WO2004002515A1

WO2004002515A1 - Diagnostics/preventives/remedies for respiratory diseases

Info

Publication number: WO2004002515A1
Application number: PCT/JP2003/008167
Authority: WO
Inventors: Atsushi Nakanishi; Hiroki Iwashita; Shigeru Morita; Tatsumi Matsumoto; Masashi Yamasaki
Original assignee: Takeda Chemical Industries, Ltd.
Priority date: 2002-06-28
Filing date: 2003-06-27
Publication date: 2004-01-08
Also published as: AU2003246085A1

Abstract

It is intended to provide diagnostics/preventives/remedies, etc. for respiratory diseases. Namely, a compound or its salt promoting the activity of a protein having an amino acid sequence which is the same or substantially the same as the amino acid sequence represented by SEQ ID NO:1, a compound or its salt promoting the expression of a gene of the above protein, an antisense nucleotide having a base sequence or a part thereof which is complementary or substantially complementary to the base sequence of a DNA encoding the above protein or its peptide fragment, an antibody against the above protein or its peptide fragment, etc. are usable as diagnostics/preventives/remedies, etc. for respiratory diseases.

Description

Specification

Diagnosis and prevention of respiratory diseases

The present invention relates to an agent for preventing or treating respiratory diseases, a diagnostic agent, and the like. Background art

Chronic obstructive pulmonary disease, chronic bronchitis, emphysema, diffuse panbronchiolitis, endogenous asthma, etc. It is thought to be.

It has been shown that smoking can be a definite etiology of chronic obstructive pulmonary disease. Smoking causes obstructive disorders and depends on the number of cigarettes. The younger the age at which smoking starts, the easier it is to progress. In addition, a dose correlation between smoking and bronchial gland hyperplasia has been confirmed. In animal studies, there are many reports that inhalation of tobacco can cause emphysema.

Pathological changes in chronic obstructive pulmonary disease (hereinafter sometimes abbreviated as C0PD) include specific abnormalities in the central airway, peripheral airway, and lung parenchyma. Central airway involvement involves changes in secretory tissue morphology, such as goblet cell hyperplasia, hyperplasia of cells in the submucosal gland, and hypertrophy. For inflammatory cells, an increase in macrophage-activated T lymphocytes has been shown in the airway mucosa. Lesions in the bronchiole region include mucus embolism in the airway lumen, goblet cell dysplasia in the airway epithelium, inflammatory cell infiltration in the airway wall, smooth muscle hypertrophy, and fibrosis. Either change results in airway narrowing. In the alveolar parenchyma, emphysema lesions defined by destruction and disappearance of the alveoli and enlargement of the air space are observed. These are thought to involve an imbalance in the protease Z antiprotease balance.

On the other hand, a microarray method in which cDNA or oligonucleotides are immobilized has been developed to comprehensively analyze gene expression, and the technology for finding disease-specific changes in gene expression has become widespread and its usefulness has been confirmed. . For example, Aifynietrix's GeneCMp system is increasingly being used to diagnose diseases such as cancer and to discover drug discovery target genes. I 2003/008167

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However, there have been no reports using this technique to find genes involved in the development or progression of emphysema.

Procollagen C-protease enhancer protein 2, PC0LCE2 has been cloned as one of the isoforms of PC0LCE1 (Genomics, 66, 264, 2000). Year) . It has been reported that PC0LCE1 has a high homology at the C-terminus to the N-terminus of tissue inhibitor of metaro protease (TIMP) (Protein Sci 8, 1636, In 1999, it has been reported that this C-terminal portion is actually cleaved after translation and acts as a meta-oral protease inhibitor (L Biol. Diem.) 275 , 1384, 2000). Disclosure of the invention

There is a need for the development of prophylactic and therapeutic agents and diagnostic agents for superior respiratory diseases with few side effects (eg, chronic obstructive pulmonary disease, etc.).

The present inventors have conducted intensive studies in order to solve the above-mentioned problems, and as a result, have found a gene whose expression is remarkably reduced in lung tissue having emphysema lesions, and based on this finding, conducted further studies based on this finding As a result, the present invention has been completed.

That is, the present invention

(1) contains a compound or a salt thereof which promotes the activity of a protein or a partial peptide thereof or a salt thereof containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1 Prevention and treatment of respiratory diseases

(2) A compound or a salt thereof that promotes the expression of a protein or a partial peptide or a salt thereof containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1 Prevention and treatment of respiratory diseases comprising

(3) Complementary or substantially complementary to the nucleotide sequence of a polynucleotide encoding a protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1 or a partial peptide thereof Base sequence or part of it Antisense polynucleotide containing,

(4) a medicine comprising the antisense polynucleotide according to the above (3),

(5) an antibody against a protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1 or a partial peptide thereof or a salt thereof;

(6) a medicament comprising the antibody according to the above (5),

(7) The medicament according to the above (6), which is an agent for preventing or treating respiratory diseases.

(8) a diagnostic agent comprising the antibody according to (5) above,

(9) The diagnostic agent according to the above (8), which is a diagnostic agent for respiratory disease,

(10) a diagnostic agent for a respiratory disease comprising a polynucleotide encoding a protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1 or a partial peptide thereof,

(11) A prophylactic / therapeutic agent for chronic obstructive pulmonary disease or bronchial asthma, comprising a compound having a procollagen C-terminal truncation promoting activity or a salt thereof, '(12) SEQ ID NO: 1 A method for screening a remedy for the prevention and treatment of respiratory diseases, which comprises using a protein having the same or substantially the same amino acid sequence as the amino acid sequence to be obtained or a partial peptide thereof or a salt thereof;

(12a) A screen for a pharmaceutical compound characterized by using a protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1 or a partial peptide thereof or a salt thereof. Eng method,

(12b) The pharmaceutical compound is a compound for preventing or treating respiratory disease, a compound used for preventing or treating respiratory disease and / or a compound having a preventive or treating effect for respiratory disease. (12a) The screening method according to the above,

(13) The screening method according to the above (12), wherein the tobacco smoke-exposed chronic obstructive pulmonary disease model mouse or the elastase-induced chronic obstructive pulmonary disease model mouse is used,

(14) Prevention of a respiratory disease characterized by containing a protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1 or a partial peptide thereof or a salt thereof · Screening kits for therapeutic agents, (15) A prophylactic / therapeutic agent for a respiratory disease obtainable by using the screening method according to (12) or the screening kit according to (14).

(15a) a pharmaceutical compound obtainable by using the screening method described in (12a) above,

(15b) The pharmaceutical compound is a compound for preventing or treating respiratory diseases, a compound used for preventing or treating respiratory diseases and / or a compound having a preventive or treating effect for respiratory diseases. The compound or a salt thereof according to the above (15a),

(15c) a prophylactic / therapeutic agent for respiratory diseases comprising the compound or salt thereof according to (15b) above,

(16) The prophylactic or therapeutic agent according to (15) above, wherein the respiratory disease is emphysema.

(17) A respiratory tract, characterized by using a polynucleotide encoding a protein or a partial peptide thereof containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1. Disease prevention and treatment screening methods,

(17a) A script of a pharmaceutical compound characterized by using a polynucleotide encoding a protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1 or a partial peptide thereof. One way,.

(17b) The pharmaceutical compound is a compound for preventing or treating respiratory disease, a compound used for preventing or treating respiratory disease and / or a compound having a preventive or treating effect for respiratory disease. (17a) the screening method described above,

(18) The screening method according to the above (17), wherein the tobacco smoke-exposed chronic obstructive pulmonary disease model mouse or elastase-induced chronic obstructive pulmonary disease model mouse is used,

(19) a respiratory disease characterized by containing a protein having an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1 or a polynucleotide encoding a partial peptide thereof Kits for screening of preventive and therapeutic agents

(20) A prophylactic or therapeutic agent for respiratory disease, which can be obtained by using the screening method according to (17) or the screening kit according to (19). (20a) a pharmaceutical compound obtainable by using the screening method according to (17a),

(2 Ob) The pharmaceutical compound is a compound for preventing or treating respiratory disease, a compound used for preventing or treating respiratory disease and / or a compound having a preventive or treating effect for respiratory disease. (20a) the compound or a salt thereof,

(20c) a prophylactic / therapeutic agent for respiratory diseases comprising the compound or salt thereof according to (20b) above,

(21) The prophylactic or therapeutic agent according to (20), wherein the respiratory disease is emphysema.

(22) a compound having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1 or a compound that promotes the activity of a partial peptide or a salt thereof in mammals, or a compound thereof; A method for preventing or treating respiratory diseases, which comprises administering an effective amount of a salt, or a compound or a salt thereof that promotes the expression of a gene of the protein,

(23) Promotes the activity of a protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1 or a partial peptide thereof or a salt thereof, or expresses a gene of the protein Prevention and treatment of respiratory diseases characterized by promoting

(24) A protein or a partial peptide thereof containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1 for producing a prophylactic or therapeutic agent for respiratory disease. It is intended to provide a compound or a salt thereof which promotes the activity of the salt, or a compound or a salt thereof which promotes the expression of a gene of the protein. BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a diagram showing a pressure-volume curve of a mouse extirpated lung obtained in Example 1. In the figure,

In (a), one group was exposed to cigarette smoke for one month (n = 8), one group was in control group (n = 7), and (b) one group was for three months. Group of mice exposed to cigarette smoke (n = l0), 1101 the control group (n = 9), (c) —Hinichi a 6 month cigarette The group of mice exposed to smoke (n = 10) and the group of 〇 indicate the control group (n = 9). ** indicates p≤0.01, and * indicates ρ≤0.05.

FIG. 2 is a diagram showing the results of the expression level of PC0LCE2 gene obtained in Example 2. In the figure, Α on the horizontal axis represents the lungs of a mouse exposed to cigarette smoke for 1 month, B represents the lungs of the control mouse, C represents the lungs of a mouse exposed to cigarette smoke for 3 months, D represents the lungs of the control mouse, and E represents the lungs of 6 months. The tobacco smoke-exposed mouse lung, F indicates the control mouse lung.

FIG. 3 is a diagram showing the results of mouse PCOLCE2 gene expression distribution obtained in Example 3.

FIG. 4 is a diagram showing a pressure-volume curve of a mouse extirpated lung obtained in Example 4. In the figure, (a) one sleep group was a group of mice (n = 6) one day after elastase administration, one bite was a control group (n = 6), and (b) one garden one was elastase administration. Model mouse group (n = 5) after 1 day, control group (n = 6), control group (n = 6), model mouse group (n = 6) 21 days after elastase administration, control Groups (n = 6) are shown. * Indicates p≤0.05, ** indicates p≤0.01.

FIG. 5 is a graph showing the time-dependent change in the compliance value of the mouse extirpated lung after administration of Erasinase obtained in Example 4. In the figure, the horizontal axis shows the number of days after elastase administration, and the vertical axis shows the compliance value.麵 indicates the elastase-administered mouse group (n = 6), and 一 indicates the control group (n = 6). * Indicates p≤0.05 and indicates p≤0.01.

FIG. 6 is a graph showing the time course of the expression level of PC PLCE2 gene in mouse lung tissue obtained after administration of elastase obtained in Example 4. In the figure, the horizontal axis shows the number of days after Erasase administration, and the vertical axis shows the amount of PCOLCE2 gene expression. — 騸 — indicates the mouse group (n = 6) to which Erasase was administered, and each bit indicates the control group (n = 6). * Indicates p≤0.05. BEST MODE FOR CARRYING OUT THE INVENTION

A protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1 used in the present invention (hereinafter referred to as the protein of the present invention or 2003/008167

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The protein used in the present invention may be referred to as a human warm-blooded animal (eg, guinea pig, rat, mouse, chicken, egret, pig, sheep, hidge, horse, monkey, etc.) cells (eg, hepatocyte, spleen, etc.). Cells, neurons, glial cells, splenic jS cells, bone marrow cells, mesangial cells, Langerhans cells, epidermal cells, epithelial cells, goblet cells, endothelial cells, smooth muscle cells, fibroblasts, fiber cells, muscle cells, adipocytes , Immune cells (eg, macrophages, T cells, B cells, natural killer cells, mast cells, neutrophils, basophils, eosinophils, monocytes), megakaryocytes, synovial cells, chondrocytes , Osteocytes, osteoblasts, osteoclasts, mammary cells, hepatocytes or stromal cells, or precursors, stem cells or cancer cells of these cells) or their cells Any tissue that does, such as the brain, parts of the brain (eg, olfactory bulb, amygdala, basal sphere, hippocampus, thalamus, hypothalamus, cerebral cortex, medulla, cerebellum), spinal cord, pituitary, stomach, knee, kidney , Liver, gonad, thyroid, gall bladder, bone marrow, adrenal gland, skin, muscle, lung, gastrointestinal tract (eg, large intestine, small intestine), blood vessels, heart, thymus, spleen, submandibular gland, peripheral blood, prostate, testicle, ovary, It may be a protein derived from placenta, uterus, bone, joint, skeletal muscle, or the like, or may be a synthetic protein.

As the amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 1, about 50% or more, preferably about 60% or more, more preferably about 50% or more of the amino acid sequence represented by SEQ ID NO: 1 Amino acid sequences having a homology of 70% or more, more preferably about 80% or more, particularly preferably about 90% or more, and most preferably about 95% or more.

The homology of amino acid sequences was determined using the homology calculation algorithm NCBI BLAST (National Center for Biotechnology Information Basic Local Search Tool). The following conditions (expected value = 10; gap allowed; matrix = BLOSUM62; Filtering = 0FF).

Examples of the protein having an amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 1 include, for example, a protein having the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1. A protein having substantially the same activity as the protein containing the amino acid sequence represented by SEQ ID NO: 1 is preferred. SEQ ID NO: protein containing an amino acid sequence substantially identical to the amino acid sequence represented by 1 Examples of the quality include a protein containing the amino acid sequence represented by SEQ ID NO: 3.

Substantially equivalent activities include, for example, protease inhibitory activity, procollagen

C-terminal cleavage promoting activity and the like. Substantially identical indicates that the properties are qualitatively (eg, physiologically or pharmacologically) identical. Therefore, the protease inhibitory activity, procollagen C-terminal cleavage promoting activity and the like are equivalent (eg, about 0.01 to 100 times, preferably about 0.1 to 10 times, more preferably 0.5 to 10 times). However, the quantitative factors such as the degree of these activities and the molecular weight of the protein may be different.

The measurement of proteases inhibitory activity and procollagen C-terminal cleavage promoting activity is performed by a method known per se, for example, J. Biol. Chem. 275 (2), 1384-90, 2000, Eur. J. Biochem. 186 Vol., U5-121, 1989, etc. or a method analogous thereto.

Specifically, the protease inhibitory activity is measured by reacting the protein of the present invention, a labeled (eg, fluorescent label) peptide substrate and a protease, and measuring the mass of the peptide group cleaved by protease activity. This reaction is performed in an appropriate buffer. 'When the labeling agent is, for example, a fluorescent substance, the peptide group mass may be measured by measuring the fluorescence intensity. The measurement of the fluorescence intensity may be performed according to a known method using a fluorescence measurement device or the like. The measurement of procollagen C-terminal cleavage promoting activity is carried out by reacting the protein of the present invention, labeled (eg, radiolabeled) procollagen, and procollagen C-terminal region cleavage proteinase (eg, BMP-1). Then, the mass of the C-terminal protein of the open collagen cleaved by the protease activity is measured. This reaction is performed in an appropriate buffer. When the labeling agent is, for example, a radioisotope, the amount of the C-terminal protein may be measured according to a known method using high performance liquid chromatography.

Examples of the protein used in the present invention include: (1) 1 or 2 or more (for example, about 1 to 100, preferably 1 to 100) in the amino acid sequence represented by SEQ ID NO: 1; An amino acid sequence in which about 30 amino acids have been deleted, preferably about 1 to 10 amino acids, and more preferably about 1 to 5 amino acids, and 2) the amino acid sequence represented by SEQ ID NO: 1. One or two or more amino acids (for example, about 1 to 100, preferably about 1 to 30, preferably about 1 to 10, and more preferably about 1 to 5) The amino acid sequence represented by SEQ ID NO: 1 or 2 or more (for example, about 1 to 100, preferably about 1 to 30, preferably about 1 to 10, More preferably, an amino acid sequence in which a number (1 to 5) of amino acids are inserted. ④ 1 or 2 or more (eg, about 1 to 100) in the amino acid sequence represented by SEQ ID NO: 1 An amino acid sequence in which about 1 to 30, preferably about 1 to 10, and more preferably a number (1 to 5) of amino acids have been substituted with another amino acid, or an amino acid obtained by combining them. So-called mutin, such as a protein containing a sequence, represented by (2) SEQ ID NO: 3 1 or 2 or more in the amino acid sequence (eg, about 1 to 100, preferably about 1 to 30, preferably about 1 to 10, more preferably number (1 to 5)) The amino acid sequence represented by SEQ ID NO: 3; 1 or 2 or more amino acids (eg, about 1 to 100, preferably about 1 to 30, preferably 1 to 1 An amino acid sequence to which about 0, more preferably a number (1 to 5) amino acids have been added; ③ one or more amino acid sequences (for example, 1 to 100) in the amino acid sequence represented by SEQ ID NO: 3 Amino acid sequence into which about, preferably about 1 to 30 amino acids, preferably about 1 to 10 amino acids, and more preferably about 1 to 5 amino acids have been inserted, represented by SEQ ID NO: 3. 1 or 2 or more in the amino acid sequence (for example, about 1 to 100, preferably about 1 to 30, and preferably 1 to So-called muteins such as proteins containing an amino acid sequence in which about 10 amino acids are substituted, and more preferably a number (1 to 5) amino acids, or an amino acid sequence combining them are also included. included.

When the amino acid sequence is inserted, deleted or substituted as described above, the position of the insertion, deletion or substitution is not particularly limited.

In the protein in the present specification, the left end is the N-terminus (amino terminus) and the right end is the C-terminus (capilloxy terminus) according to the convention of peptide labeling. The proteins used in the present invention, including the protein containing the amino acid sequence represented by SEQ ID NO: 1, include carboxyl group (-C00H), carboxylate (-C00 "), amide (- ₂ ) Or an ester (-C00R).

Here, as R in the ester, e.g., methyl, Echiru, n- propyl, isopropyl, alkyl groups such as n- butyl, for example, C ₃ _ ₈ cycloalkyl group such as cyclohexyl cyclopentyl, to sik port, for example, phenyl, one naphth> C, such as les ₆ - ₁₂ Ariru group, e.g., benzyl, C ₇ one i ₄ Ararukiru groups such as α- Nafuchiru C Bok ₂ alkyl group such as phenylene Lou alkyl or a- naphthylmethyl such phenethyl, A bivaloyloxymethyl group or the like is used.

When the protein used in the present invention has a lipoxyl group (or carboxylate) other than the C-terminus, a protein in which the carboxyl group is amidated or esterified is also included in the protein used in the present invention. In this case, as the ester J, for example, the above-mentioned C-terminal ester or the like is used.

Further, in the protein used in the present invention, the amino group at the N-terminal amino acid residue (eg, methionine residue) is protected by a protecting group (eg, a CM acyl group such as an alkanol such as a formyl group or an acetyl group). Protected, N-terminal glutamine residue generated by cleavage in vivo, pyroglutamine oxidized, Substituent on the side chain of amino acid in the molecule (for example, -0H, -SH, amino group , Imidazole, indole, guanidino, etc.) are protected with an appropriate protecting group (for example, CHW alkyl such as _CW alkanoyl such as formyl or acetyl), or a sugar chain is bonded. Complex proteins such as so-called glycoproteins are also included.

Specific examples of the protein used in 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: 3, and the like.

The partial peptide of the protein used in the present invention is the partial peptide of the protein used in the present invention described above, and is preferably any peptide having the same properties as the protein used in the present invention described above. It may be.

Specifically, for the purpose of preparing the antibody of the present invention described below, a peptide having the amino acid sequence of the 381st to 391st amino acids in the amino acid sequence represented by SEQ ID NO: 1; The amino acid sequence at positions 380-390 in the amino acid sequence shown Examples include peptides having a sequence. For example, at least 20 or more, preferably 50 or more, more preferably 70 or more, more preferably 100 or more, most preferably 2 or more of the constituent amino acid sequences of the protein used in the present invention. Peptides having 100 or more amino acid sequences are used.

In the partial peptide used in the present invention, one or more (preferably about 1 to 10, more preferably a number (1 to 5)) amino acids in the amino acid sequence are deleted. Or 1 or 2 or more (preferably, about 1 to 20; more preferably, about 1 to 10; more preferably, about 1 to 5) amino acids are added to the amino acid sequence; Or 1 or 2 or more (preferably, about 1 to 20; more preferably, about 1 to 10; more preferably, about 1 to 5) amino acids are inserted into the amino acid sequence; or In the amino acid sequence, one or more (preferably about 1 to 10, more preferably several, and still more preferably about 1 to 5) amino acids are substituted with another amino acid. Is also good.

The partial peptide used in the present invention, the C-terminus force Rupokishiru group (- C00H), Karupokishireto (- C00-), amide (- C0NH ₂₎ or ester (- C00R) may be either.

Furthermore, the partial peptides used in the present invention include those having a carbonyl group (or carboxylate) in addition to the C-terminus and the amino acid residues at the N-terminus, similarly to the proteins used in the present invention described above. The amino group of a group (eg, methionine residue) is protected by a protecting group, the glutamine residue generated by cleavage of the N-terminal side in vivo is pyroglutamine-oxidized, Also included are those in which the substituent is protected by an appropriate protecting group, and complex peptides such as so-called glycopeptides to which a sugar chain is bound.

The partial peptide used in the present invention can also be used as an antigen for producing an antibody.

As the salt of the protein or partial peptide used in the present invention, a salt with a physiologically acceptable acid (eg, an inorganic acid, an organic acid) or a base (eg, an alkali metal salt) is used. Physiologically acceptable acid addition salts are preferred. Such salts include: For example, salts with inorganic acids (eg, hydrochloric, phosphoric, hydrobromic, sulfuric) or organic acids (eg, acetic, formic, propionic, fumaric, maleic, succinic, tartaric, Salts with citric acid, malic acid, oxalic acid, benzoic acid, methanesulfonic acid, and benzenesulfonic acid are used.

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

When producing from human or mammalian tissues or cells, the human or mammalian tissues or cells are homogenized, and then extracted with an acid or the like, and the extract is subjected to reverse phase chromatography, ion exchange chromatography, or the like. Purification and isolation can be achieved by combining chromatography.

For the synthesis of the protein or partial peptide or its salt or its amide used in the present invention, a commercially available resin for protein synthesis can be usually used. Examples of such resins include chloromethyl resin, hydroxymethyl resin, benzylhydramine resin, aminomethyl resin, 4-benzyloxybenzyl alcohol resin, 4-methylbenzhydrylamine resin, PAM resin, and the like. 4-Hydroxymethylmethylphenylacetamidomethyl resin, polyacrylamide resin, 4_ (2 ', 4, dimethoxyphenylhydroxymethyl) phenoxy resin, 4- (2,, 4, dimethoxyphenylaminoethyl) Phenoxy resins and the like can be mentioned. Using such a resin, an amino acid in which the amino group and the side chain functional group are appropriately protected is condensed on the resin according to the sequence of the target protein according to various known condensation methods. At the end of the reaction, protein or partial peptide is excised from the resin, and at the same time, various protecting groups are removed.In addition, an intramolecular disulfide bond formation reaction is carried out in a highly diluted solution, and the target protein or partial peptide or their peptide Obtain the amide form.

Regarding the condensation of the protected amino acids described above, various activities that can be used for protein synthesis Activating reagents can be used, and carbodiimides are particularly preferable. Examples of the carbopimides include DCC, N, Ν'-diisopropyl carbopimide, and Ν-ethyl-N '-(3-dimethylaminoprolyl) carbopimide. Activation by these involves adding the protected amino acid directly to the resin along with a racemization inhibitor additive (eg, HOBt, HOOBt), or symmetrical acid anhydrides or HOBT esters or HOOBt. The ester can be added to the resin after activation of the protected amino acid in advance.

The solvent used for activating the protected amino acid or condensing with the resin can be appropriately selected from solvents known to be usable for the protein condensation reaction. For example, acid amides such as N, N-dimethylformamide, N, N-dimethylacetamide, N-methylpyrrolidone, halogenated hydrocarbons such as methylene chloride, chloroform, and alcohols such as trifluoroethanol. , Sulfoxides such as dimethyl sulfoxide, ethers such as pyridine, dioxane, and tetrahydrofuran; nitriles such as aceto nitrile and propionitrile; esters such as methyl acetate and ethyl acetate; or an appropriate mixture thereof. Used. The reaction temperature is appropriately selected from the range known to be usable for the protein bond formation reaction, and is usually selected appropriately from the range of about 120 ° C. (: to 50 ° C .. The activated amino acid derivative Is usually used in an excess of 1.5 to 4 times.As a result of a test using the ninhydrin reaction, if the condensation is insufficient, sufficient condensation can be carried out by repeating the condensation reaction without removing the protecting group. When sufficient condensation cannot be obtained even after repeating the reaction, acetylation of the unreacted amino acid with acetic anhydride or acetylimidazole is carried out so that the subsequent reaction is not affected. can do.

Examples of the protecting group for the amino group of the starting material include Z, Boc, t-pentyloxycarbonyl, isobornyloxycarbonyl, 4-methoxybenzyloxycarbonyl, C1-1Z, Br-Z, Damantyloxycarbonyl, trifluoroacetyl, phthaloyl, formyl, 2212 phenylsulfenyl, diphenylphosphinothioyl, Fmoc and the like are used.

Carbonyl groups are, for example, alkyl esterified (eg, methyl, ethyl, Linear, branched or cyclic alkylesters such as pill, butyl, t-butyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, 2-adamantyl, etc., aralkyl esterification (for example, benzyl ester) , 4-nitrobenzyl ester, 4-methoxybenzyl ester, 4-chlorobenzyl ester, benzylhydryl esterification), phenacyl esterification, benzyloxycarbonyl hydrazide, t-butoxycarbonyl hydrazide, trityl hydrazide Can be protected.

The hydroxyl group of serine can be protected, for example, by esterification or etherification. Examples of groups appropriately used for the esterification include a lower (C! _ ₆₎ Arukanoiru groups such Asechiru group, Aroiru group such Benzoiru group, benzyl O carboxymethyl Cal Poni group, and a group derived from carbonic acid such as ethoxycarbonyl group Is used. Examples of a group suitable for etherification include a benzyl group, a tetrahydropyranyl group, and a t-butyl group.

The protecting group of the phenolic hydroxyl group of tyrosine, for _{example, B z 1, C 1 2} -B zl, 2- nitrobenzyl, B R_Z, such as t- butyl are used.

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

Examples of the activated carbonyl group of the raw material include, for example, corresponding acid anhydrides, azides, and active esters [alcohols (eg, phenol, 2,4,5-trichlorophenol, 2,4 —Dinitrophenol, cyanomethyl alcohol, paranitrophenol, HONB, N-hydroxysuccinimide, N-hydroxyfurimide, ester with HOB t)]. As the activated amino group of the raw material, for example, a corresponding phosphoric amide is used.

Methods for removing (eliminating) protecting groups include, for example, catalytic reduction in the presence of a catalyst such as Pd-black or Pd-carbon in a stream of hydrogen, or hydrogen fluoride anhydride, methanesulfonic acid, trifluoromethane, or the like. Acid treatment with sulfonic acid, trifluoroacetic acid or a mixture thereof, diisopropylethylamine, triethylamine, Base treatment with lysine, piperazine, etc., and reduction with sodium in liquid ammonia are also used. The elimination reaction by the above-mentioned acid treatment is generally performed at a temperature of about 120 ° (: to 40 ° C). In the acid treatment, for example, anisol, phenol, thioanisole, methacrylol, paracresol, It is effective to add a cation trapping agent such as dimethinoresulfide, 1,4-butanedithiol, 1,2-ethanedithiol, etc. Also, 2,4-dinititol used as an imidazole protecting group for histidine The mouth phenyl group is removed by thiophenol treatment, and the formyl group used as an indole protecting group for tributanone is deprotected by acid treatment in the presence of 1,2-ethanedithiol, 1,4-butanedithiol and the like. In addition, it is also removed by alkali treatment with dilute sodium hydroxide solution, dilute ammonia and the like.

The protection of the functional group which should not be involved in the reaction of the raw materials, the protective group, the elimination of the protective group, the activation of the functional group involved in the reaction and the like can be appropriately selected from known groups or known means.

As another method for obtaining an amide form of a protein or a partial peptide, for example, after amidating and protecting the α-hydroxyl group of the amino acid at the carboxy terminal, a peptide (protein) chain is desired on the amino group side. And a protein or partial peptide from which only the protecting group for the α-amino group at the Ν-terminal of the peptide chain has been removed, and a protein or partial peptide from which only the protecting group for the C-terminal lipoxyl group has been removed. Is produced, and these proteins or peptides are condensed in a mixed solvent as described above. Details of the condensation reaction are the same as described above. After purifying the protected protein or peptide obtained by the condensation, all the protecting groups are removed by the above-mentioned method to obtain a desired crude protein or peptide. The crude protein or peptide is purified by using various known purification means, and the main fraction is freeze-dried to obtain an amide of the desired protein or peptide.

To obtain an ester of a protein or peptide, for example, after condensing the α-hydroxyl group of the amino acid at the carboxy terminal with a desired alcohol to form an amino acid ester, And an ester form of the protein or peptide. The partial peptide or a salt thereof used in the present invention can be produced according to a peptide synthesis method known per se, or by cleaving the protein used in the present invention with an appropriate peptidase. . As a method for synthesizing a peptide, for example, any of a solid phase synthesis method and a liquid phase synthesis method may be used. That is, the objective peptide is produced by condensing a partial peptide or amino acid that can constitute the partial peptide used in the present invention with the remaining portion, and if the product has a protecting group, removing the protecting group to produce the desired peptide. Can be. Known condensation methods and elimination of protecting groups include, for example,

The methods described in ① to ⑤ are mentioned.

ΦΜ. Bodanszky and M.A. Ondet ti, Peptide Synthes is, Interscience Publ ishers, New York (1966)

② Schroeder and Luebke, The Peptide, Academic Press, New York (1965)

(3) Nobuo Izumiya et al., Basics and experiments on peptide synthesis, Maruzen Co., Ltd. (1975)

治 Haruaki Yajima and Shunpei Sakakibara, Laboratory for Biochemical Experiments 1, Protein Chemistry IV, 205, (1977)

治 Supervised by Haruaki Yajima, Development of Continuing Drugs, Volume 14, Peptide Synthesis, Hirokawa Shoten

After the reaction, a general purification method such as solvent extraction, distillation, column chromatography, liquid chromatography, recrystallization, etc. may be combined to purify and isolate the partial peptide used in the present invention. Can be. 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. It can be converted to a free form or other salts by a method analogous thereto.

The polynucleotide encoding the protein used in the present invention may be any polynucleotide containing the above-described nucleotide sequence encoding the protein used in the present invention. Preferably it is DNA. The DNA may be any of a genomic DNA, a genomic DNA library, the above-described cell-tissue-derived cDNA, the above-described cell-tissue-derived cDNA library, and a synthetic DNA.

The vectors used for the library include bacteriophages, plasmids, And phagemid. In addition, it can be directly amplified by reverse transcriptase polymerase chain reaction (hereinafter abbreviated as RT-PCR) using a total RNA or mRNA fraction prepared from the cells and tissues described above.

Examples of the DNA encoding the protein used in the present invention include a DNA containing the nucleotide sequence represented by SEQ ID NO: 2, or a DNA that is highly stringent with the nucleotide sequence represented by SEQ ID NO: 2. Any DNA that contains a base sequence that hybridizes under the conditions and encodes a protein having substantially the same properties as the protein containing the amino acid sequence represented by SEQ ID NO: 1 described above. May be. Examples of the DNA that can be hybridized with the nucleotide sequence represented by SEQ ID NO: 2 under eight stringent conditions include, for example, about 50% or more, preferably 50% or more, of the nucleotide sequence represented by SEQ ID NO: 2. It contains a nucleotide sequence having a homology of about 60% or more, more preferably about 70% or more, more preferably about 80% or more, particularly preferably about 90% or more, and most preferably about 95% or more. DNA or the like is used. For example, a DNA containing the base sequence represented by SEQ ID NO: 4 and the like can be mentioned.

The homology of the nucleotide sequences was determined using the homology calculation algorithm NCBI BLAST (National Center for Biotechnology Information Basic Local Search Tool) under the following conditions (expected value = 10; gap allowed; filtering = 0N; match). Score = 1; mismatch score = -3).

Hybridization is performed according to a method known per se or a method analogous thereto, for example, the method described in Molecular Cloning 2nd (J. Sambrook et al., Cold Spring Harbor Lab. Press, 1989). Can be done. When a commercially available library is used, it can be performed according to the method described in the attached instruction manual. More preferably, the reaction can be performed under high stringent conditions.

Highly stringent conditions include, for example, a sodium concentration of about 19 to 40 mM, preferably about 19 to 20 mM, and a temperature of about 50 to 70 X :, preferably about 60 to 70 X. The conditions at 65 ° C are shown. In particular, when the sodium concentration is about 19 mM and the temperature is about 65 ° C, Most preferred.

More specifically, the DNA encoding the protein containing the amino acid sequence represented by SEQ ID NO: 1 includes a DNA containing the base sequence represented by SEQ ID NO: 2, and the like. As the DNA encoding the protein containing the amino acid sequence represented by the above, DNA containing the base sequence represented by SEQ ID NO: 4 or the like is used.

The polynucleotide (eg, DNA) encoding the partial peptide used in the present invention may be any polynucleotide containing the above-described nucleotide sequence encoding the partial peptide used in the present invention. Further, it may be any of genomic DNA, genomic DNA library, cDNA derived from the above-described cells and tissues, cDNA library derived from the above-described cells and tissues, and synthetic DNA.

As the DNA encoding the partial peptide used in the present invention, for example, a DNA containing a part of the DNA containing the base sequence represented by SEQ ID NO: 2, or the base sequence represented by SEQ ID NO: 2 For example, DNA containing a part of DNA encoding a protein having a nucleotide sequence that hybridizes under high stringent conditions and encoding a protein having substantially the same activity as the protein of the present invention may be used.

DNA that can hybridize with the nucleotide sequence represented by SEQ ID NO: 2 has the same meaning as described above.

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

In the description of the cloning and expression of the DNAs and partial peptides used in the present invention (hereinafter, these may be simply referred to as the proteins of the present invention in the description of the cloning and expression of the DNAs encoding them), the cloning of the DNA completely encoding As a means, DNA 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 the DNA incorporated into an appropriate vector is a part of the protein of the present invention. Alternatively, selection can be performed by hybridization with a DNA fragment encoding the entire region or a DNA fragment labeled with a synthetic DNA. The method of hybridization is, for example, molecular claw. This can be performed according to the method described in Molecular Cloning 2nd (J. Sambrook et al., Cold Spring Harbor Lab. Press, 1989). When a commercially available library is used, it can be performed according to the method described in the attached instruction manual.

The DNA base sequence can be converted using PCR, a known kit, for example, Mutan ™ -Super Express Km (Takara Shuzo Co., Ltd.), Mutan ™ -K (Takara Shuzo Co., Ltd.), ODA-LAPCR method, Gapped The method can be carried out according to a method known per se, such as the duplex method and the Kunkel method, or a method analogous thereto.

The DNA encoding the cloned protein can be used as it is depending on the purpose, or can be used after digesting with a restriction enzyme or adding a linker, if desired. The DNA may have ATG as a translation initiation codon at its 5 'end and TAA, TGA or TAG as a translation termination codon at its 3' end. Codons and translation termination codons can also be added using a suitable synthetic DNA adapter.

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

Examples of the vector include a plasmid derived from E. coli (eg, PBR322, pBR325, pUC12, pUC13), a plasmid derived from Bacillus subtilis (eg, pUB110, TP5, pC194), a plasmid derived from yeast (eg, pSHl9) , PSHI 5), bacteriophage such as λ phage, animal viruses such as retrovirus, vaccinia virus, baculovirus, etc., A1-11, pXT1, pRc / CMV, pRcZRSV, pcDNA I ZNeo or the like is used.

The promoter used in the present invention may be any promoter as long as it is appropriate for the host used for gene expression. For example, when animal cells are used as a host, SRa promoter, SV40 promoter, LTR promoter, CMV promoter, HSV-TK promoter and the like can be mentioned. Among them, it is preferable to use CMV (cytomegalovirus) promoter, SRa promoter and the like. When the host is Eshierihia genus bacterium, trp flop port mode evening -, Promo Isseki one, re cA promoter Isseki one, AP _L promoter, 1 pp promoter evening one, such as T 7 promoter one is, host Bacillus When the host is a bacterium, SP01 promoter, SP02 promoter, pen P promoter, etc., and when the host is yeast, PHO5 promoter, PGK promoter, GAP promoter, ADH promoter, etc. are preferable. When the host is an insect cell, a polyhedrin promoter, a P10 promoter and the like are preferable.

The expression vector may contain, in addition to the above, an enhancer, a splicing signal, a poly-A addition signal, a selection marker, and an SV40 replication origin (hereinafter sometimes abbreviated as SV40 ori), if desired. Can be used. Examples of the selection marker include a dihydrofolate reductase (hereinafter sometimes abbreviated as dh fr) gene (methotrexate (MTX) resistance) and an ampicillin resistance gene (hereinafter abbreviated as Amp) ), the neomycin resistance gene (hereinafter sometimes abbreviated as Ne o ^r, include G41 8-resistant) and the like. In particular, when the dh fr gene is used as a selection marker using Chinese hamster cells deficient in the dh fr gene, the target gene can be selected using a thymidine-free medium. If necessary, a signal sequence suitable for the host is added to the N-terminal of the protein of the present invention. When the host is a bacterium belonging to the genus Escherichia, a PhoA signal sequence, an OmpA signal sequence, etc., and when the host is a bacterium belonging to the genus Bacillus, a single amylase-signal sequence, subtilisin, a signal sequence, etc., and when the host is a yeast, In some cases, MFa signal sequence, SUC2 signal sequence, etc., and when the host is an animal cell, insulin signal sequence, Hi-interferon signal sequence, antibody molecule, signal sequence, etc. can be used. .

A transformant can be produced using the thus-constructed vector containing DNA encoding the protein of the present invention.

As the host, for example, Escherichia bacteria, Bacillus bacteria, yeast, insect cells, insects, animal cells, and the like are used. Specific examples of the genus Escherichia include, for example, Escherichia coli Kl 2 -DH1 CProc. Natl. Acad. Sci. USA, 60, 160 (1968)], JM103 [Nucleic Acids Research, 9] 309 (1981)), JA 221 (Journal of Molecular Biology, 120, 517 (1978)), HB 101 [Journal of Molecular Biology, 41, 459 (1969)], C600 [Genetics, 39, 440 (1954)].

As the Bacillus bacterium, for example, Bacillus subtilis MI114 [Gene, 24, 255 (1983)], 207-21 [Journal of Biochemistry, 95, 87 (1984)] and the like are used.

Examples of yeast include, for example, Saccharomyces cerevisiae AH22, AH22R—, NA87-11A, DKD-5D, 2OB-12, Schizosaccharomyces pombe NCYC 1913, NCYC 2036, Pichia pastoris Pichia pastoris) KM 71 and the like are used.

Insect cells include, for example, when the virus is Ac NPV, a cell line derived from a larva of night roth moth (Spodoptera frugiperda cell; S f cell), MG1 cell derived from the midgut of Trichoplusia ni, and egg derived from Tridioplusiani egg HigliFive ™ cells, cells derived from Mamestra brassicae or cells derived from Estigmena acrea are used. When the virus is Bm NPV, a silkworm-derived cell line (Bombyx mori N cell; BmN cell) or the like is used. As the Sf cells, for example, Sf9 cells (ATCCCRL1711), Sf21 cells (Vaughn, J 丄 et al., In Vivo, 13, 213-217, (1977)) and the like are used. As insects, for example, silkworm larvae are used [Maeda et al., Nature, Vol. 315, 592 (1985)].

Examples of animal cells include monkey cell COS-7, Vero, Chinese hamster cell CHO (hereinafter abbreviated as CHO cell), dh fr gene-deficient Chinese hamster cell CHO (hereinafter abbreviated as CHO (dh fr—) cell. ), Mouse L cells, mouse AtT-20, mouse myeloma cells, mouse ATDC5 cells, rat GH3, human FL cells, etc. are used.

For transformation of Escherichia sp., For example, Proc. Natl. Acad. Sci. USA, 69 Vol., 2110 (1972) Gene, Vol. 17, 107 (1982), and the like.

Transformation of Bacillus spp. Can be performed, for example, according to the method described in Molecular & General Genetics, vol. 168, 111 (1979).

Transformation of yeast can be performed, for example, according to the method described in Methods in Enzymology, Vol. 194, 182-187 (1991), Pro Natl. Acad. Sc. USA, Vol. 75, 1929 (1978). Can be.

To transform insect cells or insects, see, for example, Bio / Technology, 6,

47-55 (1988).

Transformation of animal cells can be performed, for example, by the method described in Cell Engineering Separate Volume 8 New Cell Engineering Experimental Protocol. 263-267 (1995) (published by Shujunsha), Virology, 52, 456 (1973). So you can do it.

Thus, a transformant transformed with the expression vector containing the DNA encoding the protein can be obtained.

When culturing a transformant whose host is a bacterium belonging to the genus Escherichia or Bacillus, a liquid medium is suitable as a medium used for culturing, and a carbon source necessary for growth of the transformant may be used therein. Nitrogen sources, inorganic substances, etc. are included. Carbon sources include, for example, glucose, dextrin, soluble starch, sucrose, etc.Nitrogen sources include, for example, ammonium salts, nitrates, copper steep liquor, peptone, casein, meat extract, soybean meal, potato inorganic or organic substances such as extract, etc. Examples of the inorganic materials are calcium chloride, sodium dihydrogen phosphate, magnesium chloride and levator only be ^5. In addition, yeast extract, vitamins, growth promoting factors and the like may be added. The pH of the medium is preferably about 5 to 8.

As a medium for cultivating a bacterium belonging to the genus Escherichia, for example, an M9 medium containing glucose and casamino acid [Miller, Journal of Experiments in Molecular

Genetics, 431-433, Cold Spring Harbor Laboratory, New York 1972]. If necessary, an agent such as 3] 3-indolylacrylic acid can be added in order to make the promoter work efficiently. When the host is a bacterium belonging to the genus Escherichia, the cultivation is usually performed at about 15 to 43 ° C for about 3 to 24 hours, and if necessary, aeration and stirring can be applied.

When the host is a bacterium belonging to the genus Bacillus, the cultivation is usually performed at about 30 to 40 for about 6 to 24 hours, and if necessary, aeration and stirring can be applied.

When culturing a transformant in which the host is yeast, the culture medium may be, for example, Burkholder's minimal medium (CBostian, KL et al., Proc. Natl. Acad. Sci. USA, 77, 4505 (1980)). And SD medium containing 0.5% casamino acid [Bitter, GA et al., Pro Natl. Acad. Sci. USA, 81, 5330 (1984)]. The pH of the medium is preferably adjusted to about 5-8. Culture is usually performed at about 20 ° C to 35 ^ for about 24 to 72 hours, and aeration and agitation are added as necessary.

When culturing a transformant whose host is insect cells or insects,

Grace's Insect Medium (Grace, T.C.C., Nature), 195, 788 (1962)) to which an additive such as immobilized 10% pepsin serum or the like is appropriately added is used. The pH of the medium is preferably adjusted to about 6.2 to 6.4. Culture is usually performed at about 27 ° C for about 3 to 5 days, and aeration and agitation are added as necessary.

When culturing a transformant in which the host is an animal cell, examples of the medium include MEM medium containing about 5 to 20% fetal bovine serum [Science, 122, 501 (1952)], DMEM medium [Virology , 8, 396 (1959)], RPM I 1640 medium [The Journal of the American Medical Association 199, 519 (1967)], 199 medium [Proceeding of the Society for the Biological Medicine, 73, 1 (1950)] Are used. Preferably, the pH is about 6-8. Culture is usually performed at about 30 to 401: about 15 to 60 hours, and aeration and stirring are added as necessary.

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

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

When extracting the protein of the present invention from the cultured cells or cells, after the culture, the cells or cells are collected by a known method, suspended in an appropriate buffer, and then subjected to ultrasound, A method of obtaining a crude protein extract by centrifugation or filtration after disrupting the cells or cells by zozyme and z or freeze-thawing or the like is appropriately used. The buffer may contain a protein denaturant such as urea or guanidine hydrochloride, or a surfactant S such as Triton X-100 ^™ . When the protein is secreted into the culture solution, after completion of the culture, the supernatant is separated from the cells or cells by a method known per se, and the supernatant is collected.

Purification of the protein contained in the culture supernatant or extract thus obtained can be carried out by appropriately combining known separation and purification methods. Methods for separating and purifying these losses include methods using solubility such as salting out and solvent precipitation, dialysis, ultrafiltration, gel filtration, and SDS-polyacrylamide gel electrophoresis. Methods that mainly use differences in molecular weight of DNA, methods that use differences in charges such as ion exchange chromatography, methods that use specific affinity such as affinity chromatography, and reversed-phase high-performance liquid chromatography. For example, a method using a difference in hydrophobicity such as isoelectric point electrophoresis and a method using a difference in isoelectric point are used. When the protein thus obtained is obtained in a free form, it can be converted to a salt by a method known per se or a method analogous thereto. The compound can be converted into a free form or another salt by an analogous method.

The protein produced by the recombinant can be arbitrarily modified or the polypeptide can be partially removed by the action of an appropriate protein-modifying enzyme before or after purification. As the protein modifying enzyme, for example, trypsin, chymotrypsin, arginyl endopeptidase, protein kinase, glycosidase and the like are used.

The presence of the protein of the present invention thus produced can be measured by enzymatic immunoassay western blotting using a specific antibody. The antibody against the protein or partial peptide or a salt thereof used in the present invention may be any of a polyclonal antibody and a monoclonal antibody as long as it can recognize the protein or partial peptide or a salt thereof used in the present invention. Yo No.

Antibodies against the protein or partial peptide used in the present invention or a salt thereof (hereinafter sometimes simply referred to as the protein of the present invention in the description of the antibody) are prepared by using the protein of the present invention as an antigen, It can be produced according to a method for producing an antibody or antiserum known per se.

[Preparation of monoclonal antibody]

(a) Preparation of monoclonal antibody-producing cells

The protein of the present invention is administered to a warm-blooded animal itself or together with a carrier or a diluent at a site capable of producing an antibody upon administration. Complete Freund's adjuvant or incomplete Freund's adjuvant may be administered in order to enhance the antibody-producing ability upon administration. Administration is usually performed once every 2 to 6 weeks, for a total of about 2 to 10 times. Examples of warm-blooded animals to be used include monkeys, rabbits, rabbits, dogs, guinea pigs, mice, rats, sheep, goats, and chickens, and mice and rats are preferably used.

When preparing monoclonal antibody-producing cells, a warm-blooded animal immunized with an antigen, for example, a mouse with an antibody titer was selected from a mouse, and the spleen or lymph node was collected 2 to 5 days after the final immunization. By fusing the antibody-producing cells contained therein with myeloma cells of the same or different species, a monoclonal antibody-producing hybridoma can be prepared. The antibody titer in the antiserum can be measured, for example, by reacting a labeled protein described below with the antiserum, and then measuring the activity of a labeling agent bound to the antibody. The fusion operation can be performed according to a known method, for example, the method of Köhler and Milstein [Nature, 256, 495 (1975)]. Examples of the fusion promoter include polyethylene glycol (PEG) and Sendai virus, but PEG is preferably used.

Examples of the myeloma cells include warm-blooded animal myeloma cells such as NS-1, P3U1, SP 2/0, and AP-1, but P3U1 is preferably used. The preferred ratio between the number of antibody-producing cells (spleen cells) and the number of myeloma cells used is about 1: 1 to 20: 1, and PEG (preferably PEG 1000 to PEG 6000) is 10 to 8%. Cell fusion can be carried out efficiently by adding at a concentration of about 0% and incubating at 20 to 40 ° C, preferably 30 to 37 ° C for 1 to 10 minutes.

Various methods can be used to screen monoclonal antibody-producing hybridomas. For example, a hybridoma culture supernatant is added to a solid phase (eg, a microplate) on which a protein antigen is directly or adsorbed together with a carrier, and then Monoclonal antibody bound to solid phase using anti-immunoglobulin antibody labeled with a radioactive substance or enzyme (anti-mouse immunoglobulin antibody is used if the cells used for cell fusion are mice) or protein A Monoclonal antibody bound to the solid phase by adding the hybridoma culture supernatant to a solid phase to which anti-immunoglobulin antibody or protein A has been adsorbed, adding a protein labeled with a radioactive enzyme, etc. And the like.

The selection of the monoclonal antibody can be performed according to a method known per se or a method analogous thereto. Usually, it can be performed in an animal cell culture medium supplemented with HAT (hypoxanthine, aminopterin, thymidine). As a medium for selection and breeding, any medium may be used as long as it can grow viable hybridomas. For example, RPMI 1640 medium containing 1 to 20%, preferably 10 to 20% fetal bovine serum, GIT medium containing 1 to 10% fetal bovine serum (Wako Pure Chemical Industries, Ltd.) Alternatively, a serum-free culture medium for hybridoma culture (SFM-101, Nissui Pharmaceutical Co., Ltd.) can be used. The culture temperature is usually 20 to 40 ° (:, preferably, about 37 ° C.) The culture time is generally 5 days to 3 weeks, preferably 1 week to 2 weeks. The antibody titer of the supernatant of the hybridoma culture medium can be measured in the same manner as in the measurement of the antibody titer in the antiserum described above.

(b) Purification of monoclonal antibodies

Monoclonal antibodies can be separated and purified by methods known per se, for example, immunoglobulin separation and purification methods (eg, salting out method, alcohol precipitation method, isoelectric point precipitation method, electrophoresis method, ion exchanger (eg, DEAE)) Absorption / desorption method, ultracentrifugation method, gel filtration method, specific purification method in which only the antibody is collected using an antigen-binding solid phase or an active adsorbent such as protein A or protein G and the bond is dissociated to obtain the antibody.) Can be performed according to (Preparation of polyclonal antibody)

The polyclonal antibody of the present invention can be produced according to a method known per se or a method analogous thereto. For example, a immunizing antigen (protein antigen) itself or a complex thereof with a carrier-protein is formed, and immunization is performed on a warm-blooded animal in the same manner as in the above-described method for producing a monoglonal antibody. The antibody can be produced by collecting an antibody-containing substance against the antibody and separating and purifying the antibody. Regarding the complex of an immunizing antigen and a carrier protein used for immunizing a warm-blooded animal, the type of carrier protein and the mixing ratio of the carrier and the hapten are determined by the antibody against the hapten immunized by cross-linking the carrier. Any material may be cross-linked at any ratio as long as it can be efficiently used.For example, serum albumin, thyroglobulin, hemocyanin, etc., in a weight ratio of about 0.1 to 2 per hapten per hapten. A method of coupling at a rate of 0, preferably about 1 to 5 is used.

Various condensing agents can be used for force coupling between the hapten and the carrier.For example, an active ester reagent containing a daltaraldehyde タル carpoimide, a maleimide active ester, a thiol group, or a dithioviridyl group is used. Can be

The condensation product is administered to a warm-blooded animal itself or together with a carrier and a diluent at a site where antibody production is possible. Complete Freund's adjuvant or incomplete Freund's adjuvant may be administered in order to enhance the antibody-producing ability upon administration. The administration is usually performed once every about 2 to 6 weeks, for a total of about 3 to 10 times.

The polyclonal antibody can be collected from blood, ascites, etc., preferably from blood of a warm-blooded animal immunized by the above method.

The measurement of the polyclonal antibody titer in the antiserum can be performed in the same manner as the measurement of the antibody titer in the antiserum described above. The polyclonal antibody can be separated and purified according to the same method for separating and purifying immunoglobulin as in the above-described method for separating and purifying a monoclonal antibody. Polynucleotide encoding the protein or partial peptide used in the present invention (eg, DNA (hereinafter, these DNAs may be abbreviated as the DNA of the present invention in the description of antisense polynucleotides)) Antisense polynucleotide having a nucleotide sequence complementary to or substantially complementary to a nucleotide sequence of As a peptide, a nucleotide having a nucleotide sequence complementary to or substantially complementary to the nucleotide sequence of the DNA of the present invention or a part thereof and having an action capable of suppressing the expression of the DNA is provided. Any antisense polynucleotide may be used, but antisense DNA is preferred.

The nucleotide sequence substantially complementary to the DNA of the present invention refers to, for example, the entire nucleotide sequence or partial nucleotide sequence of the nucleotide sequence complementary to the DNA of the present invention (that is, the complementary strand of the DNA of the present invention). And about 70% or more, preferably about 80% or more, more preferably about 90% or more, and most preferably about 95% or more. In particular, among the total nucleotide sequences of the complementary strand of the DNA of the present invention, (a) in the case of an antisense polynucleotide directed to translation inhibition, the nucleotide sequence of the portion encoding the N-terminal portion of the protein of the present invention (for example, An antisense polynucleotide having a homology of about 70% or more, preferably about 80% or more, more preferably about 90% or more, and most preferably about 95% or more with the complementary strand of Mouth) In the case of an antisense polynucleotide which directs RNA degradation by RNaseH, it is about 70% or more, preferably about 80% or more, complementary to the entire nucleotide sequence of the DNA of the present invention including introns. More preferably, antisense polynucleotides each having a homology of about 90% or more, most preferably about 95% or more are suitable.

Specifically, (1) an antisense polynucleotide containing a base sequence complementary to or substantially complementary to the base sequence of DNA containing the base sequence represented by SEQ ID NO: 2, or a part thereof An antisense polynucleotide containing a nucleotide, preferably, for example, a nucleotide sequence complementary to the nucleotide sequence of DNA containing the nucleotide sequence represented by SEQ ID NO: 2, or a part thereof (more preferably, a nucleotide sequence represented by SEQ ID NO: 2; A base sequence complementary to the base sequence of the DNA containing the base sequence represented, or an antisense polynucleotide containing a part thereof), (2) DNA containing the base sequence represented by SEQ ID NO: 4 An antisense polynucleotide containing a nucleotide sequence complementary to or substantially complementary to the nucleotide sequence of SEQ ID NO: 1, or a portion thereof, preferably, for example, represented by SEQ ID NO: 4 A base sequence complementary to the base sequence of DNA containing the base sequence, or an antisense polynucleotide containing a part thereof (more preferably, SEQ ID NO: 4 And a base sequence complementary to the base sequence of the DNA containing the base sequence represented by the following formula, or an antisense polynucleotide containing a part thereof.

An antisense polynucleotide is usually composed of about 10 to 40 bases, preferably about 15 to 30 bases.

To prevent degradation by hydrolases such as nucleases, the phosphate residue (phosphate) of each nucleotide constituting the antisense DNA may be, for example, a chemically modified phosphate residue such as phosphorothioate, methylphosphonate, or phosphorodithionate. It may be substituted by a group. In addition, the sugar (doxylipose) of each nucleotide may be substituted with a chemically modified sugar structure such as 2, -O-methylation, and the base (pyrimidine, purine) may also be chemically modified. Any one may be used as long as it hybridizes to the DNA containing the base sequence represented by SEQ ID NO: 2. These antisense polynucleotides can be produced using a known DNA synthesizer or the like.

According to the present invention, an antisense polynucleotide capable of inhibiting the replication or expression of the protein gene of the present invention is designed based on the nucleotide sequence information of the cloned or determined DNA encoding the protein. And can be synthesized. Such a nucleotide (nucleic acid) can hybridize with the RNA of the protein gene of the present invention, inhibit the synthesis or function of the RNA, or inhibit the interaction with the protein-related RNA of the present invention. Thus, the expression of the protein gene of the present invention can be regulated. Polynucleotides that are complementary to the selected sequence of the protein-related RNA of the present invention, and that can specifically hybridize with the protein-related RNA of the present invention, can be used in vivo and in vitro. It is useful for regulating and controlling gene expression, and is also useful for treating or diagnosing diseases. The term “responds” means having homology or being complementary to a nucleotide, base sequence or a specific sequence of a nucleic acid including a gene. “Corresponding” between a nucleotide, nucleotide sequence or nucleic acid and a peptide (protein) usually refers to the amino acid of the peptide (protein) specified by the sequence derived from the nucleotide (nucleic acid) sequence or its complement. I have. 5 'of protein gene End hairpin loop, 5 'end 6-base spare repeat, 5' end untranslated region, polypeptide translation initiation codon, protein coding region, ORF translation stop codon, 3 'end translation region, 3' end palindrome region , And the 3 'end hairpin loop may be selected as preferred regions of interest, but any region within the protein gene may be selected.

The relationship between the target nucleic acid and a polynucleotide complementary to at least a part of the target region can be said to be "antisense" if the relationship between the target nucleic acid and the polynucleotide that can hybridize with the target is. Antisense polynucleotides are polynucleotides that contain 2-deoxy D-reports, polynucleotides that contain D-reports, and other types of polynucleotides that are N-glycosides of purine or pyrimidine bases. Or other polymers having a non-nucleotide backbone (eg, commercially available protein nucleic acids and synthetic sequence-specific nucleic acid polymers) or other polymers containing special bonds (provided that the polymer is not contained in DNA or RNA). Pairing of bases (contains nucleotides having a configuration that allows base attachment) as described in (1). They can be double-stranded DNA, single-stranded DNA, double-stranded RNA, single-stranded RNA, or even a DNA: RNA hybrid, and can be unmodified polynucleotides (or unmodified polynucleotides). Oligonucleotides) and those with known modifications, e.g., those with labels, capped, methylated, and one or more natural nucleotides that are known in the art. , Substituted with an intramolecular nucleotide, for example, having an uncharged bond (eg, methylphosphonate, phosphotriester, phosphoramidate, olebamate, etc.), charged bond or sulfur-containing Those having a bond (for example, phosphorothioate, phosphorodithioate, etc.), for example, protein (nuclease, nuclease inhibitor, toxin) Antibodies, signal peptides, poly-L-lysine, etc.) or sugars (for example, monosaccharides), etc., which have side-chain groups, or those with interactive compounds (for example, acridine, psoralen, etc.), (Eg, metals, radioactive metals, boron, oxidizable metals, etc.), those containing alkylating agents, those with modified bonds (eg, Anomeric nucleic acids). Here, “nucleoside”, “nucleotide” and “nucleic acid” may include not only those containing purine and pyrimidine bases but also those having other modified heterocyclic bases. Such decorations may include methylated purines and pyrimidines, acylated purines and pyrimidines, or other heterocycles. Modified nucleotides and modified nucleotides may also be modified at the sugar moiety, e.g., where one or more hydroxyl groups have been replaced with halogens, aliphatic groups, etc., or ethers, amines, etc. It may have been converted to a functional group.

The antisense polynucleotide of the present invention is RNA, DNA, or a modified nucleic acid (RNA, DNA). Specific examples of the modified nucleic acid include, but are not limited to, a sulfur derivative of a nucleic acid, a thiophosphoate derivative, and a polynucleoside amide, which is resistant to degradation of an oligonucleoside amide. The antisense nucleic acid of the present invention can be preferably designed according to the following policy. That is, to make the antisense nucleic acid more stable in the cell, to make the antisense nucleic acid more cell-permeable, to have a greater affinity for the target sense strand, and if toxic Minimize the toxicity of antisense nucleic acids.

Thus, many modifications are known in the art, for example, J. Kawakami et al., Pharm Tech Japan, Vol. 8, pp. 247, 1992; Vol. 8, pp. 395, 1992; ST Crooke et al. Ed. , Ant isense Research and Applicat ions, CRC Press, 1993.

The antisense nucleic acids of the present invention may contain altered or modified sugars, bases, or bonds, and may be provided in special forms such as ribosomes or microspheres, applied by gene therapy, or added. Can be given in a prescribed form. Such additional forms include polythiones such as polylysine, which acts to neutralize the charge on the phosphate backbone, and lipids, which enhance the interaction with cell membranes and increase the uptake of nucleic acids. (Eg, phospholipids, cholesterol, etc.). Preferred lipids for addition include cholesterol and its derivatives (eg, cholesteryl chromate formate, Etc.). Such a substance can be attached to the 3 'end or 5' end of a nucleic acid, and can be attached via a base, sugar, or intramolecular nucleoside bond. Other groups include capping groups specifically located at the 3 'or 5' end of nucleic acids that prevent degradation by nucleases such as exonuclease and RNase. . Examples of such capping groups include, but are not limited to, hydroxyl-protecting groups known in the art, such as glycols such as polyethylene glycol and tetraethylene dalicol.

The inhibitory activity of the antisense nucleic acid can be examined using the transformant of the present invention, the in vivo or in vitro gene expression system of the present invention, or the in vivo or in vitro translation system of the protein of the present invention. The nucleic acid can be applied to cells by various methods known per se. Hereinafter, the protein or partial peptide of the present invention or a salt thereof (hereinafter, sometimes abbreviated as the protein of the present invention), a polynucleotide encoding the protein or partial peptide of the present invention (eg, DNA (hereinafter, referred to as the present invention) May be abbreviated as DNA))), an antibody against the protein or partial peptide of the present invention or a salt thereof (hereinafter, may be abbreviated as the antibody of the present invention), and an antisense polynucleotide of the DNA of the present invention. (Hereinafter, may be abbreviated as the antisense polynucleotide of the present invention).

Since the expression of the protein of the present invention is reduced in the lung having emphysema lesions, it can be used as a disease marker. That is, it is useful as a marker for early diagnosis in the lung, judgment of the severity of symptoms, and prediction of disease progression. Therefore, a drug containing the antisense polynucleotide of the gene encoding the protein of the present invention, a compound that inhibits the activity of the protein of the present invention or a salt thereof, or an antibody against the protein of the present invention is, for example, a respiratory tract. Diseases (eg, chronic obstructive pulmonary disease (chronic bronchitis, emphysema), diffuse panbronchiolitis, bronchial asthma, cystic fibrosis, irritable pneumonia, pulmonary fibrosis, etc.), rhinitis (eg, allergic rhinitis) , Hay fever, acute rhinitis, chronic rhinitis, hypertrophic rhinitis, atrophic rhinitis, dry rhinitis, vasomotor rhinitis, gangrene rhinitis, sinusitis, etc.), immune diseases (eg, myasthenia gravis, glomeruli) Nephritis, multiple sclerosis, siedalen syndrome, insulin resistant diabetes, rheumatoid arthritis, systemic lupus erythematosus, atopy Dermatitis, leukocyte abnormalities, spleen insufficiency or immunodeficiency associated with thymic abnormalities), inflammatory bowel disease, allergic conjunctivitis, etc. can be used as a preventive agent. Preferably, it is a prophylactic / therapeutic agent for a respiratory disease or the like, more preferably a prophylactic / therapeutic agent for a chronic obstructive pulmonary disease or the like, and further preferably a prophylactic / therapeutic agent for a pulmonary emphysema or the like.

(1) Screening of drug candidate compounds for diseases

Since the expression of the protein of the present invention is reduced in lungs having emphysema lesions, the compound or its salt that regulates (preferably enhances) the activity of the protein of the present invention may be used, for example, for respiratory diseases [eg, chronic obstructive Lung disease (chronic bronchitis, emphysema), diffuse panbronchiolitis, bronchial asthma, cystic fibrosis, irritable pneumonia, pulmonary fibrosis, etc.), rhinitis (eg, allergic rhinitis, pollinosis, acute rhinitis, Chronic rhinitis, hypertrophic rhinitis, atrophic rhinitis, dry rhinosinusitis, vasomotor rhinitis, gangrene rhinitis, sinusitis, etc., immune diseases (eg, myasthenia gravis, glomerulonephritis, multiple sclerosis) , Siedaren syndrome, insulin resistance diabetes, rheumatoid arthritis, systemic lupus erythematosus, atopic dermatitis, leukocyte abnormalities, immunodeficiency associated with splenic dysfunction or thymic abnormalities), inflammation It can be used as a prophylactic and therapeutic agent for inflammatory bowel disease, allergic conjunctivitis, etc. Preferably, it is a prophylactic / therapeutic agent for a respiratory disease or the like, more preferably a prophylactic / therapeutic agent for a chronic obstructive pulmonary disease or the like, and further preferably a prophylactic / therapeutic agent for an emphysema or the like.

Therefore, the protein of the present invention is useful as a reagent for screening a compound or a salt thereof that regulates (preferably promotes) the activity of the protein of the present invention.

That is, the present invention regulates (promotes or inhibits) the activity of the protein of the present invention (eg, a protease inhibitory activity, a procollagen C-terminal cleavage promoting activity, etc.) characterized by using the protein of the present invention. And a method for screening a compound or a salt thereof.

Specifically, (i) the protease inhibitory activity of the protein of the present invention or the C-terminal cleavage promoting activity of procollagen; and (ii) the protease inhibitory activity or procollagen c-terminal of the mixture of the protein of the present invention and a test compound. Compounds that regulate (promote or inhibit) the activity of the protein of the present invention, which are characterized by comparison with the cleavage promoting activity. Alternatively, a screening method for a salt thereof is used.

In the above screening method, for example, in the cases of (i) and (ii), the protease inhibitory activity or procollagen C-terminal cleavage promoting activity can be determined by a method known per se, for example, J. Biol. Chera. 275 (2 ), Pp. 1384-90, 2000, Eur. J. Biochem. 186, pp. 115-121, 1989, or the like, or a method analogous thereto, and compared.

Specifically, (i) the case where the protein of the present invention is reacted with a labeled (eg, fluorescent label) peptide substrate and a protease; and (ii) the case where the protein of the present invention is labeled in the presence of a test compound. (Example: fluorescent label) When the peptide substrate and the protease are reacted, the mass of the peptide group cleaved by the protease activity is measured, respectively, to regulate the activity of the protein of the present invention (promoting or inhibiting) To screen for compounds or salts thereof. This reaction is performed in an appropriate buffer. The mass of the cleaved peptide group is measured by a known method. When the labeling agent is, for example, a fluorescent substance, the fluorescence intensity may be measured for the peptide group mass. The measurement of the fluorescence intensity may be performed according to a known method using a fluorescence measurement device or the like.

The labeled peptide substrate and protease may be reacted with the protein of the present invention after being mixed with the test compound, or after contacting the peptide substrate and the protease with the protein of the present invention, Test compounds may be added. Specifically, (i) a reaction of the protein of the present invention with a labeled (eg, radiolabeled) procollagen and a procollagen C-terminal region cleaving protease (eg, BMP-1 etc.) ii) Reaction of the protein of the present invention, labeled (eg, radiolabeled) procollagen, and procollagen C-terminal region cleavage proteinase (eg, BMP-1) in the presence of a test compound Then, the mass of the C-terminal protein of procollagen cleaved by the protease activity is measured, and a compound or a salt thereof that regulates (promotes or inhibits) the activity of the protein of the present invention is screened. This reaction is performed in an appropriate buffer. The amount of the c-terminal protein of the cleaved procollagen is measured by a known method. When the labeling agent is, for example, a radioisotope, the amount of the C-terminal protein can be measured by a known method using high performance liquid chromatography or the like. It may be performed according to.

The labeled procollagen and procollagen C-terminal region cleaving protease may be mixed with a test compound and then reacted with the protein of the present invention. After contacting the domain-cleaving protease, the test compound may be added.

As the above-mentioned protein of the present invention, those produced by culturing cells having the ability to produce the protein of the present invention and the like are used. Furthermore, a culture solution of the cells, a supernatant thereof, a cell crushed product, or the like may be used.

As a cell having the ability to produce the protein of the present invention, for example, a host (transformant) transformed with a vector containing the above-described DNA encoding the protein of the present invention is used. As a host, for example, animal cells such as COS 7 cells, CHO cells, and HEK293 cells are preferably used. For the screening, for example, a transformant which secretes the protein of the present invention extracellularly or expresses it intracellularly by culturing by the above-mentioned method is preferably used. The method for culturing cells capable of expressing the protein of the present invention is the same as the above-described method for culturing the transformant of the present invention.

Test compounds include, for example, peptides, proteins, non-peptidic compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts, and the like. For example, the protease inhibitory activity or procollagen C-terminal cleavage promoting activity in the case of the above (ii) is about 20% or more, preferably 30% or more, more preferably about 50% or more, as compared with the case of the above (i). The test compound which increases the activity of the protein of the present invention by at least 10% is considered to be a compound that promotes the activity of the protein of the present invention, in which the protease inhibitory activity or procollagen C-terminal cleavage promoting activity in the above (ii) is about A test compound that reduces by 20% or more, preferably 30% or more, more preferably about 50% or more can be selected as a compound that inhibits the activity of the protein of the present invention. The compound having the activity of promoting the activity of the protein of the present invention may be a safe and low-toxic drug for enhancing the action of the protein of the present invention, for example, respiratory diseases [eg, chronic obstructive ^ J disease ( 1 man bronchitis, emphysema), diffuse panbronchiolitis, bronchial asthma, cyst Dysfibrosis, irritable pneumonia, pulmonary fibrosis, etc.), rhinitis (eg, allergic rhinitis, hay fever, acute rhinitis, chronic rhinitis, hypertrophic rhinitis, atrophic rhinitis, dry pronasitis, vasomotor rhinitis, gangrene Rhinitis, sinusitis, etc., immune diseases (eg, myasthenia gravis, glomerulonephritis, multiple sclerosis, Scheigren's syndrome, insulin resistance diabetes, rheumatoid arthritis, systemic lupus erythematosus, atopic dermatitis It is useful as a prophylactic and therapeutic agent for inflammatory bowel disease, allergic conjunctivitis, etc.)

The compound having the activity of inhibiting the activity of the protein of the present invention is a safe and low-toxic drug for suppressing the physiological activity of the protein of the present invention, for example, respiratory diseases (eg, bronchial asthma, cystic Fibrosis, pulmonary fibrosis, idiopathic interstitial pneumonia, etc.), liver disease (eg, chronic active hepatitis, cirrhosis, hepatic fibrosis, etc.), arteriosclerosis, renal sclerosis, myelofibrosis, diabetic vascular disorder, etc. It is useful as a preventive and therapeutic agent.

Compounds or salts thereof obtained using the screening method or the screening kit of the present invention include, for example, peptides, proteins, non-peptidic compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts It is a compound selected from liquids, plasma, etc. As the salt of the compound, those similar to the aforementioned salts of the peptide of the present invention are used.

Furthermore, since the expression of the gene encoding the protein of the present invention also decreases in lung tissue having emphysema lesions, a compound or a salt thereof that regulates (preferably promotes) the expression of the gene encoding the protein of the present invention may be used. For example, for example, respiratory diseases (eg, chronic obstructive pulmonary disease (chronic bronchitis, emphysema), diffuse panbronchiolitis, bronchial asthma, cystic fibrosis, irritable pneumonia, pulmonary fibrosis, etc.), Rhinitis (eg, allergic rhinitis, hay fever, acute rhinitis, chronic rhinitis, hypertrophic rhinitis, atrophic rhinitis, dry pronasitis, vasomotor rhinitis, gangrenous rhinitis, sinusitis, etc.), immunological diseases (example) , Myasthenia gravis, glomerulonephritis, multiple sclerosis, Siedalen syndrome, insulin resistance diabetes, rheumatoid arthritis, systemic lupus erythematosus, atopic dermatitis, white Spheres abnormalities, such as spleen dysfunction or thymus abnormally accompanying immunodeficiency), inflammatory bowel disease, preventing such allergic conjunctivitis - can be used as a therapeutic agent. Preferably, a prophylactic or therapeutic agent for respiratory diseases, etc., More preferably, it is a prophylactic / therapeutic agent for chronic obstructive pulmonary disease and the like, and further preferably, a prophylactic / therapeutic agent for emphysema and the like.

Therefore, the polynucleotide (eg, DNA) of the present invention is useful as a reagent for screening a compound or a salt thereof that regulates the expression of a gene encoding the protein of the present invention.

The screening methods include (iii) culturing cells having the ability to produce the protein of the present invention, and (iv) cells having the ability to produce the protein used in the present invention in the presence of the test compound. A screening method characterized by performing a comparison with the case of culturing is used.

In the above method, the expression level of the gene (specifically, the amount of the protein of the present invention or the amount of mRNA encoding the protein) in the cases (ii) and (iv) is measured and compared.

The test compound and cells having the ability to produce the protein of the present invention include those similar to those described above.

The amount of the protein is measured by a known method, for example, using an antibody that recognizes the protein of the present invention, and analyzing the protein present in a cell extract or the like according to a method such as Western analysis, ELISA, or a method analogous thereto. Can be measured. The amount of mRNA can be measured by a known method, for example, Northern hybridization using a nucleic acid containing SEQ ID NO: 2 or a part thereof as a probe, or a nucleic acid containing SEQ ID NO: 2 or a part thereof as a primer Northern hybridization using a nucleic acid containing SEQ ID NO: 4 or a part thereof, or PCR using a nucleic acid containing SEQ ID NO: 4 or a part thereof as a primer or a method analogous thereto can be used. it can.

For example, a test compound that increases the gene expression level in the case of the above (iv) by about 20% or more, preferably 30% or more, more preferably about 50% or more as compared with the case of the above (iii) As a compound that promotes the expression of a gene encoding the protein of the present invention, a test compound that inhibits about 20% or more, preferably 30% or more, more preferably about 50% or more of the protein of the present invention. Compounds that suppress the expression of encoded genes Can be selected as an object.

The screening kit of the present invention contains the protein or partial peptide used in the present invention or a salt thereof, or a cell capable of producing the protein or partial peptide used in the present invention.

The compound or a salt thereof obtained by using the screening method or the screening kit of the present invention may be a test compound described above, for example, a peptide, a protein, a non-peptidic compound, a synthetic compound, a fermentation product, a cell extract, or a plant extract. A compound selected from animal tissue extract, plasma or the like, or a salt thereof, which is a compound or a salt thereof that regulates the activity of the protein of the present invention (eg, protease inhibitory activity or procollagen C-terminal cleavage promoting activity). .

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

A compound or a salt thereof that regulates (preferably promotes) the activity of the protein of the present invention and a compound or a salt thereof that regulates (preferably promotes) the expression of a gene encoding the protein of the present invention are, for example, respiratory diseases, respectively. [Eg, chronic obstructive pulmonary disease (chronic bronchitis, emphysema), diffuse panbronchiolitis, bronchial asthma, cystic fibrosis, irritable pneumonia, pulmonary fibrosis, etc.], rhinitis (eg, allergic rhinitis, Hay fever, acute rhinitis, chronic rhinitis, hypertrophic rhinitis, atrophic rhinitis, dry rhinitis, vasomotor rhinitis, gangrene rhinitis, sinusitis, etc., immune diseases (eg, myasthenia gravis, glomerulonephritis) , Multiple sclerosis, Siedalen syndrome, insulin-resistant diabetes, rheumatoid arthritis, systemic lupus erythematosus, atopic dermatitis, leukocyte abnormalities, spleen dysfunction Or the like thymic abnormally accompanying immunodeficiency), inflammatory bowel disease, preventing such allergic conjunctivitis, it is that you use as therapeutic agents. Preferably, it is a prophylactic / therapeutic agent for a respiratory disease or the like, more preferably a prophylactic / therapeutic agent for a chronic obstructive pulmonary disease or the like, and further preferably a prophylactic / therapeutic agent for a pulmonary emphysema or the like.

When a compound or a salt thereof obtained by using the screening method or the screening kit of the present invention is used as the above-mentioned prophylactic / therapeutic agent, it can be formulated into a preparation according to a conventional method. For example, compositions for oral administration include solid or liquid dosage forms such as tablets (including sugar-coated tablets and film-coated tablets), pills, granules, powders, capsules (soft capsules and the like). ), Syrups, emulsions, suspensions and the like. Such a composition is produced by a method known per se and contains a carrier, diluent or excipient usually used in the field of pharmaceuticals. For example, lactose, starch, sucrose, magnesium stearate and the like are used as carriers and excipients for tablets. Compositions for parenteral administration include, for example, injections, suppositories, injections, intravenous injections, subcutaneous injections, intradermal injections, intramuscular injections, intravenous injections, intraarticular injections Includes dosage forms such as injections. Such injections are prepared according to a method known per se, for example, by dissolving, suspending or emulsifying the antibody or a salt thereof in a sterile aqueous or oily liquid commonly used for injections. As the aqueous liquid for injection, for example, physiological saline, isotonic solution containing glucose and other auxiliary agents and the like are used, and suitable dissolution and trapping !!, for example, alcohol (eg, ethanol), polyalcohol (Eg, propylene glycol, polyethylene glycol), nonionic surfactants (eg, polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated catalyst)), etc. Good. As the oily liquid, for example, sesame oil, soybean oil, and the like are used, and benzyl benzoate, benzyl alcohol, and the like may be used in combination as a solubilizing agent. The prepared injection is usually filled in an appropriate ampoule. Suppositories to be used for rectal administration are prepared by mixing the antibody or a salt thereof with a conventional suppository base.

The oral or parenteral pharmaceutical composition of the above 3 is conveniently prepared in a dosage unit form so as to be compatible with the dosage of the active ingredient. Examples of such dosage unit dosage forms include tablets, pills, capsules, injections (ampoules), suppositories, etc., and usually 5 to 500 mg, especially 5 for injections. 110 O mg, and other dosage forms preferably contain 10-25 O mg of the compound. Each of the above-mentioned compositions may contain another active ingredient as long as the composition does not cause an undesirable interaction with the compound.

The preparations obtained in this way are safe and of low toxicity, for example, It may be orally or parenterally administered to blood animals (eg, mice, rats, egrets, higgins, bush, pests, pests, birds, cats, dogs, monkeys, chimpanzees, etc.). it can.

The dose of the compound or a salt thereof varies depending on its action, target disease, subject to be administered, administration route, and the like.For example, a compound or a salt thereof that regulates the activity of the protein of the present invention for the purpose of treating emphysema When the compound is orally administered, generally, in an adult (with a body weight of 60 kg), the compound or a salt thereof is used in an amount of about 0.1 to 100 mg, preferably about 1.0 to 5 Omg, more preferably about 1 to 10 mg per day. Administer 0-20 mg. When administered parenterally, the single dose of the compound or a salt thereof varies depending on the administration subject, the target disease, and the like.For example, a compound that regulates the activity of the protein of the present invention for the purpose of treating emphysema Or, when the salt or its salt is administered to an adult (with a body weight of 60 kg) usually in the form of an injection, the compound or its salt is administered in an amount of about 0.01 to 30 mg, preferably about 0.1 to 2 Omg per day. It is convenient to administer preferably about 0.1 to 10 mg by intravenous injection. In the case of other animals, the dose can be administered in terms of weight per 60 kg.

(2) Quantification of the protein of the present invention, its partial peptide or its salt

An antibody against the protein of the present invention (hereinafter sometimes abbreviated as the antibody of the present invention) can specifically recognize the protein of the present invention. It can be used for quantification by San Deutsch immunoassay.

That is, the present invention

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

(ii) After reacting the test solution with the antibody of the present invention insoluble on the carrier and another labeled antibody of the present invention simultaneously or continuously, measuring the activity of the labeling agent on the insoluble carrier And a method for quantifying the protein of the present invention in a test solution. In the quantification method (ii), one of the antibodies recognizes the N-terminal of the protein of the present invention. It is desirable that the other antibody be an antibody that reacts with the C-terminal of the protein of the present invention.

In addition to 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 F (ab ') ₂ , Fab', or Fab fraction of the antibody molecule may be used.

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

As a labeling agent used in a measurement method using a labeling substance, for example, a radioisotope, an enzyme, a fluorescent substance, a luminescent substance and the like are used. As radioisotopes, for example,

^[125 1], ^[131 1], [¾] used and ^[14 c]. As the above-mentioned enzyme, those which are stable and have high specific activity are preferable, and for example, β-galactosidase, β-dalcosidase, alkaline phosphatase, peroxidase, malate dehydrogenase and the like are used. As the fluorescent substance, for example, fluorescamine, fluorescein isothiocyanate or the like is used. As the luminescent substance, for example, luminol, luminol derivative, reluciferin, lucigenin and the like are used. Further, a biotin-avidin system can be used for binding the antibody or 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 usually used for insolubilizing and 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, the test solution is reacted with the insolubilized monoclonal antibody of the present invention (primary reaction), and further reacted with another labeled monoclonal antibody of the present invention (secondary reaction). By measuring the activity of the labeling agent, the amount of the protein of the present invention in the test liquid can be determined. The primary reaction and the secondary reaction may be performed in the reverse order, may be performed simultaneously, or may be performed at staggered times. The labeling agent and the method of insolubilization can be in accordance with those described above. In addition, in the immunoassay by the sandwich method, the antibody used for the solid phase antibody or the labeling antibody is not necessarily one kind, and a mixture of two or more kinds of antibodies is used for the purpose of improving measurement sensitivity and the like. May be used.

In the method for measuring the protein of the present invention by the sandwich method of the present invention, the monoclonal antibody of the present invention used in the primary reaction and the secondary reaction is preferably an antibody having a different site to which the protein of the present invention binds. . That is, the antibody used in the primary reaction and the secondary reaction is, for example, when the antibody used in the secondary reaction recognizes the C-terminal of the protein of the present invention, the antibody used in the primary reaction is Preferably, an antibody that recognizes other than the C-terminal, for example, the N-terminal, is used.

The monoclonal 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 a nephrometry. In the competitive method, the antigen in the test solution and the labeled antigen are allowed to react competitively with the antibody, and then the unreacted labeled antigen (F) is separated from the labeled antigen (B) bound to the antibody. Then (BZF separation), the labeling amount of either B or F is measured, and the amount of antigen in the test solution is quantified. In this reaction method, a soluble antibody is used as the antibody, BZF separation is performed using polyethylene glycol, a liquid phase method using a second antibody to the antibody, a solid phase antibody is used as the first antibody, or A solid-phase method using a soluble first antibody and a solid-phased antibody as the second antibody is used.

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

In nephelometry, the amount of insoluble sediment resulting from the antigen-antibody reaction in a gel or in a solution is measured. Even when the amount of antigen in the test solution is small and only a small amount of sediment can be obtained, a laser-Nef orifice utilizing laser scattering is preferably used.

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

For example, edited by Hiro Irie "Radio Nono Atsui" (Kodansha, published in Showa 49), edited by Hiro Irie "Radio Imuno Atsue" (Kodansha, published in 1954), edited by Eiji Ishikawa et al. "Immunoassay method" (Medical Shoin, published in 1938), "Enzyme immunoassay" (ed. 2nd edition) (ed. Eiji Ishikawa et al.) (Medical Publishing, published in 1977), "Enzyme immunoassay method, edited by Eiji Ishikawa et al. (3rd edition) (Medical Shoin, published in 1962), "Methods in ENZYMOLOGYj Vol. 70 (Immunochemical Tec niues (Part A))> Ibid. Vol. 73 (Immunochemical Tec niues (Part B)), Ibid.Vol. 74 (Immunochemical Techniaues (Part C)), Ibid.Vol. 84 (Immunochemical Techniaues (Part D: Selected Immunoassays)), Ibid.Vol. 92 (Immunochemical Techniaues (Part E: Monoclonal Ant ibodies and General Immunoassay Methods)), Ibid. Vol. 121 (Immunochemical Techniaues (Part

I: Hybridoma Technology and Monoclonal Ant ibodies)) (published by Academic Press).

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

Furthermore, when an increase or decrease 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, respiratory diseases (eg, chronic obstructive Pulmonary disease (chronic bronchitis, emphysema), diffuse panbronchiolitis, bronchial asthma, cystic fibrosis, irritable pneumonia, pulmonary fibrosis, etc.), rhinitis (eg, allergy Rhinitis, hay fever, acute rhinitis, chronic rhinitis, hypertrophic rhinitis, atrophic rhinitis, dry pronasitis, vasomotor rhinitis, gangrene rhinitis, sinusitis, etc.), immune diseases (eg, myasthenia gravis, Glomerulonephritis, multiple sclerosis, siedalen syndrome, insulin resistance diabetes, rheumatoid arthritis, systemic lupus erythematosus, atopic dermatitis, leukocyte abnormalities, immunodeficiency associated with impaired splenic function or thymus, etc.), inflammatory It can be diagnosed as having bowel disease, allergic conjunctivitis, etc., 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 a body fluid or a tissue. In addition, for preparing an antibody column used for purifying the protein of the present invention, detecting the protein of the present invention in each fraction during purification, and analyzing the behavior of the protein of the present invention in test cells, etc. Can be used.

(3) Gene diagnostics

The DNA of the present invention can be used, for example, in humans or warm-blooded animals (e.g., rats, mice, guinea pigs, egrets, birds, higgies, pigs, pigs, dogs, cats, dogs, monkeys, Abnormalities (gene abnormalities) in the DNA or mRNA encoding the protein of the present invention or a partial peptide thereof in a chimpanzee or the like can be detected. Or, it is useful as a diagnostic agent for genes such as decreased expression, increased DNA or mRNA, or overexpression.

The above-described genetic diagnosis using the DNA of the present invention can be performed, for example, by the well-known Northern high predication or PCR-SSCP method (Genomics, Vol. 5, pp. 874-879 (1989), Proceedings of the National Academy). of Sciences of the United States of America, Vol. 86, pp. 2766-2770 (1989)).

For example, when overexpression or decrease is detected by Northern hybridization, or when DNA mutation is detected by PCR-SSCP method, for example, respiratory diseases [eg, chronic obstructive pulmonary disease ('| ¾ Respiratory bronchitis, emphysema), diffuse panbronchiolitis, bronchial asthma, cystic fibrosis, irritable pneumonia, pulmonary fibrosis, etc.), rhinitis (eg, allergic rhinitis, pollinosis, acute rhinitis, chronic) Rhinitis, hypertrophic rhinitis, atrophic rhinitis, before drying Rhinitis, vasomotor rhinitis, gangrenous rhinitis, sinusitis, etc., immune disorders (eg, myasthenia gravis, glomerulonephritis, multiple sclerosis, siedaren syndrome, insulin resistant diabetes, rheumatoid arthritis, systemic lupus erythema It can be diagnosed as having a high probability of being a child with autism, atopic dermatitis, leukocyte abnormalities, immunodeficiency due to spleen dysfunction or thymic abnormalities), inflammatory bowel disease, allergic conjunctivitis, etc.

(4) a drug containing an antisense polynucleotide

The antisense polynucleotide of the present invention, which complementarily binds to the DNA of the present invention and can suppress the expression of the DNA, has low toxicity, and has the protein of the present invention or the present invention in vivo. DNA function (eg, protease inhibitory activity, procollagen C-terminal truncation promoting activity, etc.) can be suppressed, so that, for example, genuine respiratory tract disease (eg, bronchial asthma, cystic fibrosis, pulmonary fibrosis, idiopathic) Prophylactic and therapeutic agents for liver disease (eg, chronic active hepatitis, cirrhosis, hepatic fibrosis, etc.), arteriosclerosis, renal sclerosis, myelofibrosis, diabetic vascular disorders, etc. Can be used.

When the antisense polynucleotide is used as the above-mentioned prophylactic / therapeutic agent, it can be formulated and administered according to a method known per se.

Further, for example, after the above-mentioned antisense polynucleotide is inserted alone or into an appropriate vector such as a retrovirus vector, an adenovirus vector, an adenovirus associated virus vector, or the like, a human or mammal can be treated in a conventional manner. It can be given orally or parenterally to animals (eg, rats, puppies, sheep, sheep, bush, puppies, cats, dogs, monkeys, etc.). The antisense polynucleotide can be administered as it is or in the form of a formulation together with a physiologically acceptable carrier such as an auxiliary agent for promoting uptake, and can be administered by a gene gun or a catheter such as a hydrogel catheter. Alternatively, they can be aerosolized and administered topically into the trachea as an inhalant.

Furthermore, for the purpose of improving pharmacokinetics, prolonging the half-life, and improving the efficiency of intracellular uptake, the antisense polynucleotide is formulated alone or together with a carrier such as liposome (injection), and is intravenously, subcutaneously, It may be administered to the respiratory tract or lung lesions.

) Dosage depends on target disease, subject, route of administration For example, when the antisense polynucleotide of the present invention is administered for the treatment of emphysema, generally, in an adult (body weight 60 kg), the antisense polynucleotide is administered daily. About 0.1 to 10 O mg is administered.

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

Similarly to the above-mentioned antisense polynucleotide, a double-stranded RNA containing a part of the RNA encoding the protein of the present invention, a lipozyme containing a part of the RNA encoding the protein of the present invention, etc. Gene expression, and the function of the protein used in the present invention or the DNA used in the present invention in vivo can be suppressed. For example, respiratory diseases (eg, bronchial asthma, cysts) Cystic fibrosis, pulmonary fibrosis, idiopathic interstitial pneumonia), liver disease (eg, chronic active hepatitis, cirrhosis, hepatic fibrosis, etc.), arteriosclerosis, renal sclerosis, myelofibrosis, diabetic It can be used as a prophylactic and therapeutic agent for vascular disorders.

The double-stranded RNA can be designed and produced based on the sequence of the polynucleotide of the present invention according to a known method (eg, Nature, 411, 494, 2001).

The lipozyme can be designed and manufactured based on the sequence of the polynucleotide of the present invention according to a known method (eg, TRENDS in Molecular Medicine, Vol. 7, pp. 221, 2001). For example, it can be produced by linking a known lipozyme to a part of the RNA encoding the protein of the present invention. A part of the RNA encoding the protein of the present invention includes a portion (RNA fragment) adjacent to the cleavage site on the RNA of the present invention which can be cleaved by a known lipozyme.

When the above-described double-stranded RNA or lipozyme is used as the above-mentioned prophylactic / therapeutic agent, it can be formulated and administered in the same manner as an antisense polynucleotide. (5) a drug containing the antibody of the present invention

The antibody of the present invention may be used, for example, for respiratory diseases [eg, chronic obstructive pulmonary disease (chronic bronchitis, emphysema), diffuse panbronchiolitis, bronchial asthma, cystic fibrosis, irritable pneumonia, pulmonary fibrosis Etc.), rhinitis (eg, allergic rhinitis, hay fever, acute rhinitis, chronic rhinitis, hypertrophic nose Inflammation, atrophic rhinitis, dry rhinitis, vasomotor rhinitis, gangrenous rhinitis, sinusitis, etc., immune disorders (eg, myasthenia gravis, glomerulonephritis, multiple sclerosis, Scheddaren syndrome, Insulin-resistant diabetes, rheumatoid arthritis, systemic lupus erythematosus, atopic dermatitis, leukocyte abnormalities, immunodeficiency associated with splenic dysfunction or thymic abnormalities), inflammatory bowel disease, allergic conjunctivitis, etc. Can be used. Or respiratory disease (eg, bronchial asthma, cystic fibrosis, pulmonary fibrosis, idiopathic interstitial pneumonia, etc.), liver disease (eg, chronic active hepatitis, cirrhosis, liver fibrosis, etc.), arteriosclerosis, It can also be used as a prophylactic and therapeutic agent for renal sclerosis, myelofibrosis, and diabetic vascular disorders.

The antibodies of the present invention can be administered as such or as a suitable 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 compositions are provided in dosage forms suitable for oral or parenteral administration.

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

Compositions for parenteral administration include, for example, injections, suppositories, etc., and injections include intravenous, subcutaneous, intradermal, intramuscular and intravenous injections. Shape. Such injections are prepared according to known methods, for example, by dissolving, suspending or emulsifying the antibody or a salt thereof in a sterile aqueous or oily liquid commonly used for injections. Examples of aqueous liquids for injection include physiological saline, isotonic solutions containing glucose and other adjuvants, and suitable solubilizing agents such as alcohol (eg, ethanol) and polyalcohol ( For example, propylene glycol, polyethylene glycol), nonionic surfactant [for example, polysorbate 80, HCO- 50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)]. As the oily liquid, for example, sesame oil, soybean oil and the like are used, and benzyl benzoate, benzyl alcohol and the like may be used in combination as a solubilizer. The prepared injection is usually filled in an appropriate ampoule. Suppositories used for rectal administration are prepared by mixing the above antibody or a salt thereof with a usual base for suppositories. The above-mentioned oral or parenteral pharmaceutical composition is conveniently prepared in a unit dosage form so as to be compatible with the dosage of the active ingredient. Examples of such dosage unit dosage forms include tablets, pills, capsules, injections (ampoules), suppositories and the like. Usually, each dosage unit dosage form is 5 to 500 mg, particularly 5 to 100 mg for injection. Other dosage forms preferably contain 10-25 Omg of the antibody. Each of the above-mentioned compositions may contain another active ingredient as long as the composition does not cause an undesirable interaction with the above antibody.

The prophylactic / therapeutic agent for the above-mentioned diseases containing the antibody of the present invention has low toxicity and can be used as it is as a liquid or as a pharmaceutical composition of an appropriate dosage form, in humans or mammals (eg, rat, porcupine, sheep, pig, etc.). It can be given orally or parenterally (eg, intravenously) to the evening, cats, cats, dogs, monkeys, etc.). The dosage varies depending on the administration subject, target disease, symptoms, administration route, and the like.For example, when used for the treatment or prevention of pulmonary emphysema in adults, the antibody of the present invention is usually administered in a single dose. 0.1 1 to 20 mg / kg body weight, preferably 0.1 to 1 OmgZ kg body weight, more preferably 0.1 to 5 mg / kg body weight, about 1 to 5 times a day, preferably 1 to 3 times a day It is convenient to administer the compound as an injection at a time. In the case of other parenteral administration and oral administration, an equivalent amount can be administered. If the symptoms are particularly severe, the dose may be increased accordingly.

Further, the antibody of the present invention may be used, for example, for respiratory diseases [eg, chronic obstructive pulmonary disease (chronic bronchitis, emphysema), diffuse panbronchiolitis, bronchial asthma, cystic fibrosis, irritable pneumonia, lung Fibrosis, etc.), rhinitis (e.g., allergic rhinitis, hay fever, acute rhinitis, chronic rhinitis, hypertrophic rhinitis, atrophic rhinitis, dry proctitis, vasomotor rhinitis, gangrene rhinitis, sinusitis, etc. ), Immune diseases (eg, myasthenia gravis, glomerulonephritis, multiple sclerosis, siedarenosis) Diagnostic group, insulin resistant diabetes mellitus, rheumatoid arthritis, systemic lupus erythematosus, atopic dermatitis, leukocyte abnormalities, immunodeficiency associated with spleen dysfunction or thymic abnormalities), inflammatory bowel disease, allergic conjunctivitis, etc. It is also useful.

(6) The “prophylactic / therapeutic agent for chronic obstructive pulmonary disease or bronchial asthma comprising a compound having a function of regulating procollagen C-terminal cleavage promoting activity or a salt thereof” of the present invention

The “compound having an effect of regulating the activity of promoting procollagen C-terminal cleavage” may be any compound having an activity of regulating the activity of promoting procollagen C-terminal cleavage. For example, chronic obstructive pulmonary disease (eg, Chronic bronchitis, emphysema), diffuse panbronchiolitis, bronchial asthma, cystic fibrosis, irritable pneumonia, immune disease (eg, myasthenia gravis, glomerulonephritis, multiple sclerosis, siedalen syndrome, insulin resistance) For the prevention and treatment of inflammatory diabetes, rheumatoid arthritis, systemic lupus erythematosus, atopic dermatitis, leukocyte abnormalities, immunodeficiency associated with spleen dysfunction or thymic abnormalities), inflammatory bowel disease, allergic conjunctivitis, etc. Can be

The prophylactic / therapeutic agent is produced in the same manner as described above.

(7) DNA transfer animal

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

That is, the present invention

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

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

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

(4) It is intended to provide a recombinant vector containing the exogenous DNA of the present invention or its mutant DNA, which can be expressed in mammals.

Non-human mammals having the exogenous DNA of the present invention or the mutant DNA thereof (hereinafter abbreviated as the DNA-transferred animal of the present invention) include unfertilized eggs, fertilized eggs, germ cells containing spermatozoa and their primordial cells, and the like. In contrast, preferably, embryo development in non-human mammal development During the raw stage (more preferably, at the single cell or fertilized egg cell stage and generally before the 8-cell stage), calcium phosphate, electric pulse, lipofection, coagulation, microinjection, particle gun, DEAE-dextran, etc. It can be produced by transferring the target DNA. Further, by the DNA transfer method, the exogenous DNA of the present invention can be transferred to somatic cells, organs of living organisms, tissue cells, and the like, and used for cell culture, tissue culture, and the like. The DNA-transferred animal of the present invention can also be produced by fusing the above-mentioned germ cells with a cell fusion method known per se.

As non-human mammals, for example, porcupines, pigs, higgins, goats, magpies, dogs, cats, guinea pigs, hamsters, mice, rats and the like are used. Above all, rodents with relatively short ontogeny and biological cycle in terms of the creation of disease animal model systems, and easy to breed rodents, especially mice (for example, pure strains such as C57BLZ6 and DBA 2 As the system, Be CSFi system, BDF system, B6D2 system, BALB / c system, ICR system, etc.) or rat (for example, Wistar, SD, etc.) are preferable.

"Mammals" in a recombinant vector that can be expressed in mammals include humans and the like in addition to the above-mentioned non-human mammals.

The exogenous DNA of the present invention refers not to the DNA of the present invention originally possessed by a non-human mammal but to the DNA of the present invention once isolated and extracted from a mammal.

As the mutant DNA of the present invention, DNA having a mutation (for example, mutation) in the base sequence of the original DNA of the present invention, specifically, addition, deletion, substitution of another base, etc. DNA that has been used is used, and abnormal DNA is also included.

The abnormal DNA refers to a DNA that expresses an abnormal protein of the present invention. For example, a DNA that expresses a protein that suppresses the function of a normal protein of the present invention is used.

The exogenous DNA of the present invention may be derived from a mammal that is the same or different from the animal of interest. When the DNA of the present invention is transferred to a target animal, a DNA construct linked to a downstream of a promoter capable of expressing the DNA in animal cells is used. It is generally advantageous to use it as a struct. For example, when the human DNA of the present invention is transferred, DNs derived from various mammals (eg, egrets, dogs, cats, guinea pigs, hamsters, rats, mice, etc.) having the DNA of the present invention having high homology to the human DNA are transferred. Microinjection of a DNA construct (eg, vector 1) with the human DNA of the present invention downstream of various promoters capable of expressing A into a fertilized egg of a target mammal, for example, a mouse fertilized egg. A DNA-transferred mammal that highly expresses the DNA of the present invention can be created.

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

Examples of the promoter which regulates the expression of DΝ are, for example, a promoter of DNA derived from ① virus (eg, Simian virus, cytomegalovirus, Moroni leukemia virus, JC virus, breast cancer virus, poliovirus, etc.). 1, ② Promoters derived from various mammals (humans, egrets, dogs, cats, guinea pigs, hamsters, rats, mice, etc.), such as albumin, insulin II, peropkin II, eras ichie, erythropoietin, endothelin, Muscle creatine kinase, glial fibrillary acidic protein, daltathione S-transferase, platelet-derived growth factor i3, keratin Kl, 10 and 1 ^ 14, collagen type I and II, cyclic AMP Dependent protein kinase / 3 I subunit, dystrophy Protein, tartrate-resistant alkaline phosphatase, atrial natriuretic factor, endothelial receptor oral synthase (generally abbreviated as Tie 2), sodium potassium adenosine 3 kinase (Na, K-ATPase), neurofilament Light chain, meta-oral thionein I and IIA, meta-oral proteinase 1 tissue inhibitor, MHC class I antigen (H-2L), H-ras, renin, dopamine] 3-hydroxylase, thyroid peroxidase (TPO), peptide Chain elongation factor la (EF-1), β Actin, ο: and] 3 myosin heavy chain, myosin light chain 1 and 2, myelin basic protein, thyroglobulin, Thy_l, immunoglobulin, heavy chain variable region (VNP), serum amyloid P component, myoglobin, troponin C For example, promoters such as smooth muscle α-actin, preproenkephalin A, and vasopressin are used. Among them, a cytomegalovirus promoter capable of high expression throughout the whole body, a human peptide chain elongation factor 1a; (EF-1a) promoter, a human and a chicken j8 actin promoter, and the like are preferable.

The vector preferably has a sequence that terminates the transcription of the messenger RNA of interest in the DNA-transferred mammal (generally called terminator). For example, DNs derived from viruses and various mammals can be used. The sequence of A can be used, and preferably, SV40 terminator of simian virus or the like is used.

In addition, the splicing signal of each DNA, the enhancer region, a part of the intron of eukaryotic DNA, etc., are added 5 'upstream of the promoter region, between the promoter region and the translation region, in order to further express the target exogenous DNA. Alternatively, it can 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 is DNA derived from liver, kidney, thyroid cells, and fibroblasts derived from humans or various mammals (eg, rabbit, dog, cat, guinea pig, hamster, rat, mouse, etc.). And all or part of the genomic DNA from commercially available genomic DNA libraries, or the complementary DNA prepared by known methods from liver, kidney, thyroid cells, and fibroblast-derived RNA. Can be done. In addition, an exogenous abnormal DNA can produce a translation region obtained by mutating the translation region of a normal protein obtained from the above cells or tissues by point mutagenesis.

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

The transfer of the exogenous DNA of the present invention at the fertilized egg cell stage is dependent on the germ cell of the target mammal. Vesicles and somatic cells. The presence of the exogenous DNA of the present invention in the germinal cells of the animal after transfer of the DNA indicates that the exogenous DNA of the present invention is retained in all of the germ cells and somatic cells of the offspring of the animal. Means to do. The offspring of such animals that have inherited the foreign DNA of the present invention have the foreign DNA of the present invention in all of their germ cells and somatic cells.

The non-human mammal to which the exogenous normal DNA of the present invention has been transferred is confirmed to stably retain exogenous DNA by mating, and can be subcultured as a DNA-bearing animal in a normal breeding environment. I can do it.

Transfer of the exogenous DNA of the present invention at the fertilized egg cell stage is ensured to be present in excess in all germ cells and somatic cells of the target mammal. Excessive presence of the exogenous DNA of the present invention in the germinal cells of the transgenic animal after the DNA transfer indicates that all the offspring of the produced animal will have excessively exogenous DNA of the present invention in all of the germinal and somatic cells. Means having. The progeny of such animals that have inherited the exogenous DNA of the present invention have an excess of the exogenous DNA of the present invention in all of their germinal and somatic cells.

By obtaining a homozygous animal having the introduced DNA on both homologous chromosomes and mating the male and female animals, all offspring can be bred to have the DNA in excess.

The non-human mammal having the normal DNA of the present invention expresses the normal DNA of the present invention at a high level, and eventually promotes the function of the endogenous normal DNA. It may develop hyperfunction and can be used as a model animal for the disease. For example, using the normal DNA-transferred animal of the present invention, elucidation of the pathological mechanism of the protein hyperactivity of the present invention and diseases associated with the protein of the present invention, and examination of treatment methods for these diseases. It is possible.

Further, since the mammal into which the exogenous normal DNA of the present invention has been transferred has an increased symptom of the released protein of the present invention, it is a prophylactic / therapeutic agent for a disease associated with the protein of the present invention. Respiratory disease (eg, chronic obstructive pulmonary disease (chronic bronchitis, emphysema), diffuse panbronchiolitis, bronchial asthma, cystic fibrosis, irritable pneumonia, pulmonary fibrosis, etc.), rhinitis (eg, Allergic rhinitis, hay fever, acute rhinitis, chronic rhinitis, hypertrophic rhinitis, Atrophic rhinitis, dry rhinitis, vasomotor rhinitis, gangrenous rhinitis, sinusitis, etc., immune disorders (eg, myasthenia gravis, glomerulonephritis, multiple sclerosis, siedalen syndrome, insulin resistance) Screening tests for preventive and therapeutic agents such as diabetes, rheumatoid arthritis, systemic lupus erythematosus, atopic dermatitis, leukocyte abnormalities, immunodeficiency associated with spleen dysfunction or thymic abnormalities), inflammatory bowel disease, allergic conjunctivitis, etc. Is also available.

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

The non-human mammal having the abnormal DNA of the present invention expresses the abnormal DNA of the present invention at a high level, and finally inhibits the function of the endogenous normal DNA by inhibiting the function of the endogenous normal DNA. It may be functionally inactive refractory and can be used as a model animal for the disease. For example, using the abnormal DNA-transferred animal of the present invention, it is possible to elucidate the pathological mechanism of the protein inactive refractory of the protein of the present invention and to consider a method for treating this disease.

Further, as a specific possibility, the abnormal DNA-highly expressing animal of the present invention is useful for inhibiting the function of a normal protein by the abnormal protein of the present invention (dominant negative effect) in the inactive refractory disease of the protein of the present invention. ) Is a model for elucidating. Further, since the mammal to which the foreign abnormal DNA of the present invention has been transferred has an increased symptom of the released protein of the present invention, the agent for preventing or treating the protein of the present invention or the functionally inactive refractory disease is used. , For example, respiratory diseases (eg, chronic obstructive pulmonary disease (chronic bronchitis, emphysema), diffuse panbronchiolitis, bronchial asthma, cystic fibrosis, irritable pneumonia, pulmonary fibrosis, etc.), rhinitis (eg, Allergic rhinitis, hay fever, acute rhinitis, chronic rhinitis, hypertrophic rhinitis, atrophic rhinitis, dry anterior rhinitis, vasomotor rhinitis, gangrene rhinitis, sinusitis, etc., immune diseases (eg, myasthenia gravis) Asthenia, glomerulonephritis, multiple sclerosis, Sheddalen syndrome, insulin resistance diabetes, 'rheumatoid arthritis, systemic lupus erythematosus, atopic dermatitis, leukocyte abnormalities, spleen dysfunction or Thymus Always such accompanying immunodeficiency), it can also be utilized in the screening test prophylactic or therapeutic agent such as an agent for the prophylaxis or treatment of such inflammatory bowel diseases, allergic conjunctivitis.

In addition, other possible uses of the above two types of DNA-transferred animals of the present invention include, for example,

① Use as a cell source for tissue culture,

(2) The protein of the present invention can be specifically analyzed by directly analyzing the DNA or RNA in the tissue of the DNA-transferred animal of the present invention or by analyzing the peptide tissue expressed by DNA. Analysis of the relationship with expressed or activating peptides, ③ culturing cells of DNA-containing tissue by standard tissue culture techniques, and using these to study the function of cells from tissues that are generally difficult to culture,

薬剤 Screening of drugs that enhance cell function by using the cells described in ③ above, and

単離 Isolation and purification of the mutant protein of the present invention and production of its antibody are considered.

Furthermore, using the DNA-transferred animal of the present invention, it is possible to examine the clinical symptoms of a disease associated with the protein of the present invention, including a functionally inactive refractory disease of the protein of the present invention. More detailed pathological findings in each organ of the protein-related disease model can be obtained, which can contribute to the development of new treatment methods and the research and treatment of secondary diseases caused by the disease.

In addition, each organ is removed from the DNA-transferred animal of the present invention, and after minced, By using the proteolytic enzyme of the present invention, it is possible to obtain free DNA transfer cells, culture them, or systematize the cultured cells. Furthermore, it is possible to examine the specificity of the protein-producing cell of the present invention, its relationship with apoptosis, differentiation or proliferation, or its signal transduction mechanism, and its abnormalities. And effective research materials for elucidating its action.

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-transferred animal of the present invention, It is possible to provide an effective and rapid screening method for the therapeutic agent for the disease by using a method or the like. Further, using the transgenic animal of the present invention or the exogenous DNA expression vector of the present invention, it is possible to examine and develop a method for treating DNA associated with the protein of the present invention.

(8) Knockout animal

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

That is, the present invention

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

(2) The embryonic stem cell according to (1), wherein the DNA is inactivated by introducing a reporter gene (eg, an i3-galactosidase gene derived from Escherichia coli).

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

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

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

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

(7) The DNA is inactivated by introducing a repo overnight gene (eg, a j8-galactosidase gene derived from Escherichia coli), and the repo overnight gene controls the promoter for the DNA of the present invention. The non-human mammal according to item (6), which can be expressed under

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

(9) the non-human mammal according to (8), wherein the rodent is a mouse; and

(10) The test compound is administered to the animal described in (7), and the reporter gene expression And a method for screening a compound or a salt thereof that promotes or inhibits the promoter activity of the DNA of the present invention, characterized by detecting the following.

A non-human mammalian embryonic stem cell in which the DNA of the present invention has been inactivated is an artificially mutated DNA of the present invention possessed by the non-human mammal, which suppresses the expression ability of the DNA. Alternatively, by substantially losing the activity of the S-encoding protein of the present invention, the DNA has substantially no ability to express the protein of the present invention (hereinafter referred to as the knockout DNA of the present invention). This may be referred to as a non-human mammalian embryonic stem cell (hereinafter abbreviated as ES cell).

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

The DNA of the present invention can be artificially mutated by, for example, deleting part or all of the DNA sequence, or inserting or substituting another DNA sequence by a genetic engineering method. With these mutations, the knockout DNA of the present invention may be prepared, for example, by shifting the codon reading frame or disrupting the function of the promoter or exon.

Specific examples of the non-human mammalian embryonic stem cells in which the DNA of the present invention has been inactivated (hereinafter abbreviated as the DNA-inactivated ES cells of the present invention or the knockout ES cells of the present invention) include: The DNA of the present invention possessed by the target non-human mammal is isolated, and its exon portion is a drug resistance gene typified by a neomycin resistance gene, a hygromycin resistance gene, or 1acZ (i3-galactosidase gene), cat (Chloramphenicol acetyltransferase gene) Insertion of a repo-specific gene, such as the representative gene, disrupts the function of exons or terminates gene transcription in the intron between exons. By inserting a DNA sequence (for example, a poly A addition signal) and preventing the synthesis of complete messenger RNA, A DNA chain having a DNA sequence constructed so as to disrupt the gene (hereinafter abbreviated as a targeting vector) is introduced into the chromosome of the animal by, for example, a homologous recombination method. Southern hybridization analysis using a DNA sequence on or near the DNA of the present invention as a probe, or preparation of DNA sequence on targeting vector and evening targeting vector The DNA sequence can be obtained by analyzing the DNA sequence of a neighboring region other than the DNA of the present invention used as a primer by PCR and selecting the knockout ES cells of the present invention.

As the ES cells from which the DNA of the present invention is inactivated by the homologous recombination method or the like, for example, those already established as described above may be used, or according to the known Evans and Kaufma method. It may be a newly established one. For example, in the case of mouse ES cells, currently, 129 ES cells are generally used, but since the immunological background is not clear, it is an alternative pure immunological and genetic background. For example, for the purpose of obtaining ES cells in which C57BLZ6 mice and C57BL / 6 mice have a low number of eggs collected by crossing with DBAZ2, BDFi mice (C57BL / 6 and DBAZ2 It can be used satisfactorily with those established using F. BD Fi mice have the advantage of high number of eggs collected and the robustness of eggs, and also have the background of C57BL / 6 mice. ES cells obtained by using C57BL / 6 mice are advantageous in that when they create a disease model mouse, their genetic background can be replaced by C57BL / 6 mice by backcrossing with C57BL / 6 mice. When ES cells are established, generally 3.5 days after fertilization Although blastocysts of the eye are used, a large number of early embryos can be obtained efficiently by collecting and culturing the 8-cell stage embryos up to the blastocysts.

Although either male or female ES cells may be used, male ES cells are generally more convenient for producing germline chimeras. It is also desirable to discriminate between males and females as soon as possible in order to reduce the complexity of culturing.

An example of a method for determining the sex of ES cells is a method of amplifying and detecting a gene in the sex-determining region on the Y chromosome by PCR. By using this method, the number of ES cells in one colony (about 50) can be reduced, compared to about 10 ⁶ cells for karyotype analysis. The primary selection of ES cells in the early stage of culture can be performed by discriminating between males and females, and the early stages of culture can be greatly reduced by enabling the selection of male cells at an early stage. Examples of the second selection include chromosomes by the G-banding method. This can be done by checking the number. The number of chromosomes in the obtained ES cells is preferably 100% of the normal number, but if it is difficult due to physical operations at the time of establishment, knock out the gene of the ES cells, and However, it is desirable to clone again into cells whose chromosome number is 2 n = 40 in mice). .

Embryonic stem cell lines obtained in this way usually have very good proliferative properties, but must be subcultured carefully since they tend to lose their ontogenetic potential. For example,

In a CO2 incubator (preferably 5% CO2, 95% air or 5% oxygen, 5%) on a suitable feeder cell such as STO fibroblasts in the presence of LIF (1 to 10,000 U / ml) Culture at about 37 ° C in carbon dioxide gas, 90% air, etc. At the time of subculture, for example, trypsin ZE DTA solution (usually 0.001-0.5% trypsin / 0.1 l) -5 mM EDTA (preferably, about 0.1% trypsin / ImM EDTA), and the cells are seeded on a freshly prepared feeder cell. Such subculture is usually performed every 113 days. At this time, it is desirable to observe the cells and, if any morphologically abnormal cells are found, discard the cultured cells.

ES cells are differentiated into various types of cells, such as parietal, visceral, and cardiac muscle, by monolayer culture up to high density or suspension culture until cell clumps are formed under appropriate conditions. [MJ Evans and Μ · H. Kaufman,

Nature, 292, 154, 1981; GR Mart in, Proc. Natl. Acad. Sci. USA 78, 7634, 1981; TC Doetschman et al., Journal of Embryology 'Ande'. Experimental 'Morphology I, Vol. 87, p. 27, 1985], and the DNA-deficient cell of the present invention obtained by differentiating the ES cell of the present invention is a cell biology of the protein of the present invention in the mouth. Is useful in technical studies.

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

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

The non-human mammal deficient in DNA expression of the present invention can be prepared, for example, by introducing the targeting vector prepared as described above into mouse embryonic stem cells or mouse egg cells, A homologous set in which a DNA sequence in which the DNA of the present invention is inactivated, which is one of the targeting vectors, replaces the DNA of the present invention on the chromosome of mouse embryonic stem cells or mouse egg cells by gene homologous recombination. By performing the replacement, the DNA of the present invention can be knocked out.

Cells in which the DNA of the present invention has been knocked out are subjected to Southern hybridization analysis using DNA sequences on or near the DNA of the present invention as a probe or DNA sequences on a targeting vector and targeting DNA. The determination can be made by PCR analysis using a mouse as a primer and a DNA sequence in the vicinity region other than the DNA of the present invention derived from the mouse used in the vector. When a non-human mammalian embryonic stem cell is used, the DNA of the present invention is inactivated by the homologous recombination to clone a follicular H follicle strain, and the cell is cultured at an appropriate time, for example, at the 8-cell stage. The chimeric embryo is injected into a human mammalian embryo or blastocyst, and the resulting chimeric embryo is implanted into the uterus of the pseudopregnant non-human mammal. The produced animal is a chimeric animal composed of both a normal cell having the DNA locus of the present invention and an artificially mutated cell having the DNA locus of the present invention.

When a part of the germ cells of the chimeric animal has the mutated DNA locus of the present invention, all tissues are artificially mutated from a population obtained by crossing such a chimeric individual with a normal individual. It can be obtained by selecting individuals composed of cells having the added DNA locus of the present invention by, for example, determining the coat color. The individual thus obtained is usually an individual having a heterozygous expression of the protein of the present invention, which is crossed with an individual having a heterozygous expression of the protein of the present invention. Can be obtained.

When an egg cell is used, for example, a transgenic non-human mammal having a targeting vector introduced into a chromosome can be obtained by injecting a DNA solution into the egg cell nucleus by a microinjection method. Compared to a genic non-human mammal, it can be obtained by selecting those having a mutation at the DNA locus of the present invention by homologous recombination of the gene.

In the individual knocked out of the DNA of the present invention in this manner, the animal individual obtained by mating is usually bred with the nuclear recognition that the DNA has been knocked out. Breeding can be carried out in the environment.

Furthermore, the germ line can be obtained and maintained according to a standard method. That is, by crossing male and female animals having the inactivated DNA, a homozygote having the inactivated DNA in both homologous chromosomes can be obtained. Animals can be obtained. The obtained homozygous animal can be efficiently obtained by rearing it in a mother animal in such a manner that there are 1 normal individual and a plurality of homozygotes. By crossing male and female heterozygous animals, homozygous and heterozygous animals having the inactivated DNA are bred and passaged.

The non-human mammalian embryonic stem cells in which the DNA of the present invention has been inactivated are extremely useful for producing the non-human mammal deficient in expressing the DNA of the present invention.

In addition, since the non-human mammal deficient in expression of the DNA of the present invention lacks various biological activities that can be induced by the protein of the present invention, it causes inactivation of the biological activity of the protein of the present invention. Since it can be a model of disease, it is useful for investigating the causes of these diseases and examining treatment methods.

(8a) A method for screening a compound having a therapeutic / preventive effect against diseases caused by DNA deficiency or damage, etc. of the present invention

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

That is, the present invention comprises administering a test compound to a non-human mammal deficient in expressing the DNA of the present invention, and observing and measuring changes in the animal. Diseases caused by, for example, respiratory diseases (eg, chronic obstructive pulmonary disease (chronic bronchitis, emphysema), diffuse panbronchiolitis, bronchial asthma, cystic fibrosis, irritable pneumonia, pulmonary fibrosis, etc.), Rhinitis (eg, allergic rhinitis, hay fever, acute rhinitis, chronic rhinitis, hypertrophic rhinitis, atrophic rhinitis, dry pronasitis, vasomotor rhinitis, gangrenous rhinitis, sinusitis, etc.), immune diseases ( Examples, myasthenia gravis, glomerulonephritis, multiple sclerosis, siedalen syndrome, insulin resistance diabetes, rheumatoid arthritis, systemic lupus erythematosus, atopic dermatitis, leukocyte abnormalities, splenic dysfunction or thymic dysthymus Such as an immune deficiency) associated with, Provided is a method for screening a compound or a salt thereof having a therapeutic / preventive effect on inflammatory bowel disease, allergic conjunctivitis, and the like.

Examples of the non-human mammal deficient in DNA expression of the present invention used in the screening method include those described above.

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

Specifically, a non-human mammal deficient in expression of the DNA of the present invention is treated with a test compound, compared with a non-treated control animal, and changes in organs, tissues, disease symptoms, etc. of the animal are used as indices. Therapeutic and prophylactic effects of test compounds can be tested.

As a method for treating a test animal with a test compound, for example, oral administration, intravenous injection and the like are used, and it can be appropriately selected according to the symptoms of the test animal, the properties of the test compound, and the like. The dose of the test compound can be appropriately selected according to the administration method, the properties of the test compound, and the like.

For example, respiratory diseases (eg, chronic obstructive pulmonary disease (chronic bronchitis, emphysema), diffuse panbronchiolitis, bronchial asthma, cystic fibrosis, irritable pneumonia, pulmonary fibrosis, etc.), rhinitis (eg, allergy) Rhinitis, hay fever, acute rhinitis, chronic rhinitis, hypertrophic rhinitis, atrophic rhinitis, dryness proprietary rhinitis, vasomotor rhinitis, gangrene rhinitis, sinusitis, etc.), immune diseases (eg, myasthenia gravis, Glomerulonephritis, multiple sclerosis, siedalen syndrome, insulin resistance diabetes, rheumatoid arthritis, systemic lupus erythematosus, atopic dermatitis, leukocyte abnormalities, immunodeficiency associated with splenic dysfunction or thymic abnormalities), inflammatory bowel When screening for a compound having a therapeutic or prophylactic effect against diseases, allergic conjunctivitis, etc., a test compound may be added to a non-human mammal deficient in DNA expression of the present invention. Administered, the difference of the test compound non-administration group and emphysema of the lungs, the differences in respiratory function is observed over time in the tissue.

In the screening method, when the test compound is administered to a test animal, the disease symptom of the test animal is improved by about 10% or more, preferably about 30% or more, more preferably about 50% or more. Test compound has therapeutic and preventive effects on the above diseases Can be selected as the compound.

The compound obtained by using the screening method is a compound selected from the above-described test compounds, and has a therapeutic / preventive effect on a disease caused by a deficiency or damage of the protein of the present invention. Safe and low toxic prophylaxis-can be used as a medicament such as a therapeutic agent. Furthermore, compounds derived from the compounds obtained by the above screening can be used in the same manner.

The compound obtained by the screening method may form a salt. Examples of the salt of the compound include physiologically acceptable acids (eg, inorganic acids, organic acids, etc.) and bases (eg, alkali metal And the like, and especially preferred are physiologically acceptable acid addition salts. Examples of such salts include salts with inorganic acids (eg, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid, etc.), and organic acids (eg, acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, Salts with succinic acid, tartaric acid, citric acid, malic acid, oxalic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid, etc. are used.

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

The preparations obtained in this way are safe and low toxic and can be used, for example, in humans or mammals (eg, rats, mice, guinea pigs, egrets, sheep, pigs, pigs, dogs, cats, dogs, monkeys). Etc.).

The dose of the compound or a salt thereof varies depending on the target disease, the subject of administration, the administration route, and the like. For example, when the compound is orally administered, it is generally used in adult (as a body weight of 60 kg) emphysema patients. Administers about 0.1 to 10 Omg, preferably about 1.0 to 5 Omg, more preferably about 1.0 to 20 mg of the compound per day. When administered parenterally, the single dose of the compound varies depending on the administration subject, target disease, and the like. For example, the compound is usually administered in the form of an injection to an adult (with a body weight of 6 O kg). When administered to patients with pulmonary emphysema, intravenous injection of about 0.01 to 3 Omg, preferably about 0.1 to 2 Omg, more preferably about 0.1 to 1 Omg of the compound per day It is convenient to administer by In the case of other animals, the dose can be administered in terms of weight per 60 kg. (8b) Method for screening for a compound that promotes or inhibits the activity of a promoter for DNA of the present invention

The present invention provides a compound which promotes or inhibits the activity of a promoter for DNA of the present invention, which comprises administering a test compound to a non-human mammal deficient in expressing the DNA of the present invention and detecting the expression of a reporter gene. Or a method for screening a salt thereof.

In the above screening method, the non-human mammal deficient in expression of the DNA of the present invention may be such that the DNA of the present invention is inactivated by introducing a reporter gene among the non-human mammals deficient in expressing the DNA of the present invention. A gene whose reporter gene 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 compounds as described above.

As the reporter gene, the same ones as described above are used, and a / 3-galactosidase gene (1 ac Z), a soluble alkaline phosphatase gene or a luciferase gene is preferable.

In a non-human mammal deficient in expression of the DNA of the present invention in which the DNA of the present invention has been replaced with a reporter gene, the reporter gene is under the control of the promoter for the DNA of the present invention, so that the substance encoded by the reporter gene may By tracing the expression, the activity of the promoter can be detected.

For example, when a part of the DNA region encoding the protein of the present invention is replaced by an i3_galactosidase gene (1 ac Z) derived from Escherichia coli, the tissue of the present invention that originally expresses the protein of the present invention J3_galactosidase is expressed instead of this protein. Therefore, for example, the present invention can be easily carried out by staining with a reagent which is a substrate of 3-galactosidase, such as 5-bromo-4-chloro-3-indolyl / 3 / 3-galactopyranoside (X-ga1). It is possible to observe the state of expression of this protein in animal organisms. Specifically, a protein-deficient mouse of the present invention or a tissue section thereof is fixed with dartalaldehyde or the like, washed with a phosphate buffered saline (PBS), and then stained with Xga1 at room temperature or at 37 ° C. After reacting for about 30 minutes to 1 hour at around ° C, wash the tissue specimen with ImM EDTA / PBS solution. Therefore, it is sufficient to stop the] S-galactosidase reaction and observe the coloration. Further, mRNA encoding 1 ac Z may be detected according to a conventional method.

The compound or a salt thereof obtained by using the above-mentioned screening method is a compound selected from the above-mentioned test compounds, and is a compound that promotes or inhibits the promoter activity for DNA of the present invention.

The compound obtained by the screening method may form a salt. Examples of the salt of the compound include physiologically acceptable acids (eg, inorganic acids) and bases (eg, alkali metals). And the like, and especially preferred are physiologically acceptable acid addition salts. Examples of such salts include inorganic acids (eg, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid, etc.). Or a salt with an organic acid (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.

The compound or a salt thereof that promotes or inhibits the promoter activity of the DNA of the present invention or the salt thereof can regulate the expression of the protein of the present invention and regulate the function of the protein. Diseases (chronic bronchitis, emphysema), diffuse panbronchiolitis, bronchial asthma, cystic fibrosis, irritable pneumonia, pulmonary fibrosis, etc.), rhinitis (eg, allergic rhinitis, hay fever, acute rhinitis, chronic rhinitis) , Hypertrophic rhinitis, atrophic rhinitis, dry rhinitis, vasomotor rhinitis, gangrene rhinitis, sinusitis, etc., immune diseases (eg, myasthenia gravis, glomerulonephritis, multiple sclerosis, siedalen syndrome) , Insulin resistance diabetes, rheumatoid arthritis, systemic lupus erythematosus, atopic dermatitis, leukocyte abnormalities, immunodeficiency associated with splenic dysfunction or thymic abnormalities ), Inflammatory bowel disease, is useful as a prophylactic or therapeutic agent such as § Rerugi one conjunctivitis.

Further, compounds derived from the compounds obtained by the above screening can be used in the same manner.

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

The preparations obtained in this way are safe and of low toxicity, for example human or It can be administered to mammals (for example, rats, mice, guinea pigs, egrets, sheep, sheep, bush, horses, horses, cats, dogs, monkeys, etc.).

The dose of the compound or a salt thereof varies depending on the disease to be treated, the subject of administration, the administration route, and the like. In a patient with emphysema weighing 60 kg, the compound is administered at about 0.1 to 100 mg, preferably about 1.0 to 50 mg, more preferably about 1.0 to 20 mg per day. When administered parenterally, the single dose of the compound varies depending on the administration subject, target disease, and the like.For example, a compound that inhibits the promoter activity against DNA of the present invention may be in the form of an injection. Normally, when administered to an adult (assuming a body weight of 60 kg) emphysema patients, the compound is administered daily at about 0.01-3 Omg, preferably about 0.1-2 Omg, more preferably about 0.1-: L per day. It is convenient to administer 0 mg by intravenous injection. In the case of other animals, the dose can be administered in terms of weight per 60 kg.

As described above, the non-human mammal deficient in expression of the DNA of the present invention is extremely useful for screening a compound or a salt thereof which promotes or inhibits the activity of the promoter of the DNA of the present invention overnight. Yes, it can greatly contribute to the investigation of the causes of various diseases caused by DNA expression deficiency of the present invention or the development of preventive and therapeutic agents.

Also, using a DNA containing the promoter region of the protein of the present invention, genes encoding various proteins are ligated downstream thereof and injected into egg cells of an animal to produce a so-called transgenic animal (transgenic animal). ) Makes it possible to specifically synthesize the protein and examine its action in living organisms. Furthermore, by linking an appropriate reporter gene to the above promoter portion and establishing a cell line that expresses the reporter gene, a low-molecular compound having an action of specifically promoting or suppressing the production ability of the protein itself of the present invention in the body. Can be used as a search system. (9) a medicine containing the protein of the present invention

Since the expression of the protein of the present invention is reduced in the lung having emphysema lesions, the protein of the present invention is defective or defective in the DNA encoding the protein of the present invention, or the protein of the present invention is defective. If the expression level of is reduced, for example, Respiratory disease (eg, chronic obstructive pulmonary disease (chronic bronchitis, emphysema), diffuse panbronchiolitis, bronchial asthma, cystic fibrosis, irritable pneumonia, pulmonary fibrosis, etc.), rhinitis (eg, a Rarginous rhinitis, hay fever, acute rhinitis, chronic rhinitis, hypertrophic rhinitis, atrophic rhinitis, dry pre-rhinitis, vasomotor rhinitis, gangrene rhinitis, sinusitis, etc., immune diseases (eg, myasthenia gravis) Disease, glomerulonephritis, multiple sclerosis, Sheddalen syndrome, insulin resistance diabetes, rheumatoid arthritis, systemic lupus erythematosus, atopic dermatitis, leukocyte abnormalities, immunodeficiency associated with spleen dysfunction or thymic abnormalities), Various diseases such as inflammatory bowel disease and allergic conjunctivitis develop.

Therefore, the protein of the present invention and the DNA of the present invention include, for example, respiratory diseases [eg, chronic obstructive pulmonary disease (chronic bronchitis, emphysema), diffuse panbronchiolitis, bronchial asthma, cystic fibrosis, Hypersensitivity pneumonia, pulmonary fibrosis, etc.), rhinitis (eg, allergic rhinitis, hay fever, acute rhinitis, chronic rhinitis, hypertrophic rhinitis, atrophic rhinitis, dry pro-rhinitis, vasomotor rhinitis, gangrene rhinitis, Sinusitis, etc., immune diseases (eg, myasthenia gravis, glomerulonephritis, multiple sclerosis, siedalen syndrome, insulin resistant diabetes, rheumatoid arthritis, systemic lupus erythematosus, atopic dermatitis, leukocyte abnormalities, It can be used as a medicine to prevent or treat spleen dysfunction or immunodeficiency due to thymic abnormalities), inflammatory bowel disease, allergic conjunctivitis, etc. Wear. Preferably, it is a prophylactic / therapeutic agent for a respiratory disease or the like, more preferably a prophylactic / therapeutic agent for a chronic obstructive pulmonary disease or the like, and further preferably a prophylactic / therapeutic agent for an emphysema or the like.

For example, when there is a patient in whom the protein of the present invention is reduced or deficient in a living body, (a) by administering the DNA of the present invention to the patient and expressing the protein of the present invention in the living body, (Mouth) inserting the DNA of the present invention into cells and expressing the protein of the present invention, and then transplanting the cells into a patient; or (8) administering the protein of the present invention to the patient. By doing so, the role of the protein of the present invention in the patient can be sufficiently or normally exerted.

When the DNA of the present invention is used as the above-mentioned prophylactic or therapeutic agent, the DNA may be used alone or after being inserted into an appropriate vector such as a retrovirus vector, an adenovirus vector, an adenovirus associated virus vector, or the like. , According to conventional means And can be administered to humans or warm-blooded animals. The DNA of the present invention can be administered as it is or in the form of a formulation together with a physiologically acceptable carrier such as an adjuvant for promoting uptake, and administered with a gene gun or a catheter such as a hydrogel catheter. When the protein of the present invention is used as the above-mentioned prophylactic / therapeutic agent, the protein purified to at least 90%, preferably 95% or more, more preferably 98% or more, and still more preferably 99% or more. It is preferred to use.

The protein of the present invention can be used, for example, in the form of tablets, capsules, elixirs, microcapsules, or the like, which are sugar-coated as required, orally, or aseptic with water or other pharmaceutically acceptable liquids. It can be used parenterally in the form of injections, such as solutions or suspensions. For example, the protein of the present invention is mixed with physiologically acceptable carriers, flavors, excipients, vehicles, preservatives, stabilizers, binders, etc. in a unit dosage form generally required for the practice of preparations. It can be manufactured by The amount of the active ingredient in these preparations is such that a suitable dosage in the specified range can be obtained.

Additives that can be incorporated into tablets, capsules, etc. include, for example, binders such as gelatin, corn starch, tragacanth, gum arabic, excipients such as crystalline cellulose, corn starch, gelatin, alginic acid, etc. Swelling agents such as magnesium stearate, sweeteners such as sucrose, lactose or saccharin, and flavoring agents such as peppermint, cocoa oil or cherry. When the unit dosage form is forcepsel, the above type of material may further contain a liquid carrier such as oil and fat. Sterile compositions for injection can be formulated according to standard pharmaceutical practice, such as by dissolving or suspending the active substance in a vehicle such as water for injection, or naturally occurring vegetable oils such as sesame oil or coconut oil. it can.

Aqueous solutions for injection include, for example, physiological saline, isotonic solutions containing glucose and other adjuvants (eg, D-sorbyl), D-manni! And suitable solubilizers, for example, alcohols (eg, ethanol), polyalcohols (eg, propylene glycol, polyethylene glycol, etc.), nonionic surfactants (polysorbate 80 ™, HCO — 5 0 Etc.). Examples of the oily liquid include sesame oil and soybean oil, and may be used in combination with benzyl benzoate, benzyl alcohol, or the like as a solubilizing agent. In addition, buffers (eg, phosphate buffer, sodium acetate buffer, etc.), soothing agents (eg, benzalkonium chloride, proforce hydrochloride, etc.), stabilizers (eg, human serum albumin, polyethylene glycol, etc.) It may be blended with preservatives (eg, benzyl alcohol, phenol, etc.), antioxidants and the like. The prepared injection liquid is usually filled in a suitable ampoule.

The vector into which the DNA of the present invention has been inserted is also formulated in the same manner as described above, and is usually used parenterally.

The preparations obtained in this way are safe and have low toxicity, for example, in warm-blooded animals (eg, humans, rats, mice, guinea pigs, egrets, birds, birds, higgies, bushus, dogs, dogs, cats). , Dogs, monkeys, chimpanzees, etc.). The dosage of the protein of the present invention varies depending on the target disease, the administration subject, the administration route, and the like. For example, when the protein of the present invention is orally administered for the purpose of treating infertility, it is generally required for an adult (body weight 6). (As 0 kg), about 0.1 to 10 Omg, preferably about 1.0 to 5 Omg, more preferably about 1.0 to 20 mg of the protein is administered per day. When administered parenterally, a single dose of the protein may vary depending on the administration subject, target disease, and the like. (As O kg), the protein per day is about 0.01 to 3 O mg, preferably about 0.1 to 2 O mg, more preferably about 0.1 to 1 O mg per day. Is conveniently administered by injection into the affected area. In the case of other animals, the dose can be administered in terms of the body weight of 60 kg.

In the present specification, bases, amino acids, and the like are represented by abbreviations based on the abbreviations by IUPAC-IUB Communication on Biochemical Nomenclature or commonly used abbreviations in the art, and examples thereof are described below. When amino acids can have optical isomers, the L-form is indicated unless otherwise specified.

DNA: Deoxyribonucleic acid

c DNA: complementary deoxylipo nucleic acid A adenine

T thymine

G Guanin

C RNA Liponucleic acid

mRNA Messenger liponucleic acid dATP Deoxyadenosine triphosphate dTTP Deoxythymidine triphosphate dGTP Deoxyguanosine triphosphate d CTP Deoxycytidine triphosphate

ATP adenosine triphosphate

EDTA ethylenediaminetetraacetic acid

SD S Sodium dodecyl sulfate

G 1 y Glycine

A la alanine

Va 1 Valine

Leu Leucine

I 1 e

S e r serine

Th r threonine

C y s Sistine

Me t Methionin

G 1 u Dalminic acid

As aspartic acid

Lys lysine

Ar g Arginine

H is histidine

Ph e feniralanin Ty r tyrosine

T r p Tribute fan

P ro

A s n asparagine

G 1 n Dal Yu Min

p G 1 u pyroglutamic acid

S e c (selenocysteine)

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

Me methyl group

Etethyl group

Bu butyl group

P h phenyl group

TC thiazolidine-1 (R) monocarboxamide group

Tosp-toluenesulfonyl

CHO Holmill

B z 1

Cl Bzl 2, 6—Si

Bom benzyloxymethyl

Z benzyloxycarponyl

B o c t—butoxycarponyl

DNP dinitrophenyl

T r t Trityl

Bum t-butoxymethyl

Fmo c N— 9 _fluorenylmethoxycarbonyl

HOB t 1-hydroxybenztriazole HO OB t: 3, 4-dihydro-3-hydroxy-4-oxo-1

1,2,3-Benzotriazine

HONB: trihydroxy-5-norpolene-2,3-dicarpoxyimide The sequence numbers in the sequence listing in the present specification show the following sequences.

[SEQ ID NO: 1]

The amino acid sequence of human procollagen C-protease enhancer protein 2 is shown.

[SEQ ID NO: 2]

This shows the base sequence of DNA encoding human procollagen C—protein-enhancer brotin 2 having the amino acid sequence represented by SEQ ID NO: 1.

[SEQ ID NO: 3]

The amino acid sequence of mouse procollagen C-protease enhancer protein 2 is shown.

[SEQ ID NO: 4]

This shows the nucleotide sequence of DNA encoding mouse procollagen C-proteinase enhancer brotin 2 having the amino acid sequence represented by SEQ ID NO: 3. [SEQ ID NO: 5]

3 shows the nucleotide sequences of primers used in Examples 2 and 3.

[SEQ ID NO: 6]

3 shows the nucleotide sequences of primers used in Examples 2 and 3.

[SEQ ID NO: 7]

3 shows the nucleotide sequences of primers used in Examples 2 and 3.

[SEQ ID NO: 8]

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

Hereinafter, the present invention will be more specifically described with reference to examples, but the present invention is not limited thereto. Example

Example 1

(1) Tobacco smoke exposure COP D model mouse production

In the C0PD model, C57BL / 6N mice (6-week-old, Japan Charls River) were inhaled with mainstream smoke generated from Kentucky Reference Cigarette 1R1 for 1 to 4 hours / day, 5 days / week for a total of 6 months. Produced. That is, the Kentucky Reference Cigarette 1R1 was attached to an evening smoke generator (SG-200, Shibata Kagaku Co., Ltd.), and mainstream smoke was collected under the conditions of 35 ml / puff, 10 puff / min, and 25 puif / cigarette. After the mainstream smoke obtained was diluted to 3% (V / V) with air, it was sent to the acrylic exposure chamber containing the mouse, and the spontaneously breathing mouse was allowed to inhale cigarette smoke for a specified period of time. . A normal mouse was used for the control group. The lung function of the mouse was evaluated using the pressure-volume curve of the isolated lung. That is, the mice were anesthetized with pentobarbital (70 mg / kg, ip) on the day after exposure to cigarette smoke for 1 month, 3 months and 6 months, and the neck was incised and the trachea was forced into the air. （(Surflow indwelling needle, 18G) was inserted. The tracheal force Nyure ventilator connecting the (Harvard Co.), under the diaphragm resection was facilities for 10 minutes ventilator by 99.995% 0 _2. The ventilation was adjusted so that the change in airway pressure at that time was 10 cm cm0. After artificial respiration, the trachea was closed with an arterial clip and degassing was performed in the lungs, and the lungs were removed. A formalin buffer solution was sequentially injected into the isolated lung at a pressure of 0 to 25 cm¾0, and the volume of the lung at every 5 cm¾0 was measured using a prethysmograph (OJgobasil) to obtain a pressure-volume curve of the removed lung. Was. The results are shown in Figure 1.

(2) Search for genes whose expression fluctuates in the lungs of C0PD model mice exposed to cigarette smoke To identify genes whose expression is fluctuated specifically in the lungs of C0PD model mice exposed to cigarette smoke, Was killed by ventalpital anesthesia, and bronchoalveolar lavage was performed, and the lungs were removed. The isolated lung was frozen in liquid nitrogen, crushed with a frozen tissue crusher, and then immersed in Isogen (Wako Pure Chemical Industries, Ltd.) equivalent to 10 times its wet weight. 1 month tobacco smoke exposure group (n = 10), its control group (n = 6), 3 month tobacco smoke exposure group (n = 8), its control group (n = 8), 6 month tobacco smoke exposure group ( n = 8) and the control group (n = 8) Using total RA extracted according to the manual as a material, gene expression analysis was performed using an oligonucleotide microarray (Mouse Genome U94A, U94B, U94C; Asymetrix).

• The experiment method was in accordance with the experiment guide (Expression analys is technical manual) of Aiymetrix. As a result, af fymetrix No. 108315_at was detected as a gene whose expression was specifically reduced in C0PD model mice exposed to cigarette smoke. When a BLAST search was performed based on this sequence, the gene was found to encode mouse procollagen C-protein-Hansa-protein 2 (PC0LCE2). Its human counterpart is human procollagen C-protease enhancer protein 2

(hPC0LCE2). Example 2

Confirmation of expression fluctuation by quantitative RT-PCR method

In order to confirm that the expression fluctuation of the PC0LCE2 gene was remarkable even at the individual level, the expression of each individual was examined by quantitative RT-PCR.

Using 200 ng of total RNA prepared from mouse lung tissue as a starting material, cDNA was synthesized by reverse transcription in a reaction mixture of 501 using TaQMan Gold RT-PCR Kit (manufactured by Applied Biosystems). After diluting the reaction solution 5 times with distilled water, use it for real-time quantitative analysis using ABI PRISM 7900 Sequence Detector (Applied Biosystems) and Quant iTect SYBR Green PCR Kit (QIAGEN). The mouse PC0LCE2 gene copy number was measured by PCR. The primers [Primer 1 (SEQ ID NO: 5) and Primer 2 (SEQ ID NO: 6)] used for the detection of the gene amount were obtained from the Primer Expression program based on the nucleotide sequence of the mouse PC0LCE2 gene [GenBank Accession Number: AF352788]. Designed using As a standard sample for calculating the copy number, total RNA extracted from mouse lung tissue was subjected to RT-PCR by using primers 3 (SEQ ID NO: 7) and primer 14 (SEQ ID NO: 8) for type I. The concentration of the amplified DNA fragment consisting of 515 base pairs was measured with a Spectrophotometer and prepared by serial dilution. Similarly, copy of GAPDH gene as housekeeping gene The number was measured. In order to eliminate non-specific amplification, samples without reverse transcriptase were treated in the same manner, and the number of gene copies per total RNA was calculated from the following formula. The expression of each control mouse lung was compared. (PC0LCE2 gene copy number in reverse transcriptase-containing sample / one copy of GAPDH gene) i (PC0LCE2 gene copy number / GAPDH gene copy number in reverse transcriptase-free sample) = (PC0LCE2 gene expression level)

The result is shown in figure 2.

From this, it was found that the expression of the mouse PC0LCE2 gene (SEQ ID NO: 4) was significantly reduced in the lungs of the C0PD model mouse (** p≤0.01). Example 3

Analysis of tissue distribution of mouse PCOLCE 2 gene product

Mouse cDNA (Mouse MTC panel I and Mouse MTC panel II) for each mouse tissue (bone marrow, lymph node, prostate, thymus, stomach, uterus, heart, brain, spleen, lung, liver, kidney, testis, day 11 embryo): ABI PRISM 7900 Sequence Detector (manufactured by Applied Biosystems) and QuantiTect (Clontech) and cigarette smoke-exposed model mouse lung and control mouse lung cDNA (mixed group treated for 1 month, 3 months and 6 months) Mouse PC0LCE gene expression distribution was examined by real-time quantitative PCR using SYBR Green PC Kit (QIAGEN). The primers [Primer 1 (SEQ ID NO: 5) and Primer 2 (SEQ ID NO: 6)] used for the detection of the gene amount were designed using the Primer Express program from the mouse PC0LCE2 gene base sequence CGenBank Accession Number: AF352788]. As a standard sample for calculating the copy number, total RNA extracted from mouse lung tissue was subjected to RT-PCR using primers (SEQ ID NO: 7) and primer 4 (SEQ ID NO: 8) with type III. The concentration of the DNA fragment consisting of 503 base pairs amplified by the above was measured using a Spectrophotometer and prepared by serial dilution. Similarly, the copy number of the GAPDH gene as a housekeeping gene was measured. In order to eliminate non-specific amplification, samples without reverse transcriptase are treated in the same manner, and the expression level is determined by calculating the number of gene copies per total RNA from the following formula. I asked.

(One copy of the PC0LCE2 gene in the sample containing reverse transcriptase / number of copies of the GAPDH gene) one (number of copies of the PC0LCE2 gene / number of copies of the GAPDH gene in the sample containing no reverse transcriptase) = (expression of the PC0LCE2 gene)

The results are shown in Figure 3.

As a result, it was revealed that the mouse PC0LCE2 gene product (niRNA) was specifically expressed in the lung. Example 4

(1) Preparation of elastase-induced COPD model mouse

In the C0PD model, C57BL / 6N mice (8-week-old, Nippon-Charles River) were administered nasal administration of bushu knee elastase solution (Wako Pure Chemical Industries) ^ imits O xL / mouse under halothane anesthesia. Produced. In addition, a physiological saline solution nasal mouse was used as a control group. Mouse lung function was evaluated using the pressure-volume curve of the isolated lung. That is, 1, 3, and 7 days after administration of elastase, the mice were anesthetized with pentobarbital (70 mg / kg intraperitoneal injection) 14 days, 21 days and 35 days later, and then the neck was incised and the tracheal force was applied to the trachea. A self-flow indwelling needle, 18G) was introduced. The tracheal force Nyure the ventilator (Harvard Co., Ltd.) consolidated and subjected to 10 minutes ventilator with 99.995% 0 ₂ under diaphragmatic resection. The ventilation was adjusted so that the airway load pressure at that time was 10 cm0. After the end of the artificial respiration, the trachea was closed with an arterial clip to perform degassing in the lung, and the lung was removed. To the isolated lung, formalin buffer was sequentially injected under a load pressure of 0 to 25 cm¾0, and the volume of the lung at intervals of 5 cm¾0 was measured using a lethysmograph (Ugobasil). A lung pressure-volume curve was determined. In addition, the compliance value was calculated as an index of the ease of lung expansion according to the following equation.

(Lung volume increase under a load pressure of 25 cm¾0 (mL)) / 25 (cmH ₂₀ ) = Compliance value The results are shown in FIGS. 4 and 5.

(2) Search for genes whose expression is fluctuated in the lungs of the C〇PD model induced by Erasasease. For clarification, mice were sacrificed by ventalpital anesthesia 1 day, 3 days, 7 days, 14 days, 21 days and 35 days after administration of Elas evening, and the lungs were removed. The removed lung was frozen in liquid nitrogen, crushed with a frozen tissue crusher, and then immersed in IS0GEN (Wako Pure Chemical Industries), which is equivalent to 10 times its wet weight. Group 1 day after administration of Elas Yuze, group 3 days after administration of Elas Yuze, group 7 days after administration of Elas Yuise, group 14 days after administration of elastase, group 21 days after administration of Elas Yuze, group 35 days after administration of elastase n = 6) and their control groups (n = 6 in each case), total RNA was prepared using IS0GEN according to the attached manual.

Using total RNA Ig prepared from mouse lung tissue as a starting material, M-MLV Reverse Transcriptase (manufactured by Invitrogen) was used to synthesize cDNA by a reverse transcription reaction in 501 reaction solutions. Using the reaction solution, the amount of mouse PC0LCE2 gene was measured by a real-time quantitative PCR method using ABI PRISM 7700 Sequence Detector I (manufactured by Applied Biosystems). The primers [Primer 1 (SEQ ID NO: 5) and Primer 2 (SEQ ID NO: 6)] used for the detection of the gene amount were obtained from the mouse PC0LCE2 gene base sequence CGenBank Accession Number: AE 352788] using the Primer Express program. Designed. Similarly, the amount of 18S liposomal RNA gene as a housekeeping gene was measured. The PC0LCE2 gene expression amount per 18S liposomal RNA gene expression amount was determined from the following equation, and the expression of each of the lungs of the C0PD model mouse and the control mouse lung induced by Erasase was compared.

(PC0CE2 gene expression level / 18S liposome RNA gene expression level) = (PC0LCE2 gene expression results The results are shown in FIG.

From this, it was found that the expression of the PC0LCE2 gene (SEQ ID NO: 4) was significantly reduced (* ≦ 0.05) in the lungs of Erasase-induced C0PD model mice. Industrial applicability

The proteins and polynucleotides of the present invention include, for example, respiratory diseases [eg, chronic obstructive pulmonary disease (chronic bronchitis, emphysema), diffuse panbronchiolitis, bronchial asthma, sac Alveolar fibrosis, irritable pneumonia, pulmonary fibrosis, etc.), rhinitis (eg, allergic rhinitis, hay fever, acute rhinitis, chronic rhinitis, hypertrophic rhinitis, atrophic rhinitis, dry pronasitis, vasomotor rhinitis, gangrene Rhinitis, sinusitis, etc., immune diseases (eg, myasthenia gravis, glomerulonephritis, multiple sclerosis, siedalen syndrome, insulin resistance diabetes, rheumatoid arthritis, systemic lupus erythematosus, atopic skin Inflammation, leukocyte abnormalities, immunodeficiency associated with spleen dysfunction or thymic abnormalities), diagnostic markers for inflammatory bowel disease, allergic conjunctivitis, etc., such as proteins, polynucleotides or antibodies against the proteins. Modulators obtained by screening using the antibody, antibodies against the protein, and the like include, for example, respiratory diseases (eg, Chronic obstructive pulmonary disease (chronic bronchitis, emphysema), diffuse panbronchiolitis, bronchial asthma, cystic fibrosis, irritable pneumonia, pulmonary fibrosis, etc.), rhinitis (eg, allergic rhinitis, pollinosis, acute) Rhinitis, chronic rhinitis, hypertrophic rhinitis, atrophic rhinitis, dry proprietary rhinitis, vasomotor rhinitis, gangrene rhinitis, sinusitis, etc., immune diseases (eg, myasthenia gravis, glomerulonephritis, multiple occurrences) Sclerosis, siedalen syndrome, insulin resistance diabetes, rheumatoid arthritis, systemic lupus erythematosus, atopic dermatitis, leukocyte abnormalities, immunodeficiency associated with splenic dysfunction or thymic abnormalities), inflammatory bowel disease, allergies It can be used as a prophylactic and therapeutic agent for conjunctivitis. Further, the antisense polynucleotide of the present invention can suppress the expression of the protein of the present invention. For example, for example, respiratory diseases [eg, chronic obstructive pulmonary disease (chronic bronchitis, emphysema), diffuse Panbronchiolitis, bronchial asthma, cystic fibrosis, irritable pneumonia, pulmonary fibrosis, etc.), rhinitis (eg, allergic rhinitis, hay fever, acute rhinitis, chronic rhinitis, hypertrophic rhinitis, atrophic rhinitis, dryness Pronasalitis, vasomotor rhinitis, gangrenous rhinitis, sinusitis, etc., immune disorders (eg, myasthenia gravis, glomerulonephritis, multiple sclerosis, Siedaren syndrome, insulin resistant diabetes, rheumatoid arthritis, Systemic lupus erythematosus, atopic dermatitis, leukocyte abnormalities, immunodeficiency associated with splenic dysfunction or thymic abnormalities), inflammatory bowel disease, allergic conjunctivitis It can be used as a preventive and therapeutic agent for diseases such as. Furthermore, various polynucleotides of the present invention include, for example, respiratory diseases (eg, chronic obstructive pulmonary disease (chronic bronchitis, emphysema), diffuse panbronchiolitis, bronchial asthma, cystic fibrosis, irritable pneumonia, Pulmonary fibrosis etc.), rhinitis (eg, allergy Rhinitis, hay fever, acute rhinitis, chronic rhinitis, hypertrophic rhinitis, atrophic rhinitis, dry pronasitis, vasomotor rhinitis, gangrene rhinitis, sinusitis, etc.), immune diseases (eg, myasthenia gravis, Glomerulonephritis, multiple sclerosis, siedalen syndrome, insulin resistance diabetes, rheumatoid arthritis, systemic lupus erythematosus, atopic dermatitis, leukocyte abnormalities, immunodeficiency associated with impaired splenic function or thymus, etc.), inflammatory It is useful for diagnosis, prevention or treatment of intestinal diseases, allergic conjunctivitis, etc., preferably respiratory diseases, etc., more preferably chronic obstructive pulmonary diseases, etc., and more preferably emphysema.

Claims

The scope of the claims

I. Contains a compound or a salt thereof which promotes the activity of a protein or a partial peptide thereof or a salt thereof containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1. Prevention and treatment of respiratory diseases.

2. It contains a compound or a salt thereof which promotes the expression of a gene or a partial peptide or a salt thereof which has the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1. Prevention and treatment of respiratory diseases.

3. A base complementary or substantially complementary to the base sequence of a polynucleotide encoding a protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1 or a partial peptide thereof Antisense polynucleotide containing the sequence or a part thereof.

4. A pharmaceutical comprising the antisense polynucleotide according to claim 3.

5. An antibody against a protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1, a partial peptide thereof, or a salt thereof.

6. A pharmaceutical comprising the antibody according to claim 5.

7. The medicament according to claim 6, which is an agent for preventing or treating respiratory diseases.

8. A diagnostic agent comprising the antibody according to claim 5.

9. The diagnostic agent according to claim 8, which is a diagnostic agent for a respiratory disease.

10. A diagnostic agent for a respiratory disease comprising a protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1 or a polynucleotide encoding a partial peptide thereof .

I I. A prophylactic / therapeutic agent for chronic obstructive pulmonary disease or bronchial asthma comprising a compound having a function of promoting c-terminal cleavage promoting activity of procollagen or a salt thereof.

12. Prevention of respiratory diseases characterized by using a protein or a partial peptide or a salt thereof containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1 · A method for screening a therapeutic agent.

1 3. Tobacco smoke-exposed chronic obstructive pulmonary disease model mouse or elastose-induced chronic 13. The screening method according to claim 12, wherein an obstructive pulmonary disease model mouse is used.

14. Prevention of respiratory disease characterized by containing a protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1 or a partial peptide thereof or a salt thereof · Kit for screening therapeutic agents.

15. A prophylactic / therapeutic agent for a respiratory disease obtainable by using the screening method according to claim 12 or the screening kit according to claim 14.

16. The preventive or therapeutic agent according to claim 15, wherein the respiratory disease is emphysema.

17. Prevention of a respiratory disease characterized by using a polynucleotide encoding a protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1 or a partial peptide thereof · How to screen therapeutic agents.

18. The screening method according to claim 17, wherein a tobacco smoke-exposed chronic obstructive pulmonary disease model mouse or an elastase-induced chronic obstructive pulmonary disease model mouse is used.

1 9. A respiratory tract characterized by containing a protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1 or a polynucleotide encoding a partial peptide thereof. Kit for screening of therapeutic and therapeutic agents for organ diseases.

20. A prophylactic / therapeutic agent for respiratory diseases obtainable by using the screening method according to claim 17 or the screening kit according to claim 19.

21. The preventive or therapeutic agent according to claim 20, wherein the respiratory disease is emphysema.

22. A compound that promotes the activity of a protein containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1 or a partial peptide or a salt thereof to a mammal; A method for preventing and treating respiratory diseases, which comprises administering an effective amount of a salt thereof, a compound that promotes the expression of a gene of the protein, or a salt thereof.

23. Promotes the activity of a protein or a partial peptide thereof or a salt thereof containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1, or a gene for the protein A method for preventing and treating respiratory diseases characterized by promoting the expression of respiratory diseases.

2 4. A protein or partial peptide containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1 for producing an agent for preventing or treating respiratory diseases Or a compound or a salt thereof which promotes the activity of a salt thereof, or a compound or a salt thereof which promotes expression of a gene of the protein.