel syndrome, inflammatory bowel disease, ulcerative colitis, Crohn's disease, reflux esophagitis, etc.) It is also useful as a prophylactic / therapeutic agent, preferably as a prophylactic / therapeutic agent for respiratory diseases and gastrointestinal diseases. When the protein of the present invention is used as the above-mentioned prophylactic / therapeutic agent, it can be formulated according to a conventional method.

For example, the protein of the present invention can be used as a tablet, capsule, elixir, microcapsule, etc., if necessary, orally coated with water or other pharmaceutically acceptable liquids. It can be used parenterally in the form of injections, such as sterile solutions or suspensions. For example, the protein of the present invention may be used together with known physiologically acceptable carriers, flavoring agents, excipients, vehicles, preservatives, stabilizers, binders, etc., in a unit dosage form generally required for the practice of the formulation. It can be manufactured by mixing with. The amount of the active ingredient in these preparations is such that a suitable dosage in the specified range can be obtained.

The dose of the protein of the present invention varies depending on the administration subject, target organ, symptoms, administration method, and the like. However, in the case of oral administration, generally, for example, in a patient (as 60 kg), about 0. It is 1 to 100 mg, preferably about 1.0 to 50 mg, more preferably about 1.0 to 20 mg. In the case of parenteral administration, the single dose varies depending on the target of administration, target organ, symptoms, administration method, etc., for example, usually in the form of injection, for example, in patients (as 60 kg), It is convenient to administer about 0.01 to 30 mg, preferably about 0.1 to 20 mg, more preferably about 0.1 to 10 mg per day by intravenous injection. In the case of other animals, the dose can be administered in terms of 60 kg. [7] Creation of an animal having the DNA of the present invention

The present invention relates to a DNA encoding an exogenous protein of the present invention (hereinafter abbreviated as the exogenous DNA of the present invention) or a mutant DNA thereof (sometimes abbreviated as the exogenous mutant DNA of the present invention). A non-human mammal having the formula:

That is, the present invention

(1) a non-human mammal having an exogenous DNA of the present invention or a mutant DNA thereof,

(2) the animal according to (1), wherein the non-human mammal is a rodent;

(3) The animal according to (2), wherein the rodent is a mouse or a rat, and

(4) A recombinant vector containing the exogenous DNA of the present invention or its mutant DNA, which can be expressed in mammals, and the like.

Non-human mammals having the exogenous DNA of the present invention or the mutant DNA thereof (hereinafter abbreviated as the DNA-transfected animal of the present invention) can be used for non-fertilized eggs, fertilized eggs, spermatozoa and germ cells including their progenitor cells. Preferably, during the stage of embryonic development in non-human mammal development (more preferably, at the stage of a single cell or fertilized egg and generally before the 8-cell stage), the calcium phosphate method, the electric pulse method, the ribofection method, It can be created by introducing the desired DNA by agglutination, microinjection, particle gun, DEAE-dextran, etc. Further, the exogenous DNA of the present invention can be introduced into somatic cells, organs of living organisms, tissue cells, and the like by the DNA introduction method, and used for cell culture, tissue culture, and the like. Can be fused with the above-mentioned germ cells by a known cell fusion method to produce the DNA-introduced animal of the present invention.

As the non-human mammal, for example, porcupine, pig, sheep, goat, goat, egret, dog, cat, guinea pig, hamster, mouse, rat and the like are used. Above all, rodents with relatively short ontogeny and biological cycle in terms of the creation of disease animal model systems and easy reproduction are particularly useful in mice (for example, pure strains such as C57BL / 6 strain and DBA2 strain). As the crossing strain, B6C3F, strain, BDF, strain, B6D2F, strain, BALB / c strain, ICR strain, etc.) or rat (eg, Wistar, SD, etc.) are preferable. "Mammals" in a recombinant vector that can be expressed in mammals include humans in addition to the above-mentioned non-human mammals.

The exogenous DNA of the present invention refers not to the DNA of the present invention originally possessed by non-human mammals, but to the DNA of the present invention once isolated and extracted from the mammal. The mutant DNA of the present invention is a DNA in which a mutation (for example, mutation) has occurred in the base sequence of the original DNA of the present invention, specifically, base addition, deletion, substitution with another base. DNA or the like that has occurred is used, and abnormal DNA is also included. As the abnormal DNA, DNA that expresses an abnormal protein of the present invention is used. For example, DNA that expresses a protein that suppresses the function of the normal protein of the present invention is used.

The exogenous DNA of the present invention may be derived from a mammal that is the same or different from the animal of interest. In introducing the DNA of the present invention into a target animal, it is generally advantageous to use the DNA as a DNA construct linked downstream of a promoter capable of being expressed in animal cells. For example, when the human DNA of the present invention is introduced, it is derived from various mammals (eg, egrets, dogs, cats, guinea pigs, hamsters, rats, mice, etc.) having the DNA of the present invention that are highly homologous 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 DNA of the present invention into a fertilized egg of a target mammal, for example, a mouse fertilized egg As a result, a mammal into which the DNA of the present invention is highly expressed can be produced.

Examples of the expression vector of the protein of the present invention include a plasmid derived from Escherichia coli, a plasmid derived from Bacillus subtilis, a plasmid derived from yeast, a bacteriophage such as λ phage, a retrovirus such as Moroni leukemia virus, a vaccinia virus or a baculovirus. For example, animal viruses such as E. coli are used. Among them, a plasmid derived from E. coli, a plasmid derived from Bacillus subtilis or a plasmid derived from yeast are preferably used.

Examples of promoters that regulate DNA expression include, for example, viruses (eg, Simian virus, cytomegalovirus, Moroni leukemia virus, Promoters of DNA derived from JC virus, breast cancer virus, poliovirus, etc., promoters derived from various mammals (human, egret, dog, cat, guinea pig, hams, etc., rat, mouse, etc.), for example, albumin, Insulin II, peroplakin II, Erasuyose, erythropoietin, endothelin, muscle creatine kinase, glial fibrillary acidic protein, daltathione S_transferase, platelet-derived growth factor i3, keratin K 1,10 And 14, collagen type I and I 1¾, cyclic AMP-dependent protein kinase — 61 subunit, dystrophin, tartrate-resistant alkaline phosphatase, atrial natriuretic factor, endothelial receptor chin mouth kinase (generally Τ ie Abbreviated as 2), sodium potassium Denosine 3 kinase (Na, K-ATPase), neurofilament light chain, meta-oral thionein I and IIA, meta-oral proteinase 1 tissue inhibitor, MHC class I antigen (H-2L): H—ras , Renin, dopamine-hydroxylase, thyroid peroxidase (TP〇), polypeptide chain elongation factor 1a (EF-1α), β-rhtin, α and / 3 myosin heavy chain, myosin Light chains 1 and 2, myelin-based protein, thyroglobulin, Thy-1, immunoglobulin, heavy-chain variable region (VNP) 'serum amyloid P component, myoglobin, troponin (:, smooth muscle α-actin, preproenke A promoter such as hualin リン or vasopressin is used, among which a cytomegalovirus promoter capable of high expression throughout the body, a human polypeptide chain extension, etc. Factor 1 flight (EF-1) promo Isseki one, such as humans and Niwatori i3 Akuchinpuromo one coater is preferred.

The vector preferably has a sequence that terminates the transcription of the messenger RNA of interest in the DNA-transfected mammal (generally called terminator). For example, it is derived from viruses and various mammals. A sequence of each DNA can be used, and preferably, Simian virus SV40, Minne, etc. is used.

In addition, translation of splicing signal of each DNA, enhancer region, part of intron of eukaryotic DNA, etc. to 5 'upstream of promoter region, promoter region, etc. for the purpose of further expressing the target foreign DNA Inter-domain or translation It is also possible to connect 3 ′ downstream of the region depending on the purpose.

The normal translation region of the protein of the present invention may be S = F kidney, kidney, thyroid cell, fibroblast derived from human or various mammals (eg, egret, dog, cat, guinea pig, hamster, rat, mouse, etc.). Complementary DNA prepared by known methods from cell-derived DNA and all or part of genomic DNA from one of various commercially available genomic DNA libraries, or from liver, kidney, thyroid cells, and fibroblast-derived RNA. It can be obtained as a raw material. In addition, a foreign abnormal DNA can produce a translation region obtained by mutating a normal polypeptide translation region obtained from the above cells or tissues by a point mutagenesis method.

The translation region can be prepared as a DNA construct that can be expressed in a DNA-transduced animal by a conventional DNA engineering technique in which the translation region is ligated downstream of the promoter and, if desired, upstream of the transcription termination site.

Introduction 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 the introduction of the DNA indicates that all the progeny of the produced animal retain the exogenous DNA of the present invention in all of the germ cells and somatic cells. Means to do. Progeny of such animals that inherit the foreign DNA of the present invention have the foreign DNA of the present invention in all of their germinal and somatic cells.

The non-human mammal to which the exogenous normal DNA of the present invention has been transferred is confirmed to stably maintain the exogenous DNA by mating, and is subcultured as an animal having the DNA in a normal breeding environment. Can be done.

The introduction of the exogenous DNA of the present invention at the fertilized egg cell stage is ensured to be present in excess in all germ cells 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 the introduction of the DNA indicates that all the offspring of the produced animal have the exogenous DNA of the present invention in all of the 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 germ cells and somatic cells. By obtaining a homozygous animal having the introduced DNA on both homologous chromosomes and mating the male and female animals, it is possible to breed and passage so that all offspring have the DNA in excess.

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 endogenous normal DNA, thereby finally obtaining the protein of the present invention. It may develop quality hyperfunction and can be used as a model animal for the disease. For example, using the normal DNA-introduced animal of the present invention, elucidation of the pathological mechanism of hyperactivity of the protein of the present invention and diseases associated with the protein of the present invention, and examination of a method for treating these diseases. It is possible. Further, since the mammal into which the exogenous normal DNA of the present invention has been introduced has an increased symptom of the released protein of the present invention, screening for a therapeutic agent for the disease associated with the protein of the present invention is performed. It can also be used for testing.

On the other hand, the non-human mammal having the foreign abnormal DNA of the present invention can be subcultured in a normal breeding environment as an animal having the DNA after confirming that the foreign DNA is stably maintained by the crossing. I can do it. Furthermore, the desired foreign DNA can be incorporated into the above-mentioned plasmid and used as a raw material. The DNA construct with the promoter can be prepared by ordinary DNA engineering techniques. Introduction of the abnormal DNA of the present invention at the fertilized egg cell stage is ensured to be present in all germinal and somatic cells of the target mammal. The presence of the abnormal DNA of the present invention in the germ cells of the produced animal after the introduction of the DNA means that all the offspring of the produced animal have the abnormal DNA of the present invention in all of the germ cells and somatic cells. The progeny of this type of animal that has inherited the exogenous DNA of the present invention has the abnormal DNA of the present invention in all of its germinal and somatic cells. By obtaining a homozygous animal having the introduced DNA on both homologous chromosomes, and crossing the male and female animals, it is possible to breed subculture so that all offspring have the DNA.

In the non-human mammal having the abnormal DNA of the present invention, the abnormal DNA of the present invention is highly expressed, and the function of the protein of the present invention is ultimately reduced by inhibiting the function of endogenous normal DNA. Inactive type refractory disease Can be used as an animal. For example, using the abnormal DNA-introduced animal of the present invention, it is possible to elucidate the pathological mechanism of the function-inactive refractory of the protein of the present invention and to examine a method for treating this disease.

Further, as a specific possibility, the abnormal DNA highly expressing animal of the present invention can be used to inhibit the function of a normal protein by the abnormal protein of the present invention in the function-inactive refractory disease of the protein of the present invention (dominant negative active protein). Action).

In addition, since the mammal into which the foreign abnormal DNA of the present invention has been introduced has an increased symptom of the released protein of the present invention, it can be used in a therapeutic drug screening test for the protein of the present invention or its function-inactive refractory disease. Is also available.

Further, as other possible uses of the above two kinds of DNA-introduced animals of the present invention, for example,

(a) use as a cell source for tissue culture,

(b) direct analysis of DNA or RNA in the tissue of the DNA-introduced animal of the present invention, or analysis of a polypeptide tissue expressed by DNA, to specifically express or express the protein of the present invention. Analysis of the relationship with the activating protein,

(c) Cells of a tissue having DNA are cultured by standard tissue culture techniques, and these are used to study the function of cells from tissues that are generally difficult to culture,

(d) screening for a drug that enhances cell function by using the cell described in (c) above; and

(e) Isolation and purification of the mutant protein of the present invention and production of its antibody can be considered. Further, using the DNA-introduced animal of the present invention, it is possible to examine clinical symptoms of diseases related to the protein of the present invention, including refractory inactivation of the protein of the present invention. It is possible to obtain more detailed pathological findings in each organ of the disease model related to the protein of the present invention, to develop new treatment methods, and to contribute to the research and treatment of secondary diseases caused by the disease. it can.

In addition, each organ is removed from the DNA-introduced animal of the present invention, cut into small pieces, and the DNA-introduced cells that have been released with a protease such as trypsin are obtained and cultured. Alternatively, it is possible to systematize the cultured cells. Furthermore, the present inventors can identify the protein-producing cells of the present invention, examine their relationship to apoptosis, differentiation or proliferation, or examine their signal transduction mechanisms, and investigate their abnormalities. It is an effective research material for elucidating the protein and its action.

Further, the use of the DNA-introduced animal of the present invention to develop a therapeutic agent for a disease associated with the protein of the present invention, including refractory inactive type of the protein of the present invention, Using a test method and a quantitative method, it is possible to provide an effective and rapid screening method for a therapeutic agent for the disease. Also, using the DNA-introduced animal of the present invention or the exogenous DNA-expressing vector of the present invention, it is possible to examine and develop a method for treating a DNA associated with the protein of the present invention.

[8] Knockout animal

The present invention provides a non-human mammalian embryonic stem cell in which the DNA of the present invention has been inactivated, and a non-human mammal deficient in expression of the DNA of the present invention.

That is, the present invention

(1) a non-human mammalian embryonic stem cell in which the DNA of the present invention has been inactivated,

(2) The embryonic stem cell according to (1), wherein the DNA is inactivated by introducing a reporter gene (eg, a β-galactosidase enzyme gene derived from E. coli).

(3) The embryonic stem cell according to (1), which is neomycin-resistant,

(4) The embryonic stem cell according to (1), wherein the non-human mammal is a rodent,

(5) The embryonic stem cell according to (4), wherein the rodent is a mouse,

(6) a DNA-deficient non-human mammal in which the DNA of the present invention has been inactivated,

(7) The DNA is inactivated by introducing a reporter gene (eg, a β-galactosidase gene derived from Escherichia coli), and

The non-human mammal according to the above (6), which can be expressed under the control of a promoter for ΝΑ,

(8) The non-human mammal according to (6), wherein the non-human mammal is a rodent,

(9) The non-human mammal according to (8), wherein the rodent is a mouse; and (10) A compound that promotes or inhibits (preferably inhibits) the promoter activity of DNA of the present invention, which comprises administering a test compound to the animal according to (7) above and detecting the expression of a reporter gene. A method for screening the salt is provided.

A non-human mammalian embryonic stem cell in which the DNA of the present invention has been inactivated is a DNA which is obtained by artificially mutating the DNA of the present invention possessed by the non-human mammal, thereby suppressing the DNA expression ability, Alternatively, the DNA substantially does not have the ability to express the protein of the present invention by substantially losing the activity of the protein of the present invention encoded by the DNA (hereinafter referred to as the knockout DNA of the present invention). In some cases, refers to embryonic stem cells of non-human mammals (hereinafter abbreviated as ES cells).

As the non-human mammal, the same one as described above is used.

The method of 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 by a genetic engineering technique. The knockout DNA of the present invention may be prepared by, for example, shifting the reading frame of a codon or disrupting the function of a promoter or exon by these mutations.

Specific examples of the non-human mammalian embryonic stem cells in which the DNA of the present invention has been inactivated (hereinafter abbreviated as the DNA-inactivated ES cells of the present invention or the knockout ES cells of the present invention) include, for example, The DNA of the present invention possessed by a non-human mammal is isolated, and its exon portion is a drug resistance gene typified by a neomycin resistance gene, a hygromycin resistance gene, lacZ (β-galactosidase gene), cat (clo DNA that disrupts exon function by inserting a reporter gene such as the ramphenicol acetyltransferase gene) or terminates gene transcription in the intron between exons. By inserting a sequence (eg, a polyA additional signal) and preventing the synthesis of the complete messenger RNA, A DNA strand having a DNA sequence constructed so as to disrupt the gene (hereinafter abbreviated as “Yuichi Getting Vector”) was introduced into the chromosome of the animal by, for example, homologous recombination, and the obtained ES was obtained. For a cell, the DNA sequence on or near the DNA of the present invention is purified. The DNA sequence on the Southern hybridization analysis or evening getter vector and the DNA sequence of the neighboring region other than the DNA of the present invention used in the evening getter vector were prepared. It can be obtained by analyzing by the PCR method using primers and selecting the knockout ES cells of the present invention.

As the ES cells from which the DNA of the present invention is inactivated by the homologous recombination method or the like, for example, those already established as described above may be used.新しく It may be newly established according to the method of ma. 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 immunological and genetically For the purpose of obtaining ES cells with a clear background, for example, BDFi mice (C57BL / 6 and C57BL / 6 mice) whose C57BL / 6 mice and C57BL / 6 have reduced the number of eggs collected by crossing with DBA / 2 It is also possible to use a mouse established with DBAZ2, etc. BDFi mice have the advantage of high number of eggs collected and robust eggs, and also have a background of C57BLZ6 mice. ES cells obtained by using C57BLZ6 mice can be used to advantage in that their genetic background can be replaced with C57BLZ6 mice by backcrossing with C57B LZ6 mice when creating disease model mice. .

In addition, when ES cells are established, 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. By using this method, the number of ES cells (about 50 cells) per colony can be reduced, compared to about 10 ⁶ cells for karyotype analysis. The primary selection of ES cells in the early stage of culture can be performed by discriminating between male and female. Can be reduced to width.

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 preferably 100% of the normal number. However, if it is difficult due to physical operations at the time of establishment, knock out the gene of the ES cells, and It is desirable to clone again into cells with a body number of 2 n = 40).

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. For example, on a suitable feeder cell such as STO fibroblasts, in the presence of LIF (1-ΙΟΟΟΟϋ / ml) in a carbon dioxide incubator (preferably 5% carbon dioxide, 95% air or 5% oxygen, Culture at about 37 ° C in 5% carbon dioxide gas and 90% air) .At the time of subculture, for example, trypsin / EDTA solution (usually 0.001 to 0.5% trypsin ZO. L to 5 mM EDTA, preferably A method is used in which cells are converted into single cells by treatment with about 0.1% trypsin / ImMEDTA and seeded on freshly prepared feeder cells. Such subculture is usually carried out 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 muscles, by monolayer culture up to high density or suspension culture until cell clumps are formed under appropriate conditions. [MJ Evans and MH Kaufman, Nature, 292, 154, 1981; GR Martin Proceedings of National Academy of Sciences, Science, Proc. Natl. Acad. Sci. USA) Vol. 78, p. 7634, 1981; TC Doetschman et al., Journal of Obemblilogi and Eximental Morphology, Vol. 87, p. 27, 1985. The DNA-deficient cells of the present invention obtained by differentiating the ES cells of the present invention are useful in the cell biology of the protein of the present invention in the mouth of in vivo.

The non-human mammal deficient in DNA expression of the present invention is distinguished from a normal animal by measuring the mRNA level of the animal using a known method and indirectly comparing the expression level. It is possible to

As the 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 evening getter vector prepared as described above into mouse embryonic stem cells or mouse egg cells. The DNA of the present invention can be knocked out by homologous recombination in which the inactivated DNA sequence replaces the DNA of the present invention on the chromosome of mouse embryonic stem cells or mouse egg cells by gene homologous recombination. it can.

Cells in which the DNA of the present invention has been knocked out can be analyzed by Southern hybridization analysis using a DNA sequence on or near the DNA of the present invention as a probe, or a DNA sequence on a evening-getting vector, and evening-getting. The determination can be made by PCR analysis using, as a primer, the DNA sequence of a neighboring region other than the DNA of the present invention derived from the mouse used in the vector. When 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 cultured at an appropriate time, for example, at the 8-cell stage. The chimeric embryo is injected into a human mammalian embryo or blastocyst, and the produced chimeric embryo is transplanted into the uterus of the pseudopregnant 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.

When a part of the germ cells of the chimeric animal has the mutated DNA locus of the present invention, all tissues are artificially obtained from the population obtained by crossing such a chimeric individual with a normal individual. It can be obtained by selecting an individual composed of cells having the DNA locus of the present invention having a mutation, for example, by judging coat color. The individual obtained in this manner is usually an individual having a heterozygous expression of the protein of the present invention, which is crossed with an individual having a heterozygous expression of the protein of the present invention. It is possible to obtain an individual with poor homo-expression.

When an egg cell is used, for example, a transgenic non-human mammal having a chromosome into which a gettering vector has been introduced can be obtained by injecting a DNA solution into the egg cell nucleus by a microinjection method. Tiger Compared to nicked non-human mammals, they can be obtained by selecting those having a mutation in the DNA locus of the present invention by gene homologous recombination.

In this way, the individual in which the DNA of the present invention has been knocked out may be subjected to rearing in an ordinary breeding environment after confirming that the DNA has been knocked out even in an animal obtained by mating. it can.

Furthermore, the germline can be obtained and maintained according to a standard 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 one homozygote are obtained. By mating male and female heterozygous animals, homozygous and heterozygous animals having the inactivated DNA are bred and subcultured.

The non-human mammalian 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.

In addition, since the non-human mammal deficient in expressing the DNA of the present invention lacks various biological activities that can be induced by the protein of the present invention, it may be caused by the inactivity of the biological activity of the protein of the present invention. It is useful for investigating the causes of these diseases and examining treatment methods, because they can serve as a model for such diseases.

[8a] A method for screening a compound having a preventive / therapeutic effect on a disease caused by DNA deficiency or damage according to the present invention

The non-human mammal deficient in DNA expression of the present invention can be used for screening for a compound having a preventive / therapeutic effect against diseases caused by DNA deficiency or damage of the present invention.

That is, the present invention is characterized in that a test compound is administered to a non-human mammal deficient in DNA expression of the present invention, and changes in the animal are observed and measured. The present invention provides a method for screening a compound or a salt thereof having a preventive / therapeutic effect on a disease to be caused.

Examples of the non-human mammal deficient in DNA expression of the present invention used in the screening method include the same ones 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.

Specifically, a non-human mammal deficient in expression of the DNA of the present invention is treated with a test compound and compared with an untreated control animal, and changes in organs, tissues, disease symptoms, etc. of the animal are indicated as an index. As a test, the preventive and therapeutic effects of the test compound can be tested.

As a method of treating a test animal with a 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.

For example, when screening for a compound having a preventive / therapeutic effect on chronic obstructive pulmonary disease or bronchial asthma, a non-human mammal deficient in expression of the DNA of the present invention is treated with coconut smoke or antigen exposure treatment, and tobacco The test compound is administered before or after the smoke or antigen exposure treatment, and changes in the airway responsiveness of the animal are measured over time. The compound obtained by using the screening method is a compound selected from the test compounds described above, and has a preventive / therapeutic effect against a disease caused by deficiency or damage of the protein of the present invention. It can be used as a medicament such as a safe and low toxic prophylactic / therapeutic agent for the disease. Further, a compound derived from the compound obtained by the above-mentioned screening can be used in the same manner.

The compound obtained by the screening method may form a salt. Examples of 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. Examples of such 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.). 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 the protein of the present invention. The preparations obtained in this way are safe and low toxic, for example, in humans or non-human mammals (eg, rats, mice, guinea pigs, egrets, higgies, bushus, dogs, dogs, cats). , Dogs, monkeys, etc.). The dose of the compound or a salt thereof varies depending on the target disease, the subject of administration, the administration route, and the like. For example, when the compound is orally administered, it is generally used for adults (with a body weight of 60 kg). In patients with chronic obstructive pulmonary disease, about 0.1 to 100 mg, preferably about 1.0 to 50 mg, more preferably about 1.0 to 20 mg of the compound is administered per day. In the case of parenteral administration, the single dose of the compound may vary depending on the administration subject, target disease, etc. For example, the compound is usually injectable in the form of an injection (typically 60 kg) in chronic obstructive pulmonary lungs of adults. When administered to a patient with a disease, it is convenient to administer about 0.01 to 30 mg, preferably about 0.1 to 20 mg, more preferably about 0.1 to 10 mg of the compound by intravenous injection per day. It is. In the case of other animals, the dose can be administered in terms of 60 kg.

[8b) Method for screening for a compound that promotes or inhibits the activity of a promoter for DNA of the present invention

The present invention relates to a method for administering a test compound to a non-human mammal deficient in expression of a DNA of the present invention, and detecting the expression of a reporter gene. Provided is a method for screening a compound or a salt thereof that inhibits the inhibition.

In the above-described screening method, the non-human mammal deficient in DNA expression of the present invention may be a non-human mammal deficient in expression of DNA of the present invention, wherein the DNA of the present invention is obtained by introducing a repo allele gene. Those inactivated and capable of expressing the repo overnight gene under the control of a promoter for the DNA of the present invention are used.

Examples of the test compound include the same compounds as described above.

As the reporter gene, the same one as described above can be used.) 3-galactosidase gene (1 ac Z), soluble alkaline phosphatase gene or The sifellanase gene and the like are preferred.

In a non-human mammal deficient in DNA expression of the present invention in which the DNA of the present invention is replaced with a repo overnight gene, since the repo overnight gene is under the control of the promoter for the DNA of the present invention, By tracing the expression of a substance encoded by the repo overnight gene, the activity of the promoter can be detected.

For example, when a part of the DNA region encoding the protein of the present invention is replaced with a β-galactosidase gene (1 ac 由来) derived from Escherichia coli, the tissue of the present invention expressing the protein of the present invention originally ] -Galactosidase is expressed instead of protein. Thus, for example, by staining with a reagent that is a substrate for 3-galactosidase (such as 5-promo-4-chloro-3-indolyl-1) 8-galactopyranoside (X-ga 1), The state of expression of the protein of the present invention in an animal organism can be easily observed. Specifically, the protein-deficient mouse of the present invention or a tissue section thereof is fixed with dartalaldehyde and the like, washed with phosphate buffered saline (PBS), and then stained with X-ga1 at room temperature or at room temperature. After reacting for about 30 minutes to 1 hour at around 7 ° C, the / 3-galactosidase reaction can be stopped by washing the tissue sample with 1 mM EDTA / PBS solution, and the coloration can be observed. . Further, mRNA encoding 1acZ 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 the DNA of the present invention.

The compound obtained by the screening method may form a salt. Examples of the salt of the compound include physiologically acceptable acids (eg, inorganic acids) and bases (eg, organic acids). And the like, and particularly preferred are physiologically acceptable acid addition salts. Examples of such 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.). Further, the compound of the present invention which inhibits the promoter activity for DNA or the salt thereof can inhibit the expression of the protein of the present invention and inhibit the function of the protein. · Respiratory diseases such as chest diseases (eg, chronic obstructive pulmonary disease (chronic bronchitis, emphysema), diffuse panbronchiolitis, bronchial asthma, cystic fibrosis, irritable pneumonia, etc.), allergic conjunctivitis, Rhinitis

(Eg, allergic rhinitis, hay fever, acute rhinitis, chronic rhinitis, hypertrophic rhinitis, atrophic rhinitis, dry pronasitis, vasomotor rhinitis, gangrene rhinitis, sinusitis, etc.), gastrointestinal diseases (eg, As preventive and therapeutic agents for irritable bowel syndrome, inflammatory bowel disease, ulcerative colitis, Crohn's disease, reflux esophagitis, etc., preferably as preventive and therapeutic agents for respiratory diseases, gastrointestinal diseases, etc. Useful.

Further, a compound derived from the compound obtained by the above screening can also be used.

A drug containing a compound or a salt thereof obtained by the screening method can be produced in the same manner as the above-mentioned drug containing a protein of the present invention or a salt thereof.

The preparations obtained in this way are safe and low toxic and can be used, for example, in humans or non-human mammals (for example, rats, mice, guinea pigs, egrets, sheep, pigs, horses, cats, cats, Dogs, monkeys, etc.). The dose of the compound or a salt thereof varies depending on the target disease, the administration subject, the administration route, and the like. For example, when the compound of the present invention that promotes the promoter activity for DNA is orally administered, generally, the adult ( In patients with chronic obstructive pulmonary disease (with a body weight of 60 kg), about 0.1 to 100 mg, preferably about 1.0 to 50 mg, more preferably about 1.0 to 20 mg of the ehine compound is administered per day. I do. When administered parenterally, the single dose of the compound varies depending on the administration subject, target disease, and the like.For example, the compound that promotes the promoter activity for DNA of the present invention is usually in the form of an injection. When administered to an adult (as 60 kg) patient with chronic obstructive pulmonary disease, about 0.01 to 30 mg, preferably about 0.1 to 20 mg, more preferably about 0.1 to 10 mg of the compound per day is administered. It is conveniently administered by intravenous injection. In the case of other animals, the dose can be administered in terms of 60 kg. On the other hand, for example, when the compound of the present invention that inhibits the promoter activity against DNA is orally administered, generally, in an adult (assuming a body weight of 60 kg) patient with chronic obstructive pulmonary disease, the compound is reduced to about 0 per day. l-100 mg, preferably about 1.0-50 mg, more preferably about 1.0-20 mg. When administered parenterally, the single dose of the compound varies depending on the administration subject, target disease, and the like.For example, a compound that inhibits promoter activity against DNA of the present invention is usually administered in the form of an injection. adult

(As 60 kg) to a patient with chronic obstructive pulmonary disease of about 0.01 to 30 mg, preferably about 0.1 to 20 mg, and more preferably about 0.1 to 1 mg of the compound per day. It is convenient to administer 10 mg by intravenous injection. In the case of other animals, the dose can be administered in terms of 60 kg.

As described above, 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 of the DNA of the present invention overnight. Yes, it can greatly contribute to the investigation of the cause of various diseases caused by DNA expression deficiency or the development of preventive and therapeutic agents of the present invention.

In addition, using a DNA containing the promoter region of the protein of the present invention, genes encoding various proteins are ligated downstream thereof and injected into egg cells of an animal to produce a so-called transgenic animal (transgenic animal). ) Makes it possible to specifically synthesize the polypeptide and examine its action in living organisms. Further, by binding an appropriate repo overnight gene to a part of the promoter and establishing a cell line in which the gene is expressed, the action of specifically promoting or suppressing the in vivo production ability of the protein of the present invention itself is achieved. It can be used as a search system for low molecular weight compounds. In the present specification, bases, amino acids, and the like are indicated by abbreviations based on the abbreviations by IUPAC-IUB Communications on Biochemical Nomenclature or commonly used abbreviations in the art, and examples thereof are described below. When amino acids can have optical isomers, the L-form is indicated unless otherwise specified.

DNA: Deoxylipo nucleic acid c DNA complementary deoxyribonucleic acid

A adenine

T thymine

G Guanin

C

R adenine (A) or guanine (G) Y cytosine (C) or thymine (T)

K, guanine (G) or thymine (T)

M adenine (A) or cytosine (C)

S guanine (G) or cytosine (C) w adenine (A) or thymine (T)

RNA liponucleic acid

mRNA messenger ribonucleic acid

d ATP Deoxyadenosine triphosphate dTTP Deoxythymidine triphosphate

dGTP Deoxyguanosine triphosphate dCTP Deoxycytidine triphosphate. ATP Adenosine triphosphate

EDTA ethylenediaminetetraacetic acid

SDS dodecyl sulfate:

G 1 y Glycine

A 1 a Alanin

Va 1 Valine

Le u leucine

I 1 e isoleucine

S e r serine

Th r threonine

Cy s

Me t Methionin G 1 u glumic acid

As aspartic acid

Lys lysine

A r g Arginine

H is histidine

P h e feniralanin

Ty r tyrosine

T r p

Pro proline

AA ss nn: Asparagine

G 1 n Glutamine

p G 1 u pyroglutamic acid

Further, substituents, protecting groups and reagents frequently used in the present specification are represented by the following symbols.

Me: methyl group

E t: ethyl group

B u: butyl group

Ph: phenyl group

TC: Thiazolidine-4 (R) one-pot lipoxamide group

To s: p-toluenesulfonyl

CHO: Formyl

B z 1: benzyl

Cl ₂ -Bzl: 2,6

Bom: benzyloxymethyl

Z: benzyloxycarponyl

C 1 Z: 2

Br-Z: 2

Bo c: t-butoxycarbonyl

DNP: dinitrophenyl T rt Trityl

Bum t-butoxymethyl

Fmo c N—9-Fluorenylmethoxycarbonyl

HOB t

HOOB t 3, 4-dihydro-1-hydroxy-1-4-oxo-1

1,2,3-benzotriazine

HONB 1-hydroxy-5-norporene-2,3-dicaroxy DCC The sequence numbers in the sequence listing in the present specification show the following sequences.

[SEQ ID NO: 1]

2 shows the amino acid sequence of mouse CLCA4 protein.

[SEQ ID NO: 2]

This shows the base sequence of DNA encoding the amino acid sequence of mouse CLCA4 protein having the amino acid sequence represented by SEQ ID NO: 1.

[SEQ ID NO: 3]

The nucleotide sequence of mouse CLCA4 gene 5 is shown.

[SEQ ID NO: 4]

The nucleotide sequence of the mouse CLCA4 gene on the 3 'side is shown.

[SEQ ID NO: 5]

3 shows the nucleotide sequence of primer 11 used in Examples 1 and 3. [SEQ ID NO: 6]

2 shows the nucleotide sequence of primer 2 used in Examples 1 and 4. [SEQ ID NO: 7]

3 shows the nucleotide sequence of primer 3 used in Examples 1 and 4.

[SEQ ID NO: 8]

2 shows the nucleotide sequence of primer 4 used in Examples 1 and 2. [SEQ ID NO: 9]

2 shows the base sequence of primer 5 used in Example 1. [SEQ ID NO: 10]

3 shows the nucleotide sequence of primer 6 used in Examples 1 and 2.

[SEQ ID NO: 11]

2 shows the base sequence of primer 7 used in Example 1.

[SEQ ID NO: 12]

2 shows the nucleotide sequence of primer 8 used in Example 1.

[SEQ ID NO: 13]

2 shows the nucleotide sequence of primer 9 used in Example 1.

[SEQ ID NO: 14]

2 shows the nucleotide sequence of primer 10 used in Example 1.

[SEQ ID NO: 15]

1 shows the nucleotide sequence of T7promoteperprimer (promoter primer 1) used in Example 1.

[SEQ ID NO: 16]

1 shows the nucleotide sequence of Ml 3 RV primer (primary) used in Example 1.

[SEQ ID NO: 17]

2 shows the amino acid sequence of mouse CLCA4 A protein.

[SEQ ID NO: 18]

The nucleotide sequence of DNA encoding the amino acid sequence of mouse CLCA4A protein having the amino acid sequence represented by SEQ ID NO: 17 is shown.

[SEQ ID NO: 19]

2 shows a partial amino acid sequence of rat CLCA4 protein.

[SEQ ID NO: 20]

This shows the base sequence of DNA encoding the partial amino acid sequence of rat CLCA4 protein having the partial amino acid sequence represented by SEQ ID NO: 19.

[SEQ ID NO: 21]

3 shows the nucleotide sequence of Primer 11 used in Example 3.

[SEQ ID NO: 22] 7 shows the nucleotide sequence of primer 12 used in Example 5.

[SEQ ID NO: 23]

13 shows the base sequence of primer 13 used in Example 5.

[SEQ ID NO: 24]

14 shows the base sequence of primer 14 used in Example 5. Transformant Escherichia coli Escherichia coli (Escherichia coli) TOPlO / pCR-Bluntll-mCLCA4 obtained in Example 1 described below has been used since February 12, 2002, 1-1 1-1 Tsukuba-Higashi, Ibaraki Pref. Deposit No.FERM BP-7887 at the National Institute of Advanced Industrial Science and Technology (AIST) with a postal code of 305-8566) as a deposit number FERM BP-7887 from January 29, 2002 at 2-317 Jusanhoncho, Yodogawa-ku, Osaka, Osaka It has been deposited with the Fermentation Research Institute (IF0) of No. 85 (zip code 532-8686) under the accession number IF0 16751.

The transformant Escherichia coli TOPlO / pCR-Bluntll-mCLCA4A obtained in Example 3 described below has been used since March 4, 2002, 1-1 1-1 Tsukuba, Higashi, Ibaraki Pref. National Institute of Advanced Industrial Science and Technology (ZIP code 305-8566). Deposited at the Patent Organisms Depositary Center as deposit number FERM BP-7934. From February 19, 2002, 2-3-17 Jusanhoncho, Yodogawa-ku, Osaka, Osaka, Japan 85 No. (postal code 532-8686) and deposited with the Fermentation Research Institute (IF0) under the accession number IF0 16764. Hereinafter, the present invention will be described more specifically with reference to Examples, but the present invention is not limited thereto. Example 1

Cloning of mouse CLCA4 gene

See the nucleotide sequence of the human CLCA4 gene (FEBS Letters, vol. 455, p. 295 (1999)) and the nucleotide sequence of the mouse gob-5 gene [Biochem. Biophys. Res. Commun., Vol. 255, p. 347 (1999)] And designed and synthesized four primers [Primer 1 (SEQ ID NO: 5), Primer 2 (SEQ ID NO: 6), Primer 3 (SEQ ID NO: 7) and Primer 4 (SEQ ID NO: 8)] . Next, from mouse colon tissue Total RNA was prepared using ISOGEN (manufactured by Wako Pure Chemical Industries, Ltd.). Furthermore, mRNA was prepared using mRNA purification kit (manufactured by Amersham Pharmacia Biotech). Using this mRNA 1 ig as a starting material, cDNA was synthesized by reverse transcription using a Marathon cDNA Amplification kit (manufactured by Clontech). After performing PCR using this cDNA as type I and a combination of Primer 1 and Primer 1 and Primer 3 and Primer 4, a DNA fragment of the expected size is amplified by agarose gel electrophoresis. Admitted. The reaction was carried out using Pyrobest DNA polymerase (Takara Shuzo Co., Ltd.) on a Thermocycler Gene Amp PCR System 9700 (PerkinElmer) at 94 ° C for 30 seconds, then at 94 for 10 seconds. The reaction cycle was repeated 35 times at 60 ° C for 30 seconds, 72 ° C for 2 minutes and 30 seconds, and finally the reaction was performed at 72 ° C for 10 minutes. It was cloned into the obtained DNA fr¾pCR_BluntII-T0P0 (manufactured by Invitrogen). Furthermore, T7 promoter primer (SEQ ID NO: 15), M13RV primer (SEQ ID NO: 16) and six synthetic primers [Primer 5 (SEQ ID NO: 9), Primer 6 (SEQ ID NO: 10), Primer 7 ( Cycle sequence reaction was performed using SEQ ID NO: 11), Primer 8 (SEQ ID NO: 12), Primer 9 (SEQ ID NO: 13), and Primer 10 (SEQ ID NO: 14)] to obtain a fluorescent DNA sequencer. The base sequence of the reaction product obtained by I-377 (PerkinElmer) was determined. As a result, clone No. 3-1 (SEQ ID NO: 3) containing a 2027 nucleotide sequence containing the mouse N-terminal sequence as a partial nucleotide sequence of the mouse CLCA4 gene and 1869 clones containing the mouse C-terminal sequence The clone No. 7-3 (SEQ ID NO: 4) into which the nucleotide sequence was inserted was obtained. Excluding the overlapping sequence of both, mouse CLCA4 was found to be composed of 2772 nucleotide sequences (SEQ ID NO: 2). Based on the nucleotide sequence represented by SEQ ID NO: 2, 924 amino acid sequences were determined (SEQ ID NO: 1), and named as mouse CLCA4 protein. Subsequently, clone No. 7-3 was digested with Hind I II to cut out a DNA fragment containing the C-terminal region of mouse CLCA4 of 1.4 Kb. This was ligated to a vector sequence containing the N-terminal region of mouse CLCA4 of clone No. 3-1 also digested with Hindi II to construct a plasmid pCR-Bluntll-mCLCA4 containing a mouse CLCA4 full-length sequence. A transformant containing this plasmid was designated as Escherichia coli TOP10 / pCR-BluntII-mCLCA4. At the amino acid level, mouse CLCA4 had 68% homology with human CLCA4 and 51% homology with mouse gob-5. Example 2

Analysis of tissue distribution of mouse CLC A4 gene product

Using Primer 4 (SEQ ID NO: 8) and Primer 1 (SEQ ID NO: 10), mouse tissues (bone marrow, eyes, lymph nodes, smooth muscle, ovary, thymus, stomach, bladder, heart) Brain, spleen, lung, liver, skeletal muscle, kidney, testis, embryos on day 7, 11, 15, 17 and colon (Mouse MTC panel I and Mouse TC panel II: Clontech) PCR was performed. The reaction was performed using Takara Ex Taq (manufactured by Takara Shuzo Co., Ltd.) on a Thermocycler GeneAmp PCR System 9600 (manufactured by Applied Biosystems) first at 94 ° C for 30 seconds, then at 94 ° C for 10 seconds and 60 ° C. The reaction was repeated for 35 cycles, with one reaction cycle consisting of 30 seconds at 72 ° C and 2 minutes at 72 ° C. The obtained reaction product was subjected to electrophoresis on a 2% agarose gel, and then stained with B. bromide to detect an amplified 800 bp band. As a result, the mouse CLCA4 gene product (mRNA) was strongly expressed in smooth muscle and large intestine, and was also expressed in skeletal muscle. Example 3

Cloning of mouse CL CA4 A gene

When cloning the mouse CLCA4 gene, it was suggested that a clone having a nucleotide sequence different from that of mouse CLCA4 was present. Therefore, using a mouse smooth muscle cDNA (Mouse MTC panel II: manufactured by Clontech) as type I, PCR was performed using a combination of primer 1 (SEQ ID NO: 5) and primer 11 (SEQ ID NO: 21), and agarose electrophoresis was performed. It was confirmed that a 2.7 Kb DNA fragment was amplified by electrophoresis. The reaction was carried out using Pyrobest DNA polymerase (Takara Shuzo Co., Ltd.) on a Thermocycler Gene Amp PCR System 9700 (Kin-Elma Co., Ltd.) for 30 seconds at 94 ° C. The reaction was repeated for 35 cycles, each cycle consisting of 30 seconds at 60 seconds and 5 minutes at 72, and the reaction was continued at 72 ° C for 10 minutes. The obtained DNA fragment was pCR-B1 until -T0P0 (Manufactured by Invitrogen). Furthermore, T7 promoter primer (SEQ ID NO: 15), 13RV primer (SEQ ID NO: 16) and six synthetic primers [Primer 1 (SEQ ID NO: 9), Primer 6 (SEQ ID NO: 10), Primer 7 (SEQ ID NO: 11), PRIMER-1 (SEQ ID NO: 12), PRIMER-1 (SEQ ID NO: 13) and PRIMER-10 (SEQ ID NO: 14) The nucleotide sequence of the reaction product obtained using DNA Sequencer-377 (manufactured by Pakinkin Elma) was determined. As a result, it was found that a protein consisting of 924 amino acids (SEQ ID NO: 17) showing homology of 94% at the amino acid level with mouse CLCA4 and 68% with human CLCA4 was encoded (SEQ ID NO: 17). No .: 18), designated as mouse CLCA4A protein. The above plasmid pCR-Bluntll-mCLCA4 was constructed. A transformant containing this plasmid pCR_BluntII-TOP0 was designated as Escherichia coli TOP10 / pCR-Bluntll-mCLCA4A. Example 4

Analysis of tissue distribution of mouse CLCA4 A gene product,

A 1020 bp DNA fragment amplified by PCR using a combination of primer 2 (SEQ ID NO: 6) and primer 3 (SEQ ID NO: 7), which are the common sequence of mouse CLCA4 and mouse CLCA4A, is derived from mouse CLCA4 by the restriction enzyme PvuII (Takara Shuzo) Using the fact that mouse CLCA4A-derived DNA was digested to 680 bp and 340 bp, the tissue distribution of each was examined. The PCR reaction was performed using Takara Ex Taq (Takara Shuzo) in a thermal cycler GeneAmp PCR System 9600 (Applied Biosystems) at 94 ° C for 30 seconds, then at 94 for 10 seconds and 6 (TC The reaction was carried out by repeating 35 cycles with 1 reaction cycle consisting of 30 seconds and 2 minutes at 72 ° C. The obtained reaction product was converted into PvuII, subjected to electrophoresis on a 2% agarose gel, and then stained with CHBr. As a result, the amplified DNA fragment derived from the large intestine was not digested, but about half of the amplified DNA fragment derived from smooth muscle was digested with PvuII, and the mouse CLCA4 gene product ( mRNA) was expressed in smooth muscle and large intestine, whereas mouse CLCA4A gene product (mRNA) was found to be expressed only in smooth muscle. Example 5

Cloning of rat CLCA4 gene

Two primers were used with reference to the nucleotide sequence of the human CLCA4 gene (FEBS Letters, vol. 455, p. 295, 1999), the nucleotide sequence of the mouse CLCA4 gene (SEQ ID NO: 2), and the nucleotide sequence of the mouse CLCA4A gene. Primer 12 (SEQ ID NO: 22) and Primer 13 (SEQ ID NO: 23)] were designed and synthesized. Next, total RNA was prepared from rat intestinal tissue using IS0GEN (manufactured by Wako Pure Chemical Industries, Ltd.). Further mRNA

mRNA was prepared using a purification kit (Amersham Pharmacia Biotech). Using this mRNA lpg as a starting material, cDNA was synthesized by a reverse transcription reaction using a Marathon cDNA Amplification kit (manufactured by Clontech). After performing PCR using this cDNA as type I and a combination of primers 12 and 13, it was confirmed that a DNA fragment of the expected size was amplified by agarose electrophoresis. The reaction was performed using Pyrobest DNA polymerase (Takara Shuzo Co., Ltd.) in a Thermocycler Gene Amp PCR System 9700 (PerkinElmer Co., Ltd.) first at 94 ° C for 30 seconds, and then at 94 ° C. The reaction cycle was repeated 35 cycles, each consisting of 10 seconds, 30 seconds at 60 ° C, and 2 minutes at 72 ° C. Finally, the reaction was performed at 72 ° C for 10 minutes. The obtained DNA fragment was cloned into pCR-Bluntll-TOP0 (Invitrogen). In addition, T7 promoter primer (SEQ ID NO: 15), M13RV primer (SEQ ID NO: 16) and three synthetic primers [Primer 12 (SEQ ID NO: 22), Primer 13 (SEQ ID NO: 23), Primer 14 (SEQ ID NO: 24)], and the nucleotide sequence of the reaction product obtained with a fluorescent DNA sequencer-377 (manufactured by PerkinElmer) was determined. As a result, a clone No. a-20 (SEQ ID NO: 20) in which 1119 nucleotide sequences were inserted as a partial nucleotide sequence of rat CLCA4 gene was obtained. Based on the nucleotide sequence represented by SEQ ID NO: 20, a partial amino acid sequence of 373 was determined (SEQ ID NO: 19). The partial amino acid sequence had 66% homology with the corresponding sequence of human CLCA4 and 81% homology with the corresponding sequences of mouse CLCA4 and mouse CLCA4A. '' Industrial applicability

The proteins and polynucleotides of the present invention include, for example, lungs, lungs with inflammation of the airways, respiratory diseases such as chest diseases [eg, chronic obstructive pulmonary disease (chronic bronchitis, emphysema), diffuse panbronchiolosis Inflammation, bronchial asthma, cystic fibrosis, irritable pneumonia, etc.), allergic conjunctivitis, rhinitis (eg, allergic rhinitis, hay fever, acute rhinitis, chronic rhinitis, hypertrophic rhinitis, atrophic rhinitis, before drying Rhinitis, vasomotor rhinitis, gangrenous rhinitis, sinusitis, etc., gastrointestinal diseases (eg, irritable bowel syndrome, inflammatory bowel disease, ulcerative colitis, Crohn's disease, reflux esophagitis, etc.) It is useful as a diagnostic marker or the like, and is an inhibitor obtained by screening using the protein, polynucleotide or an antibody against the protein, and a neutralizing antibody that inhibits the activity of the protein. For example, respiratory diseases such as lungs, lungs with inflammation of the respiratory tract, and chest disease [eg, chronic obstructive pulmonary disease (chronic bronchitis, emphysema), diffuse panbronchiolitis, bronchial asthma, cystic fibrosis Disease, irritable pneumonia, etc.), allergic conjunctivitis, rhinitis (eg, allergic rhinitis, hay fever, acute rhinitis, chronic rhinitis, hypertrophic rhinitis, atrophic rhinitis, dry prorhinitis, vascular hunting rhinitis, gangrene Prophylactic / therapeutic agents, such as rhinitis, sinusitis, etc., gastrointestinal diseases (eg, irritable bowel syndrome, inflammatory bowel disease, ulcerative colitis, Crohn's disease, reflux esophagitis, etc.), preferably respiration It can be used as a preventive and therapeutic agent for organ diseases and gastrointestinal diseases.

In addition, the antisense polynucleotide of the present invention can suppress the expression of the protein of the present invention. Examples thereof include respiratory diseases such as lungs, lungs accompanied by airway inflammation, and chest diseases (eg, chronic obstructive pulmonary disease). Diseases (chronic bronchitis, emphysema), diffuse panbronchiolitis, bronchial asthma, cystic fibrosis, irritable pneumonia, etc.), allergic conjunctivitis, rhinitis (eg, allergic rhinitis, pollinosis, acute rhinitis, chronic) Rhinitis, hypertrophic rhinitis, atrophic rhinitis, dry proctitis, vasomotor rhinitis, 'gangrene rhinitis, sinusitis, etc.), gastrointestinal disorders (eg, irritable bowel syndrome, inflammatory bowel disease, ulcerative colon) Prevention and treatment of diseases such as inflammation, Crohn's disease, reflux esophagitis, etc., preferably prevention of respiratory diseases, gastrointestinal diseases, etc. It can be used as a therapeutic agent and the like. Furthermore, various polynucleotides of the present invention may be used for respiratory diseases such as lungs, lungs with airway inflammation, and chest diseases [eg, chronic obstructive pulmonary disease (chronic bronchitis, emphysema), diffuse panbronchiole Inflammation, bronchial asthma, cystic fibrosis, irritable pneumonia, etc.), allergic conjunctivitis, rhinitis (eg, rhinorrhea, hay fever, acute rhinitis, chronic rhinitis, hypertrophic rhinitis, atrophic rhinitis, before drying Rhinitis, vasomotor rhinitis, gangrenous rhinitis, sinusitis, etc., gastrointestinal disorders (eg, irritable bowel syndrome, inflammatory bowel disease, ulcerative colitis, Crohn's disease, reflux esophagitis, etc.) Useful for diagnosis, prevention or treatment.

Claims

The scope of the claims

I. A protein containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 17, or a salt thereof.

2. A protein comprising an amino acid sequence represented by SEQ ID NO: 1 or a salt thereof.

3. A protein comprising an amino acid sequence represented by SEQ ID NO: 17 or a salt thereof.

4. A protein having an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 19, or a salt thereof.

5. A protein comprising an amino acid sequence represented by SEQ ID NO: 19 or a salt thereof.

6. The partial protein of the protein of claim 1 or a salt thereof.

7. A partial peptide of the protein according to claim 4, or a salt thereof.

8. A polynucleotide comprising a polynucleotide encoding the protein of claim 1 or a partial peptide thereof.

9. A polynucleotide containing a polynucleotide encoding the protein according to claim 4 or a partial peptide thereof.

10. The polynucleotide according to claim 8 or 9, which is 10. DNA.

II. A polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 2 or SEQ ID NO: 18.

12. The polynucleotide according to claim 10, comprising the nucleotide sequence represented by SEQ ID NO: 20.

13. A transformant containing the polynucleotide according to claim 8 or 9. 14. A transformant transformed with the recombinant vector according to claim 13.

15. The method according to claim 14, wherein the transformant according to claim 14 is cultured to produce and accumulate the protein according to claim 1 or claim 4, a partial peptide thereof, or a salt thereof, and collect this. A method for producing the protein or its partial peptide or a salt thereof according to claim 1 or 4.

16. A medicament comprising the protein according to claim 1 or 4, or a partial peptide thereof, or a salt thereof.

17. An antibody against the protein according to claim 1 or 4, or a partial peptide thereof, or a salt thereof.

18. A pharmaceutical comprising the antibody according to claim 17.

19. A diagnostic agent comprising the antibody according to claim 17.

20. An antisense polynucleotide comprising a nucleotide sequence complementary to or substantially complementary to the nucleotide sequence of the polynucleotide according to claim 8 or 9, or a part thereof.

21. A medicament comprising the antisense polynucleotide according to claim 20. 22. The activity of the protein according to claim 1 or claim 4, or a partial peptide thereof or a salt thereof, wherein the protein according to claim 1 or claim 4 or a partial peptide thereof or a salt thereof is used. A method for screening for a compound that inhibits or a salt thereof.

23. The protein according to claim 1 or claim 4, or a partial peptide thereof, or a salt thereof, characterized by containing the protein according to claim 1 or claim 4, a partial peptide thereof, or a salt thereof. A kit for screening for a compound that inhibits the activity or a salt thereof.

2 4. The protein according to claim 1 or 4, or a partial peptide thereof or a partial peptide thereof, which can be obtained by using the screening method according to claim 22 or the screening kit according to claim 23. A compound that inhibits the activity of a salt or a salt thereof.

25. A medicament comprising the compound according to claim 24 or a salt thereof.

26. A method for screening a compound or a salt thereof that inhibits the expression of the protein gene according to claim 1 or 4, characterized by using the polynucleotide according to claim 8 or 9.

27. A kit for screening a compound that inhibits the expression of the protein gene according to claim 1 or 4, or a salt thereof, which comprises the polynucleotide according to claim 8 or 9.

28. A compound that inhibits expression of the protein gene according to claim 1 or 4, which can be obtained by using the screening method according to claim 26 or the screening kit according to claim 27. Or its salt.

29. A medicament comprising the compound according to claim 28 or a salt thereof.

30. A method for screening a compound or a salt thereof that inhibits the expression of a protein according to claim 1 or claim 4, characterized by using the antibody according to claim 17.

31. A screening kit for a compound or a salt thereof that inhibits the expression of the protein according to claim 1 or 4, which comprises the antibody according to claim 17.

32. A compound that inhibits the expression of the protein according to claim 1 or 4, which can be obtained by using the screening method according to claim 30 or the screening kit according to claim 31. Or its salt.

33. A medicament comprising the compound according to claim 32 or a salt thereof.

3 4. Claim 16 which is an agent for preventing or treating respiratory diseases, rhinitis or digestive diseases.

18. The medicament according to 18, 21, 25, 29 or 33.

35. The medicament according to claim 34, which is an agent for preventing or treating chronic obstructive pulmonary disease, bronchial asthma, inflammatory bowel disease or reflux esophagitis.

36. The method according to claim 19, which is a diagnostic agent for respiratory disease, rhinitis or gastrointestinal disease.

37. The diagnostic agent according to claim 36, which is a diagnostic agent for chronic obstructive pulmonary disease, bronchial asthma, inflammatory bowel disease or reflux esophagitis.

38. Chronic obstructive pulmonary disease, bronchial asthma, inflammation characterized by administering to a mammal an effective amount of the compound according to claim 24, claim 28 or claim 32 or a salt thereof. How to prevent and treat genital bowel disease or reflux esophagitis.

39. The compound according to claim 24, claim 28 or claim 32 for producing a prophylactic or therapeutic agent for chronic obstructive pulmonary disease, bronchial asthma, inflammatory bowel disease or reflux esophagitis, or a compound thereof. Use of salt.