JP2003325187A - New protein, dna encoding the same and application of the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a new protein useful for preventing/treating pulmonary/ thoracic diseases involving lung/airway inflammation, to provide a polynucleotide encoding said protein, to provide a method for screening compounds inhibiting the activity of said protein, and to provide compounds obtained by said screening method. <P>SOLUTION: The protein and the polynucleotide are useful as diagnostic markers or the like for pulmonary/thoracic diseases involving lung/airway inflammation, rhinitis, etc. An inhibitor obtained by screening using said protein, polynucleotide or antibody, and a neutralized antibody inhibiting the activity of said protein, are usable as prophylactic/therapeutic agents for e.g. pulmonary/ thoracic diseases involving lung/airway inflammation such as chronic obstructive pulmonary diseases, rhinitis, etc. <P>COPYRIGHT: (C)2004,JPO

Description

Detailed Description of the Invention

[0001]

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

[0002]

2. Description of the Related Art Chronic obstructive pulmonary disease (chronic bronchitis, emphysema) is considered to become a major illness of respiratory diseases in the future as the smoking generation ages and the average lifespan increases. . In addition, bronchial asthma is a chronic inflammatory disease of the respiratory tract, shows airway narrowing, and has symptoms such as paroxysmal dyspnea, wheezing, and coughing. Many cells such as respiratory epithelial cells, mast cells, eosinophils, and T lymphocytes are involved in the onset and progress thereof. One of the most important features of bronchial asthma is
The airways are responsive to stimuli (airway hyperresponsiveness). This airway hyperresponsiveness is caused by airway inflammation centered on detachment of airway epithelium by chemical mediators secreted by cells infiltrating the airway such as eosinophils, but genetic factors and environmental factors are also complicatedly affected. It is believed that VCAM-1, ICAM-1 on airway epithelial cells and capillary endothelium around bronchi, when airway inflammatory response is triggered by external stimuli (allergens, exhaust products) or viral infection
Adhesive molecules such as [J.Allergy Cli
n.Immunol.), 96, 941 (1995)], cytokines and chemoattractants are produced. Th2 for patients with bronchial asthma
-Type helper T cell function is enhanced, and IL-3, IL-
4, Th2 type cytokines such as IL-5, IL-13, GM-CSF and e
Increased production of chemokines such as otaxin and RANTES. IL
-4 and IL-13 have an IgE production-inducing action, and IL-3 and IL-4 have a mast cell proliferation-inducing action. Furthermore, eosinophils differentiate and proliferate due to the action of IL-5, GM-CSF, etc., and eotaxin, RANTES
Infiltrate the respiratory tract due to [Allergy Asthma Proc.], Volume 20,
141 (1999)]. CLCA (Cl-channel, Ca ²⁺ -act derived from human
The ivated 4 DNA and protein belong to the calcium-dependent chloride channel protein family [Non-Patent Document 1 FEBS Letters
s), 455, 295 (1999)], Chronic obstructive pulmonary disease (COP
D; Chronic Obstructive Pulmonary disease), and its association with bronchial asthma is not known. On the other hand, the base sequence of the human CLCA1 gene is Genomics, 54, 20.
0 (1998), Biochemical and Biophysical Research Communications (Biochem Biop
hys Res Commun), 255, 347 (1999) and the like, and it has been reported that this gene is associated with bronchial asthma and chronic obstructive pulmonary disease (Patent Document 1 WO
01/38530 publication). The nucleotide sequence of the mouse gob-5 gene [Biochemical and Biophysical Research Communication (Biochem. Biophys. Res. Com.
mun.), 255, 347 (1999)], the nucleotide sequence of porcine CLCA1 gene [Physiological genomics (Physiol.
Genomics), Volume 3, page 101 (2000)] and the like.

[0003]

[Patent Document 1] WO 01/38530

[Non-Patent Document 1] FEBS Letters
s), 455, 295 pages, 1999

[0004]

It is desired to develop an excellent prophylactic / therapeutic agent and diagnostic agent for chronic obstructive pulmonary disease, bronchial asthma and the like with few side effects.

[0005]

As a result of intensive studies to solve the above problems, the present inventors found a novel gene belonging to the calcium-dependent chloride channel family, and completed the present invention. I arrived. That is,
The present invention includes (1) SEQ ID NO: 1 or SEQ ID NO: 1
A protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by 7, or a salt thereof,
(2) A protein consisting of the amino acid sequence represented by SEQ ID NO: 1 or a salt thereof, (3) A protein consisting of the amino acid sequence represented by SEQ ID NO: 17, or a salt thereof,
(4) A protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 19, or a salt thereof, (5) a protein containing the amino acid sequence represented by SEQ ID NO: 19, or a salt thereof salt,
(6) Partial peptide of the protein described in (1) or a salt thereof, (7) Partial peptide of the protein described in (4) or a salt thereof, (8) Code the protein described in (1) or a partial peptide thereof (9) A polynucleotide containing a polynucleotide encoding the protein or the partial peptide thereof according to (4) above,
(10) The polynucleotide according to (8) or (9) above which is DNA, (11) the polynucleotide consisting of the nucleotide sequence represented by SEQ ID NO: 2 or SEQ ID NO: 18, (12) represented by SEQ ID NO: 20. The polynucleotide according to (10) above, which comprises the base sequence shown, (13)
A recombinant vector containing the polynucleotide according to (8) or (9) above, (14) a transformant transformed with the recombinant vector according to (13) above, (15).
The transformant described in (14) above is cultivated to obtain (1) above.
Alternatively, the protein according to (4) or a partial peptide thereof or a salt thereof is produced and accumulated, and then collected, and the protein or the partial peptide thereof or a salt thereof according to the above (1) or (4) is collected. Production method, (16) A medicament comprising the protein according to (1) or (4) above or a partial peptide thereof or a salt thereof, (17) The protein according to (1) or (4) above or a partial peptide thereof. Alternatively, an antibody against a salt thereof, (18) A pharmaceutical comprising the antibody according to (17) above. (19) A diagnostic agent comprising the antibody according to (17) above, (20) a base sequence complementary or substantially complementary to the base sequence of the polynucleotide according to (8) or (9), or a base sequence thereof. An antisense polynucleotide containing a part thereof, (21) above (2
(22) A pharmaceutical comprising the antisense polynucleotide according to (0), (22) The protein according to (1) or (4) above, a partial peptide thereof or a salt thereof is used, (1) or (4) A method for screening a compound or a salt thereof which inhibits the activity of the protein or a partial peptide thereof or a salt thereof, (23) the protein or a partial peptide thereof or a salt thereof according to (1) or (4) above. A kit for screening a compound or a salt thereof that inhibits the activity of the protein according to (1) or (4) above, a partial peptide thereof, or a salt thereof, characterized in that the kit according to (22) above. It can be obtained using the screening method or the screening kit described in (23) above. (25) A compound which inhibits the activity of the protein according to (1) or (4) or a partial peptide thereof or a salt thereof, or a salt 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 which inhibits the expression of the protein gene according to (1) or (4), which comprises using the polynucleotide according to (8) or (9) above, (28) A kit for screening a compound or a salt thereof which inhibits the expression of the protein gene according to (1) or (4), which comprises the polynucleotide according to (8) or (9) above. The screening method described in (26) above or the screening kit described in (27) above can be used to obtain the above (1) or Is (4) a compound or its salt that inhibits the expression of protein gene described, (29) A pharmaceutical agent comprising the compound or its salt of the above (28) wherein, (30) (1
7) The antibody described in 1) above is used,
Or (4) a method for screening a compound or a salt thereof that inhibits the expression of the protein, (31) the protein according to (1) or (4) above, which comprises the antibody according to (17) For screening a compound or its salt that inhibits the expression of (3)
2) A compound or a salt thereof which inhibits the expression of the protein according to (1) or (4), which can be obtained by using the screening method according to (30) or the screening kit according to (31). ) Above (3
2) A pharmaceutical comprising the compound or salt thereof according to the above,
(34) Prevention of respiratory diseases, rhinitis or digestive diseases
The above therapeutic agents (16), (18), (21), (2)
5), the medicine according to (29) or (33), (35)
The drug according to (34) above, which is a prophylactic / therapeutic agent for chronic obstructive pulmonary disease, bronchial asthma, inflammatory bowel disease or reflux esophagitis, (36) a diagnostic agent for respiratory disease, rhinitis or digestive system disease. (37) The diagnostic agent according to (19), (37) The diagnostic agent according to (36) above, which is a diagnostic agent for chronic obstructive pulmonary disease, bronchial asthma, inflammatory bowel disease or reflux esophagitis, (3)
8) Chronic obstructive pulmonary disease, bronchial asthma, inflammatory bowel disease, characterized in that an effective amount of the compound or salt thereof according to (24), (28) or (32) above is administered to a mammal. Or the method of preventing / treating reflux esophagitis, (3
9) Use of the compound or salt thereof according to the above (24), (28) or (32) for producing a prophylactic / therapeutic agent for chronic obstructive pulmonary disease, bronchial asthma, inflammatory bowel disease or reflux esophagitis. And so on. Furthermore, the (40) protein has the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 17, SEQ ID NO: 1 or SEQ ID NO :.
Amino acid in which 1 or 2 or more (preferably about 1 to 30, preferably about 1 to 10 and more preferably several (1 to 5)) amino acids in the amino acid sequence represented by 17 are deleted Sequence, SEQ ID NO: 1 or SEQ ID NO:
1 or 2 or more in the amino acid sequence represented by 17 (preferably about 1 to 30, preferably about 1 to 10,
More preferably, an amino acid sequence in which several (1 to 5) amino acids are added, SEQ ID NO: 1 or SEQ ID NO: 17
An amino acid sequence in which 1 or 2 or more (preferably about 1 to 30, preferably about 1 to 10 and more preferably several (1 to 5)) amino acids are inserted into the amino acid sequence represented by 1 or 2 or more (preferably about 1 to 30, preferably about 1 to 10, more preferably number (1 to 5)) in the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 17. The protein according to (1) above, which is a protein containing an amino acid sequence in which the amino acid of) is substituted with another amino acid, or an amino acid sequence in which these are combined, (41) the poly according to (8) or (9) above. A polynucleotide that hybridizes with a nucleotide under high stringent conditions,
(42) Also provided is a diagnostic agent containing the polynucleotide according to (8) or (9) above.

[0006]

BEST MODE FOR CARRYING OUT THE INVENTION A protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 17 of the present invention, and the amino acid represented by SEQ ID NO: 19. A protein containing the same or substantially the same amino acid sequence as the sequence (hereinafter, sometimes referred to as the protein of the present invention) is a human or non-human warm-blooded animal (for example, guinea pig, rat, mouse, chicken, rabbit, Pig cells, sheep cells, bovine cells, monkey cells, etc. (eg, hepatocytes, splenocytes, nerve cells, glial cells, pancreatic β cells, bone marrow cells, mesangial cells, Langerhans cells, epidermal cells, epithelial cells, goblet cells, endothelial cells) , Smooth muscle cells, fibroblasts, fibrocytes, muscle cells, adipocytes, immune cells (eg, macrophages, T cells, B cells, natural killer) Cells, mast cells, neutrophils, basophils, eosinophils, monocytes), megakaryocytes, synovial cells, chondrocytes, osteocytes, osteoblasts, osteoclasts, mammary gland cells, hepatocytes or stroma Cells, or progenitor cells of these cells, stem cells or cancer cells, or any tissue in which these cells exist, for example, brain, various parts of the brain (eg, olfactory bulb, amygdala, basal sphere, hippocampus, thalamus, thalamus) Lower part, cerebral cortex, medulla oblongata, cerebellum), spinal cord, pituitary gland, stomach, pancreas, kidney, liver, gonad, thyroid, gallbladder, bone marrow, adrenal gland, skin, muscle, lung,
Digestive tract (eg, large intestine, small intestine), blood vessel, heart, thymus, spleen,
Submandibular gland, peripheral blood, prostate, testicles, ovaries, placenta, uterus,
It may be a protein derived from bone, joint, skeletal muscle, or the like, or may be a synthetic protein.

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 8%, with the amino acid sequence represented by SEQ ID NO: 1.
Examples thereof include amino acid sequences having a homology of 0% or more, preferably about 90% or more, more preferably about 95% or more, more preferably about 97% or more. The protein containing the amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 1 includes, for example, the amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 1 above. , A protein having substantially the same activity as the protein having the amino acid sequence represented by SEQ ID NO: 1 is preferable. An amino acid sequence which is substantially the same as the amino acid sequence represented by SEQ ID NO: 17 has about 70% or more amino acid sequence represented by SEQ ID NO: 17,
Preferably about 80% or more, preferably about 90% or more, more preferably about 95% or more, more preferably about 97%.
Examples include amino acid sequences having the above homology.
The protein containing an amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 17 includes, for example, an amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 17 above. , 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 is about 70% or more, preferably about 80% or more, preferably about 90% or more, with the amino acid sequence represented by SEQ ID NO: 19. More preferably about 95% or more,
More preferred is an amino acid sequence having a homology of about 97% or more. The protein containing the amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 19 includes, for example, the amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 19. , A protein having substantially the same activity as the protein having the amino acid sequence represented by SEQ ID NO: 19 is preferable. Examples of substantially the same activity include chloride channel activity (eg, calcium-dependent chloride channel activity, etc.) and the like. The term “substantially the same” means that those properties are qualitatively (eg, physiologically or pharmacologically) the same. Therefore, chloride channel activity is equivalent (eg, about 0.01-100).
Times, preferably about 0.1 to 10 times, more preferably 0.
However, the quantitative factors such as the degree of activity and the molecular weight of the protein may be different. To measure activity such as chloride channel activity,
The measurement can be performed according to a known method, for example, the method described in Genomics, 54, 200 (1998) or a method similar thereto.

The protein of the present invention is, for example, (1) 1 or 2 or more (preferably about 1 to 30 and preferably 1 to 10) in the amino acid sequence represented by SEQ ID NO: 1. Degree, more preferably number (1-5)
Amino acid sequence), the amino acid sequence of SEQ ID NO:
An amino acid sequence in which 1 or 2 or more (preferably about 1 to 30, preferably about 1 to 10 and more preferably several (1 to 5)) amino acids are added to the amino acid sequence represented by 1, 1 or 2 or more (preferably about 1 to 30, preferably about 1 to 10), more preferably a number (1 to 2 in the amino acid sequence represented by SEQ ID NO: 1
5) the amino acid sequence in which the amino acid is inserted, 1 or 2 or more (preferably about 1 to 30, preferably about 1 to 10) in the amino acid sequence represented by SEQ ID NO: 1, more preferably Is a so-called mutein such as a protein containing an amino acid sequence in which several (1 to 5) amino acids are substituted with other amino acids, or an amino acid sequence combining them, (2) the amino acid represented by SEQ ID NO: 17. 1 or 2 or more in the sequence (preferably,
1 to 30 amino acids, preferably 1 to 10 amino acids, more preferably several (1 to 5) amino acids are deleted, 1 in the amino acid sequence represented by SEQ ID NO: 17.
Or 2 or more (preferably about 1 to 30, preferably about 1 to 10 and more preferably a number (1 to 5)
Amino acid sequence), SEQ ID NO:
1 or 2 or more in the amino acid sequence represented by 17 (preferably about 1 to 30, preferably about 1 to 10,
More preferably, 1 or 2 or more (preferably about 1 to 30, preferably 1) in the amino acid sequence in which several (1 to 5) amino acids are inserted, or the amino acid sequence represented by SEQ ID NO: 17. A so-called mutein such as a protein containing an amino acid sequence in which about 10 to about 10, more preferably several (1 to 5) amino acids are substituted with other amino acids, or an amino acid sequence combining them,
(3) 1 in the amino acid sequence represented by SEQ ID NO: 19
Or 2 or more (preferably about 1 to 30, preferably about 1 to 10 and more preferably a number (1 to 5)
Amino acid sequence), the amino acid sequence of SEQ ID NO:
1 or 2 or more in the amino acid sequence represented by 19 (preferably about 1 to 30, preferably about 1 to 10,
More preferably, 1 or 2 or more (preferably 1 to 30 or so, preferably 1 to 10) amino acid sequence added with several (1 to 5) amino acids, or the amino acid sequence represented by SEQ ID NO: 19. An amino acid sequence in which about one, more preferably several (1 to 5) amino acids are inserted,
1 or 2 or more (preferably about 1 to 30 and preferably 1 in the amino acid sequence represented by SEQ ID NO: 19)
Also included are so-called muteins such as proteins containing an amino acid sequence in which about 10 to about 10, more preferably a few (1 to 5) amino acids are substituted with other amino acids, or an amino acid sequence combining them.

The protein in the present specification has the N-terminal (amino terminal) at the left end and the C-terminal (carboxyl terminal) at the right end according to the convention of peptide notation. SEQ ID NO: including the protein containing the amino acid sequence represented by 1, the protein of the present invention, C-terminal, is a carboxyl group (-COOH), a carboxylate (-COO ^-), an amide (-CONH ₂₎ or an It may be any of ester (-COOR). Here, R in the ester is, for example, a C _1-6 alkyl group such as methyl, ethyl, n-propyl, isopropyl and n-butyl, for example, cyclopentyl,
C _3-8 cycloalkyl group such as cyclohexyl, for example, C _6-12 aryl group such as phenyl and α-naphthyl,
For example, phenyl-C _1-2 such as benzyl and phenethyl.
Alkyl groups or C _7-14 aralkyl groups such as α-naphthyl-C _1-2 alkyl groups such as α-naphthylmethyl, pivaloyloxymethyl groups and the like are used. When the protein of the present invention has a carboxyl group (or carboxylate) in addition to the C-terminal, those in which the carboxyl group is amidated or esterified are also included in the protein of the present invention. As the ester in this case, for example, the above-mentioned C-terminal ester or the like is used. further,
The protein of the present invention includes an N-terminal amino acid residue (eg,
Amino group protecting group of the methionine residue) (e.g., formyl group, such as C _1-6 alkanoyl such as acetyl group C _1-6
Protected with an acyl group, etc., cleaved in vivo to form a glutamine residue at the N-terminal that is pyroglutamine-oxidized, a substituent on the side chain of an amino acid in the molecule (eg -OH, -SH) , An amino group, an imidazole group, an indole group, a guanidino group, etc.) are protected with an appropriate protecting group (for example, a C _1-6 acyl group such as a C _1-6 alkanoyl group such as a formyl group and an acetyl group). Also included are complex proteins such as so-called glycoproteins in which sugar chains are bound. Specific examples of the protein of the present invention include, for example, a protein containing the amino acid sequence represented by SEQ ID NO: 1, a protein containing the amino acid sequence represented by SEQ ID NO: 17, and represented by SEQ ID NO: 19. Examples thereof include proteins containing an amino acid sequence.

The partial peptide of the protein of the present invention may be any partial peptide of the above-mentioned protein of the present invention, and preferably any peptide having the same properties as the above-mentioned protein of the present invention. . For example, 20 of the constituent amino acid sequences of the protein of the present invention
Or more, preferably 50 or more, more preferably 70
Peptides having an amino acid sequence of 1 or more, more preferably 100 or more, and most preferably 200 or more are used.

The partial peptide of the present invention has one or more (preferably 1 to 10) amino acid sequences in its amino acid sequence.
1 or 2 or more (preferably 1 to 20 or so, more preferably 1 to 2 or more is deleted in the amino acid sequence, or about 1 or 2 (1 to 5) amino acids are deleted.
About 10 amino acids, more preferably several (1 to 5) amino acids are added, or 1 or 2 is added to the amino acid sequence.
1 or more (preferably about 1 to 20, more preferably about 1 to 10 and more preferably several (1 to 5)) amino acids are inserted, or 1 in the amino acid sequence is inserted.
Alternatively, two or more (preferably about 1 to 10, more preferably several, further preferably about 1 to 5) amino acids may be substituted with other amino acids. Further, the partial peptide of the present invention has a C-terminal carboxyl group (-COO
H), carboxylate (-COO ^-), may be any of an amide (-CONH ₂₎ or an ester (-COOR). Further, the partial peptide of the present invention has a carboxyl group (or carboxylate) other than the C-terminal, an N-terminal amino acid residue (eg, methionine residue), like the above-mentioned protein of the present invention. ) The amino group of which is protected by a protecting group, the glutamine residue produced by cleavage of the N-terminal side in vivo is pyroglutamine-oxidized, and the substituent on the side chain of the amino acid in the molecule is a suitable protecting group. Those which are protected by, or complex peptides such as so-called glycopeptides to which sugar chains are bound are also included. The partial peptide of the present invention can also be used as an antigen for producing an antibody. The salts of the protein or partial peptide of the present invention include physiologically acceptable acids (eg, inorganic acids, organic acids)
And salts with bases (eg, alkali metal salts) are used,
Particularly, physiologically acceptable acid addition salts are preferable. Examples of such salts include 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). Acid, tartaric acid, citric acid, malic acid, oxalic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid) and salts thereof are used.

The protein of the present invention or its partial peptide or its salt can be produced from the cells or tissues of the above-mentioned humans or warm-blooded animals by a known method for purifying the protein, or contains a DNA encoding the protein. It can also be produced by culturing a transformant. It can also be produced according to the peptide synthesis method described below. When producing from tissues or cells of humans or non-human mammals, after homogenizing tissues or cells of humans or non-human mammals, extraction is carried out with an acid or the like, and the extract is subjected to reverse phase chromatography, ion exchange chromatography. Purification and isolation can be achieved by combining chromatography such as.

For the synthesis of the protein or partial peptide of the present invention, its salt, or its amide, a commercially available resin for protein synthesis can be used. Examples of such a resin include chloromethyl resin, hydroxymethyl resin, benzhydrylamine resin, aminomethyl resin, 4-benzyloxybenzyl alcohol resin, 4-methylbenzhydrylamine resin, PAM resin, 4-hydroxymethylmethyl resin. Phenylacetamide methyl resin, polyacrylamide resin, 4- (2 ',
4'-dimethoxyphenyl-hydroxymethyl) phenoxy resin, 4- (2 ', 4'-dimethoxyphenyl-F
moc aminoethyl) phenoxy resin and the like. Using such a resin, an amino acid having an α-amino group and a side chain functional group appropriately protected is condensed on the resin according to various known condensation methods according to the sequence of the target protein. At the end of the reaction, the protein or partial peptide is cleaved from the resin and at the same time various protective groups are removed,
Further, an intramolecular disulfide bond forming reaction is carried out in a highly diluted solution to obtain the target protein or partial peptide or an amide thereof. Regarding the condensation of the above-mentioned protected amino acids, various activation reagents that can be used for protein synthesis can be used, but carbodiimides are particularly preferable. Carbodiimides include DCC, N,
N'-diisopropylcarbodiimide, N-ethyl-
N '-(3-dimethylaminoprolyl) carbodiimide or the like is used. For activation by these, the protected amino acid was added directly to the resin together with a racemization inhibitory additive (eg, HOBt, HOOBt), or the protected amino acid was previously activated as a symmetrical acid anhydride or HOBt ester or HOOBt ester. It can later be added to the resin.

The solvent used for activation of the protected amino acid and condensation with the resin can be appropriately selected from solvents known to be usable for protein condensation reaction. For example, acid amides such as N, N-dimethylformamide, N, N-dimethylacetamide and N-methylpyrrolidone, halogenated hydrocarbons such as methylene chloride and chloroform, alcohols such as trifluoroethanol,
Sulfoxides such as dimethyl sulfoxide, ethers such as pyridine, dioxane, and tetrahydrofuran, nitriles such as acetonitrile and propionitrile, esters such as methyl acetate and ethyl acetate, and appropriate mixtures thereof are used. The reaction temperature is appropriately selected from the range known to be applicable to protein bond-forming reactions, and is usually appropriately selected from the range of about -20 ° C to 50 ° C. The activated amino acid derivative is usually used in a 1.5 to 4-fold excess. As a result of the test using the ninhydrin reaction, when the condensation is insufficient, sufficient condensation can be performed by repeating the condensation reaction without removing the protecting group. When sufficient condensation cannot be obtained by repeating the reaction, it is possible to acetylate the unreacted amino acid with acetic anhydride or acetylimidazole so that the subsequent reaction is not affected.

Examples of the protective group for the amino group of the raw material include Z, Boc, t-pentyloxycarbonyl, isobornyloxycarbonyl, 4-methoxybenzyloxycarbonyl, Cl-Z, Br-Z, adamantyloxycarbonyl, Trifluoroacetyl, phthaloyl, formyl, 2-nitrophenylsulfenyl, diphenylphosphinothioyl oil, Fmoc and the like are used. The carboxyl group is, for example, alkyl esterified (for example,
Linear, branched or cyclic alkyl esterification such as methyl, ethyl, propyl, butyl, t-butyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, 2-adamantyl, etc., aralkyl esterification (eg, benzyl ester, 4-nitrobenzyl ester, 4-methoxybenzyl ester, 4-chlorobenzyl ester, benzhydryl esterification), phenacyl esterification, benzyloxycarbonylhydrazideation, t-butoxycarbonylhydrazideation, tritylhydrazideation, etc. You can The hydroxyl group of serine can be protected by, for example, esterification or etherification. Suitable groups for this esterification include, for example, lower (C _1-6 ) groups such as acetyl group.
An aroyl group such as an alkanoyl group and a benzoyl group, a group derived from carbonic acid such as a benzyloxycarbonyl group and an ethoxycarbonyl group are used. Moreover, examples of a group suitable for etherification include a benzyl group, a tetrahydropyranyl group, and a t-butyl group. Examples of the protective group for the phenolic hydroxyl group of tyrosine include Bz
1, Cl ₂ -Bzl, 2-nitrobenzyl, Br-Z,
t-Butyl or the like is used. Examples of the protecting group for imidazole of histidine include Tos and 4-methoxy-
2,3,6-trimethylbenzenesulfonyl, DNP,
Benzyloxymethyl, Bum, Boc, Trt, Fm
oc or the like is used.

The activated carboxyl group of the raw material includes, for example, corresponding acid anhydrides, azides, active esters [alcohols (for example, pentachlorophenol, 2,4,5-trichlorophenol, 2,4- Dinitrophenol, cyanomethyl alcohol, paranitrophenol, HONB, N-hydroxysuccinide, N
-Hydroxyphthalimide, ester with HOBt)]
Are used. As the activated amino group of the raw material, for example, the corresponding phosphoric acid amide is used. Examples of the method for removing (eliminating) the protecting group include Pd
-Catalytic reduction in a hydrogen stream in the presence of a catalyst such as black or Pd-carbon, or acid with anhydrous hydrogen fluoride, methanesulfonic acid, trifluoromethanesulfonic acid, trifluoroacetic acid, or a mixture thereof. Treatment, base treatment with diisopropylethylamine, triethylamine, piperidine, piperazine, etc., and reduction with sodium in liquid ammonia are also used. The elimination reaction by the acid treatment is generally carried out at a temperature of about -20 ° C to 40 ° C. In the acid treatment, for example, anisole, phenol, thioanisole, metacresol,
The addition of cation trapping agents such as para-cresol, dimethyl sulfide, 1,4-butanedithiol, 1,2-ethanedithiol is effective. Further, the 2,4-dinitrophenyl group used as the imidazole protecting group of histidine is removed by thiophenol treatment, and the formyl group used as the indole protecting group of tryptophan is the above 1,2-ethanedithiol, 1,4-butane group. In addition to deprotection by acid treatment in the presence of dithiol or the like, it is also removed by alkali treatment with dilute sodium hydroxide solution, dilute ammonia or the like.

Protection of a functional group which should not be involved in the reaction of the starting material, removal of the protective group and removal of the protective group, 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, the α-carboxyl group of the carboxy-terminal amino acid is amidated and protected, and then the peptide (protein) chain is added to the amino group side up to the desired chain length. After the extension, a protein or partial peptide in which only the N-terminal α-amino protecting group of the peptide chain is removed and a protein or partial peptide in which only the C-terminal carboxyl protecting group is removed are produced. The protein or peptide is condensed in a mixed solvent as described above. The details of the condensation reaction are the same as above. After purifying the protected protein or peptide obtained by condensation, all protecting groups can be removed by the above method to obtain the desired crude protein or peptide. This crude protein or peptide can be purified by utilizing various known purification means, and the main fraction can be lyophilized to obtain the amide of the desired protein or peptide. To obtain an ester form of a protein or peptide, for example, α- of the carboxy terminal amino acid is used.
After condensing the carboxyl group with a desired alcohol to form an amino acid ester, the desired protein or peptide ester can be obtained in the same manner as the protein or peptide amide.

The partial peptide of the present invention or a salt thereof can be produced by a known peptide synthesis method or by cleaving the protein of the present invention with an appropriate peptidase. The peptide synthesis method may be, for example, either a solid phase synthesis method or a liquid phase synthesis method. That is, the target peptide can be produced by condensing the partial peptide or amino acid that can form the partial peptide of the present invention with the residual portion and removing the protective group when the product has a protective group. Examples of known condensation methods and removal of protective groups include the methods described in the following (1) to (3). M. Bodanszky and MA Ondetti, Peptide Synthesis, Interscience Publisher
s, New York (1966) Schroeder and Luebke, The Peptid
e), Academic Press, New York (1965) Nobuo Izumiya et al., Fundamentals and experiments of peptide synthesis, Maruzen Co., Ltd.
(1975) Haruaki Yajima and Shunpei Sakakibara, Laboratory of Biochemistry 1, Protein Chemistry IV, 205, (1977) Supervised by Haruaki Yajima, Development of Pharmaceuticals, Volume 14, Peptide Synthesis, Hirokawa Shoten After that, the partial peptide of the present invention can be purified and isolated by combining usual purification methods such as solvent extraction, distillation, column chromatography, liquid chromatography, and recrystallization. When the partial peptide obtained by the above method is a free form, it can be converted into an appropriate salt by a known method or a method analogous thereto, and conversely, when a partial peptide is obtained, a known method or a method similar thereto is used. It can be converted into the educt or other salt by the method.

The polynucleotide encoding the protein of the present invention includes the above-mentioned nucleotide sequence (DNA or RNA, preferably DN) encoding the protein of the present invention.
Any material may be used as long as it contains A). The polynucleotide is DNA encoding the protein of the present invention, RNA such as mRNA, and may be double-stranded or single-stranded. When double-stranded, double-stranded DNA, double-stranded RNA or DNA: RNA
It may be a 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 publicly known experimental medicine special issue “New PCR and its applications” 15
(7), by the method described in 1997 or a method similar thereto,
The mRNA of the protein of the present invention can be quantified. The DNA encoding the protein of the present invention includes:
Any substance may be used as long as it contains the nucleotide sequence encoding the protein of the present invention. Further, it may be any of genomic DNA, genomic DNA library, cDNA derived from the cells / tissues described above, cDNA library derived from the cells / tissues described above, and synthetic DNA. The vector used for the library may be any of bacteriophage, plasmid, cosmid, phagemid and the like. In addition, the total R from the cells and tissues described above
Directly using the prepared NA or mRNA fraction
It can also be amplified by the verse Transcriptase Polymerase Chain Reaction (hereinafter abbreviated as RT-PCR method). The DNA encoding the protein of the present invention includes, for example, (1) a DNA containing the base sequence represented by SEQ ID NO: 2 or a base sequence represented by SEQ ID NO: 2 under high stringent conditions. A DNA containing a hybridizing base sequence and encoding a protein having substantially the same properties as the protein containing the amino acid sequence represented by SEQ ID NO: 1, (2) SEQ ID NO:
DNA containing the nucleotide sequence represented by 18, or the amino acid sequence represented by SEQ ID NO: 17, containing the nucleotide sequence hybridizing with the nucleotide sequence represented by SEQ ID NO: 18 under high stringent conditions DNA encoding a protein having substantially the same properties as a protein containing (3) a DNA containing the base sequence represented by SEQ ID NO: 20, or a base sequence represented by SEQ ID NO: 20 Any DNA can be used as long as it encodes a protein containing a nucleotide sequence that hybridizes under stringent conditions and having a property substantially the same as that of the protein containing the amino acid sequence represented by SEQ ID NO: 19. Good. DNA capable of hybridizing with the nucleotide sequence represented by SEQ ID NO: 2 under high stringent conditions
Is, for example, about 70% or more, preferably about 80% or more, preferably about 90% or more, further preferably about 95% or more, more preferably about 97% or more with the base sequence represented by SEQ ID NO: 2. A DNA containing a base sequence having homology with is used. The DNA that can hybridize with the base sequence represented by SEQ ID NO: 18 under high stringent conditions is, for example, about 70% or more, preferably about 80%, with the base sequence represented by SEQ ID NO: 18.
Or more, preferably about 90% or more, more preferably about 9%
A DNA containing a base sequence having a homology of 5% or more, more preferably about 97% or more is used. The DNA that can hybridize with the base sequence represented by SEQ ID NO: 20 under high stringent conditions is, for example, about 70% or more, preferably about 80% or more, with the base sequence represented by SEQ ID NO: 20. DNA containing a nucleotide sequence having a homology of preferably about 90% or more, more preferably about 95% or more, more preferably about 97% or more.
Are used.

Hybridization can be carried out by a known method or a method analogous thereto, for example, Molecular Cloning 2nd (J. Sambrook et al.
al., Cold Spring Harbor Lab. Press, 1989). When a commercially available library is used, it can be performed according to the method described in the attached instruction manual. More preferably, it can be carried out under high stringent conditions. The high stringent conditions include, for example, a sodium concentration of about 19 to 40 mM, preferably about 19 to 20 mM,
The temperature is about 50 to 70 ° C, preferably about 60 to 65 ° C. Particularly, the case where the sodium concentration is about 19 mM and the temperature is about 65 ° C. is most preferable.

More specifically, a DNA encoding a protein containing the amino acid sequence represented by SEQ ID NO: 1.
Is a DNA containing the nucleotide sequence represented by SEQ ID NO: 2, and a DNA encoding the protein containing the amino acid sequence represented by SEQ ID NO: 17 is the nucleotide sequence represented by SEQ ID NO: 18. As the contained DNA, the DNA encoding the protein containing the amino acid sequence represented by SEQ ID NO: 19, the DNA containing the base sequence represented by SEQ ID NO: 20 and the like are used. The DNA encoding the partial peptide of the present invention may be any DNA as long as it contains the nucleotide sequence encoding the partial peptide of the present invention described above. In addition, genomic DNA, genomic DNA library, cells described above
Tissue-derived cDNA, cell / tissue-derived cDNA described above
Either A library or synthetic DNA may be used. As the DNA encoding the partial peptide of the present invention, for example,
DN having a part of DNA having the nucleotide sequence represented by SEQ ID NO: 2, SEQ ID NO: 18 or SEQ ID NO: 20
A, or a base sequence represented by SEQ ID NO: 2, SEQ ID NO: 18 or SEQ ID NO: 20, containing a base sequence that hybridizes under high stringent conditions, and 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 base sequence represented by SEQ ID NO: 2, SEQ ID NO: 18 or SEQ ID NO: 20 has the same meaning as described above.
The same hybridization method and high stringent conditions as described above are used. Proteins and partial peptides of the present invention (hereinafter, in the description of cloning and expression of DNAs encoding them, these may be simply referred to as the proteins of the present invention)
As a means for cloning the DNA completely encoding DNA, DN which is amplified by the PCR method using a synthetic DNA primer having a part of the nucleotide sequence encoding the protein of the present invention or incorporated into an appropriate vector is used.
A can be selected by hybridization with a DNA fragment that encodes a part or the entire region of the protein of the present invention or that labeled with a synthetic DNA. Hybridization methods include, for example, 2nd molecular cloning (Molecular Cloning).
(J. Sambrook et al., Cold Spring Harbor Lab. Pres
s, 1989) and the like. When a commercially available library is used, it can be performed according to the method described in the attached instruction manual.

The conversion of the nucleotide sequence of DNA is carried out using PCR or a known kit such as Mutan ^™ -superExpress Km (Takara Shuzo) or Mutan ^™ -K (Takara Shuzo).
-A known method such as LAPCR method, Gapped duplex method or Kunkel method or a method similar thereto can be used. The DNA encoding the cloned protein can be used as it is, or if desired after digestion with a restriction enzyme or addition of a linker, depending on the purpose. The DNA may have ATG as a translation initiation codon at the 5'-terminal side and TAA, TGA or TAG as a translation termination codon at the 3'-terminal side. These translation initiation codon and translation termination codon can be added using an appropriate synthetic DNA adaptor.

The expression vector of the protein of the present invention is, for example, (a) a DNA fragment of interest is excised from the DNA encoding the protein of the present invention, and (b) the DNA fragment is downstream of a promoter in an appropriate expression vector. Can be manufactured by connecting to. As the vector, a plasmid derived from E. coli (eg, pBR322, pB
R325, pUC12, pUC13), a Bacillus subtilis-derived plasmid (eg, pUB110, pTP5, pC19)
4), yeast-derived plasmid (eg, pSH19, pSH1
5), bacteriophage such as λ phage, animal viruses such as retrovirus, vaccinia virus, baculovirus, etc., pA1-11, pXT1, pR
c / CMV, pRc / RSV, pcDNAI / Neo and the like are 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, SRα promoter, SV40 promoter, LTR promoter, CMV promoter, H
SV-TK promoter etc. are mentioned. Among these, CMV (cytomegalovirus) promoter, S
It is preferable to use an Rα promoter or the like. When the host is Escherichia, the trp promoter, l
ac promoter, recA promoter, λP _L promoter, lpp promoter, T7 promoter, etc., when the host is Bacillus, SPO1 promoter, SPO2 promoter, penP promoter, etc. When the host is yeast, PHO5 promoter, PGK Promoter, GAP promoter, ADH
A promoter and the like are preferable. When the host is an insect cell, polyhedrin promoter, P10 promoter and the like are preferable. In addition to the above, the expression vector may optionally include an enhancer, a splicing signal, and poly A.
Those containing an additional signal, a selection marker, an SV40 replication origin (hereinafter sometimes abbreviated as SV40ori), and the like can be used. Examples of selection markers include dihydrofolate reductase (hereinafter, dh
fr) gene [methotrexate (MTX) resistance], ampicillin resistance gene (hereinafter referred to as A
mp ^r ) and a neomycin resistance gene (hereinafter sometimes referred to as Neo ^r , G418 resistance) and the like. In particular, when the dhfr gene is used as a selection marker using Chinese hamster cells lacking the dhfr gene, the target gene can be selected by a medium containing no thymidine. In addition, a signal sequence suitable for the host is added to the N-terminal side of the protein of the present invention, if necessary. When the host is Escherichia, the PhoA signal sequence, OmpA signal sequence, etc., when the host is Bacillus, the α-amylase signal sequence, subtilisin signal sequence, etc., the host is yeast. In some cases, MFα · signal sequence, SUC2 · signal sequence, etc., and when the host is an animal cell, insulin signal sequence, α-interferon signal sequence, antibody molecule / signal sequence, etc. can be used, respectively.

Using the vector containing the DNA encoding the protein of the present invention thus constructed,
A transformant can be produced. As the host, for example, Escherichia, Bacillus, yeast, insect cells, insects, animal cells, etc. are used. Specific examples of Escherichia bacteria include, for example, Escherichia coli (Es
cherichia coli) K12 ・ DH1 [Procedures ・
Of the National Academy of Sciences of the USA (Proc. Natl. Acad. Sc
i. USA), Volume 60, 160 (1968)], JM103.
[Nucleic Acids Research
Research), Vol. 9, 309 (1981)], JA221
[Journal of Molecular Biology (Jo
urnal of Molecular Biology), 120, 517 (1)
978)], HB101 [Journal of Molecular Biology, 41, 459 (1969)], C
600 [Genetics, 39, 44
0 (1954)] and the like are used. Examples of the genus Bacillus include, for example, Bacillus subtili
s) MI114 [Gene, 24, 255 (198
3)], 207-21 [Journal of Biochemistry, Vol. 95, 87]
(1984)] and the like are used. As yeast, for example, Saccharomyces cerevisii (Saccharomyces ce
revisiae) ^AH22, AH22R ^-, NA87-11
A, DKD-5D, 20B-12, Schizosaccharomyces pombe NCYC1
913, NCYC2036, Pichia Pastoris (Pich
ia pastoris) KM71 and the like are used. As an insect cell, for example, when the virus is AcNPV, a cell line derived from a larva of a night-thief moth (Spodoptera frugiperda cell;
Sf cells), MG1 cells derived from the midgut of Trichoplusia ni, High Five ^TM cells derived from eggs of Trichoplusia ni, Mame
Cells derived from stra brassicae or cells derived from Estigmena acrea are used. When the virus is BmNPV, silkworm-derived cell lines (Bombyx mori N cells; BmN cells) and the like are used. Examples of the Sf cells include:
Sf9 cells (ATCC CRL1711), Sf21 cells (above Va
ughn, JL et al., In Vivo, 13, 213-217,
(1977)) is used. Examples of insects include silkworm larvae [Maeda et al., Nature (Na
ture), 315, 592 (1985)]. Examples of animal cells include monkey cells COS-7, Vero, Chinese hamster cells CHO (hereinafter abbreviated as CHO cells), dhfr gene-deficient Chinese hamster cells CHO (hereinafter abbreviated as CHO (dhfr ⁻ ) cells), mouse L. Cells, mouse AtT-20, mouse myeloma cells, rat GH3, human FL cells and the like are used.

To transform Escherichia, for example, Proc. Natl. Acad. Sci. USA, Volume 69, Proc. Of the National Academy of Sciences of the USA , 2110
(1972) and Gene, Volume 17, 107 (198)
It can be performed according to the method described in 2) or the like. To transform Bacillus, for example, molecular
And General Genetics (Molecular &
General Genetics), 168, 111 (1979) and the like. For transforming yeast, for example, Methods in Enzymology, 194, 182-
187 (1991), Procedures of the National Academy of Sciences of.
The USA (Proc. Natl. Acad. Sci. USA), 7
5, Volume 1929 (1978) and the like. For transforming insect cells or insects, for example, Bio / Technolog
y), 6, 47-55 (1988) and the like. To transform animal cells, see, eg, Cell Engineering Supplement 8, New Cell Engineering Experimental Protocol. 263-
267 (1995) (published by Shujunsha), Virology (Vi
rology), 52, 456 (1973). In this way, 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 of the genus Escherichia or a bacterium of the genus Bacillus, a liquid medium is suitable as a medium used for the culture, and among them, a liquid medium is necessary for the growth of the transformant. A carbon source, a nitrogen source, an inorganic substance, etc. are contained. As the carbon source, for example, glucose, dextrin, soluble starch, sucrose, etc., as the nitrogen source, for example, ammonium salts, nitrates, corn steep liquor, peptone, casein, meat extract, soybean meal, potato extract, etc. Examples of the inorganic or organic substance and the inorganic substance include calcium chloride, sodium dihydrogen phosphate, magnesium chloride and the like. Also,
Yeast extract, vitamins, growth promoting factor and the like may be added. The pH of the medium is preferably about 5-8. As a medium for culturing Escherichia bacteria, for example, M9 medium containing glucose and casamino acid [Miller, Journal of Experiments in Molar Genetics]
lecular Genetics), 431-433, Cold Spring Ha
rbor Laboratory, New York 1972] is preferred. If necessary, a drug such as 3β-indolylacrylic acid can be added to the promoter so as to work efficiently. When the host is a bacterium belonging to the genus Escherichia, the culture is usually carried out at about 15 to 43 ° C. for about 3 to 24 hours, and if necessary, aeration and stirring can be added. When the host is a bacterium of the genus Bacillus, the culture is usually carried out at about 30 to 40 ° C. for about 6 to 24 hours, and aeration and stirring can be added if necessary. When culturing a transformant whose host is yeast, examples of the medium include Burkholder (Burkho).
lder) Minimal medium [Bostian, KL et al., Proc. Natl. Aca Proc. of the National Academy of Sciences of the USA
d. Sci. USA), 77, 4505 (1980)] and 0.5.
SD medium containing% casamino acid [Bitter, GA et al.
Procedures of the National Academy of Sciences of the USA (Pr
oc. Natl. Acad. Sci. USA), 81, 5330 (19)
84)]. The pH of the medium is preferably adjusted to about 5-8. Culturing is usually performed at about 20 ° C to 35 ° C for about 24 hours.
Perform for ~ 72 hours, add aeration and agitation as needed.
When culturing a transformant whose host is an insect cell or an insect, the medium is Grace's Insect Medium (Grace, T.
CC, Nature, 195,788 (1962)) to which an additive such as immobilized 10% bovine serum is appropriately added is used. The pH of the medium is preferably adjusted to about 6.2 to 6.4. Culturing is usually performed at about 27 ° C. for about 3 to 5 days, and aeration and agitation are added if necessary. When culturing a transformant whose host is an animal cell, the medium is, for example, MEM medium containing about 5 to 20% fetal bovine serum [Science, Volume 122, 501 (1952)],
DMEM medium [Virology, 8 volumes, 39
6 (1959)], RPMI 1640 medium [The Journal of the American Medical Associatio
n) 199, 519 (1967)], 199 medium [Proceeding of the Society Medicine (Proceeding of the Society
for the Biological Medicine), 73, 1 (195
0)] is used. The pH is preferably about 6-8. Culturing is usually performed at about 30 ° C to 40 ° C for about 15 to 60 hours, and aeration and agitation are added if necessary. As described above, the protein of the present invention can be produced inside the transformant, inside the cell membrane or outside the cell.

The protein of the present invention can be separated and purified from the above culture by the following method, for example. When extracting the protein of the present invention from cultured bacterial cells or cells, after culturing, the bacterial cells or cells are collected by a known method, suspended in a suitable buffer solution, ultrasonicated,
A method of disrupting the cells or cells by lysozyme and / or freeze-thawing and then obtaining a crude protein extract by centrifugation or filtration is appropriately used. The buffer solution may contain a protein denaturing agent such as urea or guanidine hydrochloride, or a surfactant such as Triton X-100 ^™ . When the protein is secreted into the culture medium, after the culture is completed, the cells or cells are separated from the supernatant by a known method, and the supernatant is collected. The protein contained in the culture supernatant or the extract thus obtained can be purified by appropriately combining known separation / purification methods. These known separation and purification methods include
Solubility such as salting-out or solvent precipitation, dialysis, ultrafiltration, gel filtration, and SDS-polyacrylamide gel electrophoresis, etc., which mainly utilize difference in molecular weight, ion exchange chromatography , A method utilizing a specific affinity such as affinity chromatography, a method utilizing a hydrophobic difference such as reverse phase high performance liquid chromatography,
A method utilizing a difference in isoelectric points such as an isoelectric focusing method is used. When the protein thus obtained is obtained as a free form, it can be converted into a salt by a known method or a method analogous thereto, and conversely, when it is obtained as a salt, a known method or a method similar thereto is used. Depending on the method, it can be converted into the educt or other salt. In addition, the protein produced by the recombinant can be optionally modified or the polypeptide can be partially removed by acting an appropriate protein-modifying enzyme before or after the purification. Examples of the protein modifying enzyme include trypsin, chymotrypsin, arginyl endopeptidase, protein kinase, glycosidase and the like.

The presence of the protein of the present invention thus produced can be measured by an enzyme immunoassay using a specific antibody or Western blotting. The antibody against the protein or partial peptide of the present invention or a salt thereof may be either a polyclonal antibody or a monoclonal antibody as long as it is an antibody that 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 referred to as the protein of the present invention in the description of the antibody) is a known antibody using the protein of the present invention as an antigen or It can be produced according to the method for producing antiserum.

[Preparation of Monoclonal Antibody] (a) Preparation of Monoclonal Antibody-Producing Cell The protein of the present invention is administered to warm-blooded animals at a site where antibody production is possible by itself, or together with a carrier or diluent. . Complete Freund's adjuvant or incomplete Freund's adjuvant may be administered to enhance the antibody-producing ability upon administration. The administration is usually performed once every 2 to 6 weeks, about 2 to 10 times in total. Examples of warm-blooded animals used include monkeys, rabbits, dogs, guinea pigs, mice, rats, sheep, goats, and chickens, and mice and rats are preferably used. When preparing monoclonal antibody-producing cells, warm-blooded animals immunized with the antigen, for example, individuals with antibody titers are selected from mice, and spleens or lymph nodes are collected 2 to 5 days after the final immunization. A monoclonal antibody-producing hybridoma can be prepared by fusing the resulting antibody-producing cells with myeloma cells of the same or different species. The antibody titer in the antiserum can be measured, for example, by reacting the labeled protein described below with the antiserum and then measuring the activity of the labeling agent bound to the antibody. The fusion operation is a known method, for example, the method of Kohler and Milstein [Nature, 256, 495 (197).
5)]. Examples of the fusion accelerator include polyethylene glycol (PEG) and Sendai virus, and PEG is preferably used. Examples of myeloma cells include NS-1,
Examples include myeloma cells of warm-blooded animals such as P3U1, SP2 / 0 and AP-1, but P3U1 is preferably used. The preferred ratio of the number of antibody-producing cells (spleen cells) to the number of myeloma cells used is about 1: 1 to 20: 1,
PEG (preferably PEG1000-PEG6000)
Is added at a concentration of about 10 to 80%, and 20 to 40 ° C.,
Cell fusion can be efficiently performed by preferably incubating at 30 to 37 ° C. for 1 to 10 minutes. Although various methods can be used for screening a monoclonal antibody-producing hybridoma, for example, a hybridoma culture supernatant is added to a solid phase (eg, a microplate) on which a protein antigen is directly or adsorbed with a carrier, and then a radioactive substance or Anti-immunoglobulin antibody labeled with an enzyme (when the cells used for cell fusion are mice, anti-mouse immunoglobulin antibody is used) or protein A
And detecting the monoclonal antibody bound to the solid phase, adding the hybridoma culture supernatant to the solid phase adsorbing the anti-immunoglobulin antibody or protein A, adding the protein labeled with a radioactive substance or enzyme, Examples include a method of detecting a monoclonal antibody bound to a phase. Selection of the monoclonal antibody can be performed according to a known method or a method similar thereto. Normal HAT
(Hypoxanthine, aminopterin, thymidine) can be added to the medium for animal cells. As the medium for selection and breeding, any medium may be used as long as the hybridoma can grow. For example, 1 to 20
%, Preferably 10-20% fetal calf serum
I 1640 medium, GI containing 1-10% fetal bovine serum
T medium (Wako Pure Chemical Industries, Ltd.) or serum-free medium for hybridoma culture (SFM-101, Nissui Pharmaceutical Co., Ltd.)
Etc. can be used. The culture temperature is usually 20 to 4
It is 0 ° C, preferably about 37 ° C. Cultivation time is usually 5
Day to 3 weeks, preferably 1 to 2 weeks. Culturing can usually be performed under 5% carbon dioxide gas. The antibody titer of the hybridoma culture supernatant can be measured in the same manner as the above antibody titer measurement in antiserum.

(B) Purification of Monoclonal Antibody Monoclonal antibody can be separated and purified by a known method, for example, a method for separating and purifying immunoglobulin [eg, salting out method, alcohol precipitation method, isoelectric focusing method, electrophoresis method, Adsorption / desorption method using ion exchanger (eg, DEAE), ultracentrifugation method, gel filtration method, antigen binding solid phase or active adsorbent such as protein A or protein G to collect only antibody and dissociate the binding Specific Purification Method to Obtain]].

[Preparation of Polyclonal Antibody] The polyclonal antibody of the present invention can be manufactured by publicly known methods or modifications thereof. For example, an immune 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 the product and separating and purifying the antibody.
Regarding the complex of the immunizing antigen and the carrier protein used for immunizing warm-blooded animals, the type of carrier protein and the mixing ratio of the carrier and the hapten are such that the antibody is effective against the hapten immunized by crosslinking with the carrier. If possible, any kind may be cross-linked at any ratio. For example, bovine serum albumin, bovine thyroglobulin, hemocyanin, etc. may be cross-linked in a weight ratio of hapten 1.
On the other hand, a method of coupling at a ratio of about 0.1 to 20, preferably about 1 to 5 is used. Although various condensing agents can be used for coupling the hapten and carrier, glutaraldehyde, carbodiimide, maleimide active ester, active ester reagent containing thiol group, dithiopyridyl group, etc. are used. The condensation product is administered to a warm-blooded animal at a site where antibodies can be produced, by itself or together with a carrier and a diluent. Complete Freund's adjuvant or incomplete Freund's adjuvant may be administered to enhance the antibody-producing ability upon administration. The administration is usually about once every 2 to 6 weeks, about 3 to 1 in total.
It is performed about 0 times. The polyclonal antibody can be collected from the blood, ascites, etc., of the warm-blooded animal immunized by the above method, preferably from the blood. The polyclonal antibody titer in the antiserum can be measured in the same manner as the above-mentioned antibody titer in the antiserum. Separation and purification of the polyclonal antibody can be performed according to the same immunoglobulin separation and purification method as the above-described separation and purification of the monoclonal antibody.

DNAs encoding the proteins or partial peptides of the present invention (hereinafter, these DNAs are referred to as the DNAs of the present invention in the description of antisense polynucleotides).
May be abbreviated as)), or an antisense polynucleotide having a substantially complementary base sequence or a part thereof is complementary to the base sequence of the DNA of the present invention, or Any antisense polynucleotide may be used as long as it has a substantially complementary nucleotide sequence or a part thereof and has an effect of suppressing the expression of the DNA, but antisense DNA is preferable. . The term “substantially complementary nucleotide sequence to the DNA of the present invention” refers to, for example, a total nucleotide sequence or a partial nucleotide sequence of a nucleotide sequence complementary to the DNA of the present invention (that is, a complementary strand of the DNA of the present invention) and about 70 nucleotides. % Or more, preferably about 80% or more, more preferably about 90% or more, most preferably about 9% or more.
Examples include base sequences having a homology of 5% or more.
In particular, of the total base sequence of the complementary strand of the DNA of the present invention, about 70% or more with the complementary strand of the base sequence of the portion encoding the N-terminal portion of the protein of the present invention (for example, the base sequence near the start codon). Preferred are antisense polynucleotides with a homology of preferably greater than about 80%, more preferably greater than about 90%, and most preferably greater than about 95%. Specifically, it has a nucleotide sequence complementary to or substantially complementary to the nucleotide sequence of the 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 polynucleotides, preferably, eg SEQ ID NO: 2, SEQ ID NO:
18 or an antisense polynucleotide having a base sequence complementary to the base sequence of the DNA having the base sequence represented by SEQ ID NO: 20 or a part thereof (more preferably, SEQ ID NO: 2, SEQ ID NO: 18 or SEQ ID NO:
An antisense polynucleotide having a base sequence complementary to the base sequence of DNA having the base sequence represented by 20, or a part thereof, and the like.

Antisense polynucleotides are usually 1
It is composed of about 0 to 40 bases, preferably about 15 to 30 bases. In order to prevent degradation by a hydrolase such as nuclease, the phosphate residue (phosphate) of each nucleotide constituting the antisense DNA is, for example,
It may be substituted with a chemically modified phosphate residue such as phosphorothioate, methylphosphonate, and phosphorodithionate. 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 a gene of the present invention, which is capable of inhibiting the replication or expression of the gene, is a cloned or determined DN-encoding protein.
It can be designed and synthesized based on the nucleotide sequence information of A. Such an antisense polynucleotide can hybridize with RNA of the protein gene of the present invention, and the R
The NA synthesis or function can be inhibited, or the expression of the protein gene of the present invention can be regulated / controlled via the interaction with the protein-related RNA of the present invention. The polynucleotide complementary to the selected sequence of the protein-related RNA of the present invention and the polynucleotide capable of specifically hybridizing with the protein-related RNA of the present invention can be used as a protein gene of the present invention in vivo and in vitro. It is useful for regulating / controlling the expression of erythrocyte, and for treating or diagnosing diseases and the like. The term "corresponding" means having homology or being complementary to a specific sequence of nucleotides, base sequences or nucleic acids including genes. nucleotide,
"Corresponds" between a base sequence or nucleic acid and a protein
The term usually refers to the amino acids of a protein (in command) derived from the sequence of nucleotides (nucleic acid) or its complement. 5′-end hairpin loop of protein gene, 5′-end 6-base pair repeat, 5′-end untranslated region, polypeptide translation initiation codon, protein coding region, ORF translation stop codon, 3′-end untranslated region, 3 ′ The end palindromic region or the 3 ′ end hairpin loop and the like can be selected as a preferable target region, but any region in the protein gene can be selected as a target.

Regarding the relationship between the target nucleic acid and the polynucleotide complementary to at least a part of the target region, when the target nucleic acid can hybridize with the target region, the target nucleic acid is the polynucleotide of the target region. It can be said to be "antisense" to nucleotides.
Antisense polynucleotides are 2-deoxy-D-
Polynucleotides containing ribose, polynucleotides containing D-ribose, 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 Polymers are DNA or RNA
Containing a nucleotide having a configuration that allows base pairing and base attachment as found therein) and the like. They are double-stranded DNA, single-stranded DNA, 2
It may be a single-stranded RNA, a single-stranded RNA, a DNA: RNA hybrid, and further an unmodified polynucleotide (or an unmodified oligonucleotide), a known modification added thereto, such as a label known in the art. some stuff,
Capped, methylated, substituted with one or more natural nucleotides by analogs, modified with an intramolecular nucleotide, such as an uncharged bond (eg, methylphosphonate, phosphotriester, Phosphoramidates, carbamates, etc.), charged bonds or sulfur-containing bonds (eg phosphorothioates, phosphorodithioates, etc.), eg proteins (eg nucleases, nuclease inhibitors, toxins, antibodies, etc.) Signal peptide, poly-L
-Containing lysine) and sugars (eg, monosaccharides) having side chain groups, intercurrent compounds (eg, acridine, psoralen, etc.), chelate compounds (eg, metals, radioactivity) Containing a metal, boron, an oxidizing metal, etc.), containing an alkylating agent, having a modified bond (eg,
α anomeric nucleic acid). Here, "nucleoside", "nucleotide" and "nucleic acid" mean
Not only do they contain purine and pyrimidine bases, but also those with other modified heterocyclic bases. Such modifications may include methylated purines and pyrimidines, acylated purines and pyrimidines, or other heterocycles. The modified nucleotide and the modified nucleotide may also have a modified sugar moiety, for example, one or more hydroxyl groups are substituted with halogen, an aliphatic group or the like, or a functional group such as ether or amine is added. It may have been converted.

The antisense polynucleotide of the present invention comprises RNA, DNA or modified nucleic acid (RNA, D
NA). Specific examples of the modified nucleic acid include sulfur derivatives, thiophosphate derivatives, polynucleoside amides and oligonucleoside amides that are resistant to decomposition of the nucleic acid. The antisense polynucleotide of the present invention can be designed, for example, as follows. That is, the antisense polynucleotide in the cell is more stable, the cell permeability of the antisense polynucleotide is higher, the affinity for the target sense strand is higher, and the toxicity is higher. In some cases, the antisense polynucleotide is less toxic. Such modifications are for example Pharm
Tech Japan, Volume 8, 247 or 395, 1992, Antisen
Many reports have been made in se Research and Applications, CRC Press, 1993, etc. The antisense polynucleotide of the present invention has a modified or modified sugar,
It may contain a base, a bond, and can be provided in a special form such as liposome, microsphere, applied by gene therapy, or provided in an added form. Thus, as an additive form, a polycationic substance such as polylysine that acts to neutralize the charge of the phosphate group, a lipid that enhances the interaction with the cell membrane and the uptake of nucleic acid ( Examples thereof include hydrophobic ones such as phospholipid, cholesterol and the like. Preferred lipids to add include cholesterol and its derivatives (eg, cholesteryl chloroformate, cholic acid, etc.). These can be attached to the 3'-end or 5'-end of the nucleic acid, and can be attached via a base, a sugar, or an intramolecular nucleoside bond. Other groups include 3'of nucleic acid
A capping group specifically arranged at the end or the 5'end, which includes one for preventing decomposition by nucleases such as exonuclease and RNase. Examples of such a capping group include, but are not limited to, hydroxyl group-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), DNA encoding the protein or partial peptide of the present invention (hereinafter referred to as the DNA of the present invention and Abbreviated), an antibody against the protein or partial peptide of the present invention or a salt thereof (hereinafter sometimes abbreviated as an antibody of the present invention), and an antisense polynucleotide of the DNA of the present invention (hereinafter The use of (sometimes abbreviated as antisense polynucleotide) will be described. The protein of the present invention has a tissue-specific increased expression in chronic obstructive pulmonary disease and rhinitis, and thus can be used as a disease marker. Therefore, the protein of the present invention is, for example, a respiratory disease such as a lung / thoracic disease accompanied by inflammation of the lung / airway (eg, chronic obstructive pulmonary disease (chronic bronchitis, emphysema), diffuse panbronchiolitis, bronchi). Asthma, cystic fibrosis, hypersensitivity pneumonia, etc.], allergic conjunctivitis, rhinitis (eg, allergic rhinitis, hay fever, acute rhinitis, chronic rhinitis, hypertrophic rhinitis, atrophic rhinitis, dry rhinitis, vasomotility) Early diagnosis of nasal inflammation, gangrenous rhinitis, sinusitis, etc., gastrointestinal disease (eg, irritable bowel syndrome, inflammatory bowel disease, ulcerative colitis, Crohn's disease, reflux esophagitis, etc.) It is useful as a marker for determining the degree of disease and predicting disease progression. An 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 or a salt thereof that inhibits the expression of the protein gene of the present invention, Or a pharmaceutical containing an antibody against the protein of the present invention, 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, hypersensitivity pneumonia, etc.), allergic conjunctivitis, rhinitis (eg, allergic rhinitis, pollinosis, acute rhinitis, chronic rhinitis, hypertrophic rhinitis, atrophic rhinitis, Dry anterior rhinitis, vasomotor rhinitis, gangrenous rhinitis, sinusitis, etc., gastrointestinal disorders (eg, irritable bowel syndrome, inflammatory bowel disease, ulcerative colitis, Crohn's disease, reflux esophagitis, etc.) It can be used as a preventive / therapeutic agent. Preferred is a prophylactic / therapeutic agent for respiratory diseases, digestive tract diseases and the like.

[1] Screening for Drug Candidate Compounds against Diseases The protein of the present invention and the protein gene of the present invention have increased tissue-specific expression in chronic obstructive pulmonary disease and rhinitis, and mucus in the lung, respiratory tract or nasal mucosa. Compounds or salts thereof that inhibit the activity of the protein of the present invention, and compounds or salts thereof that inhibit the expression of the protein gene of the present invention, since they have a production promoting action and an alveolar wall destruction promoting action, include, for example, lung and respiratory tract. Respiratory diseases such as lung / chest diseases accompanied by inflammation of the lung [eg, chronic obstructive pulmonary disease (chronic bronchitis, emphysema), diffuse panbronchiolitis, bronchial asthma, cystic fibrosis, hypersensitivity pneumonia, etc.], Allergic conjunctivitis, rhinitis (eg, allergic rhinitis, hay fever, acute rhinitis, chronic rhinitis, hypertrophic rhinitis, atrophic rhinitis, dry anterior rhinitis, vasomotor) Rhinitis, necrotic rhinitis, sinusitis, etc.), gastrointestinal diseases (e.g., irritable bowel syndrome, inflammatory bowel disease, ulcerative colitis, Crohn's disease, etc. reflux esophagitis) prevention and such
It can be safely used as a therapeutic agent, preferably a low-toxic drug such as a prophylactic / therapeutic agent for respiratory diseases, gastrointestinal diseases and the like.
Therefore, the protein of the present invention is useful as a reagent for screening a compound or its salt that inhibits the activity of the protein of the present invention, and the polynucleotide encoding the protein inhibits the expression of the gene of the protein of the present invention. It is useful as a reagent for screening a compound or a salt thereof. That is, the present invention is
(1) A method for screening a compound or a salt thereof (hereinafter sometimes abbreviated as an inhibitor) which inhibits the activity (eg, chloride channel activity) of the protein of the present invention, which comprises using the protein of the present invention (2) A method for screening a compound or a salt thereof (hereinafter, may be abbreviated as an inhibitor) which inhibits the expression of the protein gene of the present invention, which comprises using a polynucleotide encoding the protein of the present invention, (3) The present invention provides a method for screening a compound or its salt (hereinafter sometimes abbreviated as an inhibitor) which inhibits the expression of the protein of the present invention, which comprises using the antibody of the present invention.

As a specific example of the screening method,
(I) when a cell having the ability to produce the protein of the present invention is activated with a calcium activator, and (ii) when a cell having the ability to produce the protein of the present invention and a mixture of test compounds are activated with the calcium activator. The method for screening an inhibitor is characterized in that it is compared with the case where it is modified. In the above screening method, for example, the chloride channel activity of the protein of the present invention in the cases (i) 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 mixing with the test compound, or the calcium activator has been added to cells having the ability to produce the protein of the present invention. The test compound may then 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 activating the cells having the ability to produce the protein of the present invention with a calcium activator, and A mixture of a test compound and a calcium activator may be added to cells capable of producing the protein of the present invention. As the calcium activator, ionomycin, A23187 (calcimycin) and the like are used. The chloride channel activity of the protein of the present invention can be measured by a known method, for example, the method described in Genomics, Vol. 54, p. 200 (1998) or a method analogous thereto. Examples of the test compound include peptides, proteins, non-peptidic compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts and the like, and these compounds may be novel compounds. It may be a known compound. To carry out the above-mentioned screening method, cells having the ability to produce the protein of the present invention are suspended in a buffer suitable for screening and prepared. The pH of the buffer is about 4-10.
Any phosphate buffer or borate buffer (preferably pH about 6 to 8) may be used.

Specific examples of the screening method include:
(Iii) when the cell capable of producing the protein of the present invention is cultured and (iv) when the mixture of the cell capable of producing the protein of the present invention and a test compound is cultured, The expression level (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 that recognizes the protein of the present invention, the protein present in a cell extract or the like can be analyzed by Western analysis or ELIS.
It can be measured according to a method such as Method A or a method similar thereto. The protein gene expression level of the present invention can be determined by a known method, for example, Northern blotting or Reverse.
transcription-polymerase chain reaction (RT-PC
R), real-time PCR analysis system (ABI, TaqM
an polymerase chain reaction) or the like or a method similar thereto. Examples of test compounds include peptides, proteins, non-peptidic compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts, etc. Even if these compounds are novel compounds It may be a known compound. To carry out the above-mentioned screening method, cells having the ability to produce the protein of the present invention are suspended in a buffer suitable for screening and prepared. The buffer should have a pH of about 4-10 (desirably,
Any buffer that does not inhibit the chloride channel activity of the protein of the present invention may be used, such as a phosphate buffer or a borate buffer having a pH of about 6 to 8).

Specific examples of the screening method include:
A comparison is made between (v) culturing cells having the ability to produce the protein of the present invention and (vi) culturing a mixture of cells having the ability to produce the protein of the present invention and a test compound. In the above screening method,
In the cases of (v) and (vi) using the antibody of the present invention,
The expression level of the protein of the present invention (specifically, the protein level of the present invention) is measured (eg, detection of expression, quantification of expression level, etc.) and compared. The measurement of the amount of protein of the present invention is
A known method, for example, an antibody that recognizes the protein of the present invention can be used to measure the protein present in a cell extract or the like according to a method such as Western analysis or an ELISA method or a method similar thereto. Examples of test compounds include peptides, proteins, non-peptidic compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts, etc. Even if these compounds are novel compounds It may be a known compound. As the cell having the ability to produce the protein of the present invention, for example, a host (transformant) transformed with the vector containing the DNA encoding the protein of the present invention is used. Animal cells such as CHO cells are preferably used as the host.
For the screening, for example, a transformant in which the protein of the present invention is expressed on the cell membrane by culturing by the method described above is preferably used. For example, the chloride channel activity in the case of (ii) above is about 20% or more, preferably 30% or more as compared with the case of (i) above.
A test compound that inhibits the activity of the protein of the present invention or a salt thereof can be selected as a test compound that inhibits the activity of the protein of the present invention by 50% or more, more preferably by about 50% or more. For example, above (i
The test compound which inhibits the expression level of the protein gene of the present invention in the case of v) by about 20% or more, preferably 30% or more, more preferably about 50% or more compared with the case of the above (iii) Can be selected as a compound that inhibits the expression of the protein gene or a salt thereof.
For example, a test compound that inhibits the expression level of the protein of the present invention in the case of the above (vi) by about 20% or more, preferably by 30% or more, more preferably by about 50% or more as compared with the case of the above (v). Can be selected as a compound that inhibits the expression of the protein of the present invention or a salt thereof.

The screening kit of the present invention produces the protein or partial peptide of the present invention or a salt thereof, a polypeptide encoding the protein or partial peptide of the present invention, the antibody of the present invention or the protein or partial peptide of the present invention. It contains cells having the ability. Compounds or salts thereof obtained by using the screening method or screening kit of the present invention, the test compounds described above, for example, peptides, proteins, non-peptidic compounds, synthetic compounds, fermentation products,
A compound or a salt thereof selected from cell extract, plant extract, animal tissue extract, plasma, etc., which has the activity of the protein of the present invention (eg, chloride channel activity, etc.), expression of the protein gene of the present invention, or A compound or a salt thereof which inhibits the expression of the protein of the present invention. As the salt of the compound, those similar to the salts of the protein of the present invention described above are used.

A compound or a salt thereof obtained by using the screening method or the screening kit of the present invention is, for example, a respiratory disease such as a lung / chest disease accompanied by inflammation of the lung / airway [eg, chronic obstructive pulmonary disease ( Chronic bronchitis, emphysema), diffuse panbronchiolitis, bronchial asthma, cystic fibrosis, hypersensitivity pneumonia, etc.], allergic conjunctivitis, rhinitis (eg, allergic rhinitis, pollinosis, acute rhinitis, chronic rhinitis, thickening) Rhinitis, atrophic rhinitis, dry anterior rhinitis, vasomotor rhinitis, gangrenous rhinitis, sinusitis, etc., gastrointestinal disorders (eg, irritable bowel syndrome, inflammatory bowel disease, ulcerative colitis,
Prophylactic / therapeutic agents for Crohn's disease, reflux esophagitis, etc., preferably for preventing respiratory diseases, digestive tract diseases, etc.
It is useful as a medicine such as a therapeutic agent.

The compound obtained by using the screening method or the screening kit of the present invention or a salt thereof can be safely administered per se or as a suitable drug. The drug used for the above administration contains the above compound or a salt thereof and a pharmacologically acceptable carrier, diluent or excipient, and is provided as a pharmaceutical composition suitable for oral or parenteral administration. . For example, compositions for oral administration include solid or liquid dosage forms, specifically tablets (including sugar-coated tablets, film-coated tablets), pills, granules, powders, 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 usually used in the field of formulation. For example, lactose, starch, sucrose, magnesium stearate and the like are used as carriers and excipients for tablets.

As the composition for parenteral administration, for example, injections, suppositories, etc. are used, and the injections include intravenous injection, subcutaneous injection, intradermal injection, intramuscular injection, drip injection, etc. Dosage forms of Such an injection is prepared according to a known method, for example, by dissolving, suspending or emulsifying the above-mentioned antibody or a salt thereof in a sterile aqueous or oily liquid usually used for injection. As the aqueous solution for injection, for example, physiological saline, isotonic solution containing glucose and other adjuvants, and the like are used. Suitable solubilizing agents, for example, alcohol (eg, ethanol), polyalcohol (eg, Propylene glycol, polyethylene glycol), nonionic surfactants [eg polysorbate 80,
HCO-50 (polyoxyethylene (50mol) adduct of hy
drogenated castor oil)] etc. As the oily liquid, for example, sesame oil, soybean oil and the like are used, and solubilizing agents such as benzyl benzoate and benzyl alcohol may be used in combination. The prepared injection solution is usually filled in a suitable ampoule. The suppository used for rectal administration is prepared by mixing the above antibody or a salt thereof with an ordinary suppository base.

The above-mentioned oral or parenteral pharmaceutical composition is conveniently prepared in a unit dosage form suitable for the dose of the active ingredient. Examples of the dosage form of such a dosage unit include tablets, pills, capsules, injections (ampoules), suppositories, etc., usually 5 to 500 mg per dosage unit dosage form, especially 5 to 100 mg for injections, Other dosage forms preferably contain 10-250 mg of the antibody. For example, as a composition for nasal administration, inhalants, nasal drops, aerosols and the like are used.
When the preparation obtained according to a conventional method is used as an inhalant, a known method is used to prepare a powder inhalant, an inhalation suspension, an inhalation solution or a capsule inhalation, and use an appropriate inhaler at the time of use. It can be used and applied, and a powder inhalant is particularly preferably used. The average particle size of the powder inhalant is not particularly limited, but is preferably about 0.1 to 20 μm, particularly preferably about 1 to 5 μm. The particle size of the powder inhalant is not particularly limited, but the amount of particles of about 25 μm or more is preferably about 5% or less, particularly preferably about 1% or less. When an inhalant is used, a commercially available inhaler may be used as a device to be used at the time of application, for example, VENTOLIN ROTACAPS (Glaxo Co.), Spinheller (registered trademark, Fujisawa Pharmaceutical Co., Ltd.). Co., Ltd., INTAL SPIN
CAPS; Wheatons Inc., ATROVENT AND BEROTEC INHALET
TEN; Boehringer Ingelheim, FORADIL, CENTA, BENTODISK
S; Glaxo Co., Publizer (registered trademark, Teijin Limited), Bricanyl Turbhaler (BRICANYL TURBU)
HALER; Astra), Myat Insulator (M
IAT INSUFFLATOR) and so on. As a route of administration, it is usually preferable to inhale directly into the nose, mouth, etc. using an inhaler.

Since the preparation thus obtained is safe and has low toxicity, it can be used, for example, in humans or warm-blooded animals (eg,
Mouse, rat, rabbit, sheep, pig, cow, horse,
Can be administered orally or parenterally to birds, cats, dogs, monkeys, chimpanzees, etc.). The dose of the compound or its salt that inhibits the activity of the protein of the present invention varies depending on its action, target disease, administration subject, administration route, etc., but for example, for the purpose of treating chronic obstructive pulmonary disease, Or, when the salt is orally administered, generally in an adult (weight 60 kg), the compound or the salt is administered in an amount of about 0.1 to 100 mg, preferably about 1.0 to 50 mg, more preferably about 1.0 to 20 mg per day. To do.
When administered parenterally, the single dose of the compound or a salt thereof may vary depending on the administration subject, target disease, etc., but for example, for the purpose of treating chronic obstructive pulmonary disease, the activity of the protein of the present invention is When the compound or its salt that inhibits is administered to an adult (body weight 60 kg) in the form of an injection,
About 0.01 to 30 mg of the compound or a salt thereof per day,
Preferably about 0.1 to 20 mg, more preferably about 0.1 to 10 mg.
It is convenient to administer about mg by intravenous injection. In the case of other animals, the dose converted per 60 kg of body weight can be administered.

[2] Quantification of protein of the present invention, partial peptide or salt thereof An antibody against the protein of the present invention (hereinafter sometimes abbreviated as antibody of the present invention) specifically recognizes the protein of the present invention. Therefore, it can be used for the quantification of the protein of the present invention in a test solution, particularly for the quantification by sandwich immunoassay. That is, the present invention is
(I) An antibody of the present invention is allowed to competitively react with a test solution and a labeled protein of the present invention, and the ratio of the labeled protein of the present invention bound to the antibody is measured. And a method for quantifying the protein of the present invention in a test solution, and (ii) the test solution and the antibody of the present invention insolubilized on a carrier and another labeled antibody of the present invention are simultaneously or consecutively There is provided a method for quantifying the protein of the present invention in a test solution, which comprises measuring the activity of a labeling agent on an insolubilized carrier after the reaction. In the quantification method of (ii) above, it is desirable that one antibody recognizes the N-terminal portion of the protein of the present invention and the other antibody reacts with the C-terminal portion of the protein of the present invention. In addition to the quantification of the protein of the present invention using a monoclonal antibody against the protein of the present invention (hereinafter sometimes referred to as the monoclonal antibody of the present invention), detection by tissue staining or the like can also be performed. For these purposes, the antibody molecule itself may be used, or the F of the antibody molecule may be used.
(ab ′) ₂ , Fab ′, or Fab fraction 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 corresponding to the amount of antigen (eg, protein amount) in the liquid to be measured. Any measuring method may be used as long as it is a method of detecting the amount of the complex by chemical or physical means and calculating it from a standard curve prepared using a standard solution containing a known amount of antigen. . For example, nephrometry, competitive method, immunometric method and sandwich method are preferably used, but from the viewpoint of sensitivity and specificity, the sandwich method described later is particularly preferably used. Examples of the labeling agent used in the measurement method using a labeling substance include radioisotopes (eg, [ ¹²⁵ I], [ ¹³¹ I],
[ ³ H], [ ¹⁴ C], etc., fluorescent substance [eg, cyanine fluorescent dye (eg, Cy2, Cy3, Cy5, Cy5.5, Cy7 (manufactured by Amersham Biosciences), etc.)], fluorescamine, fluorescein Isothiocyanate, etc.], enzyme (eg, β-
Galactosidase, β-glucosidase, alkaline phosphatase, peroxidase, malate dehydrogenase, etc.), luminescent substance (eg, luminol, luminol derivative, luciferin, lucigenin, etc.), biotin, lanthanide element and the like are used. Furthermore, the biotin-avidin system can be used for binding the antibody or the antigen to the labeling agent. For the insolubilization of the antigen or antibody, physical adsorption may be used, or a method using a chemical bond which is usually used for insolubilizing or immobilizing proteins or enzymes 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, an insolubilized monoclonal antibody of the present invention is reacted with a test solution (first reaction), and another labeled monoclonal antibody of the present invention is reacted (secondary reaction), followed by insolubilization. The amount of the protein of the present invention in the test liquid can be quantified by measuring the activity of the labeling agent on the carrier. The primary reaction and the secondary reaction may be carried out in the reverse order, may be carried out simultaneously, or may be carried out at different times. The labeling agent and the method of insolubilization can be the same as those described above. Further, in the immunoassay method by the sandwich method, the antibody used for the solid phase antibody or the labeling antibody does not necessarily have to be one type, and a mixture of two or more types of antibodies may be used for the purpose of improving the measurement sensitivity. May be. In the method for measuring the protein of the present invention by the sandwich method of the present invention, the monoclonal antibodies of the present invention used in the primary reaction and the secondary reaction are preferably antibodies having different binding sites for the protein of the present invention. That is, when the antibody used in the secondary reaction recognizes the C-terminal portion of the protein of the present invention, the antibody used in the primary reaction and the secondary reaction is preferably an antibody used in the primary reaction. An antibody that recognizes an N-terminal other than the C-terminal is used. The monoclonal antibody of the present invention can be used in a measurement system other than the sandwich method, for example, a competitive method, an immunometric method or nephrometry. In the competitive method, the antigen in the test liquid and the labeled antigen are reacted competitively with the antibody, and then the unreacted labeled antigen (F) and the labeled antigen (B) bound to the antibody are separated. (B / F separation), the labeled amount of either B or F is measured, and the amount of antigen in the test liquid is quantified. In this reaction method, a soluble antibody is used as an antibody, B / F separation is carried out using polyethylene glycol, a second antibody against the antibody, or the like, and a first phase
A solid-phased antibody is used as the antibody, or a solid-phased method is used in which the first antibody is soluble and the second antibody is a solid-phased antibody.

In the immunometric method, the antigen in the test liquid and the solid-phased antigen are competitively reacted with a fixed amount of the labeled antibody, and then the solid phase and the liquid phase are separated, or After reacting the antigen therein with an excess amount of the labeled antibody and then adding the immobilized antigen to bind the unreacted labeled antibody to the solid phase, the solid phase and the liquid phase are separated. Next, the labeled amount of either phase is measured to quantify the amount of antigen in the test liquid. In nephrometry, the amount of insoluble precipitate produced as a result of the antigen-antibody reaction in the gel or in the solution is measured. Even when the amount of antigen in the test liquid is small and only a small amount of precipitate can be obtained, laser nephrometry utilizing laser scattering is preferably used. In applying each of these immunological assay methods to the quantification method of the present invention, no special conditions, operations, etc. are required to be set. The protein measurement system of the present invention may be constructed by adding ordinary technical consideration to those skilled in the art to the ordinary conditions and operating methods in each method. For details of these general technical means, reference can be made to reviews, books, and the like. For example, Irie
Hiroshi "Radioimmunoassay" (Kodansha, published in 1974), Hiroshi Irie "Radioimmunoassay" edited (Kodansha, published in 1979), Eiji Ishikawa et al. "Enzyme Immunoassay" (Medical Shoin, published in 1978) ), Eiji Ishikawa et al. “Enzyme Immunoassay” (second edition) (Medical Shoin, 1982 issue), Eiji Ishikawa et al. “Enzyme Immunoassay” (3rd edition) (Medicine Shoin, 1987 issue) ), `` Methods in ENZYMOLOG
Y '' 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 D: Sele
cted Immunoassays)), ibid Vol. 92 (Immunochemical
Techniques (Part E: Monoclonal Antibodies and General
Immunoassay Methods)), ibid Vol. 121 (Immunochemi
cal Techniques (Part I: Hybridoma Technology and Mon
oclonal Antibodies)) (published by Academic Press Co., Ltd.) and so on. As described above,
By using the antibody of the present invention, the protein of the present invention can be quantified with high sensitivity. 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 / chest diseases accompanied by inflammation of the lung / airway, etc. Respiratory disease (eg, chronic obstructive pulmonary disease (chronic bronchitis, emphysema), diffuse panbronchiolitis, bronchial asthma, cystic fibrosis, hypersensitivity pneumonia, etc.), allergic conjunctivitis,
Rhinitis (eg, allergic rhinitis, hay fever, acute rhinitis, chronic rhinitis, hypertrophic rhinitis, atrophic rhinitis, dry anterior rhinitis, vasomotor rhinitis, gangrene rhinitis, sinusitis, etc.), gastrointestinal disorders (eg , Irritable bowel syndrome, inflammatory bowel disease, ulcerative colitis,
It can be diagnosed as a disease such as Crohn's disease, reflux esophagitis, or the like, or is 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 body fluid or tissue. In addition, for 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 during purification, analysis of the behavior of the protein of the present invention in a test cell, etc. Can be used.

[3] Gene Diagnostic Agent The DNA of the present invention can be used as a probe, for example, to prepare human or warm-blooded animals (eg, rat, mouse, guinea pig, rabbit, bird, sheep, pig, cow,
Since it is possible to detect an abnormality (gene abnormality) of the DNA or mRNA encoding the protein of the present invention or its partial peptide in horses, cats, dogs, monkeys, chimpanzees, etc., for example, the DNA or mRNA can be detected.
It is useful as a gene diagnostic agent for damage, mutation or decrease in expression, increase in DNA or mRNA, or excessive expression. The above-mentioned gene diagnosis using the DNA of the present invention is carried out, for example, by known Northern hybridization or PCR-SSCP method (Genomics,
Volume 5, pp. 874-879 (1989), Proceedings of the Nationa of the National Academy of Sciences of USA
lAcademy of Sciences of the United States of Ameri
ca), Vol. 86, pp. 2766-2770 (1989)) and the like. For example, when overexpression is detected by Northern hybridization or PCR-S
When a mutation in DNA is detected by the SCP method,
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, hypersensitivity Pneumonia, etc.], allergic conjunctivitis, rhinitis (eg, allergic rhinitis, pollinosis, acute rhinitis, chronic rhinitis, hypertrophic rhinitis, atrophic rhinitis, dry anterior rhinitis, vasomotor rhinitis, necrotic rhinitis, sinus sinus Inflammation, gastrointestinal disorders (eg, irritable bowel syndrome, inflammatory bowel disease, ulcerative colitis,
It can be diagnosed as being likely to have a disease such as Crohn's disease or reflux esophagitis).

[4] Medicament Containing Antisense Polynucleotide The antisense polynucleotide of the present invention capable of binding to the DNA of the present invention in a complementary manner and suppressing the expression of the DNA has low toxicity, Since the activity / function (eg, chloride channel activity) of the protein of the present invention or the DNA of the present invention can be suppressed, respiratory diseases such as lung / chest diseases accompanied by inflammation of the lung / airway [eg , Chronic obstructive pulmonary disease (chronic bronchitis, emphysema),
Diffuse panbronchiolitis, bronchial asthma, cystic fibrosis, hypersensitivity pneumonia, etc.), allergic conjunctivitis, rhinitis (eg, allergic rhinitis, pollinosis, acute rhinitis, chronic rhinitis, hypertrophic rhinitis, atrophic rhinitis, Dry anterior rhinitis, vasomotor rhinitis, gangrenous rhinitis, sinusitis, etc., gastrointestinal disorders (eg, irritable bowel syndrome, inflammatory bowel disease, ulcerative colitis, Crohn's disease, reflux esophagitis, etc.) It can be used as a prophylactic / therapeutic agent such as, preferably, a prophylactic / therapeutic agent for respiratory diseases, digestive tract diseases and the like. When the above antisense polynucleotide is used as the above-mentioned prophylactic / 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 after being inserted into an appropriate vector such as a retrovirus vector, an adenovirus vector, or an adenovirus associated virus vector, according to a conventional method. It can be orally or parenterally administered to a human mammal (eg, rat, rabbit, sheep, pig, cow, cat, dog, monkey, etc.). The antisense polynucleotide is
It can be administered as it is or in the form of a formulation with physiologically acceptable carriers such as auxiliary agents for promoting ingestion, and can be administered by a gene gun or a catheter such as a hydrogel catheter. Alternatively, it can be aerosolized and locally administered into the trachea as an inhalant. The dose of the antisense polynucleotide varies depending on the target disease, administration subject, administration route, etc., but for example, for the purpose of treating chronic obstructive pulmonary disease, the antisense polynucleotide of the present invention is inhaled into the trachea. In the case of local administration, an adult (body weight 60 kg) generally administers about 0.1 to 100 mg of the antisense polynucleotide per day. Further, the antisense polynucleotide can also be used as a diagnostic oligonucleotide probe for investigating the presence of the DNA of the present invention or the expression state thereof in tissues or cells.

The present invention further comprises a double-stranded RNA containing a part of RNA encoding the protein of the present invention, a medicine comprising the double-stranded RNA, a part of RNA encoding the protein of the present invention. There is provided a ribozyme containing: and a pharmaceutical comprising the ribozyme. These double-stranded RNA (RNAi; RNA interference method),
Ribozymes and the like can suppress the expression of the polynucleotide (eg, DNA) of the present invention in the same manner as the above antisense polynucleotides, and thus the peptide of the present invention or the polynucleotide (eg, DNA) of the present invention in vivo.
Since it can inhibit the activity and function (eg, chloride channel activity) of the lung, for example, lung / chest disease accompanied by inflammation of the lung / airway [eg, chronic obstructive pulmonary disease (chronic bronchitis, emphysema), diffuse Panbronchiolitis, bronchial asthma, cystic fibrosis, hypersensitivity pneumonia, etc.), inflammatory bowel disease, allergic conjunctivitis, rhinitis (eg, allergic rhinitis, hay fever,
Acute rhinitis, chronic rhinitis, hypertrophic rhinitis, atrophic rhinitis, dry anterior rhinitis, vasomotor rhinitis, gangrene rhinitis, sinusitis, etc.)
It can be used as a preventive / therapeutic agent. Double-stranded RNA can be prepared by a known method (eg, Nature, 411, 494,
2001), and can be designed and manufactured based on the sequence of the polynucleotide of the present invention. Ribozymes can be prepared by known methods (eg, TRENDS in Molecular Medicine, Volume 7, 2
21 page, 2001), it can be designed and manufactured based on the sequence of the polynucleotide of the present invention. For example, it can be produced by linking a known ribozyme to a part of RNA encoding the peptide of the present invention.
As a part of RNA encoding the peptide of the present invention,
RNA of the present invention that can be cleaved by known ribozymes
The part (RNA fragment) close to the above cleavage site is mentioned. The above-mentioned prevention of double-stranded RNA or ribozyme
When used as a therapeutic agent, it can be formulated and administered in the same manner as the antisense polynucleotide.

[5] Pharmaceutical containing the antibody of the present invention The antibody of the present invention having the activity of neutralizing the activity of the protein of the present invention is, for example, a respiratory organ such as lung / chest disease accompanied by inflammation of the lung / airway. Disease [eg, chronic obstructive pulmonary disease (chronic bronchitis, emphysema), diffuse panbronchiolitis, bronchial asthma,
Cystic fibrosis, hypersensitivity pneumonia, etc.], allergic conjunctivitis, rhinitis (eg, allergic rhinitis, hay fever, acute rhinitis,
Chronic rhinitis, hypertrophic rhinitis, atrophic rhinitis, dry anterior rhinitis, vasomotor rhinitis, gangrene rhinitis, sinusitis, etc., gastrointestinal disorders (eg, irritable bowel syndrome, inflammatory bowel disease, ulcerative colon) It can be used as a medicine for diseases such as inflammation, Crohn's disease, and reflux esophagitis. The preventive / therapeutic agent for the above diseases containing the antibody of the present invention has low toxicity, and as a liquid formulation as it is, or as a pharmaceutical composition in an appropriate dosage form,
It can be orally or parenterally administered to a human or non-human mammal (eg, rat, rabbit, sheep, pig, cow, cat, dog, monkey, etc.). The dosage is
Depending on the administration subject, target disease, symptom, administration route, etc., for example, when used for the treatment of adult chronic obstructive pulmonary disease, the antibody of the present invention is used as a single dose,
Usually 0.01 to 20 mg / kg body weight, preferably 0.1 to 10 mg / kg
Body weight, more preferably about 0.1-5 mg / kg body weight, 1
It is convenient to administer by intravenous injection about 1 to 5 times a day, preferably about 1 to 3 times a day. In the case of other parenteral administration and oral administration, an amount similar to this can be administered. If the symptoms are particularly severe, the dose may be increased according to the symptoms. The antibody of the present invention can be administered per se or as an appropriate pharmaceutical composition. The pharmaceutical composition used for the above administration contains the above antibody or a salt thereof and a pharmacologically acceptable carrier, diluent or excipient. Such a composition is provided as a dosage form suitable for oral or parenteral administration. That is, for example, as a composition for oral administration, a solid or liquid dosage form, specifically, tablets (including sugar-coated tablets, film-coated tablets), pills, granules, powders, capsules (soft capsules Including), syrups, emulsions, suspensions and the like. Such a composition is produced by a known method and contains a carrier, diluent or excipient usually used in the field of formulation. For example, lactose, starch, sucrose, magnesium stearate and the like are used as carriers and excipients for tablets. As the composition for parenteral administration, for example, injections, suppositories, etc. are used, and the injections are in the form of intravenous injection, subcutaneous injection, intradermal injection, intramuscular injection, drip injection, etc. Includes. Such an injection is prepared according to a known method, for example, by dissolving, suspending or emulsifying the above-mentioned antibody or a salt thereof in a sterile aqueous or oily liquid usually used for injection. As the aqueous solution for injection, for example, physiological saline, isotonic solution containing glucose and other auxiliary agents, etc. are used, and a suitable solubilizing agent such as alcohol (eg, ethanol),
Polyalcohol (eg, propylene glycol, polyethylene glycol), nonionic surfactant [eg, polysorbate 80, HCO-50 (polyoxyethylene (50mol)
adduct of hydrogenated castor oil)] and the like. As the oily liquid, for example, sesame oil, soybean oil and the like are used, and solubilizing agents such as benzyl benzoate and benzyl alcohol may be used in combination. The prepared injection solution is usually filled in a suitable ampoule. The suppository used for rectal administration is prepared by mixing the above antibody or a salt thereof with an ordinary suppository base. The oral or parenteral pharmaceutical compositions described above are conveniently prepared in dosage unit form for administration of the active ingredient. Examples of the dosage unit form include tablets, pills, capsules, injections (ampoules), suppositories, etc. 10-250m for other dosage forms
It is preferred that g of the above antibody is contained. Each composition described above may contain other active ingredients as long as the composition does not cause an unfavorable interaction with the antibody.

[6] Medicament Containing the Protein of the Present Invention The protein of the present invention (preferably a partial peptide) is a respiratory disease such as lung / chest disease accompanied by inflammation of the lung / airway [eg, chronic obstructive property]. Pulmonary diseases (chronic bronchitis, emphysema), diffuse panbronchiolitis, bronchial asthma, cystic fibrosis, hypersensitivity pneumonia, etc.], allergic conjunctivitis, rhinitis (eg, allergic rhinitis, hay fever, acute rhinitis, chronic) Rhinitis, hypertrophic rhinitis, atrophic rhinitis, dry anterior rhinitis, vasomotor rhinitis, gangrenous rhinitis, sinusitis, etc., gastrointestinal disorders (eg, irritable bowel syndrome, inflammatory bowel disease, ulcerative colitis) ,
Crohn's disease, reflux esophagitis, etc.)
Preferably, it is also useful as a prophylactic / therapeutic agent for respiratory diseases, digestive tract diseases and the like. The protein of the present invention is
When it is used as a therapeutic agent, it can be formulated according to a conventional method. For example, the protein of the present invention is orally administered as tablets, capsules, elixirs, microcapsules, etc., which are optionally sugar-coated, or aseptic with water or other pharmaceutically acceptable liquids. It can be used parenterally in the form of injection such as solution or suspension. For example, the protein of the present invention is mixed with known physiologically acceptable carriers, flavors, excipients, vehicles, preservatives, stabilizers, binders and the like in a unit dose form required for the generally accepted formulation practice. Can be manufactured. The amount of active ingredient in these preparations is such that a suitable dose within the indicated range can be obtained. The dose of the protein of the present invention is,
Although there are differences depending on the target organs, symptoms, administration method, etc., in the case of oral administration, generally, for example, in a patient (as 60 kg), about 0.1 to 100 mg, preferably about 1.0, per day.
~ 50 mg, more preferably about 1.0-20 mg. When administered parenterally, the single dose varies depending on the administration subject, target organ, symptom, administration method, etc., but usually in the form of an injection, for example, in a patient (60 kg), About 0.01 to 30 mg per day, preferably about
It is convenient to administer about 0.1 to 20 mg, more preferably about 0.1 to 10 mg by intravenous injection. In the case of other animals, the dose converted per 60 kg can be administered.

[7] Production of Animal Having DNA of the Present Invention The present invention provides D encoding the exogenous protein of the present invention.
Provided is a non-human mammal having NA (hereinafter abbreviated as exogenous DNA of the present invention) or a mutant DNA thereof (may be abbreviated as exogenous mutant DNA of the present invention). That is, the present invention includes (1) a non-human mammal having the exogenous DNA of the present invention or a mutant DNA thereof, (2) the animal according to (1) above, wherein the non-human mammal is a rodent, (3) A recombinant vector and the like containing the animal according to the above (2), wherein the rodent is a mouse or rat, and (4) containing the exogenous DNA of the present invention or a mutant DNA thereof and capable of being expressed in a mammal. A non-human mammal having the exogenous DNA of the present invention or a mutant DNA thereof (hereinafter, abbreviated as the DNA-introduced animal of the present invention) is used for embryo cells such as unfertilized eggs, fertilized eggs, sperms and their progenitor cells. Preferably, at the stage of embryogenesis in development of non-human mammals (more preferably at the stage of single cells or fertilized egg cells and generally before the 8-cell stage), the calcium phosphate method, electric pulse method, lipofection method, agglutination method, It can be produced by introducing the target DNA by the microinjection method, particle gun method, DEAE-dextran method, or the like. In addition, somatic cells,
It is also possible to introduce the foreign DNA of the present invention into an organ, tissue cell or the like of a living body, and use it for cell culture, tissue culture, etc., and further, by using these cells with the above-mentioned embryo cells by a known cell fusion method. By fusing the DN of the present invention
It is also possible to create an A-introduced animal. Examples of non-human mammals include cow, pig, sheep, goat, rabbit,
Dogs, cats, guinea pigs, hamsters, mice, rats, etc. are used. Among them, rodents that have relatively short ontogenesis and biological cycle from the viewpoint of creating a pathological animal model system and are easy to reproduce, especially mice (for example, C57BL / 6 strain and DBA2 strain as pure strains). B6C3F ₁ system, BDF ₁ system, B
6D2F ₁ system, BALB / c system, ICR system, etc.)
Alternatively, a rat (eg, Wistar, SD, etc.) and the like are preferable. Examples of the “mammal” in the 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 does not mean the DNA of the present invention originally possessed by non-human mammals, but the DNA of the present invention once isolated and extracted from mammals.
The mutant DNA of the present invention is a DNA having a mutation (eg, mutation) in the base sequence of the original DNA of the present invention,
Specifically, DNA in which a base is added, deleted, or replaced with another base is used, and abnormal DNA is also included. The abnormal DNA means a DNA that expresses the abnormal protein of the present invention, and for example, a 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 is
It may be derived from mammals of the same or different species from the target animal. When the DNA of the present invention is introduced into a target animal, it is generally advantageous to use the DNA as a DNA construct linked downstream of a promoter that can be expressed in animal cells. For example, when the human DNA of the present invention is introduced, it expresses DNA derived from various mammals (for example, rabbit, dog, cat, guinea pig, hamster, rat, mouse, etc.) having the DNA of the present invention having high homology with the human DNA. The DNA construct of the present invention is highly expressed by microinjecting a DNA construct (eg, a vector) having the human DNA of the present invention bound downstream of various promoters capable of causing the fertilized egg of a target mammal, for example, a mouse fertilized egg. It is possible to produce a DNA-introduced mammal.

The expression vector of the protein of the present invention includes Escherichia coli-derived plasmid, Bacillus subtilis-derived plasmid, yeast-derived plasmid, bacteriophage such as λ phage, retrovirus such as Moloney leukemia virus, vaccinia virus or baculovirus, etc. The animal viruses and the like are used. Of these, a plasmid derived from Escherichia coli, a plasmid derived from Bacillus subtilis, a plasmid derived from yeast, and the like are preferably used. DN above
Examples of promoters that regulate A expression include promoters of DNA derived from viruses (eg, simian virus, cytomegalovirus, Moloney leukemia virus, JC virus, breast cancer virus, poliovirus, etc.), various mammals (human, rabbit) , Dogs, cats, guinea pigs, hamsters, rats, mice, etc.), such as albumin, insulin II, uroplakin II, elastase, erythropoietin, endothelin, muscle creatine kinase, glial fibrillary acidic protein, glutathione S-transferase, platelets. Derived growth factor β, keratins K1, K10 and K1
4, collagen type I and II, cyclic AMP
-Dependent protein kinase βI subunit, dystrophin, tartrate-resistant alkaline phosphatase, atrial natriuretic factor, endothelial receptor tyrosine kinase (generally abbreviated as Tie2), sodium potassium adenosine 3 phosphatase (Na, K-ATPase),
Neurofilament light chain, metallothionein I and IIA, metalloproteinase 1 tissue inhibitor, M
HC class I antigen (H-2L), H-ras, renin,
Dopamine β-hydroxylase, thyroid peroxidase (TPO), polypeptide chain elongation factor 1α (EF-1
α), β actin, α and β myosin heavy chains, myosin light chains 1 and 2, myelin basic protein, thyroglobulin, Thy-1, immunoglobulin, H chain variable region (VN
P), serum amyloid P component, myoglobin, troponin C, smooth muscle α-actin, preproenkephalin A, vasopressin and other promoters are used. Of these, a cytomegalovirus promoter, a human polypeptide chain elongation factor 1α (EF-1α) promoter, a human and chicken β actin promoter, which can be highly expressed systemically, are preferable. The vector preferably has a sequence (generally called terminator) that terminates the transcription of the messenger RNA of interest in the mammal into which the DNA has been introduced. The simian virus SV40 terminator and the like can be used.

In addition, in order to further highly express the desired foreign DNA, the splicing signal of each DNA, the enhancer region, a part of the intron of the eukaryotic DNA, etc. are provided 5'upstream of the promoter region, between the promoter region and the translation region. Alternatively, it may be linked to the 3'downstream of the translation region depending on the purpose. The normal translation region of the protein of the present invention includes human or various mammals (for example, rabbit, dog, cat, guinea pig, hamster, rat,
Known as all or part of genomic DNA from liver, kidney, thyroid cells, fibroblast-derived DNA derived from mice etc.) and various commercially available genomic DNA libraries, or from liver, kidney, thyroid cells, fibroblast-derived RNA It is possible to obtain the complementary DNA prepared by the above method as a raw material. In addition, an exogenous abnormal DNA can be produced by mutating a translation region of a normal polypeptide 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-transfected animal by a conventional DNA engineering method in which it is linked to the downstream of the promoter and, if desired, the upstream of the transcription termination site. Exogenous D of the present invention at the fertilized egg cell stage
The introduction of NA is ensured to be present in all germinal and somatic cells of the subject mammal. The presence of the exogenous DNA of the present invention in the germinal cells of the produced animal after the introduction of DNA means that all the progeny of the produced animal retain the exogenous DNA of the present invention in all of its germinal cells and somatic cells. means. The offspring of this type of animal that inherits the exogenous DNA of the present invention has the exogenous DNA of the present invention in all of its germ cells and somatic cells. The non-human mammal to which the exogenous normal DNA of the present invention has been transferred has been confirmed to stably retain the exogenous DNA by mating, and
As a holding animal, it can be subcultured in a normal breeding environment. The introduction of the exogenous DNA of the present invention at the fertilized egg cell stage is ensured so as to be present in excess in all the germinal cells and somatic cells of the target mammal. The excessive presence of the exogenous DNA of the present invention in the germinal cells of the producing animal after the introduction of the DNA means that all the progeny of the producing animal have the exogenous DNA of the present invention in all of their germinal cells and somatic cells. Means The offspring of this type of animal that inherited the exogenous DNA of the present invention have the exogenous DNA of the present invention in excess in all of their germ cells and somatic cells. Obtain a homozygous animal having the introduced DNA on both homologous chromosomes,
By mating the male and female animals, all offspring can be bred so that they have the DNA in excess.

In the non-human mammal having the normal DNA of the present invention, the normal DNA of the present invention is highly expressed, and the function of the protein of the present invention is finally obtained by promoting the function of the endogenous normal DNA. It may develop hyperactivity and can be used as a model animal for its pathological condition.
For example, using the normal DNA-introduced animal of the present invention, it is possible to elucidate the pathological mechanism of hyperfunction of the protein of the present invention and diseases related to the protein of the present invention, and to investigate treatment methods for these diseases. It is possible. In addition, 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, it can be used in a screening test for a therapeutic agent for diseases associated with the protein of the present invention. is there. On the other hand, the non-human mammal having the exogenous abnormal DNA of the present invention can be subcultured as a DNA-carrying animal in a normal breeding environment after confirming that the exogenous DNA is stably retained by mating. Furthermore, the foreign DNA of interest can be incorporated into the above-mentioned plasmid and used as a protozoa. The DNA construct with the promoter can be prepared by a usual DNA engineering technique. The introduction of the abnormal DNA of the present invention at the fertilized egg cell stage is ensured to be present in all germinal cells and somatic cells of the target mammal. The presence of the abnormal DNA of the present invention in the germinal cells of the created animal after the introduction of the DNA means that all the progeny of the created animal have the abnormal DNA of the present invention in all of their germinal cells and somatic cells.
Is meant to have. The offspring of this type of animal that inherits the exogenous DNA of the present invention has the abnormal DNA of the present invention in all of its germ cells and somatic cells. Obtain a homozygous animal having the introduced DNA on both homologous chromosomes,
By mating the male and female animals, all offspring can be bred so that they 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 finally obtained by inhibiting the function of the endogenous normal DNA. It may become inactive refractory and can be used as a model animal for its pathological condition. For example, using the abnormal DNA-introduced animal of the present invention, it is possible to elucidate the pathological mechanism of the functionally inactive refractory disease of the protein of the present invention and to examine the treatment method for this disease. In addition, as a specific applicability, the animal with high expression of the abnormal DNA of the present invention shows the functional inhibition (dominant negative action) of the normal protein by the abnormal protein of the present invention in the functionally inactive refractory disease of the protein of the present invention. It becomes a model to elucidate. In addition, the foreign abnormal DNA of the present invention
Since the mammal into which is introduced has an increased symptom of the released protein of the present invention, it can be used for a therapeutic drug screening test for the protein of the present invention or a functionally inactive refractory disease thereof. Further, other applications of the above-mentioned two kinds of DNA-introduced animals of the present invention include, for example, use as a cell source for tissue culture, DNA or R in tissues of the DNA-introduced animals of the present invention.
Analysis of the association with a protein that is specifically expressed or activated by the protein of the present invention by directly analyzing NA or analyzing a polypeptide tissue expressed by DNA; Culturing by standard tissue culture techniques and using them to study the function of cells from tissues that are generally difficult to culture, screening for agents that enhance cell function by using the cells described above, and It is conceivable to isolate and purify the mutant protein and produce its antibody. Furthermore, by using the DNA-introduced animal of the present invention, it is possible to investigate clinical symptoms of diseases related to the protein of the present invention, including functionally inactive refractory diseases of the protein of the present invention, and More detailed pathological findings in each organ of a disease model associated with thyroid gland can be obtained, which can contribute to the development of new treatment methods and further to the study and treatment of secondary diseases caused by the disease.

Further, each organ can be taken out from the DNA-introduced animal of the present invention, and after being minced, the released DNA-introduced cells can be obtained, cultivated thereof or systematized of the cultured cells by a protease such as trypsin. It is possible. Furthermore, it is possible to investigate the specification of the protein-producing cell of the present invention, its relation to apoptosis, differentiation or proliferation, or the signal transduction mechanism in them, and to investigate their abnormalities. It becomes an effective research material for Further, in order to develop a therapeutic agent for a disease associated with the protein of the present invention, including a functionally inactive refractory disease of the protein of the present invention, using the DNA-introduced animal of the present invention, the above-mentioned test method and quantification It is possible to provide an effective and rapid screening method for the drug for treating the disease using the method. In addition, it is possible to study and develop a DNA therapeutic method for diseases associated with the protein of the present invention by using the DNA-introduced animal of the present invention or the foreign DNA expression vector of the present invention.

[8] Knockout Animal The present invention provides a non-human mammal embryonic stem cell in which the DNA of the present invention is inactivated and a DNA expression-deficient non-human mammal of the present invention. That is, the present invention provides (1) a non-human mammal embryonic stem cell in which the DNA of the present invention is inactivated,
(2) The embryonic stem cell according to (1) above, which is inactivated by introducing a reporter gene (eg, β-galactosidase gene derived from Escherichia coli), and (3) above, wherein the DNA is neomycin resistant. Embryo stem cells of (4), the non-human mammal is a rodent, the embryo stem cell of (1) above, (5) the rodent is a mouse of (4) above, (6) the present invention Non-human mammal deficient in expression of DNA, wherein (7) the DNA is inactivated by introducing a reporter gene (eg, β-galactosidase gene derived from Escherichia coli), and the reporter gene is The non-human mammal according to (6) above, which can be expressed under the control of a promoter for the DNA of the present invention,
(8) The non-human mammal according to (6) above, wherein the non-human mammal is a rodent, (9) the non-human mammal according to (8) above, wherein the rodent is a mouse, and (10) above. The DN of the present invention, which comprises administering a test compound to the animal described in (7) and detecting the expression of a reporter gene.
A method for screening a compound or its salt that promotes or inhibits (preferably inhibits) the promoter activity for A. The non-human mammal embryonic stem cell in which the DNA of the present invention is inactivated means a DNA obtained by artificially mutating the DNA of the present invention possessed by the non-human mammal.
Or the activity of the protein of the present invention encoded by the DNA is substantially lost, whereby the DNA has substantially no ability to express the protein of the present invention (hereinafter, Non-human mammalian embryonic stem cells (sometimes referred to as the knockout DNA of the present invention) (hereinafter referred to as E
(Abbreviated as S cell). As a non-human mammal,
The same as the above is used.

As a method for artificially mutating the DNA of the present invention, for example, the DNA can be prepared by a genetic engineering method.
It can be carried out by deleting a part or all of the sequence or inserting or substituting another DNA. Due to these mutations, the knockout DNA of the present invention may be prepared, for example, by shifting the reading frame of the codon or disrupting the function of the promoter or exon. Specific examples of the non-human mammalian embryonic stem cell in which the DNA of the present invention is inactivated (hereinafter abbreviated as the DNA inactivated ES cell of the present invention or the knockout ES cell of the present invention) include, for example, The DNA of the present invention contained in a non-human mammal is isolated, and its exon portion has a neomycin resistance gene, a drug resistance gene represented by a hygromycin resistance gene, lacZ (β-galactosidase gene), cat (chloramphenicol acetyl). Transferase gene) or a reporter gene typified by destroying the function of the exon, or insert a DNA sequence that terminates the transcription of the gene in the intron portion between the exons (for example, polyA addition signal), To prevent the synthesis of complete messenger RNA By introducing a DNA chain having a DNA sequence constructed so as to disrupt the gene (hereinafter abbreviated as targeting vector) into the chromosome of the animal by, for example, a homologous recombination method. DNA on Southern hybridization analysis or targeting vector using the DNA sequence on or near the DNA of the present invention as a probe
It can be obtained by selecting the knockout ES cell of the present invention by analyzing the sequence and the DNA sequence of the neighboring region other than the DNA of the present invention used for producing the targeting vector as a primer, and selecting the knockout ES cell of the present invention.

The DN of the present invention can also be obtained by the homologous recombination method or the like.
As the original ES cells for inactivating A, those already established as described above may be used, or those newly established according to the known Evans and Kaufma method may be used. For example, in the case of mouse ES cells,
Currently, 129 ES cells are generally used, but since the immunological background is not clear, we will obtain an alternative ES cell with a clear immunological genetic background. For the purpose of, for example, the BDF ₁ mouse (C57BL / 6 and DB, which has improved the number of eggs of C57BL / 6 mouse and C57BL / 6 by crossing with DBA / 2).
Those established by using F ₁ ) with A / 2 can also be favorably used. The BDF ₁ mouse has a large number of eggs and has the advantage that the eggs are strong, and since it has the C57BL / 6 mouse as a background, the ES cells obtained by using the BDF ₁ mouse are , C57BL / 6 mice backcross to reveal their genetic background
It can be advantageously used in that it can be replaced with a / 6 mouse. In addition, when ES cells are established, generally, blastocysts on the 3.5th day after fertilization are used, but in addition to this, many cells can be efficiently produced by collecting 8-cell stage embryos and culturing them up to blastocysts. The early embryos of can be obtained. Either male or female ES cells may be used, but male ES cells are usually more convenient for producing a germ line chimera. Also,
It is desirable to distinguish males and females as soon as possible in order to reduce troublesome culturing. As a method for determining the sex of ES cells, for example, a method of amplifying and detecting a gene in the sex determining region on the Y chromosome by the PCR method can be mentioned. Using this method, conventionally, for example G-banding method, requires about 10 ⁶ cells to the karyotyping, ES cell number of about 1 colony (about 50
Therefore, the primary selection of ES cells in the early stage of culture can be performed by discriminating males and females, and the early selection of male cells can significantly reduce the labor required in the early stage of culture.

The secondary selection can be performed, for example, by confirming the number of chromosomes by the G-banding method. The number of chromosomes of ES cells obtained is preferably 100% of the normal number, but if it is difficult due to physical manipulations during establishment, after knocking out the gene of ES cells, normal cells (for example, in mice, The number is 2n
= 40 cells). The embryonic stem cell line thus obtained usually has a very good proliferative property, but since it is easy to lose the ability to develop ontogeny, it is necessary to carefully subculture. For example, on a suitable feeder cell such as STO fibroblast in the presence of LIF (1-10000 U / ml) in a carbon dioxide incubator (preferably 5% carbon dioxide, 95% air or 5% oxygen, 5% It is cultured by a method such as culturing at about 37 ° C. in carbon dioxide gas, 90% air), and at the time of subculture, for example, trypsin / EDTA solution (usually 0.001-0.5% trypsin / 0.1-5 mM EDTA, Preferably, a method is employed in which about 0.1% trypsin / 1 mM EDTA) treatment is performed to form single cells, and the cells are seeded on newly prepared feeder cells. Such passage is usually carried out every 1 to 3 days. At this time, it is desirable to observe the cells and to discard the cultured cells when morphologically abnormal cells are found. ES cells are differentiated into various types of cells such as parietal muscle, visceral muscle, and myocardium by performing monolayer culture until reaching a high density or suspension culture until forming a cell clump under appropriate conditions. [MJ Evans and MH Kaufman, Nature (Natu
re) Volume 292, 154, 1981; GR Martin Proceedings of National Academy of Sciences USA (Proc. Natl. Acad. Sci. U.
SA) 78, 7634, 1981; TC Doetschman et al.
Journal of Embiology and Experimental Morphology, Vol. 87, p. 27, 1985
, The DNA expression-deficient cells of the present invention obtained by differentiating the ES cells of the present invention are useful for in vitro cell biological studies of the protein of the present invention. The non-human mammal deficient in DNA expression of the present invention is the mRN of the animal.
It is possible to distinguish from normal animals by measuring the amount of A using a known method and indirectly comparing the expression levels. As the non-human mammal, the same ones as described above are used.

In the non-human mammal deficient in DNA expression of the present invention, for example, the targeting vector prepared as described above is introduced into mouse embryonic stem cells or mouse egg cells, and the targeting vector D of the present invention is introduced.
It is possible to knock out the DNA of the present invention by performing homologous recombination in which the DNA sequence in which NA is inactivated 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. The cells in which the DNA of the present invention has been knocked out are the DNA sequences on the Southern hybridization analysis or targeting vector using the DNA sequence on or near the DNA of the present invention as a probe, and the mouse-derived cells of the present invention used for the targeting vector. It can be determined by an analysis by the PCR method using a DNA sequence in a neighboring region other than DNA as a primer. When a non-human mammal embryonic stem cell is used, a cell line in which the DNA of the present invention is inactivated by gene homologous recombination is cloned, and the cell is cloned at an appropriate time, for example, at the 8-cell stage. It is injected into an animal 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 cells having the normal DNA locus of the present invention and cells having the artificially mutated 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 mutated from the population obtained by mating such a chimeric individual with a normal individual. D of the present invention added
It can be obtained by selecting individuals composed of cells having the NA locus, for example, by judging the coat color. The individual thus obtained is usually an individual deficient in hetero expression of the protein of the present invention, and heterologous individuals deficient in hetero expression of the protein of the present invention are bred to homozygous expression of the protein of the present invention from their offspring. Defective individuals can be obtained. When using an egg cell, for example, a transgenic non-human mammal having a targeting vector introduced into its chromosome by injecting a DNA solution into the egg cell nucleus by a microinjection method can be obtained,
Compared to these transgenic non-human mammals,
It can be obtained by selecting those having a mutation in the DNA locus of the present invention by gene homologous recombination. In this way, an individual whose DNA of the present invention has been knocked out can be passaged in a normal breeding environment after confirming that the DNA of the animal individual obtained by mating is also knocked out. Furthermore, the acquisition and maintenance of the germ line may be carried out according to the conventional method. That is, by crossing male and female animals carrying the inactivated DNA, the inactivated D
A homozygous animal having NA on both homologous chromosomes can be obtained. The obtained homozygous animal can be efficiently obtained by raising the mother animal in such a state that there are 1 normal individual and 1 homozygous animal.
By mating male and female heterozygous animals, homozygous and heterozygous animals having the inactivated DNA are bred. The non-human mammal embryonic stem cell in which the DNA of the present invention is inactivated is very useful in producing the non-human mammal deficient in expression of the DNA of the present invention. Further, the non-human mammal deficient in DNA expression of the present invention,
Since it lacks various biological activities that can be induced by the protein of the present invention, it can serve as a model for diseases caused by inactivation of the biological activity of the protein of the present invention. It is useful for examination.

[8a] Method for Screening Compound Having Preventive / Therapeutic Effect on Disease Caused by DNA Deficiency or Damage of the Present Invention The non-human mammal deficient in DNA expression of the present invention is D
It can be used for screening a compound having a preventive / therapeutic effect on a disease caused by NA deficiency or damage. That is, the present invention results from a defect or damage of the DNA of the present invention, which comprises administering a test compound to a non-human mammal deficient in DNA expression of the present invention and observing and measuring the change in the animal. Provided is a method for screening a compound having a preventive / therapeutic effect against a disease or a salt thereof. 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. Examples of test compounds include peptides, proteins, non-peptidic compounds, synthetic compounds,
Examples include fermentation products, cell extracts, plant extracts, animal tissue extracts, plasma, and the like, and these compounds may be novel compounds or known compounds. Specifically, the non-human mammal deficient in DNA expression of the present invention is treated with a test compound and compared with a non-treated control animal, and tested using changes in each organ, tissue, disease symptoms, etc. of the animal as an index. The prophylactic / therapeutic effects of compounds can be tested. As a method of treating a test animal with a test compound, for example,
Oral administration, intravenous injection, etc. are used to
It can be appropriately selected depending on the properties of the test compound. Further, 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, to prevent chronic obstructive pulmonary disease and bronchial asthma
When screening a compound having a therapeutic effect, the DNA expression-deficient non-human mammal of the present invention is subjected to cigarette smoke or antigen exposure treatment, and the test compound is administered before or after the cigarette smoke or antigen exposure treatment, and the respiratory tract of the animal is treated. Changes in reactivity and the like are measured with time. The compound obtained by using the screening method is a compound selected from the above-mentioned test compounds and has a prophylactic / therapeutic effect on a disease caused by a deficiency or damage of the protein of the present invention. Safe and low toxicity prevention
It can be used as a medicine such as a therapeutic agent. Furthermore, compounds derived from the compounds obtained by the above screening can be used as well.

The compound obtained by the screening method may form a salt, and as the salt of the compound, a physiologically acceptable acid (eg, inorganic acid, organic acid, etc.) or base (eg, A salt with an alkali metal or the like) is used, and a physiologically acceptable acid addition salt is particularly preferable. Examples of such salts include salts with inorganic acids (for example, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid, etc.), or organic acids (for example, 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.) and the like salts are used. The drug containing the compound or its salt obtained by the screening method can be manufactured in the same manner as the drug containing the protein of the present invention. The formulation thus obtained is
Since it is safe and has low toxicity, it can be administered to, for example, a human or non-human mammal (eg, rat, mouse, guinea pig, rabbit, sheep, pig, cow, horse, cat, dog, monkey, etc.). . Although the dose of the compound or its salt varies depending on the target disease, administration subject, administration route, etc., for example, when the compound is orally administered, it is generally an adult (as a body weight of 60 kg) chronic obstructive pulmonary disease. In patients with
00 mg, preferably about 1.0-50 mg, more preferably about 1.0-
Administer 20 mg. When administered parenterally, the single dose of the compound varies depending on the administration subject, target disease, etc., but, for example, the compound in the form of an injection is usually an adult (as 60 kg) chronic obstructive pulmonary disease. When administered to a patient of about 0.01 to 30 mg, preferably about 0.1 to 20 m
Conveniently, g, more preferably about 0.1-10 mg, is administered by intravenous injection. In the case of other animals, the dose converted per 60 kg can be administered.

[8b] Method for Screening Compound for Promoting or Inhibiting Promoter Activity for DNA of the Present Invention The present invention provides a non-human mammal deficient in expression of the DNA of the present invention,
A method for screening a compound or a salt thereof which promotes or inhibits the activity of a promoter for the DNA of the present invention, which comprises administering a test compound and detecting the expression of a reporter gene. In the above screening method, the non-human mammal deficient in DNA expression of the present invention, among the non-human mammal deficient in DNA expression of the present invention, the DNA of the present invention is inactivated by introducing a reporter gene, A reporter gene that can be expressed under the control of a promoter for the DNA of the present invention is used. Examples of the test compound include the same ones as described above. As the reporter gene, the same ones as described above are used, and the β-galactosidase gene (lacZ), the soluble alkaline phosphatase gene, the luciferase gene and the like are preferable.

In a non-human mammal deficient in expression of the DNA of the present invention in which the DNA of the present invention is replaced with a reporter gene, the reporter gene exists under the control of the promoter for the DNA of the present invention. The activity of the promoter can be detected by tracing the expression. For example, a part of the DNA region encoding the protein of the present invention is a β derived from Escherichia coli.
-When it is replaced with the galactosidase gene (lacZ), β-galactosidase is originally expressed in the tissue in which the protein of the present invention is expressed instead of the protein of the present invention. Thus, for example, 5-bromo-4-chloro-3
-Indolyl-β-galactopyranoside (X-gal)
The expression state of the protein of the present invention in an animal body can be easily observed by staining with a reagent that serves as a substrate for β-galactosidase. Specifically, the protein-deficient mouse of the present invention or a tissue section thereof is fixed with glutaraldehyde or the like, washed with phosphate buffered saline (PBS), and then stained with X-gal at room temperature or around 37 ° C. After reacting for about 30 minutes to 1 hour, the tissue sample may be washed with a 1 mM EDTA / PBS solution to stop the β-galactosidase reaction and observe the coloration. In addition, mRNA encoding lacZ may be detected according to a conventional method.
The compound or its salt obtained by using the above screening method is a compound selected from the above-mentioned test compounds, and is a compound that promotes or inhibits the promoter activity for the DNA of the present invention.

The compound obtained by the screening method may form a salt, and as the salt of the compound, a physiologically acceptable acid (eg, inorganic acid or the like) or base (eg, organic acid or the like) is used. ) And the like are used, and physiologically acceptable acid addition salts are particularly preferable. For such salt,
For example, inorganic acids (for example, hydrochloric acid, phosphoric acid, hydrobromic acid,
Sulfates) 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, etc.) and the like are used.

Further, the compound or its salt that inhibits the promoter activity for the DNA of the present invention can inhibit the expression of the protein of the present invention and inhibit the function of the protein, and thus, for example, inflammation of the lungs and respiratory tract. Respiratory diseases such as associated lung and chest diseases [eg, chronic obstructive pulmonary disease (chronic bronchitis, emphysema), diffuse panbronchiolitis, bronchial asthma, cystic fibrosis, hypersensitivity pneumonia, etc.], allergic conjunctivitis , Rhinitis (eg, allergic rhinitis, pollinosis, acute rhinitis, chronic rhinitis, hypertrophic rhinitis, atrophic rhinitis, dry anterior rhinitis, vasomotor rhinitis, necrotizing rhinitis, sinusitis, etc.), digestive tract disease ( (Eg, irritable bowel syndrome, inflammatory bowel disease, ulcerative colitis, Crohn's disease, reflux esophagitis, etc.)
It is useful as a therapeutic agent, preferably as a medicine such as a prophylactic / therapeutic agent for respiratory diseases and digestive tract diseases. Furthermore, compounds derived from the compounds obtained by the above screening can be used as well. The drug containing the compound or its salt obtained by the screening method can be produced in the same manner as the above-mentioned drug containing the protein of the present invention or its salt. Since the preparation thus obtained is safe and has low toxicity, for example, human or non-human mammals (for example, rat, mouse, guinea pig, rabbit, sheep, pig, cow, horse, cat, dog,
Can be administered to monkeys). The dose of the compound or its salt varies depending on the target disease, the subject to be administered, the administration route, etc. However, for example, when the compound of the present invention that promotes the promoter activity for DNA is orally administered, it is generally adult (body weight In patients with chronic obstructive pulmonary disease (as 60 kg), about 0.1
~ 100 mg, preferably about 1.0-50 mg, more preferably about 1.
Administer 0 to 20 mg. When administered parenterally, the single dose of the compound varies depending on the administration subject, target disease, etc., but for example, a compound that promotes the promoter activity for the DNA of the present invention in the form of an injection is usually used in adults ( When administered to patients with chronic obstructive pulmonary disease (as 60 kg), about 0.01-30 mg, preferably about 0.1-20 m of said compound per day
Conveniently, g, more preferably about 0.1-10 mg, is administered by intravenous injection. In the case of other animals, the dose converted per 60 kg can be administered.

On the other hand, for example, when a compound which inhibits the promoter activity for the DNA of the present invention is orally administered,
Generally in an adult (as a body weight of 60 kg) patient with chronic obstructive pulmonary disease, about 0.1 to 100 mg of the compound per day,
Preferably about 1.0 to 50 mg, more preferably about 1.0 to 20 mg is administered. When administered parenterally, the single dose of the compound varies depending on the administration subject, target disease and the like, but for example, a compound that inhibits the promoter activity against the DNA of the present invention is usually in the form of an injection ( When administered to patients with chronic obstructive pulmonary disease (as 60 kg), the compound is administered by intravenous injection in an amount of about 0.01 to 30 mg, preferably about 0.1 to 20 mg, more preferably about 0.1 to 10 mg per day. It is convenient. In the case of other animals, the dose converted per 60 kg can be administered. Thus, the D of the present invention
The non-human mammal deficient in NA expression is extremely useful for screening a compound or its salt that promotes or inhibits the activity of the promoter for the DNA of the present invention,
The present invention can greatly contribute to the investigation of the cause of various diseases caused by DNA expression deficiency of the present invention or the development of preventive / therapeutic drugs. In addition, by using a DNA containing a promoter region of the protein of the present invention, genes encoding various proteins are ligated downstream thereof, and this is injected into an egg cell of an animal to obtain a so-called transgenic animal (transgenic animal). Once prepared, it is possible to specifically synthesize the polypeptide and study its action in the living body. Further, by linking an appropriate reporter gene to the above promoter part and establishing a cell line in which it is expressed,
It can be used as a search system for low-molecular compounds having an action of specifically promoting or suppressing the production ability of the protein of the present invention itself in the body.

In this specification, when abbreviations are used for bases, amino acids, etc., the IUPAC-IUB Commission
Based on the abbreviations based on on Biochemical Nomenclature or abbreviations commonly used in this field, examples are given below. When amino acids may have optical isomers, the L form is shown unless otherwise specified. DNA: deoxyribonucleic acid cDNA: complementary deoxyribonucleic acid A: adenine T: thymine G: guanine C: cytosine 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: ribonucleic acid mRNA: messenger ribonucleic acid dATP: deoxyadenosine Triphosphate dTTP: Deoxythymidine triphosphate dGTP: Deoxyguanosine triphosphate dCTP: Deoxycytidine triphosphate ATP: Adenosine triphosphate EDTA: Ethylenediaminetetraacetic acid SDS: Sodium dodecyl sulfate

[0077]   Gly: Glycine   Ala: Alanine   Val: Valine   Leu: Leucine   Ile: Isoleucine   Ser: Serine   Thr: Threonine   Cys: Cysteine   Met: Methionine   Glu: Glutamic acid   Asp: Aspartic acid   Lys: Lysine   Arg: Arginine   His: Histidine   Phe: Phenylalanine   Tyr: Tyrosine   Trp: Tryptophan   Pro: Proline   Asn: Asparagine   Gln: Glutamine   pGlu: pyroglutamic acid

The substituents, protecting groups and reagents commonly used in the present specification are represented by the following symbols. Me: methyl group Et: ethyl group Bu: butyl group Ph: phenyl group TC: thiazolidine -4 (R) - carboxamide group Tos: p-toluenesulfonyl CHO: formyl Bzl: benzyl Cl ₂ -Bzl: 2,6-dichlorobenzyl Bom: Benzyloxymethyl Z: Benzyloxycarbonyl Cl-Z: 2-Chlorobenzyloxycarbonyl Br-Z: 2-Bromobenzyloxycarbonyl Boc: t-Butoxycarbonyl DNP: Dinitrophenyl Trt: Trityl Bum: t-Butoxymethyl Fmoc. : N-9-fluorenylmethoxycarbonyl HOBt: 1-hydroxybenztriazole HOOBt: 3,4-dihydro-3-hydroxy-4-oxo-1,2,3-benzotriazine HONB: 1-hydroxy-5-no Bornene-2,3-dicarboximide DCC: N, N'-dicyclohexylcarbodiimide

The sequence numbers in the sequence listing in this specification show the following sequences. [SEQ ID NO: 1] This 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] This shows the base sequence of mouse CLCA4 gene 5 ′ side. [SEQ ID NO: 4] This shows the base sequence of mouse CLCA4 gene 3 ′ side. [SEQ ID NO: 5] This shows the base sequence of primer 1 used in Examples 1 and 3. [SEQ ID NO: 6] This shows the base sequence of primer 2 used in Examples 1 and 4. [SEQ ID NO: 7] This shows the base sequence of primer 3 used in Examples 1 and 4. [SEQ ID NO: 8] This shows the base sequence of primer 4 used in Examples 1 and 2. [SEQ ID NO: 9] This shows the base sequence of primer 5 used in Example 1. [SEQ ID NO: 10] This shows the base sequence of primer 6 used in Examples 1 and 2. [SEQ ID NO: 11] Primer 7 used in Example 1
The base sequence of is shown. [SEQ ID NO: 12] Primer 8 used in Example 1
The base sequence of is shown. [SEQ ID NO: 13] Primer 9 used in Example 1
The base sequence of is shown. [SEQ ID NO: 14] Primer 1 used in Example 1
The base sequence of 0 is shown. [SEQ ID NO: 15] T7 pro used in Example 1
motor primer (promoter primer)
The base sequence of is shown. [SEQ ID NO: 16] M13RV used in Example 1
The base sequence of a primer (primer) is shown. [SEQ ID NO: 17] This shows the amino acid sequence of mouse CLCA4A protein. [SEQ ID NO: 18] This shows the base sequence of DNA encoding the amino acid sequence of mouse CLCA4A protein having the amino acid sequence represented by SEQ ID NO: 17. [SEQ ID NO: 19] This shows the 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] Primer 1 used in Example 3
1 shows the base sequence of 1. [SEQ ID NO: 22] Primer 1 used in Example 5
2 shows the base sequence of 2. [SEQ ID NO: 23] Primer 1 used in Example 5
3 shows the nucleotide sequence of 3. [SEQ ID NO: 24] Primer 1 used in Example 5
4 shows the base sequence of 4.

Transformant Escherichia coli TOP10 / pCR-BluntI obtained in Example 1 described below
I-mCLCA4 has been located on 1st East of Tsukuba City, Ibaraki Prefecture since February 12, 2002
No. 1 at the Patent Biological Deposit Center, National Institute of Advanced Industrial Science and Technology, Central Administrative No. 6 (Zip code 305-8566)
As RM BP-7887, from January 29, 2002, to the Fermentation Research Institute (IFO) at 2-1785, Jusohon-cho, Yodogawa-ku, Osaka-shi, Osaka Prefecture (postal code 532-8686), under the contract number IFO 16751 Has been deposited. The transformant Escherichia coli TOP10 / pCR-Blun obtained in Example 3 described later.
tII-mCLCA4A has been deposited at the Patent Biological Deposit Center, National Institute of Advanced Industrial Science and Technology, Central Research Institute No. 1 (1-1, 1 Higashi, Tsukuba, Ibaraki Prefecture, from March 4, 2002).
As FERM BP-7934, from February 19, 2002, to the Fermentation Research Institute (IFO) at 2-1785, 13 Sanhonmachi, Yodogawa-ku, Osaka Prefecture, Japan (postal code 532-8686), under the contract number IFO 16764. Has been deposited.

The present invention will be described in more detail below with reference to examples, but the present invention is not limited thereto. Example 1 Cloning of mouse CLCA4 gene Nucleotide sequence of human CLCA4 gene [FEBS Letters, 455, 29
5 (1999)], nucleotide sequence of mouse gob-5 gene [Bioche
m. Biophys. Res. Commun., 255, 347 (1999)], four types of primers [primer 1 (SEQ ID NO: 5), primer 2 (SEQ ID NO: 6), primer 3 (SEQ ID NO: 6) : 7) and primer 4 (SEQ ID NO:
8)] was designed and synthesized. Next, IS from mouse colon tissue
Total RNA was prepared using OGEN (Wako Pure Chemical Industries, Ltd.). Furthermore, mRNA was prepared using mRNA purification kit (manufactured by Amersham Pharmacia Biotech). This mRNA 1μ
Marathon cDNA Amplification kit starting from g
(Clontech) was used to synthesize cDNA by reverse transcription. After performing PCR using the cDNA as a template and a combination of primer 1 and primer 2 and primer 3 and primer 4, it was confirmed by agarose electrophoresis that a DNA fragment of the expected size was amplified. The reaction is pyrobest
Using a thermal cycler Gene Amp PCRSystem 9700 (manufactured by Perkin Elmer) using DNA polymerase (manufactured by Takara Shuzo), first react at 94 ° C for 30 seconds, then at 94 ° C for 10 seconds.
Second, 30 seconds at 60 ° C, 2 minutes and 30 seconds at 72 ° C as one reaction cycle
35 cycles were repeated, and finally the reaction was carried out at 72 ° C for 10 minutes.
The obtained DNA fragment was cloned into pCR-BluntII-TOPO (Invitrogen). Furthermore, T7 promoter primer
(SEQ ID NO: 15), M13RV primer (SEQ ID NO: 16)
And 6 kinds of synthetic primers [primer 5 (SEQ ID NO: 9), primer 6 (SEQ ID NO: 10), primer 7 (SEQ ID NO: 11), primer 8 (SEQ ID NO: 1)
2), the primer 9 (SEQ ID NO: 13) and the primer 10 (SEQ ID NO: 14)] were used to carry out a cycle sequence reaction, and the base sequence of the reaction product obtained with the fluorescent DNA sequencer 377 (manufactured by Perkin Elmer) was obtained. Were determined. As a result, clone No. 3-1 (SEQ ID NO: 3) in which 2027 nucleotide sequences containing mouse N-terminal sequences were inserted as partial nucleotide sequences of mouse CLCA4 gene and 1869 nucleotides containing mouse C-terminal sequences Clone No. 7 with sequence inserted
-3 (SEQ ID NO: 4) was obtained. It was revealed that mouse CLCA4 has a nucleotide sequence of 2772 nucleotides except for the overlapping sequences of both (SEQ ID NO: 2). A 924 amino acid sequence was determined based on the nucleotide sequence represented by SEQ ID NO: 2 (SEQ ID NO: 1) and named mouse CLCA4 protein. Next, clone No.7-3 was digested with HindIII and 1.4 Kb of mouse CL
A DNA fragment containing the C-terminal region of CA4 was excised. This was also digested with HindIII to clone CL No.3-1 mouse CLCA4
The plasmid pCR-BluntII-mCLCA4 containing the mouse CLCA4 full-length sequence was constructed by ligating to the vector sequence containing the N-terminal region. Transformants containing this plasmid were transformed into Escherichia
It was named Escherichia coli TOP10 / pCR-BluntII-mCLCA4. Mouse CLCA4 is human CLC at the amino acid level.
It had 68% homology with A4 and 51% homology with mouse gob-5.

Example 2 Analysis of Tissue Distribution of Mouse CLCA4 Gene Product Using the primers 4 (SEQ ID NO: 8) and 6 (SEQ ID NO: 10), each mouse tissue (bone marrow, eye,
Lymph node, smooth muscle, ovary, thymus, stomach, bladder, heart, whole brain, spleen, lung, liver, skeletal muscle, kidney, testis, 7/11/15
・ 17th day embryo, large intestine cDNA (Mouse MTC panel I and M)
ouse MTC panel II: Clontech's) P as a mold
I did a CR. The reaction was performed using Takara Ex Taq (Takara Shuzo Co., Ltd.) with a thermal cycler GeneAmp PCR System 9600 (Applied Biosystems Co., Ltd.) at 94 ° C. for 30 seconds, then 94 ° C. for 10 seconds, 60 ° C. for 30 seconds. 35 cycles of 2 minutes at 72 ° C as one reaction cycle, and finally 10 cycles at 72 ° C.
Let react for minutes. The obtained reaction product was electrophoresed on a 2% agarose gel and then stained with ethidium 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 CLCA4A gene Upon cloning of mouse CLCA4 gene, the existence of a clone having a nucleotide sequence different from mouse CLCA4 was suggested. Therefore, mouse smooth muscle cDNA (Mouse MTC panel II:
PCR was carried out using a combination of Primer 1 (SEQ ID NO: 5) and Primer 11 (SEQ ID NO: 21) using Clontech) as a template, and 2.7 Kb of agarose gel was used for electrophoresis.
It was confirmed that the DNA fragment was amplified. The reaction is pyrobest DN
Thermal cycler using A polymerase (Takara Shuzo) Gene Amp PCR System 9700 (Perkin Elmer)
At 94 ℃ for 30 seconds, then at 94 ℃ for 10 seconds, 60 seconds
35 cycles were repeated with 30 seconds at ℃ and 5 minutes at 72 ℃ as one reaction cycle, and finally the reaction was carried out at 72 ℃ for 10 minutes. Obtained DN
The A fragment was cloned into pCR-BluntII-TOPO (Invitrogen). Furthermore, T7 promoter primer (SEQ ID NO: 1
5), M13RV primer (SEQ ID NO: 16) and 6 kinds of synthetic primers [primer 5 (SEQ ID NO: 9), primer 6 (SEQ ID NO: 10), primer 7 (SEQ ID NO :)
11), primer 8 (SEQ ID NO: 12), primer 9 (SEQ ID NO: 13) and primer 10 (SEQ ID NO: 14)], and a cycle sequence reaction is performed using a fluorescent DNA sequencer 377 (manufactured by Perkin Elmer). The base sequence of the obtained reaction product was determined. As a result, 94% at the amino acid level with mouse CLCA4, and with human CLCA4
It was revealed that a protein consisting of 924 amino acids (SEQ ID NO: 17) showing 68% homology was encoded (SEQ ID NO: 18), and named mouse CLCA4A protein. The above plasmid pCR-BluntII-mCLCA4 was constructed. The transformant containing this plasmid pCR-BluntII-TOPO was transformed into Escherichia coli TOP10 / pCR-BluntI.
It was named I-mCLCA4A.

Example 4 Analysis of Tissue Distribution of Mouse CLCA4A Gene Product Primer 2 (SEQ ID NO: 6) and Primer 3 (SEQ ID NO: 7) which are common sequences of mouse CLCA4 and mouse CLCA4A
The DNA fragment of 1020 bp amplified by PCR with the combination of
The mouse CLCA4 origin was not digested with the restriction enzyme PvuII (Takara Shuzo Co., Ltd.), whereas the mouse CLCA4A origin was digested to 680 bp and 340 bp. The PCR reaction was performed by using Takara Ex Taq (manufactured by Takara Shuzo Co., Ltd.) with a thermal cycler GeneAmp PCR System 9600 (manufactured by Applied Biosystems) at 94 ° C. for 30 seconds, and then at 94 ° C. for 10 seconds and 60 ° C. for 30 seconds. It was carried out by repeating 35 cycles with one reaction cycle of 2 minutes at 72 ° C. for 2 seconds.
The obtained reaction product was digested with PvuII, electrophoresed on a 2% agarose gel, and then stained with ethidium bromide to detect a band. 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 mouse C
It was revealed that the LCA4 gene product (mRNA) was expressed in smooth muscle and large intestine, whereas the mouse CLCA4A gene product (mRNA) was expressed only in smooth muscle.

Example 5 Cloning of rat CLCA4 gene Nucleotide sequence of human CLCA4 gene (FEBS Letters, 455 volumes, 29
Page 5, 1999), two types of primers [primer 12 (SEQ ID NO: 2) with reference to the nucleotide sequence of mouse CLCA4 gene (SEQ ID NO: 2) and the nucleotide sequence of mouse CLCA4A gene]
2) and primer 13 (SEQ ID NO: 23)] were designed and synthesized. Next, total RNA was prepared from rat intestinal tissue using ISOGEN (manufactured by Wako Pure Chemical Industries, Ltd.). Furthermore mRNA purifica
mRNA was prepared using tion kit (Amersham Pharmacia Biotech). Marathon cDNA Amplification kit (Clontech) using 1 μg of this mRNA as a starting material
Was used to synthesize cDNA by reverse transcription. After performing PCR with the combination of primer 12 and primer 13 using this cDNA as a template, it was confirmed by agarose electrophoresis that a DNA fragment of the expected size was amplified. The reaction was carried out using pyrobest DNA polymerase (manufactured by Takara Shuzo Co., Ltd.) with a thermal cycler Gene Amp PCR System 9700 (manufactured by Perkin Elmer Co., Ltd.) first at 94 ° C. for 30 seconds, and then,
Thirty-five cycles of 94 ° C. for 10 seconds, 60 ° C. for 30 seconds, and 72 ° C. for 2 minutes were repeated as one reaction cycle, and finally the reaction was performed at 72 ° C. for 10 minutes. The obtained DNA fragment was pCR-BluntII-TOPO (Invitrogen
Cloned). Furthermore, T7 promoter primer
(SEQ ID NO: 15), M13RV primer (SEQ ID NO: 16)
And three kinds of synthetic primers [primer 12 (SEQ ID NO: 22), primer 13 (SEQ ID NO: 23), primer 14 (SEQ ID NO: 24)] were used to carry out a cycle sequence reaction, and a fluorescent DNA sequencer 377 (Perkin Elmer The nucleotide sequence of the reaction product obtained in the above) was determined. As a result, clone No. a-20 (SEQ ID NO: 20) having 1119 nucleotide sequences inserted as a partial nucleotide sequence of rat CLCA4 gene was obtained. A 373 partial amino acid sequence was determined based on the nucleotide sequence represented by SEQ ID NO: 20 (SEQ ID NO: 19). The partial amino acid sequence is human CLCA.
66% with 4 corresponding sequences, mouse CLCA4 and mouse CL
It had 81% homology with the corresponding sequence of CA4A.

[0085]

INDUSTRIAL APPLICABILITY The proteins and polynucleotides of the present invention are used for 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, hypersensitivity pneumonia, etc.), allergic conjunctivitis, rhinitis (eg, allergic rhinitis, pollinosis, acute rhinitis, chronic rhinitis, hypertrophic rhinitis, atrophy) Rhinitis, dry anterior rhinitis, vasomotor rhinitis, gangrenous rhinitis, sinusitis, etc., gastrointestinal disorders (eg, irritable bowel syndrome, inflammatory bowel disease, ulcerative colitis,
Crohn's disease, reflux esophagitis, etc.) are useful as diagnostic markers, etc., and inhibitors obtained by screening using the protein, polynucleotide or antibody against the protein, neutralizing antibodies that inhibit the activity of the protein, etc. , For example, respiratory diseases such as lung / chest diseases accompanied by inflammation of the lungs / airways (eg, chronic obstructive pulmonary disease (chronic bronchitis, emphysema), diffuse panbronchiolitis, bronchial asthma, cystic fibrosis, Hypersensitivity pneumonia, etc.], allergic conjunctivitis, rhinitis (eg, allergic rhinitis, hay fever, acute rhinitis, chronic rhinitis, hypertrophic rhinitis, atrophic rhinitis, dry anterior rhinitis, vasomotor rhinitis, necrotic rhinitis, adjunct Prevention of gastrointestinal disorders (eg, irritable bowel syndrome, inflammatory bowel disease, ulcerative colitis, Crohn's disease, reflux esophagitis, etc.)
It can be used as a therapeutic agent, preferably a prophylactic / therapeutic agent for respiratory diseases, digestive tract diseases and the like. Further, the antisense polynucleotide of the present invention can suppress the expression of the protein of the present invention, and for example, respiratory diseases such as lung and chest diseases accompanied by inflammation of the lungs and respiratory tract [eg, chronic obstructive pulmonary disease] (Chronic bronchitis, emphysema), diffuse panbronchiolitis, bronchial asthma, cystic fibrosis, hypersensitivity pneumonia, etc.], allergic conjunctivitis, rhinitis (eg, allergic rhinitis, hay fever, acute rhinitis, chronic rhinitis, Hypertrophic rhinitis, atrophic rhinitis, dry anterior rhinitis, vasomotor rhinitis, gangrenous rhinitis, sinusitis, etc., gastrointestinal disease (eg, irritable bowel syndrome, inflammatory bowel disease, ulcerative colitis, clone) Disease, reflux esophagitis, etc.), and preferably as a prophylactic / therapeutic agent for diseases such as respiratory diseases and digestive tract diseases. Furthermore, various polynucleotides of the present invention are
Respiratory diseases such as lung and chest diseases accompanied by airway inflammation [eg,
Chronic obstructive pulmonary disease (chronic bronchitis, emphysema), diffuse panbronchiolitis, bronchial asthma, cystic fibrosis, hypersensitivity pneumonia, etc.], allergic conjunctivitis, rhinitis (eg, allergic rhinitis, hay fever, acute) Rhinitis, chronic rhinitis, hypertrophic rhinitis, atrophic rhinitis, dry anterior rhinitis, vasomotor rhinitis, gangrenous rhinitis,
It is useful for diagnosis, prevention or treatment of gastrointestinal tract diseases (eg, irritable bowel syndrome, inflammatory bowel disease, ulcerative colitis, Crohn's disease, reflux esophagitis, etc.).

[0086]

[Sequence list] SEQUENCE LISTING <110> Takeda Chemical Industries, Ltd. <120> New Protein, DNA and Use thereof <130> B03011 <150> JP 2002-012180 <151> 2002-01-21 <150> JP 2002-44981 <151> 2002-02-21 <150> JP 2002-61668 <151> 2002-03-07 <160> 24 <210> 1 <211> 924 <212> PRT <213> Mouse <400> 1 Met Ala Phe Ser Arg Gly Pro Val Phe Leu Leu Leu Leu Leu Tyr Leu                  5 10 15 Leu Trp Gly Ser Asp Thr Ser Leu Ile Arg Leu Asn Glu Asn Gly Tyr              20 25 30 Glu Asp Ile Ile Ile Ala Ile Asp Pro Ala Val Pro Glu Asp Thr Thr          35 40 45 Ile Ile Glu His Ile Lys Gly Met Val Thr Lys Ala Ser Thr Tyr Leu      50 55 60 Phe Glu Ala Thr Glu Lys Arg Phe Phe Phe Lys Asn Val Ser Ile Leu  65 70 75 80 Ile Pro Glu Ser Trp Lys Asp Ser Pro Gln Tyr Arg Arg Pro Lys Gln                  85 90 95 Glu Ser Tyr Lys His Ala Asp Ile Lys Val Ala Pro Pro Thr Val Glu             100 105 110 Gly Arg Asp Glu Pro Tyr Thr Arg Gln Phe Thr Gln Cys Glu Glu Lys         115 120 125 Ala Glu Tyr Ile His Phe Thr Pro Asp Phe Val Leu Gly Arg Lys Gln     130 135 140 Asp Glu Tyr Gly Asp Ser Gly Lys Val Leu Val His Glu Trp Ala His 145 150 155 160 Leu Arg Trp Gly Val Phe Asp Glu Tyr Asn Glu Asp Gln Pro Phe Tyr                 165 170 175 Ser Ala Ser Ser Lys Lys Ile Glu Ala Thr Arg Cys Ser Thr Gly Ile             180 185 190 Thr Gly Thr Asn Arg Val Tyr Ala Cys Gln Gly Gly Ser Cys Ala Met         195 200 205 Arg Arg Cys Arg Thr Asn Ser Thr Thr Lys Leu Tyr Glu Lys Asp Cys     210 215 220 Gln Phe Phe Pro Asp Lys Val Gln Ser Glu Lys Ala Ser Ile Met Phe 225 230 235 240 Met Gln Ser Ile Asp Ser Val Thr Glu Phe Cys Lys Lys Glu Asn His                 245 250 255 Asn Arg Glu Ala Pro Thr Leu His Asn Lys Lys Cys Asn Tyr Arg Ser             260 265 270 Thr Trp Glu Val Ile Ser Thr Ser Glu Asp Phe Asn Ser Ser Thr Pro         275 280 285 Met Glu Thr Ser Pro Ser Pro Pro Phe Phe Ser Leu Leu Arg Ile Ser     290 295 300 Glu Arg Ile Met Cys Leu Val Leu Asp Val Ser Gly Ser Met Thr Ser 305 310 315 320 Tyr Asp Arg Leu Asn Arg Met Asn Gln Ala Ala Lys Tyr Phe Leu Ser                 325 330 335 Gln Ile Ile Glu Asn Arg Ser Trp Val Gly Met Val His Phe Ser Ser             340 345 350 Gln Ala Thr Ile Val His Glu Leu Ile Gln Ile Asn Ser Asp Ile Glu         355 360 365 Arg Asn Gln Leu Leu Gln Thr Leu Pro Thr Ser Ala Asn Gly Gly Thr     370 375 380 Ser Ile Cys Ser Gly Ile Lys Ala Ala Phe Gln Val Phe Lys Asn Gly 385 390 395 400 Glu Tyr Gln Thr Asp Gly Thr Glu Ile Leu Leu Leu Ser Asp Gly Glu                 405 410 415 Asp Ser Thr Ala Lys Asp Cys Ile Asp Glu Val Lys Asp Ser Gly Ser             420 425 430 Ile Val His Phe Ile Ala Leu Gly Pro Ser Ala Asp Leu Ala Val Thr         435 440 445 Asn Met Ser Ile Leu Thr Gly Gly Asn His Lys Leu Ala Thr Asp Glu     450 455 460 Ala Gln Asn Asn Gly Leu Ile Asp Ala Phe Gly Ala Leu Ala Ser Glu 465 470 475 480 Asn Ala Asp Ile Thr Gln Lys Ser Leu Gln Leu Glu Ser Lys Gly Ala                 485 490 495 Ile Leu Asn Asn Ser Leu Trp Leu Asn Asp Thr Val Val Ile Asp Ser             500 505 510 Thr Leu Gly Arg Asp Thr Phe Phe Leu Val Thr Trp Ser Lys Gln Ala         515 520 525 Pro Ala Ile Tyr Leu Arg Asp Pro Lys Gly Thr Gln Thr Thr Asn Phe     530 535 540 Thr Met Asp Ser Ala Ser Lys Met Ala Tyr Leu Ser Ile Pro Gly Thr 545 550 555 560 Ala Gln Val Gly Val Trp Thr Tyr Asn Leu Glu Ala Lys Glu Asn Ser                 565 570 575 Glu Ile Leu Thr Ile Thr Val Thr Ser Arg Ala Ala Asn Ser Ser Val             580 585 590 Pro Pro Ile Thr Val Asn Ala Lys Val Asn Thr Asp Thr Asn Thr Phe         595 600 605 Pro Ser Pro Met Ile Val Tyr Ala Glu Val Leu Gln Gly Tyr Thr Pro     610 615 620 Ile Ile Gly Ala Arg Val Thr Ala Thr Ile Glu Ser Asn Ser Gly Lys 625 630 635 640 Thr Glu Glu Leu Val Leu Leu Asp Asn Gly Ala Gly Ala Asp Ala Phe                 645 650 655 Lys Asp Asp Gly Val Tyr Ser Arg Phe Phe Thr Ala Tyr Ser Val Asn             660 665 670 Gly Arg Tyr Ser Leu Lys Val Arg Ala Asp Gly Gly Arg Asn Ser Ala         675 680 685 Arg Arg Ser Leu Arg His Pro Ser Ser Arg Ala Ala Tyr Ile Pro Gly     690 695 700 Trp Val Val Asp Gly Glu Ile Gln Gly Asn Pro Pro Arg Pro Glu Thr 705 710 715 720 Thr Glu Ala Thr Gln Pro Val Leu Glu Asp Phe Ser Arg Thr Ala Ser                 725 730 735 Gly Gly Ala Phe Val Met Ser Asn Val Pro Ile Gly Pro Leu Pro Asp             740 745 750 Val Tyr Pro Pro Asn Arg Ile Thr Asp Leu Gln Ala Thr Leu Asp Gly         755 760 765 Glu Glu Ile Ser Leu Thr Trp Thr Ala Pro Gly Asp Asp Tyr Asp Val     770 775 780 Gly Arg Val Gln Gln Tyr Ile Ile Arg Thr Ser Lys Asn Ile Ile Glu 785 790 795 800 Leu Arg Asp Asn Phe Asn Asn Ser Pro Arg Val Asp Thr Thr Asn Leu                 805 810 815 Thr Pro Lys Glu Ala Asn Ser Glu Glu Thr Phe Ala Phe Lys Pro Glu             820 825 830 Asn Ile Thr Glu Glu Asn Ala Thr Tyr Ile Phe Ile Ala Ile Glu Ser         835 840 845 Val Asp Lys Ser Ser Leu Ser Ser Gly Pro Ser Asn Ile Ala Gln Val     850 855 860 Ala Leu Phe Thr Pro Gln Ala Glu Pro Asp Pro Asp Glu Ser Pro Ser 865 870 875 880 Leu Ser Gly Val Ser Val Ala Thr Ile Val Leu Ser Val Leu Gly Ala                 885 890 895 Leu Val Leu Val Cys Ile Ile Val Gly Thr Thr Ile Cys Ile Leu Lys             900 905 910 Asn Lys Arg Ser Ser Ser Ala Ala Ile Thr Lys Phe         915 920 <210> 2 <211> 2772 <212> DNA <213> Mouse <400> 2 atggctttct ccagagggcc tgttttcctc ctcctcctcc tgtacctgct ttgggggtca 60 gacacttcct tgataaggct gaatgagaat ggttatgaag acatcattat tgccattgat 120 cctgctgtgc cagaagatac aacaataatt gaacacataa agggaatggt gactaaagca 180 tctacatacc tttttgaagc tacagaaaaa agatttttct tcaaaaatgt atctatatta 240 attcctgaga gttggaaaga cagtccccag tacaggaggc caaaacagga gagttacaag 300 catgctgaca ttaaagtggc acctcccacc gttgagggca gagatgagcc ctacaccagg 360 caattcacac agtgtgaaga gaaagcagaa tacatccact tcacccctga ctttgtactg 420 gggagaaaac aagatgaata tggagattca ggaaaagtgt tggtccatga atgggcccac 480 cttcgctggg gagtatttga tgaatacaat gaagaccagc cattctacag cgcttcatca 540 aagaaaatcg aagcaacaag gtgctccaca ggtatcacgg ggacgaatag agtctatgca 600 tgccaagggg gcagctgtgc gatgagaagg tgcagaacta attccaccac aaagttatat 660 gaaaaggatt gtcaattctt ccctgataaa gttcaatcag aaaaggcttc cataatgttc 720 atgcaaagta tcgattctgt gactgaattc tgtaaaaagg aaaaccataa tcgagaggct 780 ccaactctac ataataaaaa gtgcaattac agaagcactt gggaagtaat cagcacttct 840 gaggatttta acagcagcac gcccatggag acgtcaccct ctccaccctt cttctccctg 900 ctgaggatca gtgagagaat tatgtgccta gttcttgatg tgtctgggag tatgacttct 960 tatgatcgcc tgaatcgaat gaaccaagca gcaaaatact ttctaagcca gataattgaa 1020 aataggtcct gggtagggat ggtgcacttt agtagccagg ccactattgt gcatgagctg 1080 atccaaataa acagtgacat cgagagaaac cagctcttgc aaaccttacc tacaagtgcc 1140 aatggaggga cgtccatctg ctctggaatc aaggctgcat ttcaggtatt taaaaatgga 1200 gaataccaaa cagatgggac tgagatcttg ctgttgagtg atggtgagga cagcacagcc 1260 aaagactgta ttgatgaggt gaaagacagc gggtccatag ttcatttcat tgccttggga 1320 ccatcggctg acttagctgt aacaaacatg agcattttaa caggtggaaa ccataagctt 1380 gctacagatg aagcccagaa caatggactc attgatgctt tcggggccct ggcttcagaa 1440 aatgctgata tcacccagaa gtcacttcag cttgaaagta agggtgccat actgaacaat 1500 agcctctggc tgaatgacac tgttgtaatt gacagcacct tgggaaggga cacattcttt 1560 cttgtcacct ggagcaaaca agcccctgcc atttatctca gggatcccaa aggaacacaa 1620 accaccaatt tcacaatgga ctctgcttcc aaaatggcct atctcagtat tccaggaaca 1680 gctcaggtgg gtgtttggac ttacaacctg gaagccaaag aaaactcaga gatactaacg 1740 attacagtaa cctctcgggc agcaaattct tctgtgccac caatcactgt aaatgctaag 1800 gtaaacacgg acactaacac tttccccagc ccaatgattg tgtatgctga agttctacaa 1860 ggttacactc ccatcattgg agcccgagtg acagccacca tagaatctaa cagtgggaag 1920 acagaagagc tagtccttct ggacaacggt gcaggggctg atgctttcaa ggatgatggg 1980 gtctactcca ggttcttcac agcttattca gtgaatggca ggtatagctt aaaagtccgt 2040 gcagatggag gaagaaattc tgcgaggagg agtttacgcc acccatcaag cagagctgca 2100 tacataccag gctgggtagt agatggagaa attcaaggga acccacccag acccgaaacg 2160 actgaggcta ctcagccagt cttggaggat ttcagcagga cagcatctgg aggtgccttt 2220 gtgatgtcca atgttccaat tggtccattg cctgatgtat acccaccaaa tcgaatcaca 2280 gacctccagg ccacactcga tggggaagag atcagtctaa catggacggc cccaggagat 2340 gattatgatg ttggaagagt tcaacagtat atcataagaa caagcaaaaa catcattgaa 2400 ctgagagaca attttaataa ctctcctcgg gttgatacaa ctaaccttac accaaaagaa 2460 gccaactccg aggaaacctt tgcctttaaa ccagaaaata tcacagaaga aaatgcaacg 2520 tacatattca ttgccattga aagtgtcgac aaaagcagtt tgagttcggg accatccaac 2580 atcgcacaag tggcactgtt cacccctcag gcagagcctg accctgatga aagtccaagt 2640 ttatcaggag ttagtgttgc tactattgtg ctgtctgttt tgggcgctct tgttctagtt 2700 tgtattattg taggtaccac aatttgtatc ttaaagaaca aaaggtcctc atcagcagct 2760 ataacaaaat tt 2772 <210> 3 <211> 2027 <212> DNA <213> Mouse <400> 3 gaatgatggc tttctccaga gggcctgttt tcctcctcct cctcctgtac ctgctttggg 60 ggtcagacac ttccttgata aggctgaatg agaatggtta tgaagacatc attattgcca 120 ttgatcctgc tgtgccagaa gatacaacaa taattgaaca cataaaggga atggtgacta 180 aagcatctac ataccttttt gaagctacag aaaaaagatt tttcttcaaa aatgtatcta 240 tattaattcc tgagagttgg aaagacagtc cccagtacag gaggccaaaa caggagagtt 300 acaagcatgc tgacattaaa gtggcacctc ccaccgttga gggcagagat gagccctaca 360 ccaggcaatt cacacagtgt gaagagaaag cagaatacat ccacttcacc cctgactttg 420 tactggggag aaaacaagat gaatatggag attcaggaaa agtgttggtc catgaatggg 480 cccaccttcg ctggggagta tttgatgaat acaatgaaga ccagccattc tacagcgctt 540 catcaaagaa aatcgaagca acaaggtgct ccacaggtat cacggggacg aatagagtct 600 atgcatgcca agggggcagc tgtgcgatga gaaggtgcag aactaattcc accacaaagt 660 tatatgaaaa ggattgtcaa ttcttccctg ataaagttca atcagaaaag gcttccataa 720 tgttcatgca aagtatcgat tctgtgactg aattctgtaa aaaggaaaac cataatcgag 780 aggctccaac tctacataat aaaaagtgca attacagaag cacttgggaa gtaatcagca 840 cttctgagga ttttaacagc agcacgccca tggagacgtc accctctcca cccttcttct 900 ccctgctgag gatcagtgag agaattatgt gcctagttct tgatgtgtct gggagtatga 960 cttcttatga tcgcctgaat cgaatgaacc aagcagcaaa atactttcta agccagataa 1020 ttgaaaatag gtcctgggta gggatggtgc actttagtag ccaggccact attgtgcatg 1080 agctgatcca aataaacagt gacatcgaga gaaaccagct cttgcaaacc ttacctacaa 1140 gtgccaatgg agggacgtcc atctgctctg gaatcaaggc tgcatttcag gtatttaaaa 1200 atggagaata ccaaacagat gggactgaga tcttgctgtt gagtgatggt gaggacagca 1260 cagccaaaga ctgtattgat gaggtgaaag acagcgggtc catagttcat ttcattgcct 1320 tgggaccatc ggctgactta gctgtaacaa acatgagcat tttaacaggt ggaaaccata 1380 agcttgctac agatgaagcc cagaacaatg gactcattga tgctttcggg gccctggctt 1440 cagaaaatgc tgatatcacc cagaagtcac ttcagcttga aagtaagggt gccatactga 1500 acaatagcct ctggctgaat gacactgttg taattgacag caccttggga agggacacat 1560 tctttcttgt cacctggagc aaacaagccc ctgccattta tctcagggat cccaaaggaa 1620 cacaaaccac caatttcaca atggactctg cttccaaaat ggcctatctc agtattccag 1680 gaacagctca ggtgggtgtt tggacttaca acctggaagc caaagaaaac tcagagatac 1740 taacgattac agtaacctct cgggcagcaa attcttctgt gccaccaatc actgtaaatg 1800 ctaaggtaaa cacggacact aacactttcc ccagcccaat gattgtgtat gctgaagttc 1860 tacaaggtta cactcccatc attggagccc gagtgacagc caccatagaa tctaacagtg 1920 ggaagacaga agagctagtc cttctggaca acggtgcagg ggctgatgct ttcaaggatg 1980 atggggtcta ctccaggttc ttcacagctt attcagtgaa tggcagg 2027 <210> 4 <211> 1869 <212> DNA <213> Mouse <400> 4 ctaagccaga taattgaaaa taggtcctgg gtagggatgg tgcactttag tagccaggcc 60 actattgtgc atgagctgat ccaaataaac agtgacatcg agagaaacca gctcttgcaa 120 accttaccta caagtgccaa tggagggacg tccatctgct ctggaatcaa ggctgcattt 180 caggtattta aaaatggaga ataccaaaca gatgggactg agatcttgct gttgagtgat 240 ggtgaggaca gcacagccaa agactgtatt gatgaggtga aagacagcgg gtccatagtt 300 catttcattg ccttgggacc atcggctgac ttagctgtaa caaacatgag cattttaaca 360 ggtggaaacc ataagcttgc tacagatgaa gcccagaaca atggactcat tgatgctttc 420 ggggccctgg cttcagaaaa tgctgatatc acccagaagt cacttcagct tgaaagtaag 480 ggtgccatac tgaacaatag cctctggctg aatgacactg ttgtaattga cagcaccttg 540 ggaagggaca cattctttct tgtcacctgg agcaaacaag cccctgccat ttatctcagg 600 gatcccaaag gaacacaaac caccaatttc acaatggact ctgcttccaa aatggcctat 660 ctcagtattc caggaacagc tcaggtgggt gtttggactt acaacctgga agccaaagaa 720 aactcagaga tactaacgat tacagtaacc tctcgggcag caaattcttc tgtgccacca 780 atcactgtaa atgctaaggt aaacacggac actaacactt tccccagccc aatgattgtg 840 tatgctgaag ttctacaagg ttacactccc atcattggag cccgagtgac agccaccata 900 gaatctaaca gtgggaagac agaagagcta gtccttctgg acaacggtgc aggggctgat 960 gctttcaagg atgatggggt ctactccagg ttcttcacag cttattcagt gaatggcagg 1020 tatagcttaa aagtccgtgc agatggagga agaaattctg cgaggaggag tttacgccac 1080 ccatcaagca gagctgcata cataccaggc tgggtagtag atggagaaat tcaagggaac 1140 ccacccagac ccgaaacgac tgaggctact cagccagtct tggaggattt cagcaggaca 1200 gcatctggag gtgcctttgt gatgtccaat gttccaattg gtccattgcc tgatgtatac 1260 ccaccaaatc gaatcacaga cctccaggcc acactcgatg gggaagagat cagtctaaca 1320 tggacggccc caggagatga ttatgatgtt ggaagagttc aacagtatat cataagaaca 1380 agcaaaaaca tcattgaact gagagacaat tttaataact ctcctcgggt tgatacaact 1440 aaccttacac caaaagaagc caactccgag gaaacctttg cctttaaacc agaaaatatc 1500 acagaagaaa atgcaacgta catattcatt gccattgaaa gtgtcgacaa aagcagtttg 1560 agttcgggac catccaacat cgcacaagtg gcactgttca cccctcaggc agagcctgac 1620 cctgatgaaa gtccaagttt atcaggagtt agtgttgcta ctattgtgct gtctgttttg 1680 ggcgctcttg ttctagtttg tattattgta ggtaccacaa tttgtatctt aaagaacaaa 1740 aggtcctcat cagcagctat aacaaaattt tgaaagataa catgaataaa aaagatattt 1800 ttgagttttt aaagcatatc ccatgtcatc atgaactcaa aagtaacttc aagagggtct 1860 aaaagatac 1869 <210> 5 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 5 gaatgatggc tttctccag 19 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 6 cctgccattc actgaataag 20 <210> 7 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 7 ctaagccaga taattgaaaa tag 23 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 8 gtatctttta gaccctcttg 20 <210> 9 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 9 ctattttcaa ttatctggct tag 23 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 10 cacagcttat tcagtgaatg 20 <210> 11 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 11 aagcccagaa caatggac 18 <210> 12 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 12 cgaatcacag acctccag 18 <210> 13 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 13 gaactaattc caccacaaag 20 <210> 14 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 14 agggtacact cctatcattg 20 <210> 15 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 15 aatacgactc actataggg 19 <210> 16 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 16 caggaaacag ctatgac 17 <210> 17 <211> 924 <212> PRT <213> Mouse <400> 17 Met Ala Phe Ser Arg Gly Pro Val Phe Leu Leu Phe Leu Leu Tyr Leu                  5 10 15 Leu Trp Gly Ser Asp Thr Ser Leu Ile Lys Leu Asn Gly Asn Gly Tyr              20 25 30 Glu Asp Ile Ile Ile Ala Ile Asp Pro Ala Val Pro Glu Asp Thr Thr          35 40 45 Ile Ile Glu His Ile Lys Gly Met Val Thr Lys Ala Ser Thr Tyr Leu      50 55 60 Phe Glu Ala Thr Glu Lys Arg Phe Phe Phe Lys Asn Val Ser Ile Leu  65 70 75 80 Ile Pro Glu Ser Trp Lys Asn Ser Ser Gln Tyr Arg Arg Pro Lys Gln                  85 90 95 Glu Ser Tyr Lys His Ala Asp Ile Lys Val Ala Pro Pro Ala Phe Glu             100 105 110 Gly Arg Asp Glu Pro Tyr Thr Arg Gln Phe Thr Gln Cys Glu Glu Lys         115 120 125 Ala Glu Tyr Ile His Phe Thr Pro Asp Phe Val Leu Gly Arg Lys Gln     130 135 140 Val Glu Tyr Gly Asp Ser Gly Lys Leu Leu Val His Glu Trp Ala His 145 150 155 160 Leu Arg Trp Gly Val Phe Asp Glu Tyr Asn Glu Asp Gln Pro Phe Tyr                 165 170 175 Ser Ala Ser Ser Lys Lys Ile Glu Ala Thr Arg Cys Ser Thr Gly Ile             180 185 190 Thr Gly Met Asn Arg Val His Thr Cys Gln Gly Gly Ser Cys Ile Thr         195 200 205 Arg Arg Cys Arg Thr Asn Ser Thr Thr Lys Leu Tyr Glu Lys Asp Cys     210 215 220 Gln Phe Phe Pro Asp Lys Val Gln Ser Glu Lys Ala Ser Ile Met Phe 225 230 235 240 Met Gln Ser Ile Asp Ser Val Thr Glu Phe Cys Lys Lys Glu Asn His                 245 250 255 Asn Arg Glu Ala Pro Thr Leu His Asn Glu Lys Cys Gln Trp Arg Ser             260 265 270 Thr Trp Glu Val Ile Ser Ser Ser Glu Asp Phe Asn Ser Ser Thr Pro         275 280 285 Met Glu Thr Pro Pro Ala Pro Pro Phe Phe Ser Leu Leu Arg Ile Ser     290 295 300 Glu Arg Ile Val Cys Leu Val Leu Asp Val Ser Gly Ser Met Ser Ser 305 310 315 320 Ser Asp Arg Leu Asn Arg Met Asn Gln Ala Ala Lys Tyr Phe Leu Ser                 325 330 335 Gln Ile Ile Glu Asn Arg Ser Trp Val Gly Met Val His Phe Ser Ser             340 345 350 Gln Ala Thr Ile Val His Glu Leu Ile Gln Met Asn Ser Asp Ile Glu         355 360 365 Arg Asn Lys Leu Leu Gln Thr Leu Pro Thr Ser Ala Ile Gly Gly Thr     370 375 380 Ser Ile Cys Ser Gly Ile Lys Thr Ala Phe Gln Val Phe Lys Asn Gly 385 390 395 400 Glu Tyr Gln Thr Asp Gly Thr Glu Ile Leu Leu Leu Ser Asp Gly Glu                 405 410 415 Asp Ser Thr Ala Lys Asp Cys Ile Asp Glu Val Lys Asp Ser Gly Ser             420 425 430 Ile Val His Phe Ile Ala Leu Gly Pro Ser Ala Asp Leu Ala Val Thr         435 440 445 Asn Met Ser Ile Leu Thr Gly Gly Asn His Lys Leu Ala Thr Asp Glu     450 455 460 Ala Gln Asn Asn Gly Leu Ile Asp Ala Phe Gly Ala Leu Ala Ser Glu 465 470 475 480 Asn Thr Asp Ile Thr Gln Lys Ser Leu Gln Leu Glu Ser Lys Gly Ala                 485 490 495 Ile Leu Asn Asn Ser Leu Trp Leu Asn Asp Thr Val Val Ile Asp Ser             500 505 510 Thr Val Gly Arg Asp Thr Phe Phe Leu Val Thr Trp Ser Lys Gln Ala         515 520 525 Pro Ala Ile Tyr Leu Arg Asp Pro Lys Gly Thr Gln Thr Thr Asn Phe     530 535 540 Thr Met Asp Phe Val Ser Lys Met Ala Tyr Leu Ser Ile Pro Gly Thr 545 550 555 560 Ala Glu Val Gly Val Trp Thr Tyr Asn Leu Glu Ala Lys Glu Asn Ser                 565 570 575 Glu Ile Leu Thr Ile Thr Val Thr Ser Arg Ala Ala Asn Ser Ser Val             580 585 590 Pro Pro Ile Ser Val Asn Ala Lys Val Asn Thr Asp Thr Asn Thr Phe         595 600 605 Pro Ser Pro Met Ile Val Tyr Ala Glu Val Leu Gln Gly Tyr Thr Pro     610 615 620 Ile Ile Gly Ala Arg Val Thr Ala Thr Ile Glu Ser Asn Ser Gly Lys 625 630 635 640 Thr Glu Glu Leu Val Leu Leu Asp Asn Gly Ala Gly Ala Asp Ala Phe                 645 650 655 Lys Asp Asp Gly Val Tyr Ser Arg Phe Phe Thr Ala Tyr Ser Val Asn             660 665 670 Gly Arg Tyr Ser Leu Lys Val Arg Ala Asp Gly Gly Thr Asn Ser Ala         675 680 685 Arg Arg Ser Leu Arg His Pro Ser Ser Arg Ala Ala Tyr Ile Pro Gly     690 695 700 Trp Val Val Asp Gly Glu Ile Gln Gly Asn Pro Pro Arg Pro Glu Met 705 710 715 720 Thr Glu Ala Thr Gln Pro Val Leu Glu Asn Phe Ser Arg Thr Ala Ser                 725 730 735 Gly Gly Ala Phe Val Met Ser Asn Val Pro Ile Gly Pro Leu Pro Asp             740 745 750 Gln Tyr Pro Pro Asn Arg Ile Thr Asp Leu Gln Ala Thr Leu Asp Gly         755 760 765 Glu Glu Ile Ser Leu Thr Trp Thr Ala Pro Gly Asp Asp Tyr Asp Val     770 775 780 Gly Arg Val Gln Gln Tyr Ile Ile Arg Thr Ser Glu Asn Ile Ile Asp 785 790 795 800 Leu Arg Asp Asn Phe Asn Asn Ser Leu Arg Val Asp Thr Thr Lys Leu                 805 810 815 Thr Pro Lys Glu Ala Asn Ser Lys Glu Thr Phe Ala Phe Lys Pro Glu             820 825 830 Asn Ile Ser Glu Glu Asn Ala Thr Tyr Ile Phe Ile Ala Ile Glu Ser         835 840 845 Val Asp Lys Asn Asn Leu Ser Ser Gly Pro Ser Asn Ile Ala Gln Val     850 855 860 Ala Met Phe Thr Pro Gln Ala Glu Pro Val Pro Asp Glu Ser Pro Arg 865 870 875 880 Ser Ser Gly Val Ser Ile Ser Thr Ile Val Leu Ser Val Val Gly Ser                 885 890 895 Val Val Leu Val Cys Ile Ile Val Ser Thr Thr Ile Cys Ile Leu Lys             900 905 910 Asn Lys Arg Ser Ser Ser Gly Ala Ala Thr Thr Phe         915 920 <210> 18 <211> 2772 <212> DNA <213> Mouse <400> 18 atggctttct ccagagggcc tgttttcctc ctcttcctct tgtacctgct ttgggggtca 60 gacacttcct tgataaagct gaatgggaat ggttatgaag acatcattat tgccattgat 120 cctgctgtgc cagaagatac aacaataatt gaacacataa agggaatggt gactaaagca 180 tctacatacc tttttgaagc tacagaaaaa agatttttct tcaaaaatgt gtctatatta 240 attcctgaga gttggaaaaa cagttcccag tacagaaggc caaaacagga gagttacaag 300 catgctgaca ttaaagtggc acctcccgcc ttcgagggca gagatgagcc ctacaccagg 360 cagttcacac agtgtgaaga gaaagcagaa tacattcact tcacccctga ctttgtactg 420 gggagaaaac aagttgaata tggagattca ggaaaactgt tggtccatga atgggcccac 480 cttcgctggg gagtatttga tgagtacaac gaagaccagc cattctacag tgcttcatca 540 aagaaaatcg aagcaacaag gtgctccaca ggtatcacgg gtatgaatag agtccataca 600 tgccaagggg gcagctgtat aaccagaagg tgcagaacta attccaccac aaagttatat 660 gaaaaggatt gtcagttctt cccagataaa gttcaatcag aaaaggcatc cataatgttc 720 atgcaaagta tcgattctgt gactgagttc tgtaaaaaag aaaaccataa tcgagaggct 780 ccaactctac ataatgaaaa gtgccaatgg agaagcactt gggaagtaat cagcagttct 840 gaggatttta acagcagcac gcccatggag acaccaccgg ctccaccctt cttctccctg 900 ctgaggatca gtgagagaat tgtgtgccta gttcttgatg tgtcagggag tatgagttct 960 tctgaccgtc tgaatagaat gaaccaagca gcaaaatact ttctaagcca gataattgaa 1020 aataggtcct gggtagggat ggtgcacttt agtagccagg ccactattgt gcatgagctg 1080 atccaaatga acagtgacat cgagagaaac aagctcttgc aaactttacc tacaagtgcc 1140 attggaggga cgtccatctg ctctggaatc aagactgcat ttcaggtatt taaaaatgga 1200 gaataccaaa cagatgggac tgagatcttg ctgttgagtg atggggagga cagcacagcc 1260 aaagactgta ttgacgaggt gaaagacagc gggtccatag ttcatttcat tgccttggga 1320 ccatcggctg acttagctgt aacaaacatg agcattttaa caggtggaaa ccataagctt 1380 gctacagatg aagcccagaa caatggactc attgatgctt tcggggccct ggcttcagaa 1440 aatactgata tcacccagaa gtcacttcag cttgaaagta agggtgccat actgaataat 1500 agcctctggc tgaatgacac tgttgtaatt gacagcaccg tgggaaggga cacattcttt 1560 cttgtcacct ggagcaaaca agcccctgcc atttatctca gggatcccaa aggaacacaa 1620 accaccaatt tcacaatgga ctttgtttcc aaaatggcct atctcagtat cccaggaaca 1680 gctgaggtgg gcgtttggac ttacaacctg gaagccaaag aaaactcaga gatactaaca 1740 attacagtaa cctctcgggc agcaaattct tctgtgccac caatctctgt aaatgctaag 1800 gtaaacacgg acactaacac tttccccagc ccaatgattg tgtatgctga agttctacaa 1860 gggtacactc ctatcattgg agcccgagtg acagccacca tagaatctaa cagtgggaag 1920 acagaagagc tagtcctgct ggacaacggt gcaggggctg atgctttcaa ggatgatggg 1980 gtctactcca ggttcttcac agcttattca gtgaatggca ggtatagctt aaaagtccgt 2040 gcagatggag gaacaaactc tgccaggagg agtttacgcc acccatcaag cagagctgca 2100 tacataccag gctgggtagt agatggagaa attcaaggga acccacccag acccgaaatg 2160 actgaggcta ctcagccagt cctagagaat ttcagcagga cagcatctgg aggtgccttt 2220 gtgatgtcca atgttccaat tggtccattg cctgatcagt acccaccaaa tcgaatcaca 2280 gacctccagg ccacactcga tggggaagag atcagtctaa catggacggc cccaggagat 2340 gattatgatg ttggaagagt tcaacagtat atcataagaa caagcgaaaa tatcattgat 2400 ctcagagaca attttaataa ttctcttcgg gttgatacta ctaaacttac accaaaagaa 2460 gccaactcca aagaaacctt tgcctttaaa ccagaaaata tctcagaaga aaatgcaacc 2520 tacatattca ttgccattga aagtgtcgac aaaaacaatt tgagttcggg accatccaac 2580 atcgcacaag tggcaatgtt cacccctcag gcagagcctg tccctgatga aagtccacgt 2640 tcatcaggag ttagtatttc tactattgtg ctgtctgtgg tgggctctgt tgtactagtt 2700 tgtattattg taagtaccac tatttgtatc ttaaagaaca aaaggtcctc atcaggagct 2760 gcaacaacat tt 2772 <210> 19 <211> 373 <212> PRT <213> Rat <400> 19 Ile Glu Ala Thr Arg Cys Ser Thr Gly Ile Lys Gly Met Asn Lys Ala                  5 10 15 Gln Val Cys Gln Gly Gly Ser Cys Ile Thr Arg Asn Cys Arg Arg Asn              20 25 30 Ser Thr Thr Gln Leu Tyr Glu Lys Asp Cys Gln Phe Phe Pro Asp Lys          35 40 45 Val Gln Thr Glu Lys Ser Ser Ile Met Phe Met Gln Ser Ile Asp Ser      50 55 60 Val Thr Glu Phe Cys Lys Lys Glu Asn His Asn Arg Glu Ala Pro Thr  65 70 75 80 Leu His Asn Gln Lys Cys Asp Tyr Arg Ser Thr Trp Glu Val Ile Ser                  85 90 95 Asn Ser Glu Asp Phe Lys Asn Ser Thr Pro Met Glu Met Pro Pro Ser             100 105 110 Pro Pro Phe Phe Ser Leu Leu Arg Ile Ser Glu Arg Ile Val Cys Leu         115 120 125 Val Leu Asp Val Ser Gly Ser Met Gly Ser Tyr Asp Arg Leu Asn Arg     130 135 140 Met Asn Gln Ala Ala Lys Phe Phe Leu Gln Gln Ile Leu Glu Ser Arg 145 150 155 160 Ser Trp Ala Gly Met Val His Phe His Ser Ser Ala Thr Val Lys Ser                 165 170 175 Glu Leu Ile Gln Ile Asn Ser Asp Val Glu Arg Asn Gln Leu Leu Glu             180 185 190 Thr Leu Pro Thr Ser Ala Ser Gly Gly Thr Ser Ile Cys Ser Gly Ile         195 200 205 Arg Thr Ala Phe Gln Val Phe Lys Asn Lys Gly Tyr Gln Thr Gly Gly     210 215 220 Asn Asp Ile Leu Leu Leu Ser Asp Gly Glu Asp Ser Thr Ala Lys Asp 225 230 235 240 Cys Leu Asp Glu Val Lys Asp Ser Gly Ala Val Val His Phe Ile Ala                 245 250 255 Leu Gly Lys Ala Phe Asp Gln Ser Ile Ser Asn Met Ala Asn Val Thr             260 265 270 Gly Gly Lys Gln Leu Phe Ala Thr Asp Glu Ala Gln Asn Asn Gly Leu         275 280 285 Ile Asp Ala Phe Gly Ala Leu Ala Ser Glu Asn Ala Asp Val Thr Glu     290 295 300 Lys Ser Leu Gln Leu Glu Ser Lys Gly Ala Val Leu Thr Asn Ser Arg 305 310 315 320 Trp Leu Asn Asp Thr Val Val Ile Asp Ser Thr Val Gly Lys Asp Thr                 325 330 335 Tyr Phe Leu Val Thr Trp Ser Gln Gln Ala Pro Ala Ile His Leu Arg             340 345 350 Asp Pro Lys Gly Thr Gln Ile Thr Asn Phe Thr Ile Asp Thr Ala Ser         355 360 365 Lys Met Ala Tyr Leu     370 <210> 20 <211> 1119 <212> DNA <213> Rat <400> 20 atcgaagcaa caaggtgttc cacaggtatc aagggtatga ataaagccca ggtatgccaa 60 gggggcagct gtataaccag aaattgcaga cggaattcca ccacacagtt atatgaaaaa 120 gattgtcagt tcttcccgga taaagttcaa acagaaaagt catctataat gttcatgcaa 180 agtatcgatt cagtgactga attctgtaaa aaagaaaacc ataatcgaga ggctccaact 240 ctacataatc agaagtgcga ttacagaagc acatgggaag taatcagtaa ttctgaggat 300 tttaaaaaca gcacacccat ggagatgcca ccctctccac ccttcttctc gctgctgagg 360 atcagtgaaa gaattgtgtg cctagtgctt gatgtgtctg ggagcatggg gtcttatgat 420 cgcctcaatc gaatgaacca agcagcaaaa ttcttcctac agcagatact tgaaagtagg 480 tcctgggcag ggatggtaca ctttcatagc tcggccactg ttaagagtga gctgatccaa 540 ataaacagtg atgtcgagag aaaccagctt ttggaaacct tacctacaag tgctagcgga 600 gggacatcca tctgctctgg aatcagaact gcatttcagg tatttaaaaa taaaggatac 660 caaactggtg ggaatgacat cttgctgttg agcgatgggg aggacagcac agccaaagac 720 tgccttgatg aggtgaaaga cagcggggcc gtagttcatt tcattgcctt gggaaaagca 780 tttgatcaat ctatatcaaa catggccaac gtaacaggtg gaaagcaact gtttgctaca 840 gatgaagccc agaacaatgg actcattgat gctttcgggg ccctggcttc agaaaatgct 900 gatgtcactg agaagtccct tcagcttgaa agcaagggtg ctgtgttgac caacagtcgc 960 tggctgaacg acactgtcgt tattgacagc accgtgggaa aggacacata ctttcttgtc 1020 acctggagcc aacaagcccc tgccattcat ctcagggatc ccaaaggaac acaaataaca 1080 aatttcacaa tcgacacggc ttccaaaatg gcctatctc 1119 <210> 21 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 21 tgcctttcaa aatgttgttg 20 <210> 22 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 22 aaaatcgaag caacaaggtg 20 <210> 23 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 23 gagataggcc attttggaag 20 <210> 24 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 24 aagcccagaa caatggac 18

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) A61P 1/00 A61P 1/04 4C084 1/04 11/00 4C085 11/00 11/02 4H045 11/02 11 / 06 11/06 C07K 14/47 C07K 14/47 16/18 16/18 C12N 1/15 C12N 1/15 1/19 1/19 1/21 1/21 C12P 21/02 C 5/10 C12Q 1 / 68 A C12P 21/02 G01N 33/15 Z C12Q 1/68 33/50 Z G01N 33/15 C12N 15/00 ZNAA 33/50 5/00 A F term (reference) 2G045 AA34 AA35 CB01 DA13 DA36 FB02 FB05 FB12 GC15 4B024 AA01 AA11 BA58 BA80 CA04 CA12 DA01 DA02 DA05 DA06 DA11 EA04 GA01 GA11 HA08 HA11 4B063 QA01 QA18 QQ08 QQ42 QQ53 QQ61 QR08 QR32 QR42 QR56 QR62 QS28 QS32 QS34 4B064 AG01 AG26 CA02 CA05 A01 A01 CA20 CA20 CA01 CA20 CA11 CA11 CA11 CA11 CA11 CA11 CA11 CA11 CA11 CA11 CA11 CA11 CA11 CA11 CA11 CA11 CA11 CA11 CA11 CA11 CA11 CA11 CA11 CA11 CA11 CA11 CA11 CA11 CA11 CA11 CA11 CA11 CA11 CA11 CA11 CA11 CA11 CA11 CA11 CA11 AB01 AB02 CA24 CA44 4C084 AA07 AA13 AA17 NA14 ZA34 ZA59 ZA66 ZB11 4C085 AA13 AA14 BB11 EE01 GG01 GG08 4H045 AA10 AA11 AA20 AA30 BA09 CA40 DA75 EA20 EA50 FA74

Claims (39)

[Claims] 1. A protein containing an amino acid sequence which is the same or substantially the same as the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 17, or a salt thereof. 2. A protein comprising the amino acid sequence represented by SEQ ID NO: 1 or a salt thereof. 3. A protein comprising the amino acid sequence represented by SEQ ID NO: 17 or a salt thereof. 4. A protein containing an amino acid sequence which is the same as or substantially the same as the amino acid sequence represented by SEQ ID NO: 19, or a salt thereof. 5. A protein containing the amino acid sequence represented by SEQ ID NO: 19 or a salt thereof. 6. A partial peptide of the protein according to claim 1 or a salt thereof. 7. A partial peptide of the protein according to claim 4 or a salt thereof. 8. A polynucleotide containing a polynucleotide encoding the protein according to 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 method according to claim 8 or claim 9 which is DNA.
The described polynucleotide. 11. A polynucleotide comprising the nucleotide sequence represented by SEQ ID NO: 2 or SEQ ID NO: 18. 12. The polynucleotide according to claim 10, which contains the base sequence represented by SEQ ID NO: 20. 13. A recombinant vector 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, the protein according to claim 1 or 4 or a partial peptide thereof or a salt thereof is produced and accumulated, and the collected product is collected. A method for producing the protein according to claim 1 or 4, a partial peptide thereof, or a salt thereof. 16. A medicine comprising the protein according to claim 1 or 4, a partial peptide thereof or a salt thereof. 17. An antibody against the protein according to claim 1 or 4, a partial peptide thereof, or a salt thereof. 18. A medicine comprising the antibody according to claim 17. 19. A diagnostic agent comprising the antibody according to claim 17. 20. An antisense polynucleotide containing a base sequence complementary or substantially complementary to the base sequence of the polynucleotide according to claim 8 or claim 9, or a part thereof. 21. A medicine comprising the antisense polynucleotide according to claim 20. 22. The protein according to claim 1 or 4, or a partial peptide thereof or a salt thereof is used, and the protein according to claim 1 or 4 or a partial peptide thereof or a salt thereof is used. A method for screening a compound or its salt that inhibits the activity. 23. The protein according to claim 1 or 4, or the partial peptide thereof, or a salt thereof, which comprises the protein according to claim 1 or 4 or a partial peptide thereof or a salt thereof. For screening a compound or a salt thereof that inhibits the activity of erythrocyte. 24. Inhibiting the activity of the protein according to claim 1 or 4, or a partial peptide thereof, or a salt thereof, which can be obtained by using the screening method according to claim 22 or the screening kit according to claim 23. Or a salt thereof. 25. A medicine comprising the compound according to claim 24 or a salt thereof. 26. A method for screening a compound or a salt thereof, which inhibits the expression of the protein gene according to claim 1 or 4, characterized in that the polynucleotide according to claim 8 or 9 is used. 27. A kit for screening a compound or a salt thereof which inhibits the expression of the protein gene according to claim 1 or 4, which comprises the polynucleotide according to claim 8 or 9. 28. A compound which inhibits the expression of the protein gene according to claim 1 or claim 4 or a salt thereof, which can be obtained by using the screening method according to claim 26 or the screening kit according to claim 27. 29. A medicine comprising the compound according to claim 28 or a salt thereof. 30. A method for screening a compound or a salt thereof which inhibits the expression of the protein according to claim 1 or 4, characterized in that the antibody according to claim 17 is used. 31. A kit for screening a compound or a salt thereof which inhibits the expression of the protein according to claim 1 or 4, which comprises the antibody according to claim 17. 32. A compound which inhibits the expression of the protein of claim 1 or 4, or a salt thereof, which can be obtained using the screening method of claim 30 or the screening kit of claim 31. 33. A medicine comprising the compound according to claim 32 or a salt thereof. 34. A prophylactic / therapeutic agent for respiratory diseases, rhinitis or digestive diseases, 16, 18, 21, 25, 2
The medicament according to 9 or 33. 35. The medicament according to claim 34, which is a prophylactic / therapeutic agent for chronic obstructive pulmonary disease, bronchial asthma, inflammatory bowel disease or reflux esophagitis. 36. The diagnostic agent according to claim 19, which is a diagnostic agent for respiratory diseases, rhinitis or digestive diseases. 37. A diagnostic agent for chronic obstructive pulmonary disease, bronchial asthma, inflammatory bowel disease or reflux esophagitis.
Described diagnostic agent. 38. A chronic obstructive pulmonary disease, bronchial asthma, or inflammatory condition, which comprises administering an effective amount of the compound according to claim 24, 28 or 32 or a salt thereof to a mammal. A method for preventing or treating intestinal disease or reflux esophagitis. 39. The compound according to claim 24, 28 or 32 for producing a prophylactic / therapeutic agent for chronic obstructive pulmonary disease, bronchial asthma, inflammatory bowel disease or reflux esophagitis, or a salt thereof. Use of.