WO2004101824A1 - Method of detecting chronic obstructive pulmonary disease - Google Patents

Method of detecting chronic obstructive pulmonary disease Download PDF

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Publication number
WO2004101824A1
WO2004101824A1 PCT/JP2004/006905 JP2004006905W WO2004101824A1 WO 2004101824 A1 WO2004101824 A1 WO 2004101824A1 JP 2004006905 W JP2004006905 W JP 2004006905W WO 2004101824 A1 WO2004101824 A1 WO 2004101824A1
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WIPO (PCT)
Prior art keywords
nucleic acid
family
seq
protein belonging
gene
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PCT/JP2004/006905
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French (fr)
Japanese (ja)
Inventor
Hikaru Sonoda
Yozo Hori
Jun Ishizaki
Ken-Ichi Higashino
Yoshinari Gahara
Hideki Ohta
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Shionogi & Co., Ltd.
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Publication of WO2004101824A1 publication Critical patent/WO2004101824A1/en

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6884—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids from lung
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

Definitions

  • the present invention relates to means for treating and preventing chronic obstructive pulmonary disease. More specifically, the present invention relates to a method for screening a therapeutic or preventive agent for chronic obstructive pulmonary disease, a kit used for the screening method, a pharmaceutical composition, a method for diagnosing chronic obstructive pulmonary disease, and a method for diagnosing chronic obstructive pulmonary disease.
  • the present invention relates to a kit, a method for detecting or selecting a sample derived from an individual suspected of suffering from chronic obstructive pulmonary disease, and a kit used for the detection or selection method.
  • Chronic obstructive pulmonary disease (C0PD: chronic obstructive pulmonary diseases) is a general term for diseases that indicate permanent or temporary stenosis of the small bronchi.
  • a disease that is defined as having no etiology or other more specific disease name (see Steadman Dictionary of Medicine, 5th Edition, Medical View, Inc., first print, February 20, 2002) .
  • the disease includes, for example, emphysema and chronic bronchitis. Many studies have already shown that smoking causes C0PD.
  • Emphysema is an inherently pathologically defined disease characterized by destructive changes in the airways peripheral to the terminal bronchiole and dilatation of the air space. Clinically, it is more common in smoking men after the age of 50 to 60, and may be accompanied by dyspnea on exertion, cough, and sometimes sputum, and eventually complications of respiratory failure and right heart failure. There is no doubt that smoking is the cause of the disease, but not all long-term smokers have emphysema. It has been suggested that the difference in individual susceptibility to smoking rather than the amount of smoking is more important as a factor in the onset.
  • Chronic bronchitis is defined based on the clinical finding that a cough with sputum recurs for more than 3 months and more than 2 years.
  • Pathological findings include swelling of the bronchial mucous glands, smooth muscle hypertrophy and thickening of the bronchial wall, infiltration of inflammatory cells, and edema.
  • the increased sputum volume, characteristic of this disease, may be pathologically associated with an enlarged bronchial mucous gland.
  • the Secretoglob in (SCGB) family is a group of proteins whose main members are Clara cell secretory proteins (CCS P) (Susan D. et al., Am. J. of Research and Critical Care Med., 2002, Vol. 166, 1498-1509).
  • CCS P Clara cell secretory proteins
  • the CCSP is highly expressed in the induced airway epithelium and is thought to be involved in airway inflammation.
  • the SCGB family has seven subfamilies (1A, 1B, 11D, 2A, 2B, and 3A), and the protein contained in the SCGB family 3A is, for example, human Uter oglobin-related protein (UGRP) 2.
  • UGRP2 is a secreted protein whose precursor consists of 104 amino acids.
  • As the precursor two types, one in which the amino acid sequence at position 19 is Ala and the other at Arg, have been reported. In each case, a signal peptide consisting of 20 amino acid residues (from the first position of Me to 20) Is cleaved and secreted as the mature form.
  • the present invention provides a simple and rapid method for detecting or selecting a sample derived from an individual suspected of having C0PD by measuring the expression level of a protein belonging to the SCGB family, for example, the UGRP2 gene.
  • the purpose is to provide a kit that can perform the method efficiently.
  • the present invention provides a simple and rapid method for diagnosing C0PD by measuring the expression level of a protein belonging to the SCGB family, for example, the UGRP2 gene, and a diagnostic kit capable of efficiently performing the diagnosing method.
  • the purpose is to provide.
  • the present invention also provides a method for efficiently screening for a therapeutic or prophylactic agent for C0PD by detecting or measuring the dynamics of a protein belonging to the SCGB family, for example, UGRP2 or a gene encoding the same. It is an object of the present invention to provide a screening kit that can easily and quickly perform the singing method.
  • the present invention provides a compound or a salt thereof suitably used as a therapeutic or prophylactic agent for C0PD obtained by the screening method, and a compound or a salt thereof suitable for the treatment or prevention of C0PD as an active ingredient. It is an object of the present invention to provide a pharmaceutical composition which comprises: That is, one aspect of the present invention is:
  • test sample is a sample derived from an individual suspected of suffering from chronic obstructive pulmonary disease.
  • nucleic acid encoding a protein belonging to the Secretoglobin family is a nucleic acid comprising a base sequence selected from the group consisting of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, and 15; 5) The method described,
  • the nucleic acid encoding a protein belonging to the Secretoglobin family is a nucleic acid having a sequence selected from the group consisting of SEQ ID NOs: 1, 3, 5, 6, 9, 11, 13, and 15.
  • a detection or selection kit for use in the method according to (8) comprising an antibody against a protein belonging to the Secretoglobin family or a fragment thereof;
  • a protein belonging to the Secretoglobin family In the presence of the test substance, a protein belonging to the Secretoglobin family Or a method for screening a therapeutic or preventive agent for chronic obstructive pulmonary disease, comprising detecting or measuring the dynamics of a gene encoding the protein.
  • the diagnostic method for chronic obstructive pulmonary disease is an indicator that the subject is suspected to have chronic obstructive pulmonary disease.
  • the nucleic acid encoding a protein belonging to the Secretoglobin family is a nucleic acid consisting of a sequence selected from the group consisting of SEQ ID NOs: 1, 3, 5, 6, 9, 11, 13, and 15. 22) described diagnostic kit,
  • the antibody has accession number FERM ABP-10012 or? (6)
  • Uteroglobin-related protein is Uteroglobin-related protein2, the method according to (3),
  • Uteroglobin-related protein 2 is human Uteroglobin-related protein 2, the method according to (4),
  • step (II) After the step (I), the binding between the protein and a test substance is detected, whereby the test substance binding to the protein is converted into a candidate compound for a therapeutic or preventive agent for chronic obstructive pulmonary disease. Step to select as
  • test substance detecting the presence or absence of the expression of the gene, or measuring a change in the expression of the gene, as compared to the case in the absence of the test substance, and thereby detecting a change in the expression of the gene.
  • a pharmaceutical composition comprising the compound according to (14) or a salt thereof as an active ingredient.
  • SEQ ID NO: 1, 3, 5, 7, 9, 11, 11, 13, 15 or 19 to 23, comprising a nucleic acid having a base sequence selected from the group consisting of: A diagnostic kit for use in the method of any of (18) and (20),
  • (22) a diagnostic kit for use in the method according to any of (17) to (20), which comprises an antibody against a protein belonging to the Secretoglobin family or a fragment thereof;
  • test sample (23) measuring the expression level of the gene of the protein belonging to the Secretoglobin family in each of the test sample derived from the test subject individual and the control sample derived from the normal individual, wherein the expression level in the test sample is If the expression level is higher than that in the control sample, the test sample is afflicted with chronic obstructive pulmonary disease, which indicates that the test sample is a sample derived from an individual suspected of having chronic obstructive pulmonary disease.
  • SEQ ID NO: 1, 3, 5, 7, 9, 11, 11, 13, 15 or 19 to 23, comprising a nucleic acid having a base sequence selected from the group consisting of: (24) and (26) a kit for detection or selection for use in the method of any of the above, and (28) a detection or selection kit for use in the method according to any of (23) to (26), which comprises an antibody against a protein belonging to the Secretoglobin family or a fragment thereof;
  • FIG. 1 is a graph showing the results of UGRP2 III RNA expression analysis in the lungs of C0PD model mice (smoking group), as compared to the control group.
  • FIG. 2 is a graph showing the results of analysis of the expression of UGR mRNA in the lungs of asthma model mice (asthma group) as compared to the control group.
  • the present invention is based on the surprising finding first revealed by the present inventors that UGRP2 is specifically and significantly increased in the lung tissue of a model mouse of C0PD induced by prolonged tobacco smoke exposure. It is based on.
  • Smoking activates macrophages and neutrophils to increase the release of proteolytic enzymes (factors that destroy alveoli), and also inactivates 1antitrypsin ( ⁇ ; 1AT), which has an inhibitory effect on proteolytic enzymes It is thought that this works on alveolar destruction (Crystal RG et al., The alpha-1 antitrypsin gene and its mutations. Chest 95: 196-208, 1989; Takahashi H. et al., Cathepsin L activity is increased in alveolar macrophages and bronchoalveolar lavage fluid of smokers. Am Rev Respir Dis 147: 1562-1568, 1993).
  • alAT is an important proteolytic enzyme inhibitory protein in the lung, preventing neutrophil elastase and macrophage-derived proteolytic enzymes from decomposing and destroying alveolar tissue proteins in normal individuals. Therefore, it has been pointed out that the deficiency of QIIAT is one of the predisposing factors for the development of emphysema caused by smoking, but there is no clear link between the two.
  • the present invention relates to a therapeutic or prophylactic agent for C0PD.
  • a method for screening, a kit for use in the screening method, a pharmaceutical composition, a method for diagnosing C0PD, a kit for use in the diagnostic method, a method for detecting or selecting a sample derived from an individual suspected of suffering from C0PD, and a method for detecting or selecting The present invention relates to a kit used for a selection method.
  • C0PD refers to a symptom in which at least specific high expression of a protein belonging to SCGB family 1 is observed.
  • pulmonary emphysema or chronic bronchitis is mentioned.
  • the present invention can be particularly preferably used for emphysema.
  • description of a protein may include the protein and a gene encoding the protein.
  • description of a protein may include some fragments thereof.
  • reference to a gene may include naturally occurring genes, variants and cDNAs.
  • the screening method for a therapeutic or prophylactic agent for C0PD of the present invention has one feature of detecting or measuring the kinetics of a protein belonging to SCGB family 1 or a gene encoding the protein in the presence of a test substance. I do.
  • test substances by screening the effects of test substances on the kinetics of proteins belonging to the SCGB family, which is recognized to be associated with the development of C0PD, to efficiently screen substances that are effective as therapeutic or prophylactic agents against C0PD. It is.
  • the effect of a test substance on the kinetics of a protein belonging to the SCGB family is preferably examined using the kinetics in the absence of the test substance as a control.
  • the screening method of the present invention can be used for pharmacological evaluation of a candidate compound of a therapeutic or prophylactic agent for C0PD.
  • the test substance includes a compound or a salt thereof.
  • the compound or a salt thereof is a low-molecular compound, a high-molecular compound, or a polypeptide.
  • derivatives thereof, nucleic acids or derivatives thereof, and the like may be a natural substance or a non-natural substance.
  • Derivatives of polypeptides include modified polypeptides obtained by adding a modifying group, and variant polypeptides obtained by modifying amino acid residues.
  • Examples of the derivative of the nucleic acid include a modified nucleic acid obtained by adding a modifying group, a peptide nucleic acid obtained by modifying a base, and a peptide nucleic acid.
  • the protein belonging to the SCGB family for detecting or measuring its kinetics is not particularly limited.
  • the protein described in Non-Patent Document 4 or 1 or Any variant protein having several amino acid deletions, additions, insertions or substitutions and having properties equivalent to the protein can be mentioned.
  • “equivalent properties” means that they can be specifically expressed at least in relation to the onset of C0PD.
  • the protein belonging to the SCGB family is preferably a protein belonging to SCGB family 3A, more preferably UGRP, and still more preferably UGRP2.
  • human UGRP2 is particularly preferred as UGRP2 from the viewpoint of efficiently screening a therapeutic or prophylactic agent for C0PD effective against humans according to the present invention.
  • the amino acid sequence (SEQ ID NO: 6) under CAA11863; human SCGB1D2 protein The nucleotide sequence (SEQ ID NO: 7) is under AJ224172, the amino acid sequence (SEQ ID NO: 8) is under CAA11864; the human SCGB2A1 protein is nucleotide sequence (SEQ ID NO: 9) under M224173, Amino acid sequence (SEQ ID NO: 10) below CAA11865; human SCGB2A2 protein; nucleotide sequence (SEQ ID NO: 11) below U33147; amino acid sequence (SEQ ID NO: 12) below AAC50375;
  • the nucleotide sequence (SEQ ID NO: 15) is disclosed under AF313455, and the amino acid sequence (SEQ ID NO: 16) is disclosed under AAL26215.
  • UGRP for example, as a human-derived (human SCGB3A1 protein), its base sequence (SEQ ID NO: 13) under Genebank Accession No .: AF086152, and its amino acid under AAL26217 The sequence (SEQ ID NO: 14) is disclosed. Also, as a mouse-derived product (mouse SCGB3A1 protein), its base sequence (SEQ ID NO: 17) under Gene Bank Accession No. AF313456 and its amino acid sequence (SEQ ID NO: 1) under AAL26216 8) is disclosed.
  • Human UGRP2 is expressed in lung, thymus, spleen, small intestine, lymph nodes, prostate, tongue, and trachea, showing trachea> lung> tongue> prostate> thymus, spleen, small intestine, and lymph node expression patterns It has the feature of.
  • the “kinetics of a protein belonging to the SCGB family” to be measured or detected in the screening method of the present invention includes, for example, the presence or absence of binding between the protein and its ligand; the expression of the protein, specifically, the presence or absence of the expression ⁇ ⁇ Variations in the expression and the like.
  • step (II) After the step (I), detecting the binding between the protein and the test substance, thus, a step of selecting a test substance that binds to the protein as a candidate compound for a therapeutic or prophylactic agent for C0PD
  • a substance that acts on the function of the protein through the binding to the protein can be selected. Therefore, the selected candidate compound directly and specifically binds to the protein and binds to the protein. It has the characteristic property of regulating functions.
  • the contact between the protein and the test substance is performed, for example, by mixing the protein and the test substance in a solution that does not interfere with the original function of the protein, and applying appropriate reaction conditions (for example, , Reaction temperature, reaction time, etc.).
  • appropriate reaction conditions for example, , Reaction temperature, reaction time, etc.
  • solutions such as phosphate-buffered physiological saline (PBS), HEPS buffer, and Tris buffer.
  • PBS phosphate-buffered physiological saline
  • HEPS buffer HEPS buffer
  • Tris buffer Tris buffer
  • the reaction conditions in step (I) are not particularly limited.
  • the pH is usually 6.0 to 10.0, preferably ⁇ 7.0 to 9.0, more preferably ⁇ 7.5 to 8.5, more preferably ⁇ 80, usually 10 to 50 t, preferably 20 to 40 ° C, more preferably 25 to 37 ° C, more preferably 25 ° C, usually 1 minute to 1 hour , Preferably for 3 to 30 minutes, more preferably for 5 to 20 minutes, and still more preferably for 10 minutes.
  • step (II) the binding between the protein and the test substance is detected, and the presence or absence of the binding is examined.
  • the binding is performed, for example, by measuring the radioactivity of the protein contained in the solution obtained by reacting the protein with a test substance labeled with a radioactive substance in step (I) as described above, for example. Analyze in competition with a large excess of non-radioactive analytes Can be detected. If binding is detected, the test substance used is selected as a candidate compound for a therapeutic or prophylactic agent for C0PD.
  • step (I) and step (I I) may be performed in a continuous process. Such an embodiment can be carried out, for example, by utilizing surface plasmon analysis, binding to a carrier holding a test substance, or the like.
  • step (i) sending a solution containing a test substance at a constant flow rate to a sensor chip on which the protein is immobilized [step (I )], And
  • Suitable detection means eg, optical detection (fluorescence, fluorescence polarization, etc.), combined with a mass spectrometer (matrix-assisted laser desorption ionization-time-of-flight mass spectrometer: MALDI-TOF MS, electrospray Ionization mass spectrometer: ESI-MS etc.)] to detect the binding between the protein and the test substance as optical fluctuation or mass fluctuation [Step (11)];
  • the binding between the protein and the test substance is caused by, for example, a change in an optical sensorgram or a mass sensorgram when the test substance is introduced by liquid sending.
  • a gene encoding a protein belonging to the SCGB family is introduced into a cell by, for example, transferring the protein downstream of and under the control of an expression regulatory element known as an expression regulatory region of the protein. It is preferable to use a nucleic acid construct in which the coding gene is operably linked.
  • the expression control element is described in, for example, Niimi Tetal., Molecular Endocrinology, 2001, Vol. 15, 2022136.
  • the gene encoding a protein belonging to the SCGB family includes the gene itself; a gene that hybridizes to the antisense strand of the gene under stringent conditions and encodes a protein having an action equivalent to that of the protein.
  • the nucleic acid construct can be conveniently constructed by inserting the gene encoding the expression regulatory element and a protein belonging to the SCGB family into a cloning site of a conventional expression vector.
  • the vector include a virus vector and a plasmid vector.
  • a vector used in expression of the gene when the expression of a gene encoding a protein belonging to the SCGB family is essential for its configuration, a vector used in expression of the gene, a host cell into which a nucleic acid construct is introduced, and the like. Any of them They may be derived from any species as long as expression of the genes can be achieved by the combination. Those skilled in the art can construct an expression system for the gene using any constituent material.
  • the gene encoding the protein belonging to the SCGB family is derived from eukaryotes, and that the therapeutic or prophylactic agent provided in the present invention is eukaryotes, especially mammals, especially humans
  • animal cells are suitable as host cells used for expression of the gene. Therefore, in this specification, a case where an animal cell is used for the expression of the gene will be particularly described.
  • Any vector may be used as long as it can incorporate the gene and express it in animal cells.
  • Such vectors include PCR-Bluntll-TOP0, pcDNA1.K pcDNA1.l / A, pCDM8, pREP (Invitrogen), HM6, HB6 (Roche Diagnostics), _PKK223-3 , pGEX (Amersham Biotech), pET-3, ⁇ - ⁇ pBluescriptll (registered trademark) SK (10), Bluescri ptll (registered trademark) SK (-) (Stratagene), pUC19, pTrxFus (Invitrogen) ), PUC118, PSTV28 (Yukara Bio Inc.), pMAL-c2X (New England Land Piolapo), PAGE 107 [Cytotechnology, 3 (2), 133-140 (1990); Publication), pAGE 103 [The Journal of Biochemistry, 101 (5), 13 07-1310 (1987)], pAMo,
  • the animal cell used as a host may be any appropriate cell or tissue corresponding to the vector used, and includes the following animal cells.
  • the animal cells include mouse-derived cell line L6 [skeletal muscle-derived myoblast], human-derived cell line thigh 293 (human fetal kidney cell, ATCC: CRL-1573), HeLa (melanoma cell line) , ATCC: CCL- 2) HBT5637 (leukemia cell, JP-A-63-299), BALL-1 (leukemia cell) and HCT-15 (colorectal cancer cell), mouse-derived cell line Sp2 / 0-Ag (mouse myeloid cell) , ATCC: CRL-1581) and NS0 (mouse myeloid cells), monkey-derived cell line C0S-1 [African green monkey kidney cells (SV40 transformed cells), ATCC: CRL-1650] and COS-7 [Africa Green monkey kidney cells (SV40 transformed cells), ATCC: CRL-1651], hamster-derived cell lines CHO- ⁇ (Chinese hamster ovary
  • stringent end conditions refer to conditions of high stringency, for example, conditions of 1 ⁇ SSC / 0.2% SDS.
  • lower ionic strength for example, conditions such as 0.5 X SSC, preferably 0.2 X SSC, more preferably 0.1 X SSC and / or higher temperature, for example, Washing may be performed under conditions of 50 ° C. or higher, preferably 60 ° C. or higher, more preferably 65 ° C. or higher, depending on the Tm value of the nucleic acid used.
  • mutation refers to substitution, loss, addition and insertion of a base. Such a mutation may be a naturally occurring variation, or may be a mutation artificially introduced by a conventional site-specific mutation method or the like.
  • sequence identity refers to the ability to properly align at least two sequences to be compared and to determine the same residue present in each sequence.
  • sequence identity can be determined, for example, by, for example, a homepage address ht tp:
  • the introduction of the nucleic acid construct into an animal cell may be performed by a conventional genetic method. It can be performed by a child introduction method.
  • a conventional genetic method for example, elect opening method (Cytotechnology, 3 (2), 133-140 (1990)), calcium phosphate method (Japanese Patent Laid-Open No. 2-227075), lipofection method [Proceedings of the National Academy of Sciences USA, 84, 7413 (1987); Viology, 52, 456 (1973)], DEAE dextran method, particle gun method, virus-based method, and the like.
  • the transformant into which the nucleic acid construct has been introduced can be cultured by a conventional method commonly used in the art.
  • the culture can be performed using a medium suitable for the transformed host, and a liquid medium is suitable as the medium used for the culture.
  • a medium suitable for the transformed host for example, MEM medium [Science, 130,432 (1959)], DMEM medium [Virology, 8, 396 (1959)], RPMI1640 medium [The Journal of the American Medical Association, 199, 519 (1967)], etc. Serum to which an appropriate amount of serum (FCS) has been added is used. Since those media are commercially available, such commercially available media can be used.
  • the culture can be performed under aerobic conditions such as, for example, shaking culture or deep aeration stirring culture.
  • the culture temperature, culture time, and pH of the culture solution are set in ranges suitable for various hosts.
  • the culture is usually 15 to 40, 5 hours to 7 days, PH3.0 ⁇ 9.0, can be carried out in conditions such as 5% C0 2 presence.
  • the pH can be adjusted using an inorganic or organic acid, an alkali solution, urea, calcium carbonate, ammonia, or the like. If desired, an antibiotic may be added to the medium.
  • step (A) when a stable cell line into which the nucleic acid construct has been introduced is used, step (A) may be omitted.
  • step (B) a gene encoding a protein belonging to SCGB family 1 is expressed in the cells obtained in step (A) in the presence of the test substance.
  • This step can be performed, for example, by culturing the cells obtained in step (A) using a medium containing the test substance. Culture conditions are as described above. - Subsequently, in step (C), the presence or absence of the expression of the above-described gene is detected or the fluctuation of the expression of the above-described gene is measured, as compared with the case where the test substance is not present. A test substance that results in a change in the expression of is selected as a candidate compound for a therapeutic or prophylactic agent for C0PD.
  • step (B) refers to the case where, in step (B), the gene is expressed in the cells obtained in step (A) in the absence of the test substance.
  • This serves as a standard for ascertaining changes in the expression of the gene in the presence of the test substance. Specifically, cells in such a case, or an extract of the cells, etc. are used as a control.
  • the expression of the gene is observed in the control
  • the expression of the gene is not detected in the cells contacted with the test substance or the expression level is reduced as compared with the control, that is, when the expression of the gene is reduced as compared with the control
  • the test substance is determined to be a candidate compound for a C0PD therapeutic or prophylactic agent, and the test substance is selected as a candidate compound.
  • the selected candidate compound suppresses the expression of the gene at the cellular level at the transcription level. And has an excellent effect that the expression of the gene can be specifically suppressed.
  • the detection of the presence or absence of the expression of the gene or the measurement of the fluctuation of the expression as compared to the case in the absence of the test substance can be performed, for example, as follows.
  • the expression product level from the gene in the cell after the step (B) and the expression product level in the control are determined, for example, by polyacrylamide gel electrophoresis; an antibody against the expression product of the gene Alternatively, it can be performed by Western blot analysis using a fragment thereof; by measuring with an immunoassay or the like using the antibody or a fragment thereof, and comparing.
  • nucleic acids are extracted from the cells and the control after the step (B), respectively, and the obtained nucleic acids are, for example, nucleic acids capable of detecting DiRNA of a gene encoding a protein belonging to the SCGB family, A hybridization using a nucleic acid that specifically binds to the gene or a part thereof; a PCR using a primer pair capable of specifically amplifying the gene or a part thereof.
  • the formation or the amount of the hybrid or the appearance or the amount of the amplification product can be measured and compared.
  • Examples of the antibody used in the screening method of the present invention include an expression product from the aforementioned gene, that is, an antibody against a protein belonging to the SCGB family, a fragment thereof, and the like.
  • the method for producing the antibody is described in, for example, 1992
  • Antibodies can also be produced by genetic engineering.
  • the antibody may be a polyclonal antibody or a monoclonal antibody as long as it has an ability to specifically bind to a protein belonging to the SCGB family.
  • the antibody may be, for example, an antibody capable of specifically binding to a specific partial fragment in the protein belonging to the SCGB family.
  • the partial peptide of human UGPR2, Cys_UGRP2 (46-58, CTLANPLGTLNPLK, SEQ ID NO: 2) 6) or Cys-UGRP2 (63-77, SLGIPVNHLIEGSQKC, SEQ ID NO: 27) or a monoclonal antibody against the polypeptide containing the partial peptide is preferred, and the hybrid having the accession number FERM ABP-10012 or FERM ABP-10013 is preferred. More preferred are monoclonal antibodies produced by MA.
  • the antibody fragment is obtained by purifying the obtained antibody and treating it with a peptidase or the like.
  • derivatives of the above antibodies for example, chimeric antibodies, humanized antibodies, Fab fragments, single-chain antibodies, etc.
  • Antibodies and the like modified by known techniques can also be used.
  • Such an antibody or a fragment thereof may be labeled with a conventional enzyme (for example, peroxidase or the like), a fluorescent dye, a radioactive substance, avidin or biotin, or the like.
  • the production of a polyclonal antibody that recognizes human UGRP2 can be performed using the full-length human UGRP2 polypeptide, its partial peptide Cys-UGRP2 (46-58, CTLANPLGTLNPLK, SEQ ID NO: 26) or Cys-UGRP2 (63-77, SLGIPVNHLIEGSQKC , SEQ ID NO: 27) or a polypeptide containing the partial peptide as an antigen, which can be used to produce an antibody.
  • the polypeptide or partial peptide may be used as it is as a carrier, and may be used as a carrier, for example, perforated serum albumin (BSA), keyhole lichen ethemocyanin (KLH) or pericyroglobulin.
  • BT G BT G
  • CFA complete adjuvant
  • IFA incomplete adjuvant
  • mammals used for immunization mice, rats, rabbits, goats, hamsters and the like can be used.
  • Polyclonal antibodies can be prepared, for example, according to the method of Lane et al. [Antibodies: A Laboratory Manual, Second Edition (1989) (Cold Spring Harber Laboratory Press)].
  • the antigen is administered 3 to 10 times every 1 to 2 weeks after the first administration.
  • the dose of the antigen is preferably 50 to 100 g per animal per administration.
  • Peptides used as antigens can be synthesized using genetic engineering techniques or peptide synthesizers
  • Plasma samples are collected from the fundus venous plexus on days 3 to 7 after each administration, and the reaction of the serum with the antigen used for immunization is determined by an enzyme immunoassay [“Enzyme immunoassay (ELISA)” published by Medical Shoin ( 1976); Ant ibodies: A Laboratory Manual, Second Edition (1989) (Cold Spring Harber Laboratory Press)) and the like.
  • enzyme immunoassay “Enzyme immunoassay (ELISA)” published by Medical Shoin ( 1976); Ant ibodies: A Laboratory Manual, Second Edition (1989) (Cold Spring Harber Laboratory Press)
  • Blood is collected from the immunized mammal and the antibody titer is measured. Blood is collected at the time of immunization until a sufficient antibody titer is obtained, and a serum is prepared, whereby a polyclonal antibody can be obtained.
  • Methods for separation and purification of polyclonal antibodies include centrifugation, salting out with ammonium sulfate, and prillic acid precipitation [Antibodies: A Laboratory Manual, Second Edition (1989) (Cold Spring Harber Laboratory Press)] or It can be carried out by using various chromatography such as DEA E-Sepharose column, anion exchange column, protein A column, G-column or gel filtration column alone or in combination.
  • Monoclonal antibodies that recognize human UGRP2 can be obtained, for example, by collecting spleen or lymph nodes from immunized mammals with sufficient antibody titers, and fusing the antibody-producing cells obtained therefrom with myeloma (myeloma) cells. Thus, it can be obtained by obtaining a monoclonal antibody-producing hybridoma and producing the desired antibody by a conventional method using the obtained hybridoma. .
  • a polypeptide containing the partial peptide is administered as an antigen, and the antigen is finally recruited to a mouse showing a sufficient antibody titer. Then, on days 3 to 7, the spleen is removed from the mouse.
  • the obtained spleen is shredded in a MEM medium (for example, Nissui Pharmaceutical Co., Ltd.), loosened with tweezers, and centrifuged at 1,200 rpm for 5 minutes.
  • the splenocytes in the precipitate fraction thus obtained are treated with Tris-ammonium chloride buffer (pH 7.65) for 1 to 2 minutes to remove erythrocytes.
  • the obtained spleen cells are washed three times with MEM medium.
  • the obtained splenocytes are used as antibody-producing cells.
  • Myeloma cells include cell lines derived from mice or rats. Examples of such myeloma cells include 8-azaguanine-resistant mice (derived from BALB / c) Myeloma cells P3-X63Ag8-Ul strain (hereinafter abbreviated as P3-U1) [Current Topics Microbiological cal Immunology, 81, 1 (1978)> Europian Journal of Immunology, 6, 511 (1976)], SP2 / 0_Agl4 strain ( Hereinafter, abbreviated as SP-2) [Nature, 276> 269 (1978)], P3-X63-Ag 8653 strain (hereinafter abbreviated as 653) [Journal of Immunology, 123, 1548 (1979) 3, P3-X63 -Ag8 (hereinafter abbreviated as X63) [Nature, 256, 495 (1975)].
  • P3-X63Ag8-Ul strain hereinafter abbreviated
  • 8-azaguanine medium normal medium containing 15 ⁇ ⁇ / ⁇ 8-azaguanine [1.5 mM glutamine, 5 ⁇ 1 ( ⁇ 3 ⁇ 42-mercaptoethanol, 10 g / ml genyumycin and 10% FCS (CSL RPMI1640 medium containing the same product) and culture in a normal medium 3 to 4 days before cell fusion.
  • FCS CSL RPMI1640 medium containing the same product
  • Cell fusion can be performed by a known method, for example, according to the method of Kohler and Milstein [Nature, 256, 495-497 (1975)]. Specifically, the antibody-producing cells and the myeloma cells were converted to a MEM medium or PBS (per liter; 1.83 g disodium phosphate, 0.21 g monopotassium phosphate, 7.65 g NaCl, pH 7.2). Wash and mix so that the number of antibody-producing cells is 5 to 10 times the number of myeloma cells. Centrifuge at 1,200 rpm for 5 minutes to obtain a precipitate fraction. The resulting precipitated fraction may ho Gushi cell groups, per 10 8 antibody-producing cells to the cell group, polyethylene glycol solution
  • 0.2 g of polyethylene glycol-1000 (PEG-1000), 2 ml of MEM medium, and 0.7 ml of dimethyl sulfoxide (DMS0) are added with stirring at 37 ° C, and the MEM medium is added every 1-2 minutes. Add 2 ml several times. Adjust the total volume to 50 ml with MEM medium and centrifuge at 900 rpm for 5 minutes to obtain a precipitate fraction. Precipitation fractionation HAT culture ground (normal medium, 10_ 4 M hypoxanthine, 1.5X10- 5 including M thymidine and 4X10- 7 M aminopterin) was added 100 ml, gently loosened and suspended.
  • PEG-1000 polyethylene glycol-1000
  • MEM medium 0.7 ml of dimethyl sulfoxide
  • the resulting suspension was dispensed into the wells of a 96-well culture plate at a rate of 37 ° C.
  • the antibody production by the hybridoma can be performed by a conventional method.
  • the nucleic acid (probe) used in the screening method of the present invention include a nucleic acid that specifically binds to a gene encoding a protein belonging to the SCGB family.
  • the specifically binding nucleic acid include SC
  • Such a nucleic acid can be appropriately selected based on an appropriate Tm value, a secondary structure and the like in consideration of operability at the time of use. Specifically, in humans, for example,
  • a nucleic acid having the nucleotide sequence described in SEQ ID NO: 1, 3, 5, 7, 9, 9, 11, 13, 15, or 23, and the like are preferably used.
  • a nucleic acid having the nucleotide sequence of SEQ ID NO: 17 is used.
  • Such a nucleic acid may be labeled with a conventional fluorescent dye, radioactive substance, or the like.
  • Examples of the primer pair capable of specifically amplifying a gene encoding a protein belonging to the SCGB family or a part thereof include, for example, an antisense sequence at the 5 ′ end of a nucleic acid comprising a gene encoding a protein belonging to the SCGB family.
  • a primer pair consisting of a primer and a primer corresponding to the antisense sequence at the 3 ′ end is exemplified.
  • Such a primer pair can be appropriately selected based on an appropriate Tm value, a secondary structure and the like in consideration of operability at the time of use. Specifically, it is preferable that such a primer pair be capable of amplifying all or a characteristic partial sequence of the nucleotide sequence of SEQ ID NOS: 1 to 8 in human. More specifically, for example, a primer pair selected from the group consisting of nucleic acids having the nucleotide sequences of SEQ ID NOs: 19 to 22 can be mentioned. Specific examples of the primer pair include, but are not limited to, a primer pair of SEQ ID NOS: 19 and 20, a primer pair of SEQ ID NOs: 21 and 22, and a primer pair of SEQ ID NOs: 19 and 22.
  • Primer pairs, primer pairs of SEQ ID NOS: 20 and 21, and the like In the mouse, it is preferable that the whole of the nucleotide sequence of SEQ ID NO: 17 or a characteristic partial sequence thereof can be amplified. Specifically, for example, a primer pair of SEQ ID NOs: 24 and 25 and the like can be mentioned. These primers may be primers labeled with conventional fluorescent dyes, radioactive substances and the like. The amplification product can be detected by conventional agarose gel electrophoresis or the like, by visualization with a bromide dye or a fluorescent dye, detection based on a labeled primer, or the like.
  • the “characteristic partial sequence (to nucleic acid)” refers to, for example, a nucleotide sequence of a gene other than a gene encoding a protein belonging to the SCGB family among sequences registered in a database. Refers to a partial sequence that is not substantially found.
  • the sequence identity to a sequence registered in a database is usually 20% or less, preferably 10% or less, more preferably 5% or less, and particularly preferably An array that is 0%.
  • Examples of the compound obtained by the screening method of the present invention or a salt thereof include, for example, a compound that inhibits the function of the protein by binding to a protein or the like belonging to the M SCGB family, for example, a low molecular compound, Molecular compound, the SCG
  • Antibodies against proteins belonging to the B family, evening belonging to the SCGB family Nucleic acids, such as antisense strands of nucleic acids consisting of genes encoding proteins, peptides, and the like.
  • a protein belonging to the SCGB family used in the screening method of the present invention a nucleic acid construct containing a gene encoding a protein belonging to the SCGB family, a cell into which the gene has been introduced, and a SCGB family member.
  • a kit for performing the screening method of the present invention, including an antibody of a protein belonging thereto, is provided. Such a screening kit is also included in the present invention.
  • a kit comprising an antibody against a protein belonging to the SCGB family or a fragment thereof;
  • each kit may contain the probe and / or primer pair suitably used in the screening method of the present invention, if desired. Further, if desired, the detection reagent, buffer, standard substance, and the screening method of the present invention are implemented. Instructions and the like may be included.
  • a compound or a salt thereof obtained by the screening method provides a pharmaceutical composition for use in treating or preventing C0PD, for example, emphysema or chronic bronchitis, particularly emphysema.
  • the pharmaceutical composition of the present invention contains a compound obtained by the screening method of the present invention or a salt thereof as an active ingredient. Therefore, the pharmaceutical composition of the present invention exerts a therapeutic or preventive effect on C0PD through an action on the expression of a protein belonging to the SCGB family (for example, acts to suppress the expression).
  • the content of the compound or a salt thereof in the pharmaceutical composition of the present invention can be appropriately adjusted depending on the disease to be treated, the patient's age, body weight, and the like.
  • a high molecular compound for example, 0.0001 to 1000 mg, preferably 0.001 to 100 mg
  • a polypeptide or a derivative thereof for example, 0.0001 to 1000 mg, preferably 0.001 to 100 mg, nucleic acid or
  • the derivative for example, it is desirable that the amount is 0.0001 l to 100 mg, preferably 0.0001 to 10 mg.
  • the pharmaceutical composition of the present invention may further contain various auxiliaries capable of stably retaining the compound or a salt thereof.
  • auxiliaries capable of stably retaining the compound or a salt thereof.
  • pharmaceutically acceptable auxiliaries, excipients, and binders exhibiting the property of inhibiting the active ingredient from decomposing before reaching the site to which the active ingredient is to be delivered.
  • Stabilizers, buffers, solubilizing agents, isotonic agents and the like are examples of sorbitol, sorbitol, and the like.
  • the dosage form of the pharmaceutical composition of the present invention is determined by the type of active ingredient; Government, local site, tissue; selected as appropriate according to the age, weight, etc. of the individual to be administered.
  • Examples of the administration form include subcutaneous injection, intramuscular injection, intravenous injection, topical administration and the like.
  • the dose of the pharmaceutical composition of the present invention is also appropriately selected according to the type of the active ingredient; the individual, organ, local site, or tissue to be administered; the age, body weight, etc. of the individual to be administered. .
  • the administration method is not particularly limited, but when the active ingredient is a low molecular weight compound or a high molecular weight compound, the amount of the active ingredient is, for example, 0.0001 to 1000 mg / kg body weight, preferably 0.0001 to 1000 mg / kg body weight.
  • 100 mg / kg body weight in the case of a polypeptide or a derivative thereof, for example, 0.0001 to 1000 mg / kg body weight, preferably 0.001 to 100 mg / kg, in the case of a nucleic acid or a derivative thereof, for example, 0.000001 to
  • One or more doses per day may be given to give a single dose of 100 mg / kg body weight, preferably 0.0001 to 10 mg / kg body weight.
  • the administration period is not particularly limited.
  • the pharmacological evaluation of the pharmaceutical composition of the present invention can be performed, for example, by administering the pharmaceutical composition of the present invention to the C0PD model mouse described in the Examples below, and improving the C0PD in the treated animal as compared to the non-administered animal. This can be done by a method of evaluating the case where it is seen as an index.
  • the present invention also provides a method for diagnosing C0PD.
  • One feature of the diagnostic method of the present invention is to measure the expression level of a gene of a protein belonging to the SCGB family, particularly a UGRP2 gene, in each of a test sample derived from an individual to be tested and a control sample derived from a normal individual. And Therefore, according to the diagnostic method of the present invention, an excellent effect of easily and quickly diagnosing whether or not an individual to be tested is suspected of having C0PD is exhibited.
  • an index indicating that the test subject is suspected of having C0PD is provided.
  • Examples of the individual include, but are not limited to, mammals, particularly humans, mice, mice, monkeys, and the like.
  • the test sample can be prepared, for example, from an individual's tissue, cells, blood, etc. by a conventional method. In particular, it is preferably prepared from trachea, lung, tongue, or saliva, which has a high expression level of a protein belonging to the SCGB family.
  • the control sample may be prepared from the same site as the test sample. For example, the control sample can be prepared from the same site as the test sample.
  • the expression level of the gene of the protein belonging to the SCGB family is preferably measured using the amount of the mRNA transcript of the gene or the amount of the gene product of the gene as an index.
  • each of a test sample derived from an individual to be tested and a control sample derived from a normal individual is, for example, a nucleic acid comprising a gene encoding a protein belonging to the SCGB family or Hybridization using a nucleic acid consisting of a partial sequence characteristic of the nucleic acid as a probe, a nucleic acid consisting of a gene encoding a protein belonging to the SCGB family, or a nucleic acid consisting of a partial sequence characteristic of the nucleic acid
  • PCR or the like using a primer pair that can specifically amplify, measuring the formation or the amount of the eight hybrids or the appearance or the amount of the amplification product, and comparing them, the protein belonging to the SCGB family can be identified.
  • the expression level of the gene can be evaluated.
  • a nucleic acid used as a probe can be appropriately selected based on an appropriate Tm value, a secondary structure, and the like in consideration of operability at the time of use.
  • the probe include, for example, the probe and the like that can be suitably used in the screening method of the present invention.
  • Such a nucleic acid may be labeled with a conventional fluorescent dye, radioactive substance, or the like.
  • the primer pair used for PCR includes a primer corresponding to the 5'-terminal antisense sequence of a nucleic acid comprising a gene encoding a protein belonging to the SCGB family or a nucleic acid comprising a sequence portion characteristic of the nucleic acid, and a 3'-terminal Antisense And a primer pair consisting of a primer corresponding to the sequence.
  • a pair of primers can be appropriately selected based on an appropriate Tm value, a secondary structure and the like in consideration of operability at the time of use. Specific examples include the primer pairs and the like that can be suitably used in the screening method of the present invention.
  • Such a primer may be a primer labeled with a conventional fluorescent dye, radioactive substance or the like.
  • Detection of the amplification product can be performed by conventional agarose gel electrophoresis or the like by performing visualization with a bromide reagent or a fluorescent substance, detection based on a labeled primer, or the like.
  • an antibody against the protein belonging to the SCGB family or a fragment thereof preferably a partial peptide of human UGPR2 Cys-UGRP2 (46-58, CT LANPLGTLNPLK, SEQ ID NO: 26) or Cys-UGRP2 (63- 77, SLGIPVNHLIEGSQKC, SEQ ID NO: 27) or a monoclonal antibody against a polypeptide containing the partial peptide or a fragment thereof, more preferably produced by a hybridoma having accession number FERM ABP-10012 or FERM ABP-10013 Using a monoclonal antibody or a fragment thereof, the amount of mRNA transcript is appropriately measured according to a known method, and the expression level of a gene of
  • a test sample derived from an individual to be tested and a control sample derived from a normal individual are subjected to polyacrylamide gel electrophoresis, an antibody against a protein belonging to the SCGB family or a fragment thereof,
  • it comprises a partial peptide of human UGP R2 Cys-UGRP2 (46-58, CTLANPLGTLNPLK, SEQ ID NO: 26) or Cys-UGRP2 (63-77, SLGIPVNHLIEGSQKC, SEQ ID NO: 27) or the partial peptide Western blot analysis using a monoclonal antibody against a polypeptide or a fragment thereof, more preferably a monoclonal antibody or a fragment thereof produced by a hybridoma having accession number FERM ABP-10012 or FERM ABP-10013, It was used for the imnoassay and the amount of the gene product was measured and compared.
  • the diagnostic method of the present invention can be performed simply, quickly, with a high throughput, and with high reliability, and the probe and Z or primer suitably used in the screening method of the present invention.
  • a diagnostic kit containing a monoclonal antibody produced by a hybridoma that is ABP-10013 or a fragment thereof is provided.
  • the diagnostic kit of the present invention may further contain a detection reagent, a buffer, a control sample, instructions for the diagnostic method of the present invention, and the like. Further, the present invention provides a method for detecting or selecting a sample derived from an individual suspected of having C0PD.
  • a sample derived from an individual suspected of suffering from C0PD can be detected or selected easily and quickly.
  • the detection or selection method of the present invention includes a step of measuring the expression level of the gene of the protein belonging to the SCGB family in each of a test sample derived from an individual to be tested and a control sample derived from a normal individual. If the expression level in the test sample is higher than the expression level in the control sample, this is an indicator that the test sample is a sample derived from an individual suspected of having C0PP.
  • the expression level of a gene of a protein belonging to the SCGB family is preferably measured using the amount of the mRNA transcript of the gene or the amount of the gene product of the gene as an index.
  • the expression level of the protein gene can be measured in the same manner as in the case of the diagnostic method of the present invention.
  • a detection containing the probe and / or the primer pair described in the description of the diagnostic method for performing the detection or selection method of the present invention simply, quickly and with a high throughput.
  • a selection kit, or an antibody against a protein belonging to the SCGB family or a fragment thereof preferably a partial peptide of human UGPR2 Cys-UGRP2 (46-58, CTLANPLGTLNPLK, SEQ ID NO: 26) or Cys-UGRP2 (63-77, SLGIPVNHL IEGSQKC, SEQ ID NO: 27) or a monoclonal antibody against a polypeptide containing the partial peptide or a fragment thereof, and more preferably, accession number FERM ABP-10012 or FERM ABP-10013.
  • a detection or selection kit containing a monoclonal antibody or a fragment thereof produced by a hybridoma is provided.
  • the present invention further provides a partial peptide of human UGPR2, Cys-UGRP2 (46-58, CTLANPLGTLNPLK, SEQ ID NO: 26) or Cys-UGRP2 (63-77, SLGIPVNHLIEGSQKC, SEQ ID NO:
  • a candidate compound for a therapeutic or prophylactic agent for C0PD can be screened simply and quickly, and
  • the pharmacological evaluation of the candidate compound for the therapeutic or prophylactic agent can be performed. Further, according to the screening kit of the present invention, the screening can be performed simply, quickly, and with a high throughput. Further, according to the pharmaceutical composition of the present invention, C0PD is effective. It can be effectively treated or prevented. Further, according to the method for diagnosing C0PD of the present invention, C0PD can be diagnosed simply and quickly. Further, according to the diagnostic kit of the present invention, the diagnosis can be performed simply, quickly and with a high throughput. Furthermore, according to the method for detecting or selecting a sample derived from an individual suffering from C0PD of the present invention, a sample derived from an individual suffering from C0PD can be detected or selected easily and quickly.
  • the detection or selection can be performed easily and quickly with a high throughput.
  • the present invention will be described in more detail by way of examples, but the present invention is not limited to only these examples.
  • the method described in Molecular Cloning A Laboratory Manual, Second Edition (1989) (Cold Spring Harbor Laboratory Press) was used as a genetic manipulation technique.
  • Example 1 Expression of UGRP2 mRNA in C0PD model mouse 1
  • the C0PD model mouse was prepared by subjecting Balb / c mice (Charles River Japan, Japan, 6-week-old, male) to whole-body exposure to cigarette smoke (mainstream smoke), which is the primary cause of C0PD development. (Smoking group) That is, during the rearing of mice, the mainstream smoke of 20 highlights (trade name, manufactured by Japan Tobacco Inc.) was used for 20 minutes per day using a mainstream smoke exposure device (manufactured by MIPS). Over the whole body. Exposure to tobacco smoke was carried out every day for four months except weekends and holidays. Exposure continued until the day before dissection. The breeding conditions were breeding temperature 24 ⁇ 1, humidity 55 ⁇ 5%, lighting period 12 hours (8:00 to 20:00), and free feeding. , '
  • mice bred in the same manner except that the whole body was not exposed to mainstream smoke were used as a control group.
  • lung function evaluation and group Weaving evaluation was performed. As a result, it was found that respiratory function deteriorated and lung tissue destruction increased in the smoking group.
  • Deterioration of respiratory function due to tobacco smoke exposure was measured on the day after final exposure to mainstream smoke, and total lung volume and quasi-static lung compliance were measured. The increase in those values due to tobacco smoke exposure was compared between the smoking group and the control group. The difference was determined, and the obtained value was evaluated as an index.
  • the average alveolar diameter was measured under a microscope, and the increase in lung tissue destruction due to tobacco smoke exposure was evaluated using the difference between the smoking group and the control group as an index.
  • Lungs were excised from the mice of the smoking group and the control group, and IS0GEN (trade name) was obtained from the obtained lungs.
  • RNA was treated with DNase I using an RNeasy mini kit (trade name, manufactured by QIAGEN) according to the attached manual. Using 2.5 g of total RNA obtained in this way, MessageAmp aRNA
  • RNA Amplified RNA (aRNA) was synthesized according to the manual of Kit (trade name, manufactured by Ambion). Using the synthesized aRNA as probe type II, using the Atlas Glass Fluorescent Labeling Kit (trade name, CLONTECH (currently BD BIOSCIENCE)) or the Atlas PowerScript Fluorescent Labeling Kit (trade name, current BD BIOSCIENCE), According to the manual, Cy-3 dCTP or Cy-5 dCTP (manufactured by Amersham Bioscience) was incorporated with a Random 9mer primer, and the cDNA was fluorescently labeled. Using this as a probe, hybridization with a DNA chip was performed using an Automated Slide Processor (trade name, manufactured by Amersham Bioscience).
  • Hybridization was performed at 48 ° C for 12 hours with the probe after pretreatment according to the protocol for Type 7 slides.
  • Hybridizaton Buffer Ver.2 (trade name, manufactured by Amersham BioSigns) was used as a buffer. After washing, read with Microarray System Generation III Scanner (trade name, manufactured by Molecular Dynamics) and analyze with analysis software (trade name: Lucidea Automated Spotfinder (produced by Molecular Dynamics) and trade name: GeneSpring (silicon) The data were analyzed using the method of Genenetex Corporation)].
  • UGRP2 was found as a gene whose expression level was increased in the lungs of mice in the smoking group (onset of C0PD) compared to the lungs of mice in the control group.
  • the expression profile of the UGRP2 gene in the lungs of C0PD model mice was examined by RT-PCR.
  • RNA was extracted from the lungs of the mice of the smoking group and the control group of Example 1 using IS0GEN (trade name, manufactured by Nippon Gene) according to the attached manual. This RNA was obtained from QIAGEN under the trade name of RNeasy mini kit according to the attached manual. After eI treatment, it was recovered. From the obtained total RNA2.5, cDNA was synthesized using Superscript (registered trademark) First-strand Synthesis System for RT-PCR (trade name, manufactured by Invitrogen) according to the attached manual.
  • Superscript registered trademark
  • First-strand Synthesis System for RT-PCR (trade name, manufactured by Invitrogen) according to the attached manual.
  • primers for real-time quantitative PCR were selected using probe search software Primer Express (trade name, manufactured by ABI). . Primers were requested to be synthesized by Kokusai Reagent. Using these primers, real-time quantitative PCR was performed using SYBR Green I (manufactured by QIAGEN) using the synthesized cDNA as type I, and the amount of mRNA was quantified.
  • Applied Biosysteis (trade name: SYBR Green PCR Master Mix) is used. After reacting at 95 ° C for 10 minutes, 40 cycles of 95 ° C for 10 seconds and 60 ° C for 60 seconds are performed. And quantified. PCR and fluorescence measurements were performed using ABI's ABI PRISM 5700 Sequence Detection System (trade name). The results are shown in Figure 1.
  • the graph in FIG. 1 shows the relative amount with respect to the expression level in the control group of mice. In real-time quantitative PCR, it was confirmed that only one kind of the target UGRP2 mRNA was amplified by electrophoresis of the amplification product and by preparing a melting curve.
  • the expression profile of the UGRP2 gene in the lungs of asthma model mice was examined by RT-PCR.
  • mice in the asthma group and the control group were obtained as follows.
  • OVA egg white albumin
  • Alum aluminum hydroxide gel
  • the whole body was immunized (day 0 and day 14) and further sprayed with a 1% 0VA solution nebulized with a nebulizer (Omron) three times for 30 minutes (day 25, day 30 and day 36).
  • Eye prepared (asthma group).
  • the control group had saline injected intraperitoneally and sprayed similarly.
  • methacholine-induced airway hyperreactivity was measured in mice of both groups using Whole Body Unrestrained PlethysmograpH (manufactured by BAXCO), and asthma symptoms were confirmed in mice of the asthma group using Penh values as indicators.
  • the mesacholine solution (6, 12, and 24 mg / ml) was atomized with a nebulizer and sprayed for 2 minutes. The Penh value was measured one minute after spraying and averaged over four minutes.
  • RNA was extracted from lungs of mice in the asthma group and the control group using IS0GEN (trade name, manufactured by Nippon Gene Co., Ltd.) according to the attached manual.
  • the RNA was treated with DNase I using RNeasy mini kit (trade name, manufactured by QIAG EN) according to the attached manual, and collected.
  • CDNA was synthesized from 2.5 g of the obtained total RNA using a Superscript for RT-PCR (registered trademark) First-strand Synthesis System (trade name, manufactured by Invitrogen) according to the attached manual.
  • mouse UGRP2 SEQ ID NO:
  • primers for real-time quantitative PCR were selected using probe search software Primer Express (trade name, manufactured by ABI). Primers were requested to be synthesized by Nisshinbo. Using this primer, real-time quantitative PCR using SYBR Green I was performed using the synthesized cDNA as type II to quantify the amount of mRNA.
  • reaction reagent a product name of Applied Biosystems: SYBR Green PCR Master
  • the mixture was reacted at 95 "C for 10 minutes, and quantified by performing 40 cycles of 95 ° C for 10 seconds and at 60 seconds.
  • PCR and fluorescence measurement were performed using ABI's ABI PRISM 5700 Sequence. The results were shown in Fig. 2. The results are shown in Fig. 2.
  • the graph in Fig. 2 shows the relative amount with respect to the expression level in the control group of mice. In real-time quantitative PCR, it was confirmed that only one kind of the target UGRP2 mRNA was amplified by electrophoresis of the amplification product and by preparing a melting curve.
  • the mixture of cDNA fragments amplified by this reaction is further designated as ⁇ , and primer pairs 5′-GCGCCATGAAGCTGGCCG-3 ′ and 5′-TCAGCCAAACACTGTCAGGG-3 ′ (SEQ ID NO: 1)
  • a cDNA fragment corresponding to the entire translation region of the human UGRP2 gene was synthesized.
  • the composition of the reaction solution in the reaction was as follows: 1/50 of the above single-stranded cDNA or PCR reaction solution was used as type III, 1/200 amount of Ex Taq (Yukara), and 1 primer pair. M and dNTPs were added in a volume of 200, and the buffer attached to the enzyme was added to make a liquid volume of 501.
  • the PCR reaction is repeated at 94 ° C for 3 minutes, followed by a cycle of 94 ° C for 30 seconds, 50 ° C for 30 seconds, 72 at 1 minute 25 times, and finally an extension reaction at 72 at 1 minute Was.
  • the 320 bp cDNA fragment obtained as a result of the PCR reaction was collected after agarose electrophoresis, and cloned into pCR2.T0P0 vector (manufactured by Invitrogen).
  • a Human 12-Lane MTN Blot (Clontech; lane 1: brain, lane 2: heart, lane 3: skeletal muscle, lane 4: colon, lane 5: thymus, lane 6: spleen , Lane 7: Kidney, Lane 8: Liver, Lane 9: Small intestine, Lane 10: Placenta, Lane 11: Lung, Lane, 12: Peripheral blood lymphocyte) and Human 12- Lane MTN Blot-II ( Manufactured by Clontech; Lane 1: adrenal gland, lane 2: bladder, lane 3: bone marrow, lane 4: brain (whole), lane 5: lymph node, lane 6: prostate, lane 7: spinal cord, lane 8: stomach, lane Northern plot analysis was performed on (9: thyroid, lane 10: tongue, lane 11: trachea, lane 12: uterus).
  • GMC buffer Hybridization buffer
  • a hybridization buffer consisting of 0.5 M phosphate buffer (pH 7.2), 1% serum albumin, ImM EDTA, and 7% SDS, and incubated at 65 ° C for 1 hour.
  • the nylon membrane was transferred to a solution in which a 32 P-labeled probe was added to a GMC buffer, and hybridization was performed at 65 ° C. for 16 hours.
  • the nylon membrane was rinsed with 2XSSC (1XSSC is a mixed aqueous solution of 15mM sodium citrate and 150mM sodium chloride) at room temperature for 5 minutes, then 1XSSC at 50 ° C for 30 minutes and 0.5XSSC at 50.
  • 2XSSC 1XSSC is a mixed aqueous solution of 15mM sodium citrate and 150mM sodium chloride
  • Hybridomas producing monoclonal antibodies against UGRP were produced by cell fusion technology.
  • Cys_UGRP2 46-58, CTLANPLGTLNPLK A conjugate in which SEQ ID NO: 26) and Cys-UGRP2 (63-77, SLGIPVNHLIEGSQKC, SEQ ID NO: 27) were bound to maleimidated Keyhole le Li immediately et Hemocyanin (KLH, Pierce) was used.
  • a / J Jms Sic mice were subcutaneously and intraperitoneally immunized subcutaneously and intraperitoneally with an emulsion mixed with Freund's complete adjuvant (FCA) for the first time and incomplete Freund's adjuvant for the second and subsequent times.
  • FCA Freund's complete adjuvant
  • Partial blood was collected after the second immunization, and antibody production was performed by the DELFIA method. That is, on a plate on which a goat anti-mouse IgG Fc-specific antibody (ICN) was immobilized, the above peptide labeled with biotin, Streptavidin Eu-labeled (Perkin Elmer) and an antibody solution were incubated at room temperature for 2 hours or 4 t. After the reaction overnight, washing was performed, an enhancing reagent was added, and evaluation was performed by measuring time-resolved fluorescence with Walac ARV0 (manufactured by Pakinkin Elma).
  • ICN goat anti-mouse IgG Fc-specific antibody
  • a booster (boost) was performed. Three days later, spleen cells were prepared from the spleen removed under sterile conditions, and cell fusion with myeloma cells was performed using 50% polyethylene glycol. Was done. The fused cells were seeded on a 96-well plate in HAT medium and cultured for 7 to 9 days. After confirming that a colony was formed, the supernatant was collected and the antibody-producing ability was evaluated by the DELFIA method described above. The antibody-producing hybridomas were cloned by the limiting dilution method and UG2-6A9 (accession number FERM
  • the UGRP2 assay uses an enzyme-linked immunosorbent assay (ELISA) using a horseradish peroxidase (HRP) -labeled antibody.
  • ELISA enzyme-linked immunosorbent assay
  • HRP horseradish peroxidase
  • Antibodies were obtained by culturing eight hybridoma cells in a serum-free medium (Invitrogen) and using the culture supernatant as MAPS II Protein A kit (
  • UGRP2 a recombinant UGRP2 protein expressed by using a wheat germ development system II: Wheat germel 1-free protein in synthes is core kit (manufactured by Toyobo) for wheat ij is used. Those who express According to the attached manual, expression is performed using a gene with a FLAG tag gene added to the 3 'side of the UGR gene. The expressed protein is purified by affinity purification using an anti-FLAG antibody gel (manufactured by SIGMA).
  • the antibody that recognizes one of the epitopes is diluted to an appropriate concentration with 50 mM Tris-HCl buffered saline (TBS) and spread on a 96-well plate (Nunc) at 4 ° C. Physically adsorb by standing overnight. After washing, blocking is performed by adding a solution containing serum albumin, and an antibody solid phase plate is prepared.
  • Antibodies that recognize the other epitope are labeled with HRP using the hinge method. The antibody is digested with pepsin to prepare F (ab ') 2, and then reduced with 2 mercaptoethylamine at a final concentration of 10 mM for 90 minutes to prepare Fal]'.
  • HRP is reacted with 50-fold molar amount of Sulfo-H MCS (manufactured by Dojindo Laboratories) to produce maleimidated HRP.
  • Sulfo-H MCS manufactured by Dojindo Laboratories
  • the above Fab ′ and maleimidated HRP are reacted in equimolar amounts to prepare an HRP-labeled Fab ′ (HRP-labeled antibody).
  • the labeled antibody is purified by HPLC using a TSKgel G2000SWXL column.
  • ELISA In ELISA, first, standard UGRP2 or a sample containing UGRP2 is added to an antibody solid-phase plate and reacted. After the reaction, wash the plate and add HRP-labeled antibody. After the reaction, wash the plate and add Colorburst Blue solution (tetramethylbenzidine solution, ALerCHEK). After color development, add an equal volume of 0.18N sulfuric acid solution to stop the enzyme reaction. Measure absorbance at 450 nm with Walac ARV0 SX (PerkinElmer) and create a standard curve from the absorbance of standard UGRP2 to determine the sample concentration
  • SEQ ID NO: 19 shows a sequence of a human SCGB3A1 (UGRP2) RT-PCR forward primer.
  • SEQ ID NO: 20 shows the sequence of human SCGB3A1 (UGRP2) RT-PCR lipase primer.
  • SEQ ID NO: 21 shows a sequence of a human SCGB3A1 (UGRP2) RT-PCR forward primer.
  • SEQ ID NO: 22 shows the sequence of a human SCGB3A1 (UGRP2) RT-PCR reverse primer.
  • SEQ ID NO: 23 shows the sequence of a human SCGB3A1 (UGRP2) Northern analysis probe.
  • SEQ ID NO: 24 shows the sequence of mouse SCGB3A1 (UGRP2) RT-PCR forward primer.
  • SEQ ID NO: 25 shows a sequence of a mouse SCGB3A1 (UGRP2) RT-PCR reverse primer.

Abstract

A method of readily and speedily detecting or screening a specimen derived from individuals suspected of infection with COPD by measuring the expression level of gene coding for protein belonging to SCGB family, for example, UGRP2; and a kit with which the method can be effectively carried out. In particular, a method of detecting or screening a specimen derived from individuals suspected of infection with chronic obstructive pulmonary disease, comprising the step of measuring the expression level of gene coding for protein belonging to Secretoglobin family in each of a test sample derived from test subject individuals and a control sample derived from normal individuals, wherein when the expression level in the test sample is higher than that in the control sample, this provides an index expressing that the test sample is one derived from individuals suspected of infection with chronic obstructive pulmonary disease. There is further provided a kit for detection or screening to be used in the above method, comprising a nucleic acid capable of detecting a nucleic acid coding for protein belonging to Secretoglobin family.

Description

慢性閉塞性肺疾患の検出方法 技術分野  Method for detecting chronic obstructive pulmonary disease
本発明は、 慢性閉塞性肺疾患の治療、 予防等のための手段に関する。 より詳し くは、 本発明は、 慢性閉塞性肺疾患の治療剤又は予防剤のスクリーニング方法、 該スクリーニング方法に用いるキット、 医薬組成物、 慢性閉塞性肺疾患の診断方 法、 該診断方法に用いるキット、 慢性閉塞性肺疾患に罹患している疑いがある個 体由来の試料の検出又は選別方法、 並びに該検出又は選別方法に用いるキットに 関する。 背景技術  The present invention relates to means for treating and preventing chronic obstructive pulmonary disease. More specifically, the present invention relates to a method for screening a therapeutic or preventive agent for chronic obstructive pulmonary disease, a kit used for the screening method, a pharmaceutical composition, a method for diagnosing chronic obstructive pulmonary disease, and a method for diagnosing chronic obstructive pulmonary disease. The present invention relates to a kit, a method for detecting or selecting a sample derived from an individual suspected of suffering from chronic obstructive pulmonary disease, and a kit used for the detection or selection method. Background art
慢性閉塞性肺疾患（C0PD： chroni c obs t ruc t ive pulmonary d i seases)とは、 一 般に小気管支の永続的あるいは一時的狭窄を示す病気の総称で、 努力呼気流が低 下し、 特に病因や他のより特異的病名が付けられないものをいうと定義される疾 患である （ステッドマン医学大辞典第 5版、 （株） メジカルビユー社、 2002年 2 月 20 日第 1刷発行参照） 。 当該疾患には、 例えば、 肺気腫や慢性気管支炎が含 まれる。 喫煙が C0PD の原因となることはすでに多くの調査研究で明らかにされ ている。  Chronic obstructive pulmonary disease (C0PD: chronic obstructive pulmonary diseases) is a general term for diseases that indicate permanent or temporary stenosis of the small bronchi. A disease that is defined as having no etiology or other more specific disease name (see Steadman Dictionary of Medicine, 5th Edition, Medical View, Inc., first print, February 20, 2002) . The disease includes, for example, emphysema and chronic bronchitis. Many studies have already shown that smoking causes C0PD.
肺気腫は本来病理学的に定義された疾患であり、 終末細気管支より末梢の気道 の破壊性変化と気腔の拡張を特徴とする。 臨床的には 50〜60 歳台以後の喫煙男 性に多く、 労作時呼吸困難、 咳、 場合によっては喀痰を伴うこともあり、 最終的 には呼吸不全、 右心不全を合併してくる。 喫煙が本疾患の原因であることに異論 はないが、 逆に長期喫煙者がすべて肺気腫になるわけではない。 喫煙量よりも喫 煙に対する個体の感受性の差が発症の要因として重要であることが示唆されてい る （泉 孝英ら、 「喫煙の肺気腫の発症 ·進展に及ぼす影響に関する臨床的 ·基 礎的研究- C0PD登録症例 m例における喫煙と C0PD発症の関連性に関する検討 」 平成 5年度喫煙科学研究財団研究年報： 288-293参照） 。 Emphysema is an inherently pathologically defined disease characterized by destructive changes in the airways peripheral to the terminal bronchiole and dilatation of the air space. Clinically, it is more common in smoking men after the age of 50 to 60, and may be accompanied by dyspnea on exertion, cough, and sometimes sputum, and eventually complications of respiratory failure and right heart failure. There is no doubt that smoking is the cause of the disease, but not all long-term smokers have emphysema. It has been suggested that the difference in individual susceptibility to smoking rather than the amount of smoking is more important as a factor in the onset. (Takahide Izumi, et al., "Clinical and Basic Study on the Effect of Smoking on the Onset and Progression of Emphysema-Examination of the Relationship between Smoking and the Onset of C0PD in m C0PD Registered Cases") Annual report: 288-293).
慢性気管支炎は喀痰を伴った咳が 3か月以上、 しかも 2年以上の期間にわたつ て反復するという臨床所見に基づいて定義される。 病理学的には気管支粘液腺の 腫大、 平滑筋肥大と気管支壁の肥厚 ·炎症細胞の浸潤 ·浮腫などの炎症所見がみ られる。 本症の特徴である喀痰量増加は病理学的に気管支粘液腺の腫大と対応す ると考えられる。 喫煙刺激が気管支腺組織の肥大をもたらし、 気道分泌亢進の一 因となることが指摘されている （吉良枝郎ら、 「呼吸器病学的、 気道構成上及び 呼吸器防御系からみた長期喫煙の指標」 昭和 63年度喫煙科学研究財団研究年報： 354- 359参照） 。  Chronic bronchitis is defined based on the clinical finding that a cough with sputum recurs for more than 3 months and more than 2 years. Pathological findings include swelling of the bronchial mucous glands, smooth muscle hypertrophy and thickening of the bronchial wall, infiltration of inflammatory cells, and edema. The increased sputum volume, characteristic of this disease, may be pathologically associated with an enlarged bronchial mucous gland. It has been pointed out that smoking stimulation causes bronchial gland tissue hypertrophy and contributes to an increase in airway secretion (Erashiro Kira et al., "Long term smoking from the viewpoint of respiratory pathology, airway composition and respiratory defense system." Indicators of the 1988 Annual Report of the Smoking Science Research Foundation: 354-359).
しかしながら、 喫煙による C0PD の発症にどのような因子が関係しているかに ついては未だ不明である。  However, it is still unclear what factors are involved in the onset of C0PD from smoking.
一方、 Secretoglob in (SCGB) ファミリーとはクララ細胞分泌タンパク質 （CCS P ; Cl ara ce l l secretory pro te in) を主要なメンバーとする一群のタンパク質 である (Susan D. e t al . , Am. J. of Respi ratory and Cr i t i cal Care Med. , 2 002, Vo l . 166, 1498-1509 参照） 。 該 CCSP は誘導気道上皮内で多く発現されて おり、 気道の炎症に関与しているものと考えられている。  On the other hand, the Secretoglob in (SCGB) family is a group of proteins whose main members are Clara cell secretory proteins (CCS P) (Susan D. et al., Am. J. of Research and Critical Care Med., 2002, Vol. 166, 1498-1509). The CCSP is highly expressed in the induced airway epithelium and is thought to be involved in airway inflammation.
SCGB ファミリーには 7つのサブファミリー （1A、 1B、 1 1D、 2A、 2B、 3A) があり、 SCGBファミリー 3Aに含まれるタンパク質としては、 例えば、 ヒト Uter oglobin-rel ated protein (UGRP) 2が知られている。 ヒト UGRP2は前駆体が 104 個のアミノ酸からなる分泌型タンパク質である。 該前駆体としては、 アミノ酸配 列の 19位が Alaであるものと、 Argであるものの 2種類が報告されており、 い ずれも 20アミノ酸残基からなるシグナルペプチド （1位の Me tから 20位の Al a まで） が切断され、 成熟型として分泌される。  The SCGB family has seven subfamilies (1A, 1B, 11D, 2A, 2B, and 3A), and the protein contained in the SCGB family 3A is, for example, human Uter oglobin-related protein (UGRP) 2. Have been. Human UGRP2 is a secreted protein whose precursor consists of 104 amino acids. As the precursor, two types, one in which the amino acid sequence at position 19 is Ala and the other at Arg, have been reported. In each case, a signal peptide consisting of 20 amino acid residues (from the first position of Me to 20) Is cleaved and secreted as the mature form.
例えば、 ヒト UGRP2の核酸配列の検出方法及びそのキット、 核酸配列、 組換え 体発現システム及び形質転換細胞、 ポリペプチド、 抗体及びそれを用いた検出キ ット、 抗体作出方法、 遺伝子及びその断片などが報告されており、 肺癌等の一般 的な肺疾患の予防 '治療等への適用が報告されている （国際公開第 98/33926 号 パンフレツト参照） 。 For example, a method for detecting the nucleic acid sequence of human UGRP2 and its kit, nucleic acid sequence, recombination An in vivo expression system and transformed cells, polypeptides, antibodies and detection kits using them, methods for producing antibodies, genes and fragments thereof, etc., have been reported.Prevention and treatment of general lung diseases such as lung cancer Application has been reported (see WO 98/33926 pamphlet).
しかしながら、 ヒト UGRP2と COPDとの関係については具体的な開示はない。 , また、' 喫煙による COPD の発症モデルとしては、 マウスにおいて長期間の煙草 煙暴露で COPD病態 （特に肺気腫、 肺組織 （肺胞） の破壊） が惹起されることが 報告されている (Haut amaki R. D. et al .， Sc ience, 1997, Vol . 277, 2002-200 4参照） 。 発明の開示  However, there is no specific disclosure of the relationship between human UGRP2 and COPD. In addition, as a model for the onset of COPD by smoking, it has been reported that long-term exposure to cigarette smoke in mice induces COPD pathology (especially emphysema and destruction of lung tissue (alveoli)) (Haut amaki RD et al., Science, 1997, Vol. 277, 2002-2004). Disclosure of the invention
本発明は、 SCGBファミリーに属するタンパク質、 例えば、 UGRP2の遺伝子の発 現レベルを測定することによる、 C0PD に罹患している疑いがある個体由来の試 料の簡便かつ迅速な検出又は選別方法、 該方法を効率的に行いうるキットを提供 することを目的とする。  The present invention provides a simple and rapid method for detecting or selecting a sample derived from an individual suspected of having C0PD by measuring the expression level of a protein belonging to the SCGB family, for example, the UGRP2 gene. The purpose is to provide a kit that can perform the method efficiently.
また、 本発明は、 SCGBファミリーに属するタンパク質、 例えば、 UGRP2の遺伝 子の発現レベルを測定することによる、 C0PD の簡便かつ迅速な診断方法、 該診 断方法を効率的に行いうる診断用キットを提供することを目的とする。  In addition, the present invention provides a simple and rapid method for diagnosing C0PD by measuring the expression level of a protein belonging to the SCGB family, for example, the UGRP2 gene, and a diagnostic kit capable of efficiently performing the diagnosing method. The purpose is to provide.
また、 本発明は、 SCGBファミリーに属するタンパク質、 例えば、 UGRP2又はそ れをコードする遺伝子の動態を検出又は測定することによる、 C0PD の治療剤又 は予防剤の効率的なスクリーニング方法、 前記スクリ.一二ング方法を簡便かつ迅 速に行いうるスクリーニング用キットを提供することを目的とする。  The present invention also provides a method for efficiently screening for a therapeutic or prophylactic agent for C0PD by detecting or measuring the dynamics of a protein belonging to the SCGB family, for example, UGRP2 or a gene encoding the same. It is an object of the present invention to provide a screening kit that can easily and quickly perform the singing method.
また、 本発明は、 前記スクリーニング方法により得られる、 C0PD の治療剤又 は予防剤として好適に使用される化合物又はその塩、 C0PD の治療又は予防に好 適な前記化合物又はその塩を有効成分とする医薬組成物を提供することを目的と する。 即ち、 本発明の一つの局面は、 Further, the present invention provides a compound or a salt thereof suitably used as a therapeutic or prophylactic agent for C0PD obtained by the screening method, and a compound or a salt thereof suitable for the treatment or prevention of C0PD as an active ingredient. It is an object of the present invention to provide a pharmaceutical composition which comprises: That is, one aspect of the present invention is:
( 1 ) 被検対象の個体由来の被検試料及び正常個体由来の対照試料それぞれにお ける Secretoglobinファミリーに属するタンパク質の遺伝子の発現レベルを測定 するステップを含み、 ここで、 被検試料における発現レベルが、 対照試料におけ る発現'レベルよりも高くなる場合、 該被検試料が、 慢性閉塞性肺疾患に罹患して いる疑いがある個体由来の試料であることの指標となる、 慢性閉塞性肺疾患に罹 患している疑いがある個体由来の試料の検出又は選別方法、  (1) measuring the expression level of a gene of a protein belonging to the Secretoglobin family in each of a test sample derived from an individual to be tested and a control sample derived from a normal individual, wherein the expression level in the test sample is included. Is higher than the expression level in the control sample, it indicates that the test sample is a sample derived from an individual suspected of suffering from chronic obstructive pulmonary disease. A method for detecting or selecting a sample from an individual suspected of having a lung disease,
(2) 発現レベルが、 Secretoglobin ファミリーに属するタンパク質の遺伝子の mRNA転写物の量を指標として測定される、 前記 （1) 記載の方法、  (2) The method according to (1), wherein the expression level is measured using an amount of an mRNA transcript of a gene of a protein belonging to the Secretoglobin family as an index.
(3) 発現レベルが、 Secretoglobin ファミリ一に属するタンパク質の遺伝子の 遺伝子産物の量を指標として測定される、 前記 （1) 記載の方法、  (3) The method according to (1), wherein the expression level is measured using an amount of a gene product of a gene of a protein belonging to the Secretoglobin family 1 as an index.
(4) 慢性閉塞性肺疾患が肺気腫である、 前記 （1) 〜 （3) いずれか記載の方 法、  (4) The method according to any one of (1) to (3), wherein the chronic obstructive pulmonary disease is emphysema.
(5) Secretoglobin ファミリ一に属するタンパク質をコードする核酸を検出し うる核酸を用いて測定される、 前記 （2) 記載の方法、  (5) the method according to (2), wherein the measurement is performed using a nucleic acid capable of detecting a nucleic acid encoding a protein belonging to the Secretoglobin family 1;
(6) Secretoglobin ファミリーに属するタンパク質をコードする核酸が、 配列 番号： 1、 3、 5、 7、 9、 1 1、 13及び 15からなる群より選ばれる塩基配 列からなる核酸である、 前記 （5) 記載の方法、  (6) the nucleic acid encoding a protein belonging to the Secretoglobin family is a nucleic acid comprising a base sequence selected from the group consisting of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, and 15; 5) The method described,
(7) 配列番号： 19、 20、 21、 22及び 23からなる群より選ばれる少な くとも 1つの核酸を用いて測定される、.前記 （6) 記載の方法、  (7) the method according to (6), wherein the measurement is performed using at least one nucleic acid selected from the group consisting of SEQ ID NOs: 19, 20, 21, 22, and 23;
(8) Secretoglobin ファミリーに属するタンパク質に対する抗体又はその断片 を用いて測定される、 前記 （3) 記載の方法、  (8) The method according to (3), wherein the measurement is performed using an antibody against a protein belonging to the Secretoglobin family or a fragment thereof.
(9) 前記抗体又はその断片が、 配列番号 26又は 27のアミノ酸配列を有する ぺプチド又は該ぺプチドを含むポリぺプチドに対するモノクローナル抗体又はそ の断片である、 前記 （8) 記載の方法、 (1 0) 前記抗体が受領番号 FERM ABP- 10012又は FERM ABP- 10013であるハイブ リドーマにより産生されるものである、 前記 （9) 記載の方法、 (9) The method according to (8), wherein the antibody or a fragment thereof is a monoclonal antibody against a peptide having the amino acid sequence of SEQ ID NO: 26 or 27 or a polypeptide containing the peptide or a fragment thereof. (10) The method according to (9), wherein the antibody is produced by a hybridoma having accession number FERM ABP-10012 or FERM ABP-10013.
(1 1) Secretoglobin ファミリーに属するタンパク質をコードする核酸を検出 しうる核酸を含有してなる、 前記 （5) 記載の方法に使用するための検出又は選 別用キッ卜、  (11) A detection or selection kit for use in the method according to (5), comprising a nucleic acid capable of detecting a nucleic acid encoding a protein belonging to the Secretoglobin family,
(1 2う Secretoglobin ファミリ一に属するタンパク質をコードする核酸が、 配 列番号： 1、 3、 5、 6、 9、 1 1、 1 3及び 1 5からなる群より選ばれる配列 からなる核酸である、 前記 （1 1) 記載の検出又は選別用キット、  (12) The nucleic acid encoding a protein belonging to the Secretoglobin family is a nucleic acid having a sequence selected from the group consisting of SEQ ID NOs: 1, 3, 5, 6, 9, 11, 13, and 15. The kit for detection or selection according to the above (11),
(1 3) 配列番号： 1 9、 20、 2 1、 22及び 23からなる群より選ばれる少 なくとも 1つの核酸を含有してなる、 前記 （1 2) 記載の検出又は選別用キット  (13) SEQ ID NO: 19, 20, 21 or 22, the detection or selection kit according to (12), comprising at least one nucleic acid selected from the group consisting of:
(14) Secretoglobin ファミリーに属するタンパク質に対する抗体又はその断 片を含有してなる、 前記 （8) 記載の方法に使用するための検出又は選別用キッ 卜、 (14) a detection or selection kit for use in the method according to (8), comprising an antibody against a protein belonging to the Secretoglobin family or a fragment thereof;
(1 5) 前記抗体又はその断片が、 配列番号 26又は 27のアミノ酸配列を有す るべプチド又は該ぺプチドを含むポリべプチドに対するモノクローナル抗体又は その断片である、 前記 （14) 記載の検出又は選別用キット、  (15) The detection according to (14), wherein the antibody or a fragment thereof is a monoclonal antibody or a fragment thereof against a peptide having the amino acid sequence of SEQ ID NO: 26 or 27 or a polypeptide containing the peptide. Or a sorting kit,
(1 6) 前記抗体が受領番号 FERM ABP-10012又は FERM ABP-10013であるハイブ リドーマにより産生されるものである、 前記 （1 5) 記載の検出又は選別用キッ 卜、  (16) The kit for detection or selection according to (15), wherein the antibody is produced by a hybridoma having accession number FERM ABP-10012 or FERM ABP-10013.
(1 7) 配列番号 26又は 27のアミノ酸配列を有するペプチド又は該ペプチド を含むポリぺプチドに対するモノクローナル抗体、  (17) a peptide having the amino acid sequence of SEQ ID NO: 26 or 27 or a monoclonal antibody against a polypeptide comprising the peptide,
(1 8) 受領番号 FERM ABP- 10012又は FERM ABP- 10013であるハイプリドーマに より産生される抗体、  (18) an antibody produced by a hybridoma having accession number FERM ABP-10012 or FERM ABP-10013,
(1 9) 受領番号 FERM ABP-10012又は FERM ABP- 10013であるハイプリドーマ、 (1 9) A hybridoma with a receipt number FERM ABP-10012 or FERM ABP-10013,
(20) 被検物質の存在下、 Secretoglobin ファミリーに属するタンパク質若し くは該タンパク質をコードする遺伝子の動態を検出又は測定することを特徴とす る、 慢性閉塞性肺疾患治療剤又は予防剤のスクリーニング方法、 (20) In the presence of the test substance, a protein belonging to the Secretoglobin family Or a method for screening a therapeutic or preventive agent for chronic obstructive pulmonary disease, comprising detecting or measuring the dynamics of a gene encoding the protein.
(21) 被検対象の個体由来の被検試料及び正常個体由来の対照試料それぞれに おける SecretogloMnファミリーに属するタンパク質の遺伝子の発現レベルを測 定するステップを含み、 ここで、 被検試料における発現レベルが、 対照試料にお ける発現レベルよりも高くなる場合、 該被検対象の個体が、 慢性閉塞性肺疾患に 罹患している疑いがあることの指標となる、 慢性閉塞性肺疾患の診断方法、  (21) measuring the expression level of the gene of the protein belonging to the SecretogloMn family in each of the test sample derived from the test subject individual and the control sample derived from the normal individual, wherein the expression level in the test sample is included. If the expression level is higher than the expression level in a control sample, the diagnostic method for chronic obstructive pulmonary disease is an indicator that the subject is suspected to have chronic obstructive pulmonary disease. ,
(22) Secretoglobin ファミリーに属するタンパク質をコードする核酸を検出 しうる核酸を含有してなる、 前記 （21) 記載の方法に使用するための診断用キ ッ卜、  (22) A diagnostic kit for use in the method according to (21), comprising a nucleic acid capable of detecting a nucleic acid encoding a protein belonging to the Secretoglobin family,
(23) Secretoglobin ファミリーに属するタンパク質をコードする核酸が、 配 列番号： 1、 3、 5、 6、 9、 1 1、 13及び 1 5からなる群より選ばれる配列 からなる核酸である、 前記 （22) 記載の診断用キット、  (23) The nucleic acid encoding a protein belonging to the Secretoglobin family is a nucleic acid consisting of a sequence selected from the group consisting of SEQ ID NOs: 1, 3, 5, 6, 9, 11, 13, and 15. 22) described diagnostic kit,
(24) 配列番号： 19、 20、 21、 22及び 23からなる群より選ばれる少 なくとも 1つの核酸を含有してなる、 前記 （23) 記載の診断用キット、  (24) The diagnostic kit according to (23), which comprises at least one nucleic acid selected from the group consisting of SEQ ID NOs: 19, 20, 21, 22, and 23.
(25) Secretoglobin ファミリーに属するタンパク質に対する抗体又はその断 片を含有してなる、 前記 （21) 記載の方法に使用するための診断用キット、 (25) a diagnostic kit for use in the method according to (21), comprising an antibody against a protein belonging to the Secretoglobin family or a fragment thereof;
(26) 前記抗体又はその断片が、 配列番号 26又は 27のアミノ酸配列を有す るぺプチド又は該ぺプチドを含むポリべプチドに対するモノクロ一ナル坊体又は その断片である、 前記 （25) 記載の診断用キット、 ならびに (26) The above (25), wherein the antibody or a fragment thereof is a monoclonal antibody against a peptide having the amino acid sequence of SEQ ID NO: 26 or 27 or a polypeptide containing the peptide, or a fragment thereof. Diagnostic kit, and
(27) 該抗体が受領番号 FERM ABP- 10012又は？6 ¾1 ABP-10013であるハイプリ ドーマにより産生されるものである、 前記 （26) 記載の診断用キット に関する。  (27) The antibody has accession number FERM ABP-10012 or? (6) The diagnostic kit according to (26), which is produced by a hybridoma that is ABP-10013.
さらに、 本発明の第二の局面は、  Furthermore, the second aspect of the present invention
(1) 被検物質の存在下、 Secretoglobin ファミリーに属するタンパク質若し くは該タンパク質をコードする遺伝子の動態を検出又は測定することを特徴とす る、 慢性閉塞性肺疾患治療剤又は予防剤のスクリーニング方法、 (1) detecting or measuring the dynamics of a protein belonging to the Secretoglobin family or a gene encoding the protein in the presence of a test substance. Screening method for a therapeutic or prophylactic agent for chronic obstructive pulmonary disease,
(2) Secretoglobin ファミリーに属するタンパク質が、 SecretogloMn ファ ミリ一 3Aに属するタンパク質である、 前記 （1) 記載の方法、  (2) The method according to (1), wherein the protein belonging to the Secretoglobin family is a protein belonging to SecretogloMn family 3A.
(3) Secretoglobinファミリー 3Aに属するタンパク質が、 Uteroglobin-rela ted proteinである、 前記 （2) 記載の方法、  (3) The method according to (2), wherein the protein belonging to Secretoglobin family 3A is Uteroglobin-related protein.
(4) Uteroglobin - related proteinが、 Uteroglobin-related protein2 ¾ る、 前記 （3) 記載の方法、 · (4) Uteroglobin-related protein is Uteroglobin-related protein2, the method according to (3),
(5) Uteroglobin-related protein2が、 ヒ卜 Uteroglobin - related protein 2である、 前記 （4) 記載の方法、 (5) Uteroglobin-related protein 2 is human Uteroglobin-related protein 2, the method according to (4),
(6) (I) Secretoglobin ファミリーに属するタンパク質と、 被検物質とを 接触させるステップ、 並びに  (6) (I) contacting a protein belonging to the Secretoglobin family with a test substance, and
(I I) 前記ステップ （I) の後、 該タンパク質と被検物質との結合を検出し、 それにより、 該タンパク質と結合する被検物質を、 慢性閉塞性肺疾患治療剤又は 予防剤の候補化合物として選択するステップ  (II) After the step (I), the binding between the protein and a test substance is detected, whereby the test substance binding to the protein is converted into a candidate compound for a therapeutic or preventive agent for chronic obstructive pulmonary disease. Step to select as
を含む、 前記 （1) 〜 （5) いずれか記載の方法、 The method according to any one of the above (1) to (5),
(7) (A) Secretoglobin ファミリーに属するタンパク質をコードする遺伝 子を細胞に導入するステップ、  (7) (A) a step of introducing a gene encoding a protein belonging to the Secretoglobin family into a cell,
(B) 被検物質の存在下、 前記ステップ （A) で得られた細胞において前記遺伝 子を発現させるステップ、 並びに  (B) expressing the gene in the cell obtained in the step (A) in the presence of a test substance; and
(C) 被検物質の非存在下の場合と比較して、 前記遺伝子の発現の有無を検出し 、 又は前記遺伝子の発現の変動を測定し、 それにより、 該遺伝子の発現の変化を もたらす被検物質を慢性閉塞性肺疾患治療剤又は予防剤の候補化合物として選択 するステップ  (C) detecting the presence or absence of the expression of the gene, or measuring a change in the expression of the gene, as compared to the case in the absence of the test substance, and thereby detecting a change in the expression of the gene. Selecting the test substance as a candidate compound for a therapeutic or prophylactic agent for chronic obstructive pulmonary disease
を含む、 前記 （1) 〜 （5) いずれか記載の方法、 The method according to any one of the above (1) to (5),
(8) 該遺伝子の発現の変化が該遺伝子の発現の減少である前記 （7) 記載の 方法、 (9) 慢性閉塞性肺疾患が肺気腫である前記 （1) 〜 （8) いずれか記載の方 法、 (8) The method according to the above (7), wherein the change in the expression of the gene is a decrease in the expression of the gene. (9) The method according to any one of (1) to (8) above, wherein the chronic obstructive pulmonary disease is emphysema.
(10) Secretoglobin ファミリ一に属するタンパク質を含有してなる前記 （ 1) 〜 （6) 及び （9) いずれかに記載の方法に使用するためのキット、  (10) A kit for use in the method according to any one of (1) to (6) and (9), which comprises a protein belonging to the Secretoglobin family 1;
(1 1) , Secretoglobin ファミリーに属するタンパク質をコードする遺伝^"を 含む核酸構築物を含有してなる前記 （1) 〜 （5) 及び （7) 〜 （9) いずれか に記載の方法に使用するためのキット、  (11) The method according to any one of (1) to (5) and (7) to (9) above, which comprises a nucleic acid construct containing a gene ^ "encoding a protein belonging to the Secretoglobin family. Kit for the
(12) Secretoglobin ファミリーに属するタンパク質をコードする遺伝子が 導入された細胞を含有してなる前記 （1) 〜 （5) 及び （7) 〜 （9) いずれか に記載の方法に使用するためのキット、  (12) A kit for use in the method according to any one of (1) to (5) and (7) to (9), comprising a cell into which a gene encoding a protein belonging to the Secretoglobin family has been introduced. ,
(13) Secretoglobin ファミリ一に属するタンパク質に対する抗体又はその 断片を含有してなる前記 （1) 〜 （9) いずれかに記載の方法に使用するための キッ卜、  (13) A kit for use in the method according to any one of (1) to (9) above, which comprises an antibody against a protein belonging to the Secretoglobin family 1 or a fragment thereof.
(14) 前記 （1) 〜 （9) いずれかに記載の方法により得られる化合物又は その塩、  (14) The compound or a salt thereof obtained by the method according to any one of (1) to (9),
(15) 前記 （14) に記載の化合物又はその塩を有効成分として含有してな る医薬組成物、 '  (15) A pharmaceutical composition comprising the compound according to (14) or a salt thereof as an active ingredient.
(16) 慢性閉塞性肺疾患の治療又は予防に用いる、 前記 （15) 記載の医薬 組成物、  (16) The pharmaceutical composition according to (15), which is used for treating or preventing chronic obstructive pulmonary disease.
(17) 被検対象の個体由来の被検試料及び正常個体由来の対照試料それぞれ における Secretoglobinファミリ一に属するタンパク質の遺伝子の発現レベルを 測定するステップを含み、 ここで、 被検試料における発現レベルが、 対照試料に おける発現レベルよりも高くなる場合、 該被検対象の個体が、 慢性閉塞性肺疾患 に罹患している疑いがあることの指標となる、 慢性閉塞性肺疾患の診断方法、 (17) measuring the expression level of the gene of a protein belonging to the Secretoglobin family in each of the test sample derived from the test subject and the control sample derived from the normal individual, wherein the expression level in the test sample is A method for diagnosing chronic obstructive pulmonary disease, wherein the expression level is higher than that in a control sample, which indicates that the subject is suspected of having chronic obstructive pulmonary disease.
(18) 発現レベルが、 Secretoglobin ファミリーに属するタンパク質の遺伝 子の mRNA転写物の量を指標として測定される、 前記 （17) 記載の方法、 (19) 発現レベルが、 Secretoglobin ファミリーに属するタンパク質の遺伝 子の遺伝子産物の量を指標として測定される、 前記 （17) 記載の方法、 (18) The method according to (17), wherein the expression level is measured using an amount of mRNA transcript of a gene of a protein belonging to the Secretoglobin family as an index. (19) The method according to (17), wherein the expression level is measured using an amount of a gene product of a gene of a protein belonging to the Secretoglobin family as an index.
(20) 慢性閉塞性肺疾患が肺気腫である前記 （17) 〜 （19) いずれか記 載の方法、  (20) The method according to any one of (17) to (19) above, wherein the chronic obstructive pulmonary disease is emphysema.
(21) 配列番号： 1、 3、 5、 7、 9、 1 1、 13、 1 5及び 19〜 23か らなる群より選ばれる塩基配列からなる核酸を含有してなる、 前記 （17) 〜 （ 18) 及び （20) いずれかに記載の方法に使用するための診断用キット、 (21) SEQ ID NO: 1, 3, 5, 7, 9, 11, 11, 13, 15 or 19 to 23, comprising a nucleic acid having a base sequence selected from the group consisting of: A diagnostic kit for use in the method of any of (18) and (20),
(22) Secretoglobin ファミリーに属するタンパク質に対する抗体又はその 断片を含有してなる、 前記 （17) 〜 （20) いずれかに記載の方法に使用する ための診断用キット、 (22) a diagnostic kit for use in the method according to any of (17) to (20), which comprises an antibody against a protein belonging to the Secretoglobin family or a fragment thereof;
(23) 被検対象の個体由来の被検試料及び正常個体由来の対照試料それぞれ における Secretoglobinファミリーに属するタンパク質の遺伝子の発現レベルを 測定するステップを含み、 ここで、 被検試料における発現レベルが、 対照試料に おける発現レベルよりも高くなる場合、 該被検試料が、 慢性閉塞性肺疾患に罹患 している疑いがある個体由来の試料であることの指標となる、 慢性閉塞性肺疾患 に罹患している疑いがある個体由来の試料の検出又は選別方法、  (23) measuring the expression level of the gene of the protein belonging to the Secretoglobin family in each of the test sample derived from the test subject individual and the control sample derived from the normal individual, wherein the expression level in the test sample is If the expression level is higher than that in the control sample, the test sample is afflicted with chronic obstructive pulmonary disease, which indicates that the test sample is a sample derived from an individual suspected of having chronic obstructive pulmonary disease. Method of detecting or selecting a sample from an individual suspected of doing,
(24) 発現レベルが、 Secretoglobin ファミリーに属するタンパク質の遺伝 子の inRNA転写物の量を指標として測定される、 前記 （23) 記載の方法、  (24) The method according to (23), wherein the expression level is measured using an amount of an inRNA transcript of a gene of a protein belonging to the Secretoglobin family as an index.
(25) 発現レベルが、 Secretoglobin ファミリーに属するタンパク質の遺伝 子の遺伝子産物の量を指標として測定される、 前記 （23) 記載の方法、  (25) The method according to (23), wherein the expression level is measured using an amount of a gene product of a gene of a protein belonging to the Secretoglobin family as an index.
(26) 慢性閉塞性肺疾患が肺気腫である前記 （23) 〜 （25) いずれか記 載の方法、  (26) The method according to any one of (23) to (25) above, wherein the chronic obstructive pulmonary disease is emphysema.
(27) 配列番号： 1、 3、 5、 7、 9、 1 1、 13、 1 5及び 19〜 23か らなる群より選ばれる塩基配列からなる核酸を含有してなる、 前記 （23) 〜 （ 24) 及び （26) いずれかに記載の方法に使用するための検出又は選別用キッ ト、 並びに (28) Secretoglobin ファミリーに属するタンパク質に対する抗体又はその 断片を含有してなる、 前記 （23) 〜 （26) いずれかに記載の方法に使用する ための検出又は選別用キット、 (27) SEQ ID NO: 1, 3, 5, 7, 9, 11, 11, 13, 15 or 19 to 23, comprising a nucleic acid having a base sequence selected from the group consisting of: (24) and (26) a kit for detection or selection for use in the method of any of the above, and (28) a detection or selection kit for use in the method according to any of (23) to (26), which comprises an antibody against a protein belonging to the Secretoglobin family or a fragment thereof;
に関する。 図面の簡単な説明 About. BRIEF DESCRIPTION OF THE FIGURES
図 1は、 対照群と比較した、 C0PDモデルマウス （喫煙群） の肺における UGRP2 IIIRNAの発現解析の結果を示すグラフである。  FIG. 1 is a graph showing the results of UGRP2 III RNA expression analysis in the lungs of C0PD model mice (smoking group), as compared to the control group.
図 2は、 対照群と比較した、 喘息モデルマウス （喘息群） の肺における UGR mRNAの発現解析の結果を示すグラフである。 発明を実施するための最良の形態  FIG. 2 is a graph showing the results of analysis of the expression of UGR mRNA in the lungs of asthma model mice (asthma group) as compared to the control group. BEST MODE FOR CARRYING OUT THE INVENTION
本発明は、 長期間の煙草煙暴露により誘発された C0PD のモデルマウスの肺組 織において UGRP2が特異的に有意に増加しているという、 本発明者らにより初め て明らかにされた驚くべき知見に基づくものである。  The present invention is based on the surprising finding first revealed by the present inventors that UGRP2 is specifically and significantly increased in the lung tissue of a model mouse of C0PD induced by prolonged tobacco smoke exposure. It is based on.
喫煙はマクロファージゃ好中球を活性化してタンパク質分解酵素 （肺胞を破壊 する因子） の放出を増加させ、 さらにタンパク質分解酵素阻害作用を持つ 1 ァ ンチトリプシン（ α; 1AT)を不活性化することにより、 肺胞破壊性に働くと考えら れている (Crystal R. G. et al. , The alpha - 1 antitrypsin gene and its muta tions. Chest 95:196-208, 1989； Takahashi H. et al., Cathepsin L activity is increased in alveolar macrophages and bronchoalveolar lavage fluid of smokers. Am Rev Respir Dis 147:1562-1568, 1993) 。 alAT は肺における重 要なタンパク質分解酵素阻害タンパク質であり、 正常人では好中球エラスターゼ やマクロファージ由来のタンパク質分解酵素が肺胞組織夕ンパク質を分解し組織 破壊するのを防いでいる。 それゆえ、 QIIAT の欠損が喫煙による肺気腫の発生の 素因の 1つであることが指摘されているが、 両者の明らかな関連性については見 いだされていない （福地義之助ら、 「喫煙の老化に及ぼす影響に関する研究-喫 煙の肺機能に及ぼす影響と遺伝的素因に関する研究 - α ΐ-アンチトリプシンの遺 伝子表現正常亜型 ( Μ)による喫煙の影響について」 平成 5年度喫煙科学研究財 団研究年報： 357- 363) 。 また、 アレルギー素因や白血球抗原 (HLA)と喫煙による 肺気腫発生についても検討されたが、 関連性は認められなかった （泉 孝英ら、Smoking activates macrophages and neutrophils to increase the release of proteolytic enzymes (factors that destroy alveoli), and also inactivates 1antitrypsin (α; 1AT), which has an inhibitory effect on proteolytic enzymes It is thought that this works on alveolar destruction (Crystal RG et al., The alpha-1 antitrypsin gene and its mutations. Chest 95: 196-208, 1989; Takahashi H. et al., Cathepsin L activity is increased in alveolar macrophages and bronchoalveolar lavage fluid of smokers. Am Rev Respir Dis 147: 1562-1568, 1993). alAT is an important proteolytic enzyme inhibitory protein in the lung, preventing neutrophil elastase and macrophage-derived proteolytic enzymes from decomposing and destroying alveolar tissue proteins in normal individuals. Therefore, it has been pointed out that the deficiency of QIIAT is one of the predisposing factors for the development of emphysema caused by smoking, but there is no clear link between the two. (Fukuchi Yoshinosuke et al., “Study on the effects of smoking on aging-Studies on the effects of smoking on pulmonary function and genetic predisposition-Normal subtype of α 表現 -antitrypsin gene expression (Μ About the effect of smoking due to)), Annual Report of the Smoking Science Research Foundation 1993: 357-363). Allergic predisposition, leukocyte antigen (HLA), and emphysema caused by smoking were also examined, but no association was found (Takahide Izumi et al.,
「喫煙の肺気腫の発症 ·進展に及ぼす影響に関する臨床的 ·基礎的研究 -C0PD (慢 性気管支炎/肺気腫）の発症素因としてのアレルギー素因に関する検討」 平成 3年 度喫煙科学研究財団研究年報： 258- 261；泉 孝英ら、 「喫煙の肺気腫の発症 ·進 展に及ぼす影響に関する臨床的 ·基礎的研究- C0PD (慢性気管支炎/肺気腫）の発症 素因としての HLA抗原分布に関する検討」 平成 4年度喫煙科学研究財団研究年報"Clinical and basic research on the effects of smoking on the development and progression of pulmonary emphysema -A study on allergic predisposition as a predisposition to the development of C0PD (Chronic Bronchitis / Pulmonary Emphysema)" Annual Report of the Smoking Science Research Foundation, 1991: 258 -261; Takahide Izumi et al., "Clinical and basic study on the effects of smoking on the development and progression of emphysema-Examination on the distribution of HLA antigens as a predisposition to the development of C0PD (chronic bronchitis / emphysema)" Annual report of Science Research Foundation
： 258-261) 。 : 258-261).
これらは、 喫煙と肺気腫発症との関係についての報告であるが、 一般に C0PD の発症にどのような因子が関係しているかについては未だ不明である。  These are reports of the relationship between smoking and the development of emphysema, but it is still unknown what factors generally contribute to the development of C0PD.
本発明において初めて C0PDモデルマウスの肺組織における UGRP2の特異的な 増加が見出された。 C0PD発症メカニズムと UGRP2 の特異的高発現との関係の詳 細は未だ不明であるが、 UGRP2 の特異的高発現が喫煙による C0PD の発症の素因 であるか、 若しくは C0PD の発症を促進している可能性がある。 また、 肺気腫発 症については、 喫煙量よりも喫煙に対する個体の感受性の差が発症の要因として 重要であることが示唆されているが、 UGRP2 が喫煙に対する個体の感受性の差と 何らかの関係を有する可能性がある。  In the present invention, for the first time, a specific increase in UGRP2 in lung tissue of a C0PD model mouse was found. Although the details of the relationship between the mechanism of C0PD development and the specific high expression of UGRP2 are still unknown, the specific high expression of UGRP2 is a predisposition to the development of C0PD due to smoking or promotes the development of C0PD there is a possibility. In addition, it has been suggested that the difference in individual sensitivity to smoking is more important as a factor in the occurrence of emphysema than the amount of smoking, but UGRP2 may have some relationship with the difference in individual sensitivity to smoking. There is.
なお、 後述の実施例に記載のように、 オボアルブミンで誘発された喘息のモデ ルマウスでは UGRP2の特異的な発現増加は見られず、 従って、 UGRP2の特異的な 発現増加は、 喫煙により誘発される C0PD の発症に特異的なものであると考えら れる。 以下、 本発明を詳細に説明する。 本発明は、 C0PD の治療剤又は予防剤のスク リーニング方法、 該スクリーニング方法に用いるキット、 医薬組成物、 C0PD の 診断方法、 該診断方法に用いるキット、 C0PD に罹患している疑いがある個体由 来の試料の検出又は選別方法、 並びに該検出又は選別方法に用いるキットに関す るものであるが、 本明細書において C0PDとは、 少なくとも SCGBファミリ一に属 するタンパク質の特異的な高発現が認められる症状をいう。 具体的には、 例えば 、 肺気-腫又は慢性気管支炎が挙げられる。 本発明は、 肺気腫に対し特に好適に使 用されうる。 As described in the Examples below, no specific increase in UGRP2 expression was observed in ovalbumin-induced asthmatic model mice, and thus the specific increase in UGRP2 expression was induced by smoking. It is considered to be specific to the onset of C0PD. Hereinafter, the present invention will be described in detail. The present invention relates to a therapeutic or prophylactic agent for C0PD. A method for screening, a kit for use in the screening method, a pharmaceutical composition, a method for diagnosing C0PD, a kit for use in the diagnostic method, a method for detecting or selecting a sample derived from an individual suspected of suffering from C0PD, and a method for detecting or selecting The present invention relates to a kit used for a selection method. In the present specification, C0PD refers to a symptom in which at least specific high expression of a protein belonging to SCGB family 1 is observed. Specifically, for example, pulmonary emphysema or chronic bronchitis is mentioned. The present invention can be particularly preferably used for emphysema.
なお、 本明細書において、 特に指示のない限り、 遺伝子組換え技術、 細胞での 組換えタンパク質の生産技術、 発現タンパク質の分離精製法、 分析法及び免疫学 的手法等は公知の方法が採用される。 また、 本明細書において、 タンパク質との 記載は、 該タンパク質及び該タンパク質をコードする遺伝子を含む場合がある。 また、 タンパク質との記載は、 その一部の断片を含む場合がある。 さらに、 遺伝 子との記載は、 天然の遺伝子、 バリアント及び cDNAを含む場合がある。 本発明の C0PD の治療剤又は予防剤のスクリーニング方法は、 被検物質の存在 下、 SCGB ファミリ一に属するタンパク質若しくは該タンパク質をコードする遺 伝子の動態を検出又は測定することを 1つの特徴とする。  In this specification, unless otherwise specified, known methods are used for gene recombination techniques, techniques for producing recombinant proteins in cells, methods for separating and purifying expressed proteins, analytical methods, and immunological techniques. You. Further, in this specification, description of a protein may include the protein and a gene encoding the protein. In addition, the description of a protein may include some fragments thereof. Further, reference to a gene may include naturally occurring genes, variants and cDNAs. The screening method for a therapeutic or prophylactic agent for C0PD of the present invention has one feature of detecting or measuring the kinetics of a protein belonging to SCGB family 1 or a gene encoding the protein in the presence of a test substance. I do.
すなわち、 C0PDの発症との関連が認められる SCGBファミリーに属するタンパ ク質の動態に対する被検物質による影響を調べることにより、 C0PD に対する治 療剤又は予防剤として有効な物質のスクリーニングを効率的に行うものである。 SCGB ファミリーに属するタンパク質の動態に対する被検物質による影響は、 被 検物質の非存在下における該動態を対照として調べるのが好適である。  In other words, by screening the effects of test substances on the kinetics of proteins belonging to the SCGB family, which is recognized to be associated with the development of C0PD, to efficiently screen substances that are effective as therapeutic or prophylactic agents against C0PD. It is. The effect of a test substance on the kinetics of a protein belonging to the SCGB family is preferably examined using the kinetics in the absence of the test substance as a control.
また、 本発明のスクリーニング方法は、 C0PD に対する治療剤又は予防剤の候 補化合物の薬理評価に用いることができる。  Further, the screening method of the present invention can be used for pharmacological evaluation of a candidate compound of a therapeutic or prophylactic agent for C0PD.
前記被検物質としては、 化合物又はその塩が挙げられる。 なお、 本明細書にお いては、 前記化合物又はその塩は、 低分子化合物、 高分子化合物、 ポリペプチド 又はその誘導体、 核酸又はその誘導体等を含む。 かかる被検物質は、 天然物質で あってもよく、 非天然物質であってもよい。 ポリペプチドの誘導体としては、 修 飾基を付加して得られた修飾ポリぺプチド、 アミノ酸残基を改変することにより 得られたバリアントポリペプチド等が挙げられる。 また、 核酸の誘導体としては 、 修飾基を付加して得られた修飾核酸、 塩基を改変することにより得られたパリ アント核酸、 ペプチド核酸等が挙げられる。 The test substance includes a compound or a salt thereof. In the present specification, the compound or a salt thereof is a low-molecular compound, a high-molecular compound, or a polypeptide. Or derivatives thereof, nucleic acids or derivatives thereof, and the like. Such a test substance may be a natural substance or a non-natural substance. Derivatives of polypeptides include modified polypeptides obtained by adding a modifying group, and variant polypeptides obtained by modifying amino acid residues. Examples of the derivative of the nucleic acid include a modified nucleic acid obtained by adding a modifying group, a peptide nucleic acid obtained by modifying a base, and a peptide nucleic acid.
本発明において、 その動態を検出又は測定する SCGB ファミリーに属するタン パク質としては、 特に限定されるものではないが、 例えば、 前記非特許文献 4に 記載されるタンパク質、 或いはそのアミノ酸配列において 1若しくは数個のアミ ノ酸の欠失、 付加、 挿入又は置換を有し、 かつ該タンパク質と同等の性質を有す る任意のバリアン卜タンパク質が挙げられる。 本明細書において 「同等の性質」 とは、 少なくとも C0PD の発症との関連において特異的に発現されうることをい 。  In the present invention, the protein belonging to the SCGB family for detecting or measuring its kinetics is not particularly limited. For example, the protein described in Non-Patent Document 4 or 1 or Any variant protein having several amino acid deletions, additions, insertions or substitutions and having properties equivalent to the protein can be mentioned. As used herein, “equivalent properties” means that they can be specifically expressed at least in relation to the onset of C0PD.
スクリーニングの有効性の向上の観点から、 SCGB ファミリーに属するタンパ ク質としては、 好ましくは SCGBファミリー 3Aに属するタンパク質であり、 より 好ましくは UGRPであり、 さらに好ましくは UGRP2である。 特に、 本発明により 、 ヒトに対して有効な C0PD の治療剤又は予防剤のスクリーニングを効率的に行 う観点からは、 UGRP2としてはヒト UGRP2が特に好適である。  From the viewpoint of improving the effectiveness of screening, the protein belonging to the SCGB family is preferably a protein belonging to SCGB family 3A, more preferably UGRP, and still more preferably UGRP2. In particular, human UGRP2 is particularly preferred as UGRP2 from the viewpoint of efficiently screening a therapeutic or prophylactic agent for C0PD effective against humans according to the present invention.
SCGB ファミリーに属するタンパク質としては、 例えば、 ヒト SCGB1A1 タンパ ク質にっき、 それをコードする遺伝子の塩基配列 （配列番号： 1 ) がジーンバン ク （GenBank) ァクセッション番号： X13197 の下に、 また、 そのアミノ酸配列 （ 配列番号： 2 ) がジーンバンクァクセッション番号： CAA31584 の下に開示され ている。 また、 同様に、 ヒト SCGB1C1タンパク質にっき、 塩基配列 （配列番号： As proteins belonging to the SCGB family, for example, the base sequence of the gene encoding human SCGB1A1 protein (SEQ ID NO: 1) is shown under GenBank Accession No .: X13197. The amino acid sequence (SEQ ID NO: 2) is disclosed under Genebank Accession Number: CAA31584. Similarly, the nucleotide sequence of human SCGB1C1 protein (SEQ ID NO:
3 ) が NM— 145651の下に、 アミノ酸配列 （配列番号： 4 ) が NP一 663626の下に； ヒト SCGB1D1 タンパク質にっき、 塩基配列 （配列番号： 5 ) が AJ224171 の下に3) is under NM-145651, the amino acid sequence (SEQ ID NO: 4) is under NP-1666366; the human SCGB1D1 protein has its nucleotide sequence (SEQ ID NO: 5) under AJ224171
、 アミノ酸配列 （配列番号： 6 ) が CAA11863の下に； ヒト SCGB1D2タンパク質 にっき、 塩基配列 （配列番号： 7 ) が AJ224172 の下に、 アミノ酸配列 （配列番 号： 8 ) が CAA11864の下に； ヒト SCGB2A1 タンパク質にっき、 塩基配列 （配列 番号： 9 ) が M224173の下に、 アミノ酸配列 （配列番号： 1 0 ) が CAA11865の 下に； ヒト SCGB2A2 タンパク質にっき、 塩基配列 （配列番号： 1 1 ) が U33147 の下に、 アミノ酸配列 （配列番号： 1 2 ) が AAC50375 の下に； ヒト SCGB3A2夕 ンパヶ質につき、 塩基配列 （配列番号： 1 5 ) が AF313455 の下に、 アミノ酸配 列 （配列番号： 1 6 ) が AAL26215の下に、 それぞれ開示されている。 The amino acid sequence (SEQ ID NO: 6) under CAA11863; human SCGB1D2 protein The nucleotide sequence (SEQ ID NO: 7) is under AJ224172, the amino acid sequence (SEQ ID NO: 8) is under CAA11864; the human SCGB2A1 protein is nucleotide sequence (SEQ ID NO: 9) under M224173, Amino acid sequence (SEQ ID NO: 10) below CAA11865; human SCGB2A2 protein; nucleotide sequence (SEQ ID NO: 11) below U33147; amino acid sequence (SEQ ID NO: 12) below AAC50375; For human SCGB3A2 protein, the nucleotide sequence (SEQ ID NO: 15) is disclosed under AF313455, and the amino acid sequence (SEQ ID NO: 16) is disclosed under AAL26215.
特に、 UGRP としては、.例えば、 ヒト由来のもの （ヒト SCGB3A1 タンパク質） として、 ジーンバンクァクセッション番号： AF086152 の下に、 その塩基配列 （ 配列番号： 1 3 ) が、 AAL26217 の下に、 そのアミノ酸配列 （配列番号： 1 4 ) が開示されている。 また、 マウス由来のもの （マウス SCGB3A1タンパク質） とし て、 ジーンバンクァクセッション番号： AF313456 の下に、 その塩基配列 （配列 番号： 1 7 ) が、 AAL26216 の下に、 そのアミノ酸配列 （配列番号： 1 8 ) が開 示されている。  In particular, as UGRP, for example, as a human-derived (human SCGB3A1 protein), its base sequence (SEQ ID NO: 13) under Genebank Accession No .: AF086152, and its amino acid under AAL26217 The sequence (SEQ ID NO: 14) is disclosed. Also, as a mouse-derived product (mouse SCGB3A1 protein), its base sequence (SEQ ID NO: 17) under Gene Bank Accession No. AF313456 and its amino acid sequence (SEQ ID NO: 1) under AAL26216 8) is disclosed.
ヒト UGRP2は、 肺、 胸腺、 脾臓、 小腸、 リンパ節、 前立腺、 舌、 気管において 発現が見られ、 気管 >肺 >舌>前立腺 >胸腺、 脾臓、 小腸、 リンパ節の発現パ夕 ーンを示すという特徴を有する。  Human UGRP2 is expressed in lung, thymus, spleen, small intestine, lymph nodes, prostate, tongue, and trachea, showing trachea> lung> tongue> prostate> thymus, spleen, small intestine, and lymph node expression patterns It has the feature of.
本発明のスクリーニング方法における測定又は検出対象の 「SCGB ファミリー に属するタンパク質の動態」 としては、 例えば、 該タンパク質とそのリガンドと の結合の有無；該タンパク質の発現、 具体的には、 該発現の有無ゃ該発現の変動 等が挙げられる。 '  The “kinetics of a protein belonging to the SCGB family” to be measured or detected in the screening method of the present invention includes, for example, the presence or absence of binding between the protein and its ligand; the expression of the protein, specifically, the presence or absence of the expression変 動 Variations in the expression and the like. '
本発明のスクリーニング方法としては、 測定又は検出対象の 「SCGB ファミリ 一に属するタンパク質の動態」 に応じて、 大きく 2つの実施態様がある。  As the screening method of the present invention, there are roughly two embodiments according to the “kinetics of a protein belonging to SCGB family 1” to be measured or detected.
本発明のスクリーニング方法の第 1の実施態様としては、 （ I ) SCGB フアミ リーに属するタンパク質と、 被検物質とを接触させるステップ、 並びに  As a first embodiment of the screening method of the present invention, (I) a step of contacting a protein belonging to the SCGB family with a test substance; and
( I I ) 前記ステップ （I ) の後、 該タンパク質と被検物質との結合を検出し、 それにより、 該タンパク質と結合する被検物質を、 C0PD 治療剤又は予防剤の候 補化合物として選択するステップ (II) After the step (I), detecting the binding between the protein and the test substance, Thus, a step of selecting a test substance that binds to the protein as a candidate compound for a therapeutic or prophylactic agent for C0PD
を含む方法が挙げられる。 かかる方法によれば、 前記タンパク質への結合を介し て当該タンパク質の機能に作用する物質を選択することができるため、 選択され た候補化合物は、 直接的かつ特異的に前記タンパク質に結合してその機能を調節 するという特徴的な性質を有する。 And a method including: According to such a method, a substance that acts on the function of the protein through the binding to the protein can be selected. Therefore, the selected candidate compound directly and specifically binds to the protein and binds to the protein. It has the characteristic property of regulating functions.
前記ステップ （I ) において、 前記タンパク質と被検物質との接触は、 例えば 、 該タンパク質の本来の機能を妨げない溶液中において、 前記タンパク質と被検 物質とを混合し、 適切な反応条件 （例えば、 反応温度、 反応時間等） の下、 維持 する (すなわち、 両者を反応させる） ことにより行なわれうる。  In the step (I), the contact between the protein and the test substance is performed, for example, by mixing the protein and the test substance in a solution that does not interfere with the original function of the protein, and applying appropriate reaction conditions (for example, , Reaction temperature, reaction time, etc.).
前記 「タンパク質の本来の機能を妨げない溶液」 としては、 例えば、 リン酸緩 衝化生理的食塩水 （P B S ) 、 H E P E Sバッファー、 T r i sバッファ一等の 溶液が挙げられる。  Examples of the “solution that does not interfere with the original function of the protein” include solutions such as phosphate-buffered physiological saline (PBS), HEPS buffer, and Tris buffer.
ステップ （I ) における反応条件としては、 特に限定されないが、 例えば、 前 記溶液中、 通常、 pH6. 0〜10. 0、 好ましくは ρΗ7. 0〜9· 0、 より好ましくは. ρΗ7. 5 〜8· 5、 さらに好ましくは ρΗ8· 0で、 通常、 10〜50t、 好ましくは 20〜40°C、 よ り好ましくは 25〜37°C、 さらに好ましくは 25°C、 通常、 1分間〜 1時間、 好まし くは 3〜30分間、 より好ましくは 5〜20分間、 さらに好ましくは 10分間維持す ること等が挙げられる。 より具体的には、 10mM Hepes、 80mM KCK 5 %グリセ口 ール、 lOmM DTT、 0. lmg/ml polydldC, 50ng/ml herr ing sperm DNA、 lmg/ml BSA 、 10% ret iculocyte lysate中、 前記条件下に維持することが挙げられる。  The reaction conditions in step (I) are not particularly limited. For example, in the above solution, the pH is usually 6.0 to 10.0, preferably ρΗ7.0 to 9.0, more preferably ρΗ7.5 to 8.5, more preferably ρΗ80, usually 10 to 50 t, preferably 20 to 40 ° C, more preferably 25 to 37 ° C, more preferably 25 ° C, usually 1 minute to 1 hour , Preferably for 3 to 30 minutes, more preferably for 5 to 20 minutes, and still more preferably for 10 minutes. More specifically, in 10 mM Hepes, 80 mM KCK 5% glycerol, lOmM DTT, 0.1 mg / ml polydldC, 50 ng / ml herring sperm DNA, lmg / ml BSA, in 10% ret iculocyte lysate, the above conditions To keep it below.
ついで、 ステップ （I I ) において、 前記タンパク質と被検物質との結合を検 出し、 該結合の有無を調べる。  Next, in step (II), the binding between the protein and the test substance is detected, and the presence or absence of the binding is examined.
結合は、 例えば、 前記のようにしてステップ （I ) で前記タンパク質と、 例え ば、 放射性物質により標識した被検物質とを反応させた後の溶液に含まれる該夕 ンパク質の放射活性を、 大過剰の非放射性被検物質との競合下において解析する ことにより検出することができる。 結合が検出された場合、 使用された被検物質 を C0PD治療剤又は予防剤の候補化合物として選択する。 The binding is performed, for example, by measuring the radioactivity of the protein contained in the solution obtained by reacting the protein with a test substance labeled with a radioactive substance in step (I) as described above, for example. Analyze in competition with a large excess of non-radioactive analytes Can be detected. If binding is detected, the test substance used is selected as a candidate compound for a therapeutic or prophylactic agent for C0PD.
また、 ステップ （I ) とステップ （I I ) を連続した工程により実施してもよ い。 かかる態様は、 例えば、 表面プラズモン解析、 被検物質を保持した担体への バインディングアツセィ等を利用することにより実施することができる。  Further, step (I) and step (I I) may be performed in a continuous process. Such an embodiment can be carried out, for example, by utilizing surface plasmon analysis, binding to a carrier holding a test substance, or the like.
例え-ば、 表面プラズモン解析を利用する場合、 例えば、 下記ステップ： 一 前記タンパク質を固定化したセンサーチップに対し、 被検物質を含有した溶 液を一定の流速で送液するステップ 〔ステップ （I ) 〕 、 並びに  For example, when surface plasmon analysis is used, for example, the following steps: (i) sending a solution containing a test substance at a constant flow rate to a sensor chip on which the protein is immobilized [step (I )], And
一 適切な検出手段 〔例えば、 光学的検出 （蛍光度、 蛍光偏向度等） 、 質量分析 計との組み合わせ （マトリックス支援レーザー脱離イオン化—飛行時間型質量分 析計： MALDI- TOF MS、 エレクトロスプレー ·イオン化質量分析計： ES I- MS 等） 〕 により、 光学的変動又は質量の変動として、 前記タンパク質と、 被検物質との 結合を検出するステップ 〔ステップ （1 1 ) 〕 ；  (I) Suitable detection means [eg, optical detection (fluorescence, fluorescence polarization, etc.), combined with a mass spectrometer (matrix-assisted laser desorption ionization-time-of-flight mass spectrometer: MALDI-TOF MS, electrospray Ionization mass spectrometer: ESI-MS etc.)] to detect the binding between the protein and the test substance as optical fluctuation or mass fluctuation [Step (11)];
を含む方法により実施することができる。 表面プラズモン解析を利用する一般的 方法の詳細については、 例えば、 「生体物質相互作用のリアルタイム解析実験法 Biacore を中心に」 永田和宏 半田 宏共編： シュプリンガー ·フエアラーク東 京，全頁（1998)を参照すればよい。 Can be carried out by a method including: For details of the general method using surface plasmon analysis, see, for example, "Focusing on Biacore, a Real-Time Experimental Method for Interaction of Biological Substances," Kazuhiro Nagata, Hiroshi Handa, ed. do it.
なお、 表面プラズモン解析を利用する場合、 前記タンパク質と被検物質との間 の結合は、 送液による被検物質の導入を行った際の、 例えば、 光学的センサーグ ラム又は質量センサーグラムの変動を検出することにより検出されることになる 本発明のスクリーニング方法の第 2の実施態様としては、  In the case where surface plasmon analysis is used, the binding between the protein and the test substance is caused by, for example, a change in an optical sensorgram or a mass sensorgram when the test substance is introduced by liquid sending. As a second embodiment of the screening method of the present invention to be detected by detection,
(A) SCGB ファミリーに属するタンパク質をコードする遺伝子を細胞に導入す るステップ、  (A) introducing a gene encoding a protein belonging to the SCGB family into cells,
( B ) 被検物質の存在下、 前記ステップ （A) で得られた細胞において前記遺伝 子を発現させるステップ、 並びに ( C ) 被検物質の非存在下の場合と比較して、 前記遺伝子の発現の有無を検出し 、 又は前記遺伝子の発現の変動を測定し、 それにより、 該遺伝子の発現の変化を もたらす被検物質を C0PD治療剤又は予防剤の候補化合物として選択するステツ プ (B) expressing the gene in the cells obtained in the step (A) in the presence of a test substance; and (C) detecting the presence or absence of the expression of the gene or measuring the fluctuation of the expression of the gene as compared to the case in the absence of the test substance, and thereby detecting the change in the expression of the gene. Steps for selecting a test substance as a candidate compound for a C0PD therapeutic or prophylactic agent
を含む方法が挙げられる。 And a method including:
前記ステップ （A) において、 SCGB ファミリーに属するタンパク質をコード する遺伝子を細胞に導入するには、 例えば、 該タンパク質の発現調節領域として 知られる発現調節性エレメントの下流かつその支配下に、 前記タンパク質をコー ドする遺伝子が動作可能に連結されてなる核酸構築物を用いて行うのが好適であ る。 前記発現調節性エレメントとしては、 例えば、 Ni imi T e t al .， Mo l ecu l ar Endocr ino logy, 2001 , Vo l. 15, 202卜 2136に記載されている。  In the step (A), a gene encoding a protein belonging to the SCGB family is introduced into a cell by, for example, transferring the protein downstream of and under the control of an expression regulatory element known as an expression regulatory region of the protein. It is preferable to use a nucleic acid construct in which the coding gene is operably linked. The expression control element is described in, for example, Niimi Tetal., Molecular Endocrinology, 2001, Vol. 15, 2022136.
なお、 SCGB ファミリーに属するタンパク質をコードする遺伝子には、 当該遺 伝子そのもの；前記遺伝子のアンチセンス鎖にストリンジェントな条件下にハイ ブリダィズし、 かつ前記タンパク質と同等の作用を有するタンパク質をコードす る遺伝子；前記遺伝子の塩基配列において、 少なくとも 1塩基の変異を有し、 か つ前記タンパク質と同等の作用を有するタンパク質をコードする遺伝子；前記遺 伝子の塩基配'列と少なくとも 60 %、 好ましくは 80 %以上、 より好ましくは 90 % 以上の配列同一性を有し、 かつ前記タンパク質と同等の作用を有するタンパク質 をコードする遺伝子等も含まれる。  The gene encoding a protein belonging to the SCGB family includes the gene itself; a gene that hybridizes to the antisense strand of the gene under stringent conditions and encodes a protein having an action equivalent to that of the protein. A gene having a mutation of at least one base in the base sequence of the gene and encoding a protein having an action equivalent to that of the protein; at least 60% of the base sequence of the gene, preferably Also includes a gene encoding a protein having a sequence identity of 80% or more, more preferably 90% or more, and having the same action as the above protein.
前記核酸構築物は、 前記発現調節性エレメント及び SCGB ファミリーに属する タンパク質をコードする遺伝子を慣用の発現ベクターのクローニング部位に挿入 することにより簡便に構築されうる。 当該ベクターとしては、 例えば、 ウィルス ベクター、 プラスミドベクター等が挙げられる。  The nucleic acid construct can be conveniently constructed by inserting the gene encoding the expression regulatory element and a protein belonging to the SCGB family into a cloning site of a conventional expression vector. Examples of the vector include a virus vector and a plasmid vector.
なお、 本発明の種々の態様において、 その構成上、 SCGB ファミリーに属する タンパク質をコードする遺伝子の発現を必須とする場合、 当該遺伝子の発現にお いて用いられるベクター、 核酸構築物を導入する宿主細胞等は、 それらの任意の 組み合わせにより、 前記遺伝子の発現が達成されうるのであれば、 それらはどの ような生物種由来のものであってもよい。 当業者であれば、 任意の構成材料を用 いて前記遺伝子の発現系を構築することができる。 SCGB ファミリーに属する夕 ンパク質をコードする遺伝子が真核生物に由来するものである点、 及び本発明に おいて提供される治療剤又は予防剤等が真核生物、 中でも哺乳動物、 特にヒ卜に おいて好適に使用されうる点を考慮すれば、 前記遺伝子の発現に使用される宿主 細胞としては動物細胞であるのが好適である。 よって、 本明細書においては、 特 に前記遺伝子の発現に動物細胞を使用する場合について説明する。 In various embodiments of the present invention, when the expression of a gene encoding a protein belonging to the SCGB family is essential for its configuration, a vector used in expression of the gene, a host cell into which a nucleic acid construct is introduced, and the like. Any of them They may be derived from any species as long as expression of the genes can be achieved by the combination. Those skilled in the art can construct an expression system for the gene using any constituent material. The fact that the gene encoding the protein belonging to the SCGB family is derived from eukaryotes, and that the therapeutic or prophylactic agent provided in the present invention is eukaryotes, especially mammals, especially humans In view of the fact that the gene can be suitably used in the above, animal cells are suitable as host cells used for expression of the gene. Therefore, in this specification, a case where an animal cell is used for the expression of the gene will be particularly described.
使用されるベクターとしては、 前記遺伝子を組み込んで動物細胞で発現できる ベクターであればいずれでもよい。 そのようなベクターとしては、 PCR- Bluntll- T0P0、 pcDNAl. K pcDNAl.l/A即、 pCDM8、 pREP (インビトロジェン社製） 、 HM6 、 HB6 (ロシュダイァグノスティックス社製） 、_ PKK223-3, pGEX (アマシャムバ ィォテク社製） 、 pET- 3、 ρΕΤ-ΙΚ pBluescriptll (登録商標) SK (十）、 Bluescri ptll (登録商標） SK (-) (ストラタジーン社製） 、 pUC19、 pTrxFus (インビトロ ジェン社製） 、 pUC118、 PSTV28 (夕カラバイオ社製） 、 pMAL- c2X (ニューイング ランドパイオラポ社製） 、 PAGE 107 [Cytotechnology, 3 (2), 133-140 (1990) ；特開 平 3— 2297 9号公報〕 、 pAGE 103 [The Journal of Biochemistry, 101 (5) , 13 07-1310 (1987)] 、 pAMo、 pAMoA [The Journal of Biologycal Chemistry, 268(30 ),22782-22787 (1993)] 、 pAMoPRSA (特開平 5— 336 963号公報) 、 AS3-3 (特開平 2— 22707 5号公報） 等が挙げられる。 かかるベクターには、 任意 にポリ A付加シグナル、 複製開始起点、 抗生物質耐性遺伝子等が含まれていても よい。  Any vector may be used as long as it can incorporate the gene and express it in animal cells. Such vectors include PCR-Bluntll-TOP0, pcDNA1.K pcDNA1.l / A, pCDM8, pREP (Invitrogen), HM6, HB6 (Roche Diagnostics), _PKK223-3 , pGEX (Amersham Biotech), pET-3, ρΕΤ-ΙΚ pBluescriptll (registered trademark) SK (10), Bluescri ptll (registered trademark) SK (-) (Stratagene), pUC19, pTrxFus (Invitrogen) ), PUC118, PSTV28 (Yukara Bio Inc.), pMAL-c2X (New England Land Piolapo), PAGE 107 [Cytotechnology, 3 (2), 133-140 (1990); Publication), pAGE 103 [The Journal of Biochemistry, 101 (5), 13 07-1310 (1987)], pAMo, pAMoA [The Journal of Biologycal Chemistry, 268 (30), 22722-22787 (1993)], pAMoPRSA (JP-A-5-336963) and AS3-3 (JP-A-2-227707). Such a vector may optionally contain a polyA addition signal, an origin of replication, an antibiotic resistance gene, and the like.
宿主に用いる動物細胞としては、 使用するベクターに対応する適当な細胞又は 組織であればよく、 以下のような動物細胞等が挙げられる。 当該動物細胞として は、 例えば、 マウス由来株細胞の L6 〔骨格筋由来筋芽細胞〕 、 ヒト由来株細胞の 腿 293 (ヒト胎児腎細胞、 ATCC： CRL-1573) 、 HeLa (子宮穎部癌細胞、 ATCC： CCL- 2) HBT5637 (白血病細胞、 特開昭 6 3— 2 9 9 ) 、 BALL- 1 (白血病細胞） 及び HCT - 15 (大腸癌細胞） 、 マウス由来株細胞の Sp2/0- Ag (マウス骨髄種細胞、 ATCC : CR L - 1581) 及び NS0 (マウス骨髄種細胞） 、 サル由来株細胞の C0S-1 〔アフリカミド リザル腎細胞 (SV40形質転換細胞） 、 ATCC： CRL-1650] 及び COS - 7 〔アフリカミド リザル腎細胞 （SV40形質転換細胞） 、 ATCC： CRL- 1651〕 、 ハムスター由来株細胞の CHO-ΚΓ (チャイニーズハムスター卵巣細胞、 ATCC： CCL-61) 及び BHK- 21 (C - 13) ( シシリアンハムスター仔腎細胞、 ATCC : CCL- 10) 、 マウス由来株細胞の PC12 (副腎 褐色細胞腫、 ATCC： CRL-1721) 及び YB2/0 (マウス骨髄種細胞、 ATCC： CRL-1662) 等が挙げられる。 The animal cell used as a host may be any appropriate cell or tissue corresponding to the vector used, and includes the following animal cells. Examples of the animal cells include mouse-derived cell line L6 [skeletal muscle-derived myoblast], human-derived cell line thigh 293 (human fetal kidney cell, ATCC: CRL-1573), HeLa (melanoma cell line) , ATCC: CCL- 2) HBT5637 (leukemia cell, JP-A-63-299), BALL-1 (leukemia cell) and HCT-15 (colorectal cancer cell), mouse-derived cell line Sp2 / 0-Ag (mouse myeloid cell) , ATCC: CRL-1581) and NS0 (mouse myeloid cells), monkey-derived cell line C0S-1 [African green monkey kidney cells (SV40 transformed cells), ATCC: CRL-1650] and COS-7 [Africa Green monkey kidney cells (SV40 transformed cells), ATCC: CRL-1651], hamster-derived cell lines CHO-ΚΓ (Chinese hamster ovary cells, ATCC: CCL-61) and BHK-21 (C-13) (Sicilian hamster) Offspring kidney cells, ATCC: CCL-10), mouse-derived cell lines PC12 (adrenal pheochromocytoma, ATCC: CRL-1721) and YB2 / 0 (mouse myeloid cells, ATCC: CRL-1662).
なお、 本明細書に用いられる 「ストリンジエンドな条件」 とは、 高ストリンジ エンシーな条件、 例えば、 1 X SSC/0. 2 % SDS の条件等をいう。 ここで、 ストリン ジエンシーを向上させるため、 より低イオン強度、 例えば、 0. 5 X SSC、 好ましく は 0. 2 X SSC、 より好ましくは 0. 1 X SSC等の条件及び/又はより高温、 例えば、 用いられる核酸の Tm値により異なるが、 50°C以上、 好ましくは 60°C以上、 より 好ましくは 65°C以上等の条件下で洗浄等を行ってもよい。  As used herein, “stringent end conditions” refer to conditions of high stringency, for example, conditions of 1 × SSC / 0.2% SDS. Here, in order to improve the stringency, lower ionic strength, for example, conditions such as 0.5 X SSC, preferably 0.2 X SSC, more preferably 0.1 X SSC and / or higher temperature, for example, Washing may be performed under conditions of 50 ° C. or higher, preferably 60 ° C. or higher, more preferably 65 ° C. or higher, depending on the Tm value of the nucleic acid used.
本明細書に用いられる 「変異」 とは、 塩基の置換、 失、 付加及び挿入をいう 。 かかる変異は、 天然に存在するバリエーションであってもよく、 慣用の部位特 異的変異方法等により人為的に導入された変異であってもよい。  As used herein, “mutation” refers to substitution, loss, addition and insertion of a base. Such a mutation may be a naturally occurring variation, or may be a mutation artificially introduced by a conventional site-specific mutation method or the like.
本明細書に用いられる 「配列同一性」 とは、 比較対象の少なくとも 2つの配列 について、 適切に整列化させ、 それぞれの配列に存在する同一の残基を決定して As used herein, "sequence identity" refers to the ability to properly align at least two sequences to be compared and to determine the same residue present in each sequence.
、 適合部位の数を決定し、 ついで、 比較対象の配列領域内の残基の総数で、 前記 適合部位の数を割、 得られた数値に 1 0 0をかけることにより算出されうる値を いう。 かかる配列同一性は、 具体的には、 例えば、 ホームページアドレス ht tp :A value that can be calculated by determining the number of matching sites, then dividing the number of matching sites by the total number of residues in the sequence region to be compared, and multiplying the obtained numerical value by 100. . Such sequence identity can be determined, for example, by, for example, a homepage address ht tp:
〃www. ncbi . nlm. nih. gov/BLAST/において、 一般に利用可能である B L A S Tァ ルゴリズム等により算出されうる。 Gov / BLAST / at www.ncbi.nlm.nih.gov/BLAST/, which can be calculated using the BLAST algorithm that is generally available.
ステップ （A) における、 前記核酸構築物の動物細胞への導入は、 慣用の遺伝 子導入法により行なわれうる。 例えば、 エレクト口ポレーシヨン法 〔Cytotechno logy,3 (2), 133-140 (1990)) 、 リン酸カルシウム法 （特開平 2— 2270 7 5号 公報) 、 リポフエクシヨン法 [Proceedings of the National Academy of Scien ces USA, 84,7413(1987)； Vi lology, 52, 456 (1973)] 、 DEAE デキストラン法、 パー ティクルガン法、 ウィルスを利用した方法等により行なわれうる。 In the step (A), the introduction of the nucleic acid construct into an animal cell may be performed by a conventional genetic method. It can be performed by a child introduction method. For example, elect opening method (Cytotechnology, 3 (2), 133-140 (1990)), calcium phosphate method (Japanese Patent Laid-Open No. 2-227075), lipofection method [Proceedings of the National Academy of Sciences USA, 84, 7413 (1987); Viology, 52, 456 (1973)], DEAE dextran method, particle gun method, virus-based method, and the like.
前記核酸構築物が導入された形質転換体は、 当該分野において周知慣用の常法 により培養することができる。 形質転換した宿主に適した培地を用いて行うこと ができ、 培養に使用される培地としては液体培地が適当である。 例えば、 MEM培 地 [Science, 130,432 (1959)] 、 DMEM培地 [Virology, 8, 396(1959)) 、 RPMI1640 培地 [The Journal of the American Medical Association, 199， 519 (1967)〕 等 にゥシ胎仔血清 （FCS) を適量添加したもの等が用いられる。 なお、 それらの培 地は市販されているので、 そのような市販培地を用いることができる。  The transformant into which the nucleic acid construct has been introduced can be cultured by a conventional method commonly used in the art. The culture can be performed using a medium suitable for the transformed host, and a liquid medium is suitable as the medium used for the culture. For example, MEM medium [Science, 130,432 (1959)], DMEM medium [Virology, 8, 396 (1959)], RPMI1640 medium [The Journal of the American Medical Association, 199, 519 (1967)], etc. Serum to which an appropriate amount of serum (FCS) has been added is used. Since those media are commercially available, such commercially available media can be used.
培養は、 例えば、 振盪培養又は深部通気攪拌培養等の好気的条件で行うことが できる。 培養温度、 培養時間及び培養液の pH は、 各種宿主に適した範囲に設定 される。 例えば、 培養は、 通常、 15〜40で、 5時間〜 7日間、 pH3.0〜9.0、 5%C0 2存在下等の条件で行なわれうる。 pHの調整は、 無機酸又は有機酸、 アルカリ溶 液、 尿素、 炭酸カルシウム、 アンモニア等を用いて行いうる。 また、 所望により 抗生物質を培地に添加してもよい。 The culture can be performed under aerobic conditions such as, for example, shaking culture or deep aeration stirring culture. The culture temperature, culture time, and pH of the culture solution are set in ranges suitable for various hosts. For example, the culture is usually 15 to 40, 5 hours to 7 days, PH3.0～9.0, can be carried out in conditions such as 5% C0 2 presence. The pH can be adjusted using an inorganic or organic acid, an alkali solution, urea, calcium carbonate, ammonia, or the like. If desired, an antibiotic may be added to the medium.
なお、 本実施態様においては、 前記核酸構築物が導入された安定な細胞系を用 いる場合、 ステップ （A) は、 省略してもよい。  In the present embodiment, when a stable cell line into which the nucleic acid construct has been introduced is used, step (A) may be omitted.
ついで、 ステップ （B) において、 被検物質の存在下、 前記ステップ （A) で 得られた細胞において SCGB ファミリ一に属するタンパク質をコードする遺伝子 を発現させる。  Next, in step (B), a gene encoding a protein belonging to SCGB family 1 is expressed in the cells obtained in step (A) in the presence of the test substance.
当該工程は、 例えば、 被検物質を含有した培地を用いて、 前記ステップ （A) で得られた細胞を培養することにより行なわれうる。 培養条件は前記の通りであ る。 - 続いて、 ステップ （C ) において、 被検物質の非存在下の場合と比較して、 前 記遺伝子の発現の有無を検出し、 又は前記遺伝子の発現の変動を測定し、 それに より、 該遺伝子の発現の変化をもたらす被検物質を C0PD 治療剤又は予防剤の候 補化合物として選択する。 This step can be performed, for example, by culturing the cells obtained in step (A) using a medium containing the test substance. Culture conditions are as described above. - Subsequently, in step (C), the presence or absence of the expression of the above-described gene is detected or the fluctuation of the expression of the above-described gene is measured, as compared with the case where the test substance is not present. A test substance that results in a change in the expression of is selected as a candidate compound for a therapeutic or prophylactic agent for C0PD.
なお、 「被検物質の非存在下の場合」 とは、 ステップ （B ) において、 被検物 質の非存在下にステップ （A) で得られた細胞において前記遺伝子を発現させた 場合をいい、 被検物質の存在下における前記遺伝子の発現の変化を把握するため の基準となる。 具体的には、 かかる場合の細胞、 又は該細胞の抽出液等を対照と して用いる。  Note that “in the absence of the test substance” refers to the case where, in step (B), the gene is expressed in the cells obtained in step (A) in the absence of the test substance. This serves as a standard for ascertaining changes in the expression of the gene in the presence of the test substance. Specifically, cells in such a case, or an extract of the cells, etc. are used as a control.
前記遺伝子の発現の有無を検出し、 又はその発現の変動を測定した結果、 対照 と比較して該遺伝子の発現の変化が認められた時、 例えば、 対照において前記遺 伝子の発現が見られた場合に、 被検物質と接触させた細胞において前記遺伝子の 発現が検出されないか、 又は対照に比べて発現レベルが低下した時、 すなわち、 対照と比較して前記遺伝子の発現が減少した時、 該被検物質が C0PD治療剤又は 予防剤の候補化合物であると判定され、 該被検物質を候補化合物として選択する 選択された候補化合物は、 細胞レベルにおいて前記遺伝子の発現を転写レベル で抑制するという特徴的な性質を有するものと考えられ、 前記遺伝子の発現を特 異的に抑制することができるという優れた効果を発揮する。  As a result of detecting the presence or absence of the expression of the gene or measuring the fluctuation of the expression, when a change in the expression of the gene is observed as compared with the control, for example, the expression of the gene is observed in the control When the expression of the gene is not detected in the cells contacted with the test substance or the expression level is reduced as compared with the control, that is, when the expression of the gene is reduced as compared with the control, The test substance is determined to be a candidate compound for a C0PD therapeutic or prophylactic agent, and the test substance is selected as a candidate compound. The selected candidate compound suppresses the expression of the gene at the cellular level at the transcription level. And has an excellent effect that the expression of the gene can be specifically suppressed.
被検物質の非存在下の場合と比較した前記遺伝子の発現の有無の検出、 又はそ の発現の変動の測定は、 例えば、 以下のようにして行うことができる。 タンパク 質レベルでは、 例えば、 前記ステップ （B ) を経た後の細胞における前記遺伝子 からの発現産物レベルと、 対照における発現産物レベルとを、 例えば、 ポリアク リルアミドゲル電気泳動により ；前記遺伝子の発現産物に対する抗体又はその断 片を用いたウェスタンブロット解析により ；前記抗体又はその断片を用いたィム ノアッセィ等により測定し、 比較することにより行うことができる。 また、 核酸 レベルでは、 例えば、 前記ステップ （B ) を経た後の細胞及び対照からそれぞれ 核酸を抽出し、 得られた核酸を、 例えば、 SCGB ファミリーに属するタンパク質 をコードする遺伝子の DiRNA を検出しうる核酸、 すなわち、 前記遺伝子又はその 一部の配列に特異的に結合する核酸を用いたハイブリダィゼーシヨン；前記遺伝 子又はその一部の配列を特異的に増幅しうるプライマー対を用いた PCR等に供しThe detection of the presence or absence of the expression of the gene or the measurement of the fluctuation of the expression as compared to the case in the absence of the test substance can be performed, for example, as follows. At the protein level, for example, the expression product level from the gene in the cell after the step (B) and the expression product level in the control are determined, for example, by polyacrylamide gel electrophoresis; an antibody against the expression product of the gene Alternatively, it can be performed by Western blot analysis using a fragment thereof; by measuring with an immunoassay or the like using the antibody or a fragment thereof, and comparing. Also, nucleic acids At the level, for example, nucleic acids are extracted from the cells and the control after the step (B), respectively, and the obtained nucleic acids are, for example, nucleic acids capable of detecting DiRNA of a gene encoding a protein belonging to the SCGB family, A hybridization using a nucleic acid that specifically binds to the gene or a part thereof; a PCR using a primer pair capable of specifically amplifying the gene or a part thereof.
、 ハイプリッドの形成若しくはその量又は増幅産物の出現若しくはその量を測定 し、 比較することにより行うことができる。 The formation or the amount of the hybrid or the appearance or the amount of the amplification product can be measured and compared.
なお、 本発明のスクリーニング方法において使用される前記抗体としては、 前 記遺伝子からの発現産物、 すなわち、 SCGB ファミリーに属するタンパク質に対 する抗体、 その断片等が挙げられる。 前記抗体の作製方法は、 例えば、 1992 年 Examples of the antibody used in the screening method of the present invention include an expression product from the aforementioned gene, that is, an antibody against a protein belonging to the SCGB family, a fragment thereof, and the like. The method for producing the antibody is described in, for example, 1992
、 ジョン ·ワイリ一&サンズ社 （John We i ly & Sons, Inc) 発行、 ジョン · E · コリガン （John E. Co l igan ) 編集、 カレント ·プロトコルズ ·イン ·ィムノロ ジー （Current Pro t oco l s in I匪 uno l ogy ) に記載の方法により、 前記 SCGB フ アミリーに属するタンパク質の全部又は一部を用いて、 ゥサギやマウス等を免疫 することにより、 容易に作製され得る。 また、 遺伝子工学的に抗体を作製するこ ともできる。 前記抗体は、 前記 SCGB ファミリーに属するタンパク質に特異的に 結合する能力を有するものであれば、 ポリクローナル抗体であってもよく、 モノ クローナル抗体であってもよい。 前記抗体は、 例えば、 前記 SCGB ファミリーに 属するタンパク質中における特定の部分断片に特異的に結合しうる抗体であって もよく、 ヒト UGPR2の部分ペプチド Cys_UGRP2 (46- 58、 CTLANPLGTLNPLK, 配列番 号： 2 6 )若しくは Cys-UGRP2 (63-77, SLGIPVNHLIEGSQKC, 配列番号： 2 7 )又は 該部分べプチドを含むポリぺプチドに対するモノクローナル抗体が好ましく、 受 領番号 FERM ABP- 10012又は FERM ABP- 10013であるハイプリド一マにより産生さ れるモノクローナル抗体がより好ましい。 また、 得られた抗体を精製後、 ぺプチ ダーゼ等により処理することにより、 抗体の断片が得られる。 さらに、 前記抗体 の誘導体、 例えば、 キメラ抗体、 ヒト化抗体、 Fab フラグメント、 単鎖抗体等や 公知技術により修飾された抗体等を使用することもできる。 かかる抗体又はその 断片は、 慣用の酵素 （例えば、 ペルォキシダーゼ等） 、 蛍光色素、 放射性物質、 アビジン若しくはビォチン等で標識されていてもよい。 Published by John Weily & Sons, Inc. Edited by John E. Coligan, Current Protocols in Immunology The method can be easily prepared by immunizing a egret, a mouse, or the like with all or a part of the protein belonging to the SCGB family by the method described in "I. Antibodies can also be produced by genetic engineering. The antibody may be a polyclonal antibody or a monoclonal antibody as long as it has an ability to specifically bind to a protein belonging to the SCGB family. The antibody may be, for example, an antibody capable of specifically binding to a specific partial fragment in the protein belonging to the SCGB family. The partial peptide of human UGPR2, Cys_UGRP2 (46-58, CTLANPLGTLNPLK, SEQ ID NO: 2) 6) or Cys-UGRP2 (63-77, SLGIPVNHLIEGSQKC, SEQ ID NO: 27) or a monoclonal antibody against the polypeptide containing the partial peptide is preferred, and the hybrid having the accession number FERM ABP-10012 or FERM ABP-10013 is preferred. More preferred are monoclonal antibodies produced by MA. The antibody fragment is obtained by purifying the obtained antibody and treating it with a peptidase or the like. Furthermore, derivatives of the above antibodies, for example, chimeric antibodies, humanized antibodies, Fab fragments, single-chain antibodies, etc. Antibodies and the like modified by known techniques can also be used. Such an antibody or a fragment thereof may be labeled with a conventional enzyme (for example, peroxidase or the like), a fluorescent dye, a radioactive substance, avidin or biotin, or the like.
ヒト UGRP2を認識するポリクローナル抗体の作製は、 ヒト UGRP2のポリべプチ ドの全長、 その部分ペプチド Cys-UGRP2 (46-58、 CTLANPLGTLNPLK, 配列番号： 2 6)若しくは Cys-UGRP2(63-77, SLGIPVNHLIEGSQKC, 配列番号： 27)又は該部分 ぺプチドを含むポリぺプチドを抗原として哺乳動物に投与することにより抗体を 作製することができる。 坊原としては、 前記ポリペプチド又は部分ペプチドをそ のまま用いてもよく、 担体、 例えば、 ゥシ血清アルブミン （BSA) 、 スカシガイ のへモシァニン （keyhole li即 et hemocyanin； KLH) やゥシチログロブリン （BT G) 等に結合したものを用いてもよい。 また、 抗原による免疫反応を高めるため に、 例えば、 フロインドの完全アジュバント （CFA) 及び不完全アジュバント （I FA) を投与してもよい。 免疫に用いられる哺乳動物としてはマウス、 ラット、 ゥ サギ、 ャギ、 ハムスター等を用いることができる。  The production of a polyclonal antibody that recognizes human UGRP2 can be performed using the full-length human UGRP2 polypeptide, its partial peptide Cys-UGRP2 (46-58, CTLANPLGTLNPLK, SEQ ID NO: 26) or Cys-UGRP2 (63-77, SLGIPVNHLIEGSQKC , SEQ ID NO: 27) or a polypeptide containing the partial peptide as an antigen, which can be used to produce an antibody. The polypeptide or partial peptide may be used as it is as a carrier, and may be used as a carrier, for example, perforated serum albumin (BSA), keyhole lichen ethemocyanin (KLH) or pericyroglobulin. (BT G) or the like may be used. Further, in order to enhance the immune response by the antigen, for example, Freund's complete adjuvant (CFA) and incomplete adjuvant (IFA) may be administered. As mammals used for immunization, mice, rats, rabbits, goats, hamsters and the like can be used.
ポリクローナル抗体は、 例えば、 レーンらの方法 [Antibodies： A Laboratory Manual, Second Edition(1989) (Cold Spring Harber Laboratory Press)] 等に 従って作製することができる。  Polyclonal antibodies can be prepared, for example, according to the method of Lane et al. [Antibodies: A Laboratory Manual, Second Edition (1989) (Cold Spring Harber Laboratory Press)].
哺乳動物に抗原を 1回目の投与後、 1〜2 週間毎に 3〜10 回投与することで免 疫された哺乳動物を得て、 それらの哺乳動物から血清を採取し、 精製することで 作製できる。  After the first dose of antigen to mammals, 3 to 10 doses are given every 1 to 2 weeks to obtain immunized mammals, and serum is collected from those mammals and purified. it can.
前記抗原の投与は、 1 回目の投与の後 1〜2週間おきに 3〜10回行う。 該抗原 の投与量は 1 回当たり動物 1 匹に対し、 50〜100 gが好ましい。 ペプチドを用 いる場合は、 適当な担体に共有結合させたものを抗原とするのが望ましい。 抗原 とするぺプチドは、 遺伝子工学的手法やべプチド合成機で合成することができる The antigen is administered 3 to 10 times every 1 to 2 weeks after the first administration. The dose of the antigen is preferably 50 to 100 g per animal per administration. When a peptide is used, it is desirable to use the antigen covalently bound to an appropriate carrier as the antigen. Peptides used as antigens can be synthesized using genetic engineering techniques or peptide synthesizers
。 各投与後、 3〜7 日目に眼底静脈叢より採血し、 該血清が免疫に用いた抗原と 反応することを酵素免疫測定法 〔 「酵素免疫測定法 （ELISA法） 」 医学書院刊 （ 1976 年） ； Ant ibodies :A Laboratory Manual, Second Edi t ion (1989) (Co l d Spr i ng Harber Laboratory Press) ) 等に記載の方法で確認することができる。 . Blood is collected from the fundus venous plexus on days 3 to 7 after each administration, and the reaction of the serum with the antigen used for immunization is determined by an enzyme immunoassay [“Enzyme immunoassay (ELISA)” published by Medical Shoin ( 1976); Ant ibodies: A Laboratory Manual, Second Edition (1989) (Cold Spring Harber Laboratory Press)) and the like.
免疫された哺乳動物から採血し、 抗体価を測定する。 十分な抗体価が得られる までに免疫された時点で採血し、 血清を調製することによりポリクローナル抗体 を得ることができる。 ポリクローナル抗体の分離、 精製方法としては、 遠心分離 、 硫酸アンモニゥムによる塩析、 力プリル酸沈殿 〔Ant ibodies : A Laboratory Ma nual , Second Ed i t ion (1989) (Col d Spr ing Harber Laboratory Press) ] 又は DEA E-セファロ一スカラム、 陰イオン交換カラム、 プロテイン Aカラム、 G-カラムあ るいはゲル濾過カラム等の各種クロマトグラフィーを単独又は組み合わせること で行うことができる。  Blood is collected from the immunized mammal and the antibody titer is measured. Blood is collected at the time of immunization until a sufficient antibody titer is obtained, and a serum is prepared, whereby a polyclonal antibody can be obtained. Methods for separation and purification of polyclonal antibodies include centrifugation, salting out with ammonium sulfate, and prillic acid precipitation [Antibodies: A Laboratory Manual, Second Edition (1989) (Cold Spring Harber Laboratory Press)] or It can be carried out by using various chromatography such as DEA E-Sepharose column, anion exchange column, protein A column, G-column or gel filtration column alone or in combination.
ヒト UGRP2を認識するモノクローナル抗体は、 例えば、 十分な抗体価が得られ た免疫哺乳動物から、 脾臓又はリンパ節を採取し、 それらから得られた抗体産生 細胞を骨髄腫 （ミエローマ） 細胞と融合させて、 モノクローナル抗体産生ハイブ リドーマを得、 得られたハイプリドーマを用いて慣用の方法により目的の抗体を 生産させることにより得られうる。 .  Monoclonal antibodies that recognize human UGRP2 can be obtained, for example, by collecting spleen or lymph nodes from immunized mammals with sufficient antibody titers, and fusing the antibody-producing cells obtained therefrom with myeloma (myeloma) cells. Thus, it can be obtained by obtaining a monoclonal antibody-producing hybridoma and producing the desired antibody by a conventional method using the obtained hybridoma. .
具体的には、 ヒト UGRP2のポリぺプチドの全長、 その部分べプチド Cys-UGRP2 (46-58、 CTLANPLGTLNPLK, 配列番号： 2 6 )若しくは Cys- UGRP2 (63- 77、 SLGIPVN HLIEGSQKC、 配列番号： 2 7 )又は該部分ペプチドを含むポリペプチドを抗原とし て投与して、 十分な抗体価を示したマウスに、 前記抗原を最終揆与する。 その後 、 3〜7日目に、 マウスから、 脾臓を摘出する。 得られた脾臓を、 MEM培地 （例え ば、 日水製薬社製） 中で細断し、 ピンセットでほぐし、 l, 200rpm で 5 分間遠心 分離する。 得られた沈殿分画中の脾細胞を Tr i s-塩化アンモニゥム緩衝液 （pH7. 65) で 1〜2分間処理して、 赤血球を除去する。 その後、 得られた脾細胞を、 MEM 培地で 3回洗浄する。 得られた脾細胞を抗体産生細胞として用いる。  Specifically, the full length of the human UGRP2 polypeptide, its partial peptide Cys-UGRP2 (46-58, CTLANPLGTLNPLK, SEQ ID NO: 26) or Cys-UGRP2 (63-77, SLGIPVN HLIEGSQKC, SEQ ID NO: 2) 7) Or a polypeptide containing the partial peptide is administered as an antigen, and the antigen is finally recruited to a mouse showing a sufficient antibody titer. Then, on days 3 to 7, the spleen is removed from the mouse. The obtained spleen is shredded in a MEM medium (for example, Nissui Pharmaceutical Co., Ltd.), loosened with tweezers, and centrifuged at 1,200 rpm for 5 minutes. The splenocytes in the precipitate fraction thus obtained are treated with Tris-ammonium chloride buffer (pH 7.65) for 1 to 2 minutes to remove erythrocytes. Then, the obtained spleen cells are washed three times with MEM medium. The obtained splenocytes are used as antibody-producing cells.
骨髄腫細胞としては、 マウス又はラットから由来の株化細胞が挙げられる。 か かる骨髄腫細胞としては、 例えば、 8-ァザグァニン耐性マウス （BALB/c 由来） 骨髄腫細胞 P3- X63Ag8- Ul株 （以下、 P3-U1 と略す） [Current Topics Microbio logycal Immunology, 81, 1 (1978)> Europian Journal of Immunology, 6， 511 (1976) 〕 、 SP2/0_Agl4株 （以下、 SP- 2と略す） [Nature, 276> 269 (1978)] 、 P3-X63-Ag 8653株 (以下、 653と略す) [Journal of Immunology, 123, 1548(1979)3 、 P3-X 63 - Ag8 (以下、 X63 と略す） 〔Nature， 256, 495 (1975)〕 等が挙げられる。 これら の細胞株は、 8 -ァザグァニン培地 （15^§/ιηΙ 8-ァザグァニンを含む正常培地 〔 1.5mM グルタミン、 5Χ1(Γ¾ 2-メルカプトエタノール、 10 g/ml ジェン夕マイ シン及び 10%FCS (CSL社製） を含む RPMI1640培地） 〕 で継代し、 細胞融合を行 う 3〜4 日前に正常培地で培養する。 細胞融合には、 そのようにして調製した細 胞を 2X107個以上用いる。 Myeloma cells include cell lines derived from mice or rats. Examples of such myeloma cells include 8-azaguanine-resistant mice (derived from BALB / c) Myeloma cells P3-X63Ag8-Ul strain (hereinafter abbreviated as P3-U1) [Current Topics Microbiological cal Immunology, 81, 1 (1978)> Europian Journal of Immunology, 6, 511 (1976)], SP2 / 0_Agl4 strain ( Hereinafter, abbreviated as SP-2) [Nature, 276> 269 (1978)], P3-X63-Ag 8653 strain (hereinafter abbreviated as 653) [Journal of Immunology, 123, 1548 (1979) 3, P3-X63 -Ag8 (hereinafter abbreviated as X63) [Nature, 256, 495 (1975)]. These cell lines were cultured in 8-azaguanine medium (normal medium containing 15 ^ § / ιηΙ 8-azaguanine [1.5 mM glutamine, 5Χ1 (Γ¾2-mercaptoethanol, 10 g / ml genyumycin and 10% FCS (CSL RPMI1640 medium containing the same product) and culture in a normal medium 3 to 4 days before cell fusion.For cell fusion, use 2 × 10 7 or more cells prepared in this manner.
細胞融合は、 既知の方法で行なわれ得、 例えば、 ケーラーとミルスタインの方 法 [Nature, 256,495-497 (1975)〕 に従って行なわれうる。 具体的には、 前記抗体 産生細胞と前記骨髄腫細胞とを、 MEM培地又は PBS (1 リットル当たり ； 1.83gリ ン酸ニナトリウム、 0.21g リン酸一カリウム、 7.65g NaCl、 pH7.2) で洗浄し、 骨髄腫細胞の細胞数に対し抗体産生細胞 5〜10 倍になるよう混合し、 l，200rpm で 5分間遠心分離した後、 沈殿分画を得る。 得られた沈澱画分の細胞群をよくほ ぐし、 該細胞群に対し抗体産生細胞 108個当たり、 ポリエチレングリコール溶液Cell fusion can be performed by a known method, for example, according to the method of Kohler and Milstein [Nature, 256, 495-497 (1975)]. Specifically, the antibody-producing cells and the myeloma cells were converted to a MEM medium or PBS (per liter; 1.83 g disodium phosphate, 0.21 g monopotassium phosphate, 7.65 g NaCl, pH 7.2). Wash and mix so that the number of antibody-producing cells is 5 to 10 times the number of myeloma cells. Centrifuge at 1,200 rpm for 5 minutes to obtain a precipitate fraction. The resulting precipitated fraction may ho Gushi cell groups, per 10 8 antibody-producing cells to the cell group, polyethylene glycol solution
〔2gポリエチレングリコール- 1000 (PEG- 1000) 、 2ml MEM培地、 0.7ml ジメチ ルスルホキシド （DMS0) 〕 を 0.2〜lmlを 37°Cで攪拌しながら添加し、 更に 1〜2 分間毎に MEM培地 〜 2mlを数回添加する。 MEM培地で全量を 50mlになるように 調製し、 900rpmで 5分間遠心分離した後、 沈殿分画を得る。 沈殿分画に HAT培 地 （正常培地、 10_4M ヒポキサンチン、 1.5X10—5Mチミジン及び 4X10— 7Mアミノ プテリンを含む） 100mlを添加し、 ゆるやかにほぐし、 懸濁する。 0.2 g of polyethylene glycol-1000 (PEG-1000), 2 ml of MEM medium, and 0.7 ml of dimethyl sulfoxide (DMS0) are added with stirring at 37 ° C, and the MEM medium is added every 1-2 minutes. Add 2 ml several times. Adjust the total volume to 50 ml with MEM medium and centrifuge at 900 rpm for 5 minutes to obtain a precipitate fraction. Precipitation fractionation HAT culture ground (normal medium, 10_ 4 M hypoxanthine, 1.5X10- 5 including M thymidine and 4X10- 7 M aminopterin) was added 100 ml, gently loosened and suspended.
得られた懸濁液を 96穴培養用プレートの各穴に 100 1穴ずつ分注し、 37°C、 The resulting suspension was dispensed into the wells of a 96-well culture plate at a rate of 37 ° C.
5%C02存在下で？〜 14日間培養する。 酵素免疫測定法 〔Antibodies:A Laborator y Manual, Second Edition (1989) (Cold Spring Harber Laboratory Press)] 等に 記載の方法で、 培養上清に産生された抗体のうち、 UGRP2の部分ペプチド Cys-UG5% C0 2 in the presence of? Incubate for ~ 14 days. Enzyme immunoassay [Antibodies: A Laboratory Manual, Second Edition (1989) (Cold Spring Harber Laboratory Press)] Among the antibodies produced in the culture supernatant by the method described, UGRP2 partial peptide Cys-UG
RP2 (46-58、 CTLANPLGTLNPLK, 配列番号： 2 6 )若しくは Cys- UGRP2 (63- 77、 SLGIRP2 (46-58, CTLANPLGTLNPLK, SEQ ID NO: 26) or Cys-UGRP2 (63-77, SLGI
PVNHLIEGSQKC, 配列番号： 2 7 )に特異的に反応する抗体を産生する八イブリド 一マを選択する。 このようにして得られたハイプリドーマは、 UG2- 6A9及び UG3-One antibody producing an antibody that specifically reacts with PVNHLIEGSQKC, SEQ ID NO: 27) is selected. The thus obtained hybridomas are UG2-6A9 and UG3-
1B12と命名 ·表示され、 それぞれ受領番号 FERM ABP- 10012及び FERM ABP-10013 として '2 0 0 4年 4月 2 1日に独立行政法人産業技術総合研究所 特許生物寄託 センター （郵便番号 305-8566 日本国 茨城県つくば巿東 1丁目 1番地 1 中 央第 6 ) に寄託されている。 Named 1B12 and displayed as receipt numbers FERM ABP-10012 and FERM ABP-10013, respectively, on April 21, 2004, National Institute of Advanced Industrial Science and Technology, Patent Organism Depositary (Postal code 305-8566 Deposited at Tsukuba East 1-chome, Ibaraki Pref.
前記ハイプリドーマによる抗体の生産は、 慣用の方法により行なわれうる。 また、 本発明のスクリーニング方法において使用される核酸 （プローブ） とし ては、 前記 SCGB ファミリーに属するタンパク質をコードする遺伝子に特異的に 結合する核酸等が挙げられる。 前記特異的に結合する核酸としては、 例えば、 SC The antibody production by the hybridoma can be performed by a conventional method. Examples of the nucleic acid (probe) used in the screening method of the present invention include a nucleic acid that specifically binds to a gene encoding a protein belonging to the SCGB family. Examples of the specifically binding nucleic acid include SC
GB ファミリーに属するタンパク質をコードする遺伝子からなる核酸、 前記 SCGB ファミリーに属するタンパク質をコードする遺伝子に特徴的な部分配列からなる 核酸等が挙げられる。 かかる核酸は、 使用時における操作性を考慮し、 適切な T m値、 二次構造等に基づき、 適宜選択されうる。 具体的には、 ヒトでは、 例えばA nucleic acid comprising a gene encoding a protein belonging to the GB family; a nucleic acid comprising a partial sequence characteristic of the gene encoding a protein belonging to the SCGB family; Such a nucleic acid can be appropriately selected based on an appropriate Tm value, a secondary structure and the like in consideration of operability at the time of use. Specifically, in humans, for example,
、 配列番号： 1、 3、 5、 7、 9、 1 1、 1 3、 1 5又は 2 3に記載の塩基配列 を有する核酸等が好適に使用される。 また、 マウスでは、 配列番号： 1 7に記載 の塩基配列を有する核酸等が使用される。 かかる核酸は、 慣用の蛍光色素、 放射 性物質等で標識されていてもよい。 A nucleic acid having the nucleotide sequence described in SEQ ID NO: 1, 3, 5, 7, 9, 9, 11, 13, 15, or 23, and the like are preferably used. In a mouse, a nucleic acid having the nucleotide sequence of SEQ ID NO: 17 is used. Such a nucleic acid may be labeled with a conventional fluorescent dye, radioactive substance, or the like.
前記 SCGB ファミリーに属するタンパク質をコードする遺伝子又はその一部を 特異的に増幅しうるプライマー対としては、 例えば、 前記 SCGB ファミリーに属 するタンパク質をコードする遺伝子からなる核酸の 5'末端のアンチセンス配列 に対応するプライマーと 3'末端のアンチセンス配列に対応するプライマーとか らなるプライマー対；前記 SCGB ファミリーに属するタンパク質をコードする遺 伝子に特徴的な部分配列からなる核酸の 5'末端のアンチセンス配列に対応する プライマーと 3'末端のアンチセンス配列に対応するプライマ—とからなるプラ イマ一対等が挙げられる。 かかるプライマー対は、 使用時における操作性を考慮 し、 適切な Tm値、 二次構造等に基づき、 適宜選択されうる。 具体的には、 かか るプライマー対としては、 ヒトでは、 前記配列番号： 1〜8に記載の塩基配列の 全部又はその特徴的な部分配列を増幅可能なものであるのが好適である。 より具 体的には、 例えば、 配列番号： 1 9〜2 2に記載の塩基配列を有する核酸からな る群より選ばれたプライマー対等が挙げられる。 プライマー対の具体例としては 、 特に限定はないが、 配列番号： 1 9と 2 0とのプライマー対、 配列番号： 2 1 と 2 2とのプライマー対、 配列番号： 1 9と 2 2とのプライマ一対、 配列番号： 2 0と 2 1とのプライマー対等が挙げられる。 また、 マウスでは、 前記配列番号 ： 1 7に記載の塩基配列の全部又はその特徴的な部分配列を増幅可能なものであ るのが好適である。 具体的には、 例えば、 配列番号： 2 4と 2 5とのプライマ一 対等が挙げられる。 それらのプライマーは、 慣用の蛍光色素、 放射性物質等で標 識されたプライマ一であってもよい。 増幅産物の検出は、 慣用のァガロースゲル 電気泳動等において、 臭化工チジゥムゃ蛍光色素による可視化、 標識プライマー に基づく検出等を行.うこと等により行なわれうる。 Examples of the primer pair capable of specifically amplifying a gene encoding a protein belonging to the SCGB family or a part thereof include, for example, an antisense sequence at the 5 ′ end of a nucleic acid comprising a gene encoding a protein belonging to the SCGB family. A primer pair consisting of a primer corresponding to the above and a primer corresponding to the 3′-end antisense sequence; the 5′-end antisense of a nucleic acid consisting of a partial sequence characteristic of a gene encoding a protein belonging to the SCGB family Corresponding to an array A primer pair consisting of a primer and a primer corresponding to the antisense sequence at the 3 ′ end is exemplified. Such a primer pair can be appropriately selected based on an appropriate Tm value, a secondary structure and the like in consideration of operability at the time of use. Specifically, it is preferable that such a primer pair be capable of amplifying all or a characteristic partial sequence of the nucleotide sequence of SEQ ID NOS: 1 to 8 in human. More specifically, for example, a primer pair selected from the group consisting of nucleic acids having the nucleotide sequences of SEQ ID NOs: 19 to 22 can be mentioned. Specific examples of the primer pair include, but are not limited to, a primer pair of SEQ ID NOS: 19 and 20, a primer pair of SEQ ID NOs: 21 and 22, and a primer pair of SEQ ID NOs: 19 and 22. Primer pairs, primer pairs of SEQ ID NOS: 20 and 21, and the like. In the mouse, it is preferable that the whole of the nucleotide sequence of SEQ ID NO: 17 or a characteristic partial sequence thereof can be amplified. Specifically, for example, a primer pair of SEQ ID NOs: 24 and 25 and the like can be mentioned. These primers may be primers labeled with conventional fluorescent dyes, radioactive substances and the like. The amplification product can be detected by conventional agarose gel electrophoresis or the like, by visualization with a bromide dye or a fluorescent dye, detection based on a labeled primer, or the like.
本明細書において、 「 （核酸に） 特徴的な部分配列」 とは、 例えば、 データべ ースに登録された配列のうち、 前記 SCGB ファミリーに属するタンパク質をコー ドする遺伝子以外の遺伝子の塩基配列には実質的に見出されない部分配列をいい 、 例えば、 データベースに登録された配列との配列同一性が、 通常、 20%以下、 好ましくは 10%以下、 より好ましくは 5 %以下、 特に好ましくは 0%である配列 をい 。  In the present specification, the “characteristic partial sequence (to nucleic acid)” refers to, for example, a nucleotide sequence of a gene other than a gene encoding a protein belonging to the SCGB family among sequences registered in a database. Refers to a partial sequence that is not substantially found.For example, the sequence identity to a sequence registered in a database is usually 20% or less, preferably 10% or less, more preferably 5% or less, and particularly preferably An array that is 0%.
本発明のスクリーニング方法により得られる化合物又はその塩としては、 例え ば、 M記 SCGB ファミリーに属するタンパク質等に結合することにより、 該タン パク質の機能を阻害する化合物、 例えば、 低分子化合物、 高分子化合物、 該 SCG Examples of the compound obtained by the screening method of the present invention or a salt thereof include, for example, a compound that inhibits the function of the protein by binding to a protein or the like belonging to the M SCGB family, for example, a low molecular compound, Molecular compound, the SCG
B ファミリーに属するタンパク質に対する抗体、 該 SCGB ファミリーに属する夕 ンパク質をコードする遺伝子からなる核酸のアンチセンス鎖等の核酸、 ペプチド 等が挙げられる。 _ また、 本発明のスクリーニング方法に用いられる前記 SCGB ファミリーに属す るタンパク質、 該 SCGB ファミリーに属するタンパク質をコードする遺伝子を保 持してなる核酸構築物、 該遺伝子が導入された細胞、 SCGB ファミリ一に属する タンパク質の抗体等を含む、 本発明のスクリーニング方法を行うためのキットが 提供される。 かかるスクリーニング用キットも本発明に含まれる。 Antibodies against proteins belonging to the B family, evening belonging to the SCGB family Nucleic acids, such as antisense strands of nucleic acids consisting of genes encoding proteins, peptides, and the like. _ Further, a protein belonging to the SCGB family used in the screening method of the present invention, a nucleic acid construct containing a gene encoding a protein belonging to the SCGB family, a cell into which the gene has been introduced, and a SCGB family member. A kit for performing the screening method of the present invention, including an antibody of a protein belonging thereto, is provided. Such a screening kit is also included in the present invention.
本発明のスクリーニング用キットとしては、 具体的には、  As the screening kit of the present invention, specifically,
一 SCGBファミリ一に属するタンパク質を含有してなるキット、 A kit containing a protein belonging to one SCGB family,
- SCGB ファミリ一に属するタンパク質をコードする遺伝子を含む核酸構築物 を含有してなるキット、 -A kit containing a nucleic acid construct containing a gene encoding a protein belonging to the SCGB family 1;
一 SCGB ファミリーに属するタンパク質をコードする遺伝子が導入された細胞 を含有してなるキット、 A kit containing cells into which a gene encoding a protein belonging to the SCGB family has been introduced;
一 SCGB ファミリーに属するタンパク質に対する抗体又はその断片を含有して なるキッ卜、 A kit comprising an antibody against a protein belonging to the SCGB family or a fragment thereof;
― ヒト UGPR2の部分ペプチド Cys-UGRP2 (46-58、 CTLANPLGTLNPLK, 配列番号： 2 6 )若しくは Cys-UGRP2 (63-77, SLGIPVNHLIEGSQKC, 配列番号： 2 7 )又は該部 分ペプチドを含むポリペプチドに対するモノクローナル抗体又はその断片を含有 してなるキット、  -Human UGPR2 partial peptide Cys-UGRP2 (46-58, CTLANPLGTLNPLK, SEQ ID NO: 26) or Cys-UGRP2 (63-77, SLGIPVNHLIEGSQKC, SEQ ID NO: 27) or monoclonal for the polypeptide containing the partial peptide A kit comprising the antibody or a fragment thereof,
一 受領番号 FERM ABP- 10012及び FERM ABP- 10013であるハイプリドーマにより 産生されるモノクローナル抗体又はその断片を含有してなるキット (I) a kit containing a monoclonal antibody produced by a hybridoma having a receipt number of FERM ABP-10012 or FERM ABP-10013 or a fragment thereof;
等が挙げられる。 And the like.
また、 各キットには、 所望により、 本発明のスクリーニング方法に好適に使用 されうる前記プローブ及び 又はプライマー対を含有させてもよい。 さらには、 所望により検出用試薬、 緩衝液、 標準物質、 本発明のスクリーニング方法を実施 するための説明書等を含有させてもよい。 Further, each kit may contain the probe and / or primer pair suitably used in the screening method of the present invention, if desired. Further, if desired, the detection reagent, buffer, standard substance, and the screening method of the present invention are implemented. Instructions and the like may be included.
本発明のスクリーニング用キットによれば、 前記スクリーニング方法を簡便か つ迅速に、 高処理量で行うことができるという優れた効果が発揮される。 本発明のスクリーニング方法により得られた化合物又はその塩は、 その発症に ADVANTAGE OF THE INVENTION According to the screening kit of this invention, the outstanding effect that the said screening method can be performed simply and quickly with a high throughput is exhibited. Compounds or salts thereof obtained by the screening method of the present invention
SCGBファミリーに属するタンパク質の発現が関与する C0PDに対して治療又は予 防作用を発揮しうる。 したがって、 前記スクリーニング方法により得られた化合 物又はその塩により、 C0PD、 例えば、 肺気腫又は慢性気管支炎、 特に肺気腫の治 療又は予防に用いるための医薬組成物が提供される。 It can exert a therapeutic or preventive effect on C0PD involving expression of a protein belonging to the SCGB family. Therefore, a compound or a salt thereof obtained by the screening method provides a pharmaceutical composition for use in treating or preventing C0PD, for example, emphysema or chronic bronchitis, particularly emphysema.
本発明の医薬組成物は、 本発明のスクリーニング方法により得られた化合物又 はその塩を有効成分として含有することに 1つの特徴がある。 したがって、 本発 明の医薬組成物は、 C0PD に対し、 SCGB ファミリーに属するタンパク質の発現に 対する作用 （例えば、 該発現に対して抑制的に働く） を介して治療又は予防効果 を発揮する。  One feature of the pharmaceutical composition of the present invention is that it contains a compound obtained by the screening method of the present invention or a salt thereof as an active ingredient. Therefore, the pharmaceutical composition of the present invention exerts a therapeutic or preventive effect on C0PD through an action on the expression of a protein belonging to the SCGB family (for example, acts to suppress the expression).
本発明の医薬組成物中における前記化合物又はその塩の含有量は、 治療目的の 疾患、 患者の年齢、 体重等により適宜調節することができ、 治療上有効量であれ ぱよく、 低分子化合物又は高分子化合物の場合、 例えば、 0. 0001〜1000mg、 好ま しくは 0. 001〜100mg、 ポリペプチド又はその誘導体の場合、 例えば、 0. 0001〜1 000mg、 好ましくは 0. 001〜100mg、 核酸又はその誘導体の場合、 例えば、 0. 0000 l〜100mg、 好ましくは 0. 0001〜10mgであることが望ましい。  The content of the compound or a salt thereof in the pharmaceutical composition of the present invention can be appropriately adjusted depending on the disease to be treated, the patient's age, body weight, and the like. In the case of a high molecular compound, for example, 0.0001 to 1000 mg, preferably 0.001 to 100 mg, and in the case of a polypeptide or a derivative thereof, for example, 0.0001 to 1000 mg, preferably 0.001 to 100 mg, nucleic acid or In the case of the derivative, for example, it is desirable that the amount is 0.0001 l to 100 mg, preferably 0.0001 to 10 mg.
本発明の医薬組成物は、 前記化合物又はその塩を安定に保持しうる種々の助剤 をさらに含有してもよい。 具体的には、 有効成分の送達対象となる部位に到達す るまでの間に、 有効成分が分解することを抑制する性質を呈する薬学的に許容さ れうる助剤、 賦形剤、 結合剤、 安定剤、 緩衝剤、 溶解補助剤、 等張剤等が挙げら れる。  The pharmaceutical composition of the present invention may further contain various auxiliaries capable of stably retaining the compound or a salt thereof. Specifically, pharmaceutically acceptable auxiliaries, excipients, and binders exhibiting the property of inhibiting the active ingredient from decomposing before reaching the site to which the active ingredient is to be delivered. , Stabilizers, buffers, solubilizing agents, isotonic agents and the like.
本発明の医薬組成物の投与形態は、 有効成分の種類；投与対象となる個体、 器 官、 局所部位、 組織.；投与対象となる個体の年齢、 体重等に応じて、 適宜選択さ れる。 前記投与形態としては、 皮下注射、 筋肉内注射、 静脈内注射、 局所投与等 が挙げられる。 The dosage form of the pharmaceutical composition of the present invention is determined by the type of active ingredient; Government, local site, tissue; selected as appropriate according to the age, weight, etc. of the individual to be administered. Examples of the administration form include subcutaneous injection, intramuscular injection, intravenous injection, topical administration and the like.
また、 本発明の医薬組成物の投与量も、 有効成分の種類；投与対象となる個体 、 器官、 局所部位、 組織；投与対象となる個体の年齢、 体重等に応じて、 適宜選 択される。 投与方法としては、 特に限定されないが、 有効成分が、 低分子化合物 又は高分子化合物である場合、 前記有効成分の量として、 例えば、 0. 0001〜1000 mg/kg体重、 好ましくは 0. 001〜100mg/kg体重、 ポリぺプチド又はその誘導体の 場合、 例えば、 0. 0001〜1000mg/kg体重、 好ましくは 0. 001〜100mg/kg体重、 核 酸又はその誘導体の場合、 例えば、 0. 00001〜100mg/kg体重、 好ましくは 0. 0001 〜10mg/kg体重の 1回投与量となるように、 1日につき、 1回若しくは複数回投. 与すること等が挙げられる。 投与期間も特に限定されるものではない。  The dose of the pharmaceutical composition of the present invention is also appropriately selected according to the type of the active ingredient; the individual, organ, local site, or tissue to be administered; the age, body weight, etc. of the individual to be administered. . The administration method is not particularly limited, but when the active ingredient is a low molecular weight compound or a high molecular weight compound, the amount of the active ingredient is, for example, 0.0001 to 1000 mg / kg body weight, preferably 0.0001 to 1000 mg / kg body weight. 100 mg / kg body weight, in the case of a polypeptide or a derivative thereof, for example, 0.0001 to 1000 mg / kg body weight, preferably 0.001 to 100 mg / kg, in the case of a nucleic acid or a derivative thereof, for example, 0.000001 to One or more doses per day may be given to give a single dose of 100 mg / kg body weight, preferably 0.0001 to 10 mg / kg body weight. The administration period is not particularly limited.
本発明の医薬組成物の薬理評価は、 例えば、 後述の実施例に記載の C0PD モデ ルマウスに、 本発明の医薬組成物を投与し、 非投与動物に比べ、 投与動物におい て、 C0PD の改善が見られた場合を指標として評価する方法により行うことがで きる。 また、 本発明により、 C0PD の診断方法が提供される。 本発明の診断方法は、 被検対象の個体由来の被検試料及び正常個体由来の対照試料それぞれにおける S CGBファミリーに属するタンパク質の遺伝子、 特に UGRP2の遺伝子の発現レベル を測定することを 1つの特徴とする。 したがって、 本発明の診断方法によれば、 被検対象の個体が C0PD に罹患している疑いがあるか否かを簡便かつ迅速に診断 することができるという優れた効果が発揮される。  The pharmacological evaluation of the pharmaceutical composition of the present invention can be performed, for example, by administering the pharmaceutical composition of the present invention to the C0PD model mouse described in the Examples below, and improving the C0PD in the treated animal as compared to the non-administered animal. This can be done by a method of evaluating the case where it is seen as an index. The present invention also provides a method for diagnosing C0PD. One feature of the diagnostic method of the present invention is to measure the expression level of a gene of a protein belonging to the SCGB family, particularly a UGRP2 gene, in each of a test sample derived from an individual to be tested and a control sample derived from a normal individual. And Therefore, according to the diagnostic method of the present invention, an excellent effect of easily and quickly diagnosing whether or not an individual to be tested is suspected of having C0PD is exhibited.
本発明の診断方法においては、 被検試料における発現レベルが、 対照試料にお ける発現レベルよりも高くなる場合、 該被検対象の個体が、 C0PD に罹患してい る疑いがあることの指標となる。 前記個体としては、 特に限定されるものではないが、 哺乳動物、 特にヒト、 マ ウス、 マウス、 サル等が挙げられる。 In the diagnostic method of the present invention, when the expression level in the test sample is higher than the expression level in the control sample, an index indicating that the test subject is suspected of having C0PD is provided. Become. Examples of the individual include, but are not limited to, mammals, particularly humans, mice, mice, monkeys, and the like.
被検試料は、 例えば、 個体の組織、 細胞、 血液等から慣用の手法により調製さ れうる。 特に SCGB ファミリーに属するタンパク質の発現量が高い、 気管、 肺、 舌、 あるいは唾液から調製するのが好ましい。 対照試料は、 被検試料と同様の部 位から調製されたものであればよく、 例えば、 被検試料と同様にして同部位より 調製することができる。  The test sample can be prepared, for example, from an individual's tissue, cells, blood, etc. by a conventional method. In particular, it is preferably prepared from trachea, lung, tongue, or saliva, which has a high expression level of a protein belonging to the SCGB family. The control sample may be prepared from the same site as the test sample. For example, the control sample can be prepared from the same site as the test sample.
SCGBファミリーに属する夕ンパク質の遺伝子の発現レベルは、 該遺伝子の mRN A転写物の量又は該遺伝子の遺伝子産物の量を指標として測定するのが好適であ る。  The expression level of the gene of the protein belonging to the SCGB family is preferably measured using the amount of the mRNA transcript of the gene or the amount of the gene product of the gene as an index.
mRNA転写物の量を指標とする場合、 被検対象の個体由来の被検試料及び正常 個体由来の対照試料のそれぞれを、 例えば、 前記 SCGB ファミリーに属するタン パク質をコードする遺伝子からなる核酸若しくは該核酸に特徴的な部分配列から なる核酸をプローブとして用いたハイブリダィゼーシヨン、 あるいは前記 SCGB ファミリーに属するタンパク質をコードする遺伝子からなる核酸若しくは該核酸 に特徴的な部分配列からなる核酸をそれぞれ特異的に増幅しうるプライマー対を 用いた PCR等に供し、 八イブリッドの形成若しくはその量又は増幅産物の出現若 しくはその量を測定し、 比較することにより、 SCGB ファミリーに属するタンパ ク質の遺伝子の発現レベルを評価することができる。  When the amount of mRNA transcript is used as an index, each of a test sample derived from an individual to be tested and a control sample derived from a normal individual is, for example, a nucleic acid comprising a gene encoding a protein belonging to the SCGB family or Hybridization using a nucleic acid consisting of a partial sequence characteristic of the nucleic acid as a probe, a nucleic acid consisting of a gene encoding a protein belonging to the SCGB family, or a nucleic acid consisting of a partial sequence characteristic of the nucleic acid By subjecting it to PCR or the like using a primer pair that can specifically amplify, measuring the formation or the amount of the eight hybrids or the appearance or the amount of the amplification product, and comparing them, the protein belonging to the SCGB family can be identified. The expression level of the gene can be evaluated.
プローブとして用いられる核酸は、 使用時における操作性を考慮し、 適切な T m値、 二次構造等に基づき、 適宜選択されうる。 プローブとしては、 例えば、 本 発明のスクリーニング方法において好適に使用されうる前記プローブ等が挙げら れる。 かかる核酸は、 慣用の蛍光色素、 放射性物質等で標識されていてもよい。  A nucleic acid used as a probe can be appropriately selected based on an appropriate Tm value, a secondary structure, and the like in consideration of operability at the time of use. Examples of the probe include, for example, the probe and the like that can be suitably used in the screening method of the present invention. Such a nucleic acid may be labeled with a conventional fluorescent dye, radioactive substance, or the like.
PCRに用いられるプライマー対としては、 SCGBファミリーに属するタンパク質 をコードする遺伝子からなる核酸若しくは該核酸に特徴的な配列部分からなる核 酸の 5'末端のアンチセンス配列に対応するプライマーと 3'末端のアンチセンス 配列に対応するプライマ一とからなるプライマ一対等が挙げられる。 かかるブラ イマ一対は、 使用時における操作性を考慮し、 適切な Tm値、 二次構造等に基づ き、 適宜選択されうる。 具体的には、 本発明のスクリーニング方法において好適 に使用されうる前記プライマー対等が挙げられる。 かかるプライマーは、 慣用の 蛍光色素、 放射性物質等で標識されたプライマーであってもよい。 増幅産物の検 出は、 慣用のァガロースゲル電気泳動等において、 臭化工チジゥムゃ蛍光物質に よる可視化、 標識プライマーに基づく検出等を行うこと等により行なわれうる。 また、 本態様においては、 前記 SCGB ファミリーに属するタンパク質に対する 抗体又はその断片、 好ましくはヒト UGPR2の部分ペプチド Cys- UGRP2 (46-58、 CT LANPLGTLNPLK, 配列番号： 2 6 )若しくは Cys- UGRP2 (63- 77、 SLGIPVNHLIEGSQKC 、 配列番号： 2 7 )又は該部分ペプチドを含むポリペプチドに対するモノクロ一 ナル抗体又はその断片、 さらに好ましくは受領番号 FERM ABP-10012又は FERM A BP-10013 であるハイプリドーマにより産生されるモノクローナル抗体又はその 断片を用いて公知の方法に従って適宜 mRNA転写物の量を測定し、 SCGBファミリ 一に属するタンパク質の遺伝子の発現レベルを評価することができる。 The primer pair used for PCR includes a primer corresponding to the 5'-terminal antisense sequence of a nucleic acid comprising a gene encoding a protein belonging to the SCGB family or a nucleic acid comprising a sequence portion characteristic of the nucleic acid, and a 3'-terminal Antisense And a primer pair consisting of a primer corresponding to the sequence. Such a pair of primers can be appropriately selected based on an appropriate Tm value, a secondary structure and the like in consideration of operability at the time of use. Specific examples include the primer pairs and the like that can be suitably used in the screening method of the present invention. Such a primer may be a primer labeled with a conventional fluorescent dye, radioactive substance or the like. Detection of the amplification product can be performed by conventional agarose gel electrophoresis or the like by performing visualization with a bromide reagent or a fluorescent substance, detection based on a labeled primer, or the like. Further, in this embodiment, an antibody against the protein belonging to the SCGB family or a fragment thereof, preferably a partial peptide of human UGPR2 Cys-UGRP2 (46-58, CT LANPLGTLNPLK, SEQ ID NO: 26) or Cys-UGRP2 (63- 77, SLGIPVNHLIEGSQKC, SEQ ID NO: 27) or a monoclonal antibody against a polypeptide containing the partial peptide or a fragment thereof, more preferably produced by a hybridoma having accession number FERM ABP-10012 or FERM ABP-10013 Using a monoclonal antibody or a fragment thereof, the amount of mRNA transcript is appropriately measured according to a known method, and the expression level of a gene of a protein belonging to the SCGB family can be evaluated.
遺伝子産物の量を指標とする場合、 被検対象の個体由来の被検試料及び正常個 体由来の対照試料のそれぞれを、 ポリアクリルアミドゲル電気泳動、 前記 SCGB ファミリーに属するタンパク質に対する抗体又はその断片、 好ましくはヒ卜 UGP R2 の部分ペプチド Cys- UGRP2 (46-58、 CTLANPLGTLNPLK, 配列番号： 2 6 )若しく は Cys-UGRP2 (63-77, SLGIPVNHLIEGSQKC, 配列番号： 2 7 )又は該部分ペプチド を含むポリペプチドに対するモノクローナル抗体又はその断片、 さらに好ましく は受領番号 FERM ABP- 10012又は FERM ABP- 10013であるハイブリドーマにより産 生されるモノクローナル抗体又はその断片を用いたウエスタンブロット解析、 前 記抗体又はその断片を用いたィムノアッセィ等に供し、 遺伝子産物量を測定し、 比較することにより、 SCGB ファミリーに属するタンパク質の遺伝子の発現レべ ルを評価することができる。 さらに、 本発明によれば、 本発明の診断方法を、 簡便、 迅速に、 高処理量で、 高い信頼性で行いうる、 本発明のスクリーニング方法において好適に使用されう る前記プローブ及び Z又はプライマー対を含有した診断用キット、 又は前記 SCG B ファミリーに属するタンパク質に対する抗体若しくはその断片、 好ましくはヒ ト UGPR2 の部分ペプチド Cys-UGR (46-58、 CTLANPLGTLNPLK, 配列番号： 2 6 ) 若しくは Cys-UGRP2 (63-77, SLGIPVNHLIEGSQKC, 配列番号： 2 7 )又は該部分べ プチドを含むポリぺプチドに対するモノクローナル抗体又はその断片、 さらに好 ましくは受領番号 FERM ABP- 10012又は？81«1 ABP- 10013であるハイブリドーマに より産生されるモノクローナル抗体又はその断片を含有した診断用キットが提供 される。 When the amount of the gene product is used as an index, a test sample derived from an individual to be tested and a control sample derived from a normal individual are subjected to polyacrylamide gel electrophoresis, an antibody against a protein belonging to the SCGB family or a fragment thereof, Preferably, it comprises a partial peptide of human UGP R2 Cys-UGRP2 (46-58, CTLANPLGTLNPLK, SEQ ID NO: 26) or Cys-UGRP2 (63-77, SLGIPVNHLIEGSQKC, SEQ ID NO: 27) or the partial peptide Western blot analysis using a monoclonal antibody against a polypeptide or a fragment thereof, more preferably a monoclonal antibody or a fragment thereof produced by a hybridoma having accession number FERM ABP-10012 or FERM ABP-10013, It was used for the imnoassay and the amount of the gene product was measured and compared. It is possible to assess the expression levels of the genes of click quality. Furthermore, according to the present invention, the diagnostic method of the present invention can be performed simply, quickly, with a high throughput, and with high reliability, and the probe and Z or primer suitably used in the screening method of the present invention. A diagnostic kit containing a pair, or an antibody against a protein belonging to the SCG B family or a fragment thereof, preferably a partial peptide of human UGPR2 Cys-UGR (46-58, CTLANPLGTLNPLK, SEQ ID NO: 26) or Cys-UGRP2 (63-77, SLGIPVNHLIEGSQKC, SEQ ID NO: 27) or a monoclonal antibody against a polypeptide containing the partial peptide or a fragment thereof, more preferably, the accession number FERM ABP-10012 or? 81 «1 A diagnostic kit containing a monoclonal antibody produced by a hybridoma that is ABP-10013 or a fragment thereof is provided.
本発明の診断用キットには、 検出用試薬、 緩衝液、 対照試料、 本発明の診断方 法の説明書等をさらに含有させてもよい。 さらに、 本発明によれば、 C0PD に罹患している疑いがある個体由来の試料の 検出又は選別方法が提供される。  The diagnostic kit of the present invention may further contain a detection reagent, a buffer, a control sample, instructions for the diagnostic method of the present invention, and the like. Further, the present invention provides a method for detecting or selecting a sample derived from an individual suspected of having C0PD.
本発明の検出又は選別方法によれば、 C0PD に罹患している疑いがある個体由 来の試料を簡便かつ迅速に検出又は選別することができる。  According to the detection or selection method of the present invention, a sample derived from an individual suspected of suffering from C0PD can be detected or selected easily and quickly.
本発明の検出又は選別方法は、 被検対象の個体由来の被検試料及び正常個体由 来の対照試料それぞれにおける前記 SCGB ファミリーに属するタンパク質の遺伝 子の発現レベルを測定するステップを含み、 ここで、 被検試料における発現レべ ルが、 対照試料における発現レベルよりも高くなる場合、 該被検試料が、 C0PP に罹患している疑いがある個体由来の試料であることの指標となる。  The detection or selection method of the present invention includes a step of measuring the expression level of the gene of the protein belonging to the SCGB family in each of a test sample derived from an individual to be tested and a control sample derived from a normal individual. If the expression level in the test sample is higher than the expression level in the control sample, this is an indicator that the test sample is a sample derived from an individual suspected of having C0PP.
本発明の検出又は選別方法においては、 SCGB ファミリーに属するタンパク質 の遺伝子の発現レベルは、 該遺伝子の mRNA転写物の量又は該遺伝子の遺伝子産 物の量を指標として測定するのが好適である。 SCGB ファミリーに属するタンパ ク質の遺伝子の発現レベルの測定は、 本発明の前記診断方法の場合と同様にして 行うことができる。 In the detection or selection method of the present invention, the expression level of a gene of a protein belonging to the SCGB family is preferably measured using the amount of the mRNA transcript of the gene or the amount of the gene product of the gene as an index. Tampa belonging to SCGB family The expression level of the protein gene can be measured in the same manner as in the case of the diagnostic method of the present invention.
また、 本発明によれば、 本発明の検出又は選別方法を、 簡便、 迅速に、 高処理 量で行うための、 前記診断方法の説明において記載したプローブ及び 又はブラ イマ—-対を含有した検出又は選別用キット、 又は前記 SCGB ファミリーに属する タンパク質に対する抗体若しくはその断片、 好ましくは、 ヒト UGPR2の部分ぺプ チド Cys-UGRP2 (46-58、 CTLANPLGTLNPLK, 配列番号： 2 6 )若しくほ Cys- UGRP2 (6 3-77、 SLGIPVNHL IEGSQKC, 配列番号： 2 7 )又は該部分ペプチドを含むポリぺプ チドに対するモノクローナル抗体又はその断片、 さらに好ましくは受領番号 FER M ABP-10012 又は， FERM ABP-10013 であるハイブリドーマにより産生されるモノ クローナル抗体又はその断片を含有した検出又は選別用キットが提供される。 Further, according to the present invention, a detection containing the probe and / or the primer pair described in the description of the diagnostic method for performing the detection or selection method of the present invention simply, quickly and with a high throughput. Or a selection kit, or an antibody against a protein belonging to the SCGB family or a fragment thereof, preferably a partial peptide of human UGPR2 Cys-UGRP2 (46-58, CTLANPLGTLNPLK, SEQ ID NO: 26) or Cys-UGRP2 (63-77, SLGIPVNHL IEGSQKC, SEQ ID NO: 27) or a monoclonal antibody against a polypeptide containing the partial peptide or a fragment thereof, and more preferably, accession number FERM ABP-10012 or FERM ABP-10013. A detection or selection kit containing a monoclonal antibody or a fragment thereof produced by a hybridoma is provided.
本発明はさらに、 ヒト UGPR2の部分ペプチド Cys-UGRP2 (46- 58、 CTLANPLGTLNP LK、 配列番号： 2 6 )若しくは Cys- UGRP2 (63-77、 SLGIPVNHLIEGSQKC, 配列番号The present invention further provides a partial peptide of human UGPR2, Cys-UGRP2 (46-58, CTLANPLGTLNPLK, SEQ ID NO: 26) or Cys-UGRP2 (63-77, SLGIPVNHLIEGSQKC, SEQ ID NO:
： 2 7 )又は該部分ペプチドを含むポリペプチドに対するモノクローナル抗体、 受領番号 FERM ABP- 10012又は FERM ABP- 10013であるハイブリドーマにより産生 される抗体、 及び受領番号 FERM ABP- 10012又は FERM ABP- 10013であるハイプリ ドーマを提供する。 かかるモノクローナル抗体及びハイプリドーマは、 前記した とおりに調製される。 27) or a monoclonal antibody against a polypeptide containing the partial peptide, an antibody produced by a hybridoma having accession number FERM ABP-10012 or FERM ABP-10013, and an accession number FERM ABP-10012 or FERM ABP-10013. Provide Hypri-Dorma. Such monoclonal antibodies and hybridomas are prepared as described above.
本発明の C0PD治療剤又は予防剤のスクリーニング方法によれば、 C0PD治療剤 又は予防剤の候補化合物を簡便かつ迅速にスクリーニングすることができ、 またAccording to the method for screening a therapeutic or prophylactic agent for C0PD of the present invention, a candidate compound for a therapeutic or prophylactic agent for C0PD can be screened simply and quickly, and
、 該治療剤又は予防剤の候補化合物の薬理評価を行うことができる。 また、 本発 明のスクリーニング用キットによれば、 前記スクリーニングを簡便、 迅速に、 高 処理量で行うことができる。 さらに、 本発明の医薬組成物によれば、 C0PD を効 果的に治療又は予防することができる。 また、 本発明の C0PD の診断方法によれ ば、 C0PD を簡便かつ迅速に診断することができる。 また、 本発明の診断用キッ トによれば、 前記診断を、 簡便、 迅速に、 高処理量で行うことができる。 さらに 、 本発明の C0PDに罹患している個体由来の試料の検出又は選別方法によれば、 C 0PD に罹患している個体由来の試料を簡便かつ迅速に検出又は選別することがで きる。 また、 本発明の検出又は選別用キットによれば、 前記検出又は選別を簡便 かつ迅速に、 高処理量で行うことができる。 本発明を、 実施例によりさらに詳細に説明するが、 本発明は、 かかる実施例の みに限定されるものではない。 また、 以下の実施例において、 遺伝子操作手法と して、 特に断らない限り、 Molecular Cloning： A Laboratory Manual, Second Ed ition(1989) (Cold Spring Harbor Laboratory Press) に記載された方法を用い た。 実施例 1 C0PDモデルマウスにおける UGRP2 mRNAの発現 1 The pharmacological evaluation of the candidate compound for the therapeutic or prophylactic agent can be performed. Further, according to the screening kit of the present invention, the screening can be performed simply, quickly, and with a high throughput. Further, according to the pharmaceutical composition of the present invention, C0PD is effective. It can be effectively treated or prevented. Further, according to the method for diagnosing C0PD of the present invention, C0PD can be diagnosed simply and quickly. Further, according to the diagnostic kit of the present invention, the diagnosis can be performed simply, quickly and with a high throughput. Furthermore, according to the method for detecting or selecting a sample derived from an individual suffering from C0PD of the present invention, a sample derived from an individual suffering from C0PD can be detected or selected easily and quickly. Further, according to the detection or selection kit of the present invention, the detection or selection can be performed easily and quickly with a high throughput. The present invention will be described in more detail by way of examples, but the present invention is not limited to only these examples. In the following examples, unless otherwise specified, the method described in Molecular Cloning: A Laboratory Manual, Second Edition (1989) (Cold Spring Harbor Laboratory Press) was used as a genetic manipulation technique. Example 1 Expression of UGRP2 mRNA in C0PD model mouse 1
(1) C0PDモデルマウスの作製  (1) Preparation of C0PD model mouse
C0PDモデルマウスは、 Balb/c系マウス （日本チャールズリバ一育成供給、 6週 齢、雄） に対し、 C0PD の発症に関わる第一原因である煙草煙 (主流煙）の全身曝露 を行って作製した （喫煙群） 。 すなわち、 マウスを飼育する間に、 主流煙曝露装 置 （M I P S社製） を用いて 1日あたり 20 本のハイライト （商品名、 日本たば こ産業 （株） 製） の主流煙を 20 分間かけて全身曝露した。 なお、 煙草煙曝露は 土日休日を除き、 4 ヶ月間毎日行った。解剖前日まで曝露を続けた。 飼育条件は 、 飼育温度 24±1 、 湿度 55±5%、 照明期間 12時間 （8時から 20時） とし、 自由摂餌とした。 ， '  The C0PD model mouse was prepared by subjecting Balb / c mice (Charles River Japan, Japan, 6-week-old, male) to whole-body exposure to cigarette smoke (mainstream smoke), which is the primary cause of C0PD development. (Smoking group) That is, during the rearing of mice, the mainstream smoke of 20 highlights (trade name, manufactured by Japan Tobacco Inc.) was used for 20 minutes per day using a mainstream smoke exposure device (manufactured by MIPS). Over the whole body. Exposure to tobacco smoke was carried out every day for four months except weekends and holidays. Exposure continued until the day before dissection. The breeding conditions were breeding temperature 24 ± 1, humidity 55 ± 5%, lighting period 12 hours (8:00 to 20:00), and free feeding. , '
主流煙の全身曝露を行わないこと以外は同様にして飼育したマウスを対照群と した。 喫煙群及び対照群のマウスについて、 以下のようにして肺機能評価及び組 織学的評価を行った。 その結果、 喫煙群において呼吸機能が悪化し、 肺組織破壊 が増加していることが分かつた。 Mice bred in the same manner except that the whole body was not exposed to mainstream smoke were used as a control group. For the mice in the smoking group and the control group, lung function evaluation and group Weaving evaluation was performed. As a result, it was found that respiratory function deteriorated and lung tissue destruction increased in the smoking group.
(肺機能評価）  (Lung function evaluation)
煙草煙曝露による呼吸機能の悪化を、 主流煙の最終曝露の翌日、 全肺気量及び 準静肺コンプライアンスの測定を行い、 煙草煙曝露による、 それらの値の増加を 喫煙群と対照群との差として求め、 得られた値を指標として評価した。  Deterioration of respiratory function due to tobacco smoke exposure was measured on the day after final exposure to mainstream smoke, and total lung volume and quasi-static lung compliance were measured.The increase in those values due to tobacco smoke exposure was compared between the smoking group and the control group. The difference was determined, and the obtained value was evaluated as an index.
(組織学的評価）  (Histological evaluation)
煙草煙による肺組織破壊を、 主流煙の最終曝露の翌日、 肺組織切片を作成し、 Lung tissue destruction due to tobacco smoke was determined the day after the final exposure to mainstream smoke by making lung tissue sections,
HE染色後、 顕微鏡下で平均肺胞径を測定し、 煙草煙曝露による肺組織破壊の増 加を喫煙群と対照群との差を指標として評価した。 After HE staining, the average alveolar diameter was measured under a microscope, and the increase in lung tissue destruction due to tobacco smoke exposure was evaluated using the difference between the smoking group and the control group as an index.
(2) C0PDにおいて発現が変動する遺伝子の検索 (2) Search for genes whose expression fluctuates in C0PD
主流煙の最終曝露の翌日、 喫煙群及び対照群のマウスから肺を摘出し、 当該肺 から全 RNAを抽出し、 両群での mRNAの発現プロファイルを比較することで、 発 現が変動する遺伝子を検索した。  On the day after the final exposure to mainstream smoke, the lungs were removed from the mice in the smoking group and the control group, total RNA was extracted from the lungs, and the gene whose expression fluctuated by comparing the mRNA expression profiles in both groups was determined. Searched.
arrayTAG (LION社製） の添付のプロトコールに従い、 PCR法によりマウス cDN A断片 10000種を調製し、 MAチップ作製システム Microarray System Generati on III Spotter (商品名、 Molecular Dynamics 社製） を用いて、 Type7スライド グラス （商品名、 アマシャム バイオサイエンス社製） にスポットして DNAチップ を作製した。  According to the protocol attached to arrayTAG (manufactured by LION), 10,000 kinds of mouse cDNA fragments were prepared by PCR, and Type 7 slides were prepared using the MA chip preparation system Microarray System Generion on III Spotter (trade name, manufactured by Molecular Dynamics). A DNA chip was prepared by spotting on a glass (trade name, manufactured by Amersham Bioscience).
この DNAチップを用いて C0PDモデルマウスでの遺伝子発現変動解析を実施した Using this DNA chip, gene expression fluctuation analysis was performed in C0PD model mouse
。 喫煙群及び対照群のマウスから肺を摘出し、 得られた肺から、 IS0GEN (商品名. Lungs were excised from the mice of the smoking group and the control group, and IS0GEN (trade name) was obtained from the obtained lungs.
、 日本ジーン社製） を用い、 添付のマニュアルに従って全 RNAを調製した。 得ら れた RNAをキアゲン （QIAGEN) 社製の商品名： RNeasy mini kit にて添付マニュ アルに従い DNase I処理した。これで得た全 RNA2.5 gを用い、 MessageAmp aRNA, Manufactured by Nippon Gene Co., Ltd.) according to the attached manual. The obtained RNA was treated with DNase I using an RNeasy mini kit (trade name, manufactured by QIAGEN) according to the attached manual. Using 2.5 g of total RNA obtained in this way, MessageAmp aRNA
Kit (商品名、 Ambion社製） のマニュアルに従い増幅 RNA (aRNA) を合成した。 合成した aRNAをプローブの錶型とし、 Atlas Glass Fluorescent Labelling Kit 〔商品名、 CLONTECH (現 BD BIOSCIENCE) 社製〕 もしくは At las PowerScript Fl uorescent Labeling Kit (商品名、 現 BD BIOSCIENCE社製） を用いて、 マ二ユア ルに従い Random 9merプライマーにより Cy- 3 dCTP又は Cy- 5 dCTP (アマシャム 'バイオサイエンス社製） を取り込ませ cDNA を蛍光標識した。 これをプローブ とし、 Automated Slide Processor (商品名、 アマシャム バイオサイエンス社製） を用いて DNAチップとのハイブリダィゼ一シヨンを実施した。 ハイブリダィゼーシ ョンは、 Type 7スライドのプロトコールに従い、 前処理の後にプローブと 48°Cで 1 2 時間行った。 バッファーには Hybridizaton Buffer Ver.2 (商品名、 アマシャム バイオサインス社製） を用いた。 洗浄後、 Microarray System Generation III Sc anner (商品名、 Molecular Dynamics 社製） により読み取りを行い、 解析コンビ ユー夕—ソフト 〔商品名： Lucidea Automated Spotf inder (Molecular Dynamics 社製） 及び商品名： GeneSpring (シリコンジエネテックス社製） 〕 を用いてデー 夕解析を実施した。 ' Amplified RNA (aRNA) was synthesized according to the manual of Kit (trade name, manufactured by Ambion). Using the synthesized aRNA as probe type II, using the Atlas Glass Fluorescent Labeling Kit (trade name, CLONTECH (currently BD BIOSCIENCE)) or the Atlas PowerScript Fluorescent Labeling Kit (trade name, current BD BIOSCIENCE), According to the manual, Cy-3 dCTP or Cy-5 dCTP (manufactured by Amersham Bioscience) was incorporated with a Random 9mer primer, and the cDNA was fluorescently labeled. Using this as a probe, hybridization with a DNA chip was performed using an Automated Slide Processor (trade name, manufactured by Amersham Bioscience). Hybridization was performed at 48 ° C for 12 hours with the probe after pretreatment according to the protocol for Type 7 slides. Hybridizaton Buffer Ver.2 (trade name, manufactured by Amersham BioSigns) was used as a buffer. After washing, read with Microarray System Generation III Scanner (trade name, manufactured by Molecular Dynamics) and analyze with analysis software (trade name: Lucidea Automated Spotfinder (produced by Molecular Dynamics) and trade name: GeneSpring (silicon) The data were analyzed using the method of Genenetex Corporation)]. '
その結果、 対照群のマウス肺と比較して喫煙群 （C0PD 発症） のマウス肺におい て発現量の増加が認められた遺伝子として UGRP2が見出された。  As a result, UGRP2 was found as a gene whose expression level was increased in the lungs of mice in the smoking group (onset of C0PD) compared to the lungs of mice in the control group.
以上の結果より、 UGRP2の発現を指標として、 C0PDが診断できることが示唆さ れる。 実施例 2 C0PDモデルマウスにおける UGRP2 mRNAの発現解析 2  The above results suggest that C0PD can be diagnosed using UGRP2 expression as an index. Example 2 Expression analysis of UGRP2 mRNA in C0PD model mouse 2
RT-PCRにより、 C0PDモデルマウスの肺における UGRP2遺伝子の発現プロファ ィルを調べた。  The expression profile of the UGRP2 gene in the lungs of C0PD model mice was examined by RT-PCR.
RT-PCR には、 配列番号： 24と 2 5のプライマー対を用いた。 具体的には、 実施例 1の喫煙群及び対照群のマウスの肺より、 IS0GEN (商品名、 日本ジーン社 製） を用いて、 添付のマニュアルに従い、 全 RNAを抽出した。 この RNAをキアゲ ン （QIAGEN) 社製の商品名： RNeasy mini kitにて添付のマニュアルに従い DNas e I処理した後、 回収した。 得られた全 RNA2.5 より RT-PCR用 Superscript ( 登録商標） First- strand Synthesis System (商品名、 インビトロジェン社製） を用いて添付のマニュアルに従い、 cDNAを合成した。 次に、 マウス UGRP2(配列 番号： 1 7) の配列をもとに、 プローブ検索ソフト Primer Express (商品名、 AB I社製） によりリアルタイム定量 PCR用プライマー （配列番号 24及び 2 5) を 選択した。 プライマーは、 国際試薬社に合成を依頼した。 このプライマーを用い て、 合成した cDNAを錡型として SYBR Green I 〔キアゲン （QIAGEN) 社製〕 によ るリアルタイム定量 PCRを行い、 mRNA量を定量した。 For RT-PCR, primer pairs of SEQ ID NOS: 24 and 25 were used. Specifically, total RNA was extracted from the lungs of the mice of the smoking group and the control group of Example 1 using IS0GEN (trade name, manufactured by Nippon Gene) according to the attached manual. This RNA was obtained from QIAGEN under the trade name of RNeasy mini kit according to the attached manual. After eI treatment, it was recovered. From the obtained total RNA2.5, cDNA was synthesized using Superscript (registered trademark) First-strand Synthesis System for RT-PCR (trade name, manufactured by Invitrogen) according to the attached manual. Next, based on the sequence of mouse UGRP2 (SEQ ID NO: 17), primers for real-time quantitative PCR (SEQ ID NOs: 24 and 25) were selected using probe search software Primer Express (trade name, manufactured by ABI). . Primers were requested to be synthesized by Kokusai Reagent. Using these primers, real-time quantitative PCR was performed using SYBR Green I (manufactured by QIAGEN) using the synthesized cDNA as type I, and the amount of mRNA was quantified.
反応試薬としては、 Applied Biosysteis 社の商品名： SYBR Green PCR Master Mixを使用し、 95°Cで 10分間反応させた後、 95°Cで 10秒、 60°Cで 60秒の 40 サイクルを行って定量した。 PCR、 及び蛍光測定は、 ABI社の ABI PRISM 5700 Se quence Detection System (商品名） を用いて行った。 結果を図 1に示す。 図 1 のグラフは、 対照群のマウスにおける発現量を 1とした相対量で示している。 な お、 リアルタイム定量 PCRでは、 増幅産物の電気泳動と融解曲線を作製すること により目的の UGRP2 mRNAの一種類のみが増幅されていることを確認した。  As a reaction reagent, Applied Biosysteis (trade name: SYBR Green PCR Master Mix) is used. After reacting at 95 ° C for 10 minutes, 40 cycles of 95 ° C for 10 seconds and 60 ° C for 60 seconds are performed. And quantified. PCR and fluorescence measurements were performed using ABI's ABI PRISM 5700 Sequence Detection System (trade name). The results are shown in Figure 1. The graph in FIG. 1 shows the relative amount with respect to the expression level in the control group of mice. In real-time quantitative PCR, it was confirmed that only one kind of the target UGRP2 mRNA was amplified by electrophoresis of the amplification product and by preparing a melting curve.
図 1に示すように、 C0PD モデルマウス （喫煙群） の肺において、 対照群と比 ベて、 UGRP2 の発現が増強されることが確認された。 当該結果より、 対照群に比 ベ、 C0PD モデルマウスの肺において UGRP2 遺伝子の発現が約 4倍に増強される ことが分かる。 参考例 1 喘息モデルマウスにおける UGRP2 mRNAの発現解析  As shown in FIG. 1, it was confirmed that UGRP2 expression was enhanced in the lungs of C0PD model mice (smoking group) compared to the control group. The results show that the expression of the UGRP2 gene in the lungs of C0PD model mice was enhanced about 4-fold as compared to the control group. Reference Example 1 UGRP2 mRNA expression analysis in asthma model mouse
RT-PCR により、 喘息モデルマウスの肺における UGRP2 遺伝子の発現プロファ ィルを調べた。  The expression profile of the UGRP2 gene in the lungs of asthma model mice was examined by RT-PCR.
喘息群及び対照群のマウスを以下のようにして得た。 喘息モデルマウスを、 Ba lb/c 系マウス （日本チャールズリパー育成供給、 9週齢、 雌） に対し、 卵白ァ ルブミン （OVA) 水酸化アルミニウムゲル （Alum) 懸濁液.を 1 回腹腔内注射し て全身免疫し （0 日目及び 14 日目） 、 さらにネブライザ一 （オムロン社製） に て霧化した 1%0VA溶液を 30分間 3回噴霧して （25日目、 30日目及び 36日目） 作製した （喘息群） 。 対照群は生理食塩水を腹腔内注射し同様に噴霧したものと した。 Mice in the asthma group and the control group were obtained as follows. A single intraperitoneal injection of egg white albumin (OVA) aluminum hydroxide gel (Alum) suspension into an asthma model mouse against a Ba lb / c mouse (Japanese Charles Lipper rearing supply, 9 weeks old, female) And The whole body was immunized (day 0 and day 14) and further sprayed with a 1% 0VA solution nebulized with a nebulizer (Omron) three times for 30 minutes (day 25, day 30 and day 36). Eye) prepared (asthma group). The control group had saline injected intraperitoneally and sprayed similarly.
最終チャレンジの翌日、 Whole Body Unrestrained PlethysmograpH (BAXCO 社 製） を'用いてメサコリン誘発気道過敏性を両群のマウスで測定し、 Penh 値を指 標に喘息症状を喘息群のマウスで確認した。 なお、 メサコリン溶液 （6、 12 及び 24mg/ml) はネブライザ一にて霧化して 2分間噴霧した。 Penh値は噴霧 1分後か ら測定を開始し、 4分間の測定値を平均して得た。  The day after the final challenge, methacholine-induced airway hyperreactivity was measured in mice of both groups using Whole Body Unrestrained PlethysmograpH (manufactured by BAXCO), and asthma symptoms were confirmed in mice of the asthma group using Penh values as indicators. The mesacholine solution (6, 12, and 24 mg / ml) was atomized with a nebulizer and sprayed for 2 minutes. The Penh value was measured one minute after spraying and averaged over four minutes.
RT-PCR には、 配列番号： 24と 2 5とのプライマー対を用いた。 具体的には 、 喘息群及び対照群のマウスの肺より、 IS0GEN (商品名、 日本ジーン社製） を用 いて、 添付のマニュアルに従い、 全 RNAを抽出した。 この RNAをキアゲン （QIAG EN) 社製の商品名： RNeasy mini kit にて添付のマニュアルに従い DNase I処理 した後、 回収した。 得られた全 RNA2.5 gより RT-PCR用 Superscript (登録商 標） First- strand Synthesis System (商品名、 インビトロジェン社製） を用い て添付のマニュアルに従い、 cDNA を合成した。 次に、 マウス UGRP2 (配列番号 For RT-PCR, primer pairs of SEQ ID NOS: 24 and 25 were used. Specifically, total RNA was extracted from lungs of mice in the asthma group and the control group using IS0GEN (trade name, manufactured by Nippon Gene Co., Ltd.) according to the attached manual. The RNA was treated with DNase I using RNeasy mini kit (trade name, manufactured by QIAG EN) according to the attached manual, and collected. CDNA was synthesized from 2.5 g of the obtained total RNA using a Superscript for RT-PCR (registered trademark) First-strand Synthesis System (trade name, manufactured by Invitrogen) according to the attached manual. Next, mouse UGRP2 (SEQ ID NO:
： 1 7) の配列をもとに、 プローブ検索ソフト Primer Express (商品名、 ABI社 製） によりリアルタイム定量 PCR用プライマー （配列番号： 24及び 2 5) を選 択した。 プライマーは、 日清紡社に合成を依頼した。 このプライマーを用いて、 合成した cDNAを铸型として SYBR Green I によるリアルタイム定量 PCRを行い 、 mRNA量を定量した。 : Based on the sequence of 17), primers for real-time quantitative PCR (SEQ ID NOS: 24 and 25) were selected using probe search software Primer Express (trade name, manufactured by ABI). Primers were requested to be synthesized by Nisshinbo. Using this primer, real-time quantitative PCR using SYBR Green I was performed using the synthesized cDNA as type II to quantify the amount of mRNA.
反応試薬としては、 Applied Biosystems 社の商品名： SYBR Green PCR Master As a reaction reagent, a product name of Applied Biosystems: SYBR Green PCR Master
Mixを使用し、 95"Cで 10分間反応させた後、 95°Cで 10秒、 で 60秒の 40 サイクルを行って定量した。 PCR、 及び蛍光測定は、 ABI社の ABI PRISM 5700 Se quence Detection System (商品名） を用いて行った。 結果を図 2に示す。 図 2 のグラフは、 対照群のマウスにおける発現量を 1とした相対量で示している。 な お、 リアルタイム定量 PCRでは、 増幅産物の電気泳動と融解曲線を作製すること により目的の UGRP2 mRNAの一種類のみが増幅されていることを確認した。 The mixture was reacted at 95 "C for 10 minutes, and quantified by performing 40 cycles of 95 ° C for 10 seconds and at 60 seconds. PCR and fluorescence measurement were performed using ABI's ABI PRISM 5700 Sequence. The results were shown in Fig. 2. The results are shown in Fig. 2. The graph in Fig. 2 shows the relative amount with respect to the expression level in the control group of mice. In real-time quantitative PCR, it was confirmed that only one kind of the target UGRP2 mRNA was amplified by electrophoresis of the amplification product and by preparing a melting curve.
図 2に示すように、 喘息モデルマウス （喘息群） の肺においては、 対照群と比 ベて、 UGRP2の有意な発現変動は検出されなかった。 実施例 3 ヒト正常組織における UGRP2 mRNAの発現解析  As shown in FIG. 2, no significant change in the expression of UGRP2 was detected in the lungs of asthma model mice (asthma group) as compared to the control group. Example 3 Expression analysis of UGRP2 mRNA in normal human tissues
NCBI のデータベースより得られた UGRP2 遺伝子配列 （配列番号： 1 3 ) に基 づき、 プライマー対 5' -GCGCCATGAAGCTGGCCG-3'及び 5' -ATGCTCCAGTCTCGGCTCAG-3 ' (配列番号： 1 9及び 2 0 ) を合成し、 ヒト肺由来全 RNA (クロンテック社製 ) より逆転写酵素 XL (夕カラ社製） を用いて合成した 1本鎖 cDNAを铸型 として、 PCR反応を行った。  Based on the UGRP2 gene sequence (SEQ ID NO: 13) obtained from the NCBI database, primer pairs 5'-GCGCCATGAAGCTGGCCG-3 'and 5'-ATGCTCCAGTCTCGGCTCAG-3' (SEQ ID NOs: 19 and 20) were synthesized. Then, a single-stranded cDNA synthesized from human lung-derived total RNA (Clontech) using reverse transcriptase XL (Yukara) was used as a type I PCR reaction.
この反応により増幅された cDNA断片混合物をさらに铸型として、 プライマー 対 5' - GCGCCATGAAGCTGGCCG- 3'及び 5' - TCAGCCAAACACTGTCAGGG- 3' (配列番号： 1 The mixture of cDNA fragments amplified by this reaction is further designated as 铸, and primer pairs 5′-GCGCCATGAAGCTGGCCG-3 ′ and 5′-TCAGCCAAACACTGTCAGGG-3 ′ (SEQ ID NO: 1)
9及び 2 2 ) を用いた PCR反応により、 ヒト UGRP 2遺伝子の全翻訳領域に相当す る cDNA断片を合成した。 該反応における反応液の組成は、 上記 1本鎖 cDNAある いは PCR反応液の 50分の 1量を铸型として使用し、 Ex Taq (夕カラ社製） 1/200 量、 プライマー対を 1 M、 dNTPs を 200 ^ Μ、 及び酵素に添付されたバッファー を加え、 50 1の液量とした。 PCR反応は、 94°Cで 3分の後、 94°Cで 30秒、 50°C で 30秒、 72 で 1分のサイクルを 25回繰り返し、 最後に 72 で 1分の伸長反 応を行った。 PCR反応の結果、 得られた 320bpの cDNA断片をァガロース電気泳 動後、 回収し、 pCR2.卜 T0P0 ベクター （インビトロジェン社製） にクローン化後By the PCR reaction using 9 and 22), a cDNA fragment corresponding to the entire translation region of the human UGRP2 gene was synthesized. The composition of the reaction solution in the reaction was as follows: 1/50 of the above single-stranded cDNA or PCR reaction solution was used as type III, 1/200 amount of Ex Taq (Yukara), and 1 primer pair. M and dNTPs were added in a volume of 200, and the buffer attached to the enzyme was added to make a liquid volume of 501. The PCR reaction is repeated at 94 ° C for 3 minutes, followed by a cycle of 94 ° C for 30 seconds, 50 ° C for 30 seconds, 72 at 1 minute 25 times, and finally an extension reaction at 72 at 1 minute Was. The 320 bp cDNA fragment obtained as a result of the PCR reaction was collected after agarose electrophoresis, and cloned into pCR2.T0P0 vector (manufactured by Invitrogen).
、 M13フォワード 'プライマー及び M13 リバース ·プライマー （インビトロジェ ン社製） と BigDye Terminator vl . 0 Cyc l e Sequenc ing Ready Reac t i on Ki t ( アプライド ·バイオシステムズ社製） によりシーケンシング反応後、 ABI PRI SMABI PRI SM
310 (アプライド ·バイオシステムズ社製） にて塩基配列を決定することにより310 (manufactured by Applied Biosystems)
、 回収した DNA断片が目的物であることを確認した。 この DNA断片を [ひ- 32P] dC TP (3000Ci/mmoK lOmCi/ml) (アマシャム 'バイオサイエンス社製）を用いて Re diprime II DNA Labeling system (アマシャム ·バイオサイエンス社製）を使用 して、 その処方に従い 32P標識した。 この 32 P標識プローブを用いて、 Human 12- Lane MTN Blot (クロンテック社製； レーン 1 ：脳、 レーン 2 ：心臓、 レーン 3 ：骨格筋、 レーン 4 :結腸、 レーン 5 :胸腺、 レーン 6 :脾臓、 レーン 7 :腎臓 、 レ ン 8 ：肝臓、 レーン 9 ：小腸、 レーン 1 0 ：胎盤、 レーン 1 1 ：肺、 レー ン、 1 2 ：末梢血リンパ球） 及び Human 12- Lane MTN Blot-ΙΠ (クロンテック社 製； レーン 1 :副腎、 レーン 2 :膀胱、 レーン 3 :骨髄、 レーン 4 :脳 （全体） 、 レーン 5 : リンパ節、 レーン 6 :前立腺、 レーン 7 :脊髄、 レーン 8 :胃、 レ ーン 9 ：甲状腺、 レーン 1 0 ：舌、 レーン 1 1 ：気管、 レーン 12 ：子宮）に対 してノーザンプロット解析を行った。 これらのナイロン膜を、 0.5Mリン酸バッ ファー（pH7.2)、 1 %ゥシ血清アルブミン、 ImM EDTA、 7 %SDS からなるハイブ リダィゼーシヨン ·バッファー （GMCバッファー） に浸し、 65°Cで 1時間、 予備 ハイブリダィゼーシヨンを行った後、 32P標識プローブを GMCバッファーに加え た液にナイロン膜を移し、 65°Cで 16 時間ハイブリダィゼーシヨンを行った。 引 続きこのナイロン膜を 2XSSC (1XSSCは 15mMクェン酸ナトリウムと 150mM塩化 ナトリゥムの混合水溶液） で室温にて 5分間濯いだ後、 1 XSSCで 50°Cにて 30分 間、 0.5XSSCで 50 にて 30分間洗浄した。 風乾後、 このナイロン膜をフジ写真 フィルム社のイメージング ·プレートに密着させ、 同社イメージ ·アナライザー BAS2000 で画像解析を行った。その結果、 肺、 気管、 舌などの組織において強い 発現が観察された。 実施例 4 抗 UGRP2モノクローナル抗体の作製 It was confirmed that the recovered DNA fragment was the desired product. The DNA fragment [shed - 32 P] dC Use TP (3000Ci / mmoK lOmCi / ml ) Re diprime II DNA Labeling system ( manufactured by Amersham Biosciences) using (Amersham 'Biosciences), was 32 P-labeled according to its formulation. Using this 32 P-labeled probe, a Human 12-Lane MTN Blot (Clontech; lane 1: brain, lane 2: heart, lane 3: skeletal muscle, lane 4: colon, lane 5: thymus, lane 6: spleen , Lane 7: Kidney, Lane 8: Liver, Lane 9: Small intestine, Lane 10: Placenta, Lane 11: Lung, Lane, 12: Peripheral blood lymphocyte) and Human 12- Lane MTN Blot-II ( Manufactured by Clontech; Lane 1: adrenal gland, lane 2: bladder, lane 3: bone marrow, lane 4: brain (whole), lane 5: lymph node, lane 6: prostate, lane 7: spinal cord, lane 8: stomach, lane Northern plot analysis was performed on (9: thyroid, lane 10: tongue, lane 11: trachea, lane 12: uterus). These nylon membranes are immersed in a hybridization buffer (GMC buffer) consisting of 0.5 M phosphate buffer (pH 7.2), 1% serum albumin, ImM EDTA, and 7% SDS, and incubated at 65 ° C for 1 hour. After the preliminary hybridization, the nylon membrane was transferred to a solution in which a 32 P-labeled probe was added to a GMC buffer, and hybridization was performed at 65 ° C. for 16 hours. Subsequently, the nylon membrane was rinsed with 2XSSC (1XSSC is a mixed aqueous solution of 15mM sodium citrate and 150mM sodium chloride) at room temperature for 5 minutes, then 1XSSC at 50 ° C for 30 minutes and 0.5XSSC at 50. For 30 minutes. After air-drying, the nylon membrane was brought into close contact with the Fuji Photo Film Imaging Plate, and image analysis was performed using the company's Image Analyzer BAS2000. As a result, strong expression was observed in tissues such as lung, trachea, and tongue. Example 4 Preparation of anti-UGRP2 monoclonal antibody
UGRP に対するモノクローナル抗体を産生するハイプリドーマを細胞融合技術 により作製した。  Hybridomas producing monoclonal antibodies against UGRP were produced by cell fusion technology.
免疫原として 2種類の UGRP2部分べプチド Cys_UGRP2 (46-58、 CTLANPLGTLNPLK 、 配列番号： 2 6 )と Cys-UGRP2 (63-77、 SLGIPVNHLIEGSQKC, 配列番号： 2 7 )を マレイミド化 Keyho l e Li即 e t Hemocyanin (KLH、 ピアス社製）と結合させたコン ジュゲートを用いた。 A/J Jms S i c マウスの皮下と腹腔に初回はフロイント完全 アジュパンド （FCA) 、 2回目以降はフロイント不完全アジュパンドと混和した 乳化液を皮下及び腹腔に免疫した。 2回目の免疫以降は部分採血を行い、 抗体の 産生を DELFIA法により行った。 即ち、 ャギ抗マウス IgG Fc特異抗体（ICN社製） を固相したプレート上にて、 ピオチン標識した上記ペプチド、 Eu 標識ストレブ トァビジン （パーキンエルマ一社製） 及び抗体溶液を室温 2時間あるいは 4tに て終夜反応後、 洗浄を行い、 増強試薬を添加し、 時間分解蛍光を Wal l ac ARV0 ( パ一キンエルマ一社製） ,にて測定することにより評価を行つた。 Two types of UGRP2 partial peptides Cys_UGRP2 (46-58, CTLANPLGTLNPLK A conjugate in which SEQ ID NO: 26) and Cys-UGRP2 (63-77, SLGIPVNHLIEGSQKC, SEQ ID NO: 27) were bound to maleimidated Keyhole le Li immediately et Hemocyanin (KLH, Pierce) was used. A / J Jms Sic mice were subcutaneously and intraperitoneally immunized subcutaneously and intraperitoneally with an emulsion mixed with Freund's complete adjuvant (FCA) for the first time and incomplete Freund's adjuvant for the second and subsequent times. Partial blood was collected after the second immunization, and antibody production was performed by the DELFIA method. That is, on a plate on which a goat anti-mouse IgG Fc-specific antibody (ICN) was immobilized, the above peptide labeled with biotin, Streptavidin Eu-labeled (Perkin Elmer) and an antibody solution were incubated at room temperature for 2 hours or 4 t. After the reaction overnight, washing was performed, an enhancing reagent was added, and evaluation was performed by measuring time-resolved fluorescence with Walac ARV0 (manufactured by Pakinkin Elma).
4回目の免疫を実施してから 2週間経過後にブースター （追加免疫） を行い、 さらに 3日後に無菌下で摘出した脾臓より脾細胞を調製し、 50 %ポリエチレング リコールによりミエローマ細胞との細胞融合を行った。 HAT 培地にて 96 穴プレ 一卜に融合細胞を撒き、 7〜9 日間培養した。 コロニーが形成された事を確認し た後、 上清を採取し、 上記の DELFIA.法にて抗体産生能を評価した。 抗体産生ハ イブリドーマは限界希釈法によりクロ一ニングを行い、 UG2- 6A9 (受領番号 FERM Two weeks after the fourth immunization, a booster (boost) was performed. Three days later, spleen cells were prepared from the spleen removed under sterile conditions, and cell fusion with myeloma cells was performed using 50% polyethylene glycol. Was done. The fused cells were seeded on a 96-well plate in HAT medium and cultured for 7 to 9 days. After confirming that a colony was formed, the supernatant was collected and the antibody-producing ability was evaluated by the DELFIA method described above. The antibody-producing hybridomas were cloned by the limiting dilution method and UG2-6A9 (accession number FERM
ABP-10012) と UG3-1B12 (受領番号 FERM ABP-10013) のモノクローナル抗体産 生八イブリドーマを得た。 実施例 5 UGRP2測定法 ABP-10012) and UG3-1B12 (receipt number FERM ABP-10013) were produced. Example 5 UGRP2 measurement method
UGRP2 測定法は西洋ヮサビペルォキシダーゼ （HRP) 標識抗体を用いた酵素免 疫測定法 （EL ISA法） を利用する。 抗体は、 八イブリドーマ細胞を無血清培地 （ インビトロジェン社） にて培養し、 その培養上清から MAPS I I Pro t e in A ki t ( The UGRP2 assay uses an enzyme-linked immunosorbent assay (ELISA) using a horseradish peroxidase (HRP) -labeled antibody. Antibodies were obtained by culturing eight hybridoma cells in a serum-free medium (Invitrogen) and using the culture supernatant as MAPS II Protein A kit (
BIO- RAD社）を用いァフィ二ティー精製する。 標準品の UGRP2 は、 コムギ胚芽発 システム ¾：禾 ij用した Wheat germ eel 1-f ree pro te in synthes i s core ki t (東 洋紡社製） を用い発現させたリコンビナント UGRP2タンパクを用いる。 発現の方 法は添付のマニュアルに従い、 UGR 遺伝子の 3'側に FLAGタグ遺伝子を付加し た遺伝子を利用し発現させる。 発現タンパクの精製には抗 FLAG抗体ゲル （SIGMA 社製） を用いたァフィ二ティー精製にて行う。 (BIO-RAD). As a standard UGRP2, a recombinant UGRP2 protein expressed by using a wheat germ development system II: Wheat germel 1-free protein in synthes is core kit (manufactured by Toyobo) for wheat ij is used. Those who express According to the attached manual, expression is performed using a gene with a FLAG tag gene added to the 3 'side of the UGR gene. The expressed protein is purified by affinity purification using an anti-FLAG antibody gel (manufactured by SIGMA).
2 種類の抗体のうち、 一方のェピトープを認識する抗体は 50mM トリス塩酸緩 衝生理食塩水 （TBS) にて適当な濃度に希釈し、 96穴プレート （Nunc社製） に撒 き 4°Cで終夜静置する事により物理的に吸着させる。 洗浄後、 ゥシ血清アルブミ ンを含む溶液を添加することによりブロッキングを行い、 抗体固相プレートを作 製する。 もう一方のェピトープを認識する抗体は、 ヒンジ法を用いて HRPで標識 する。 抗体をペプシンにて消化し F (ab' ) 2を調製後、 終濃度 10mMの 2 メルカプ トェチルァミンで 90分間還元して Fal]'を調製する。 HRPは 50倍モルの Sul fo-H MCS (同仁化学研究所社製） と反応させマレイミド化 HRPを作製する。 上記 Fab' とマレイミド化 HRPを等モル反応させ、 HRP標識 Fab'を調製する （HRP標識抗体 ) 。 標識抗体の精製には TSKgel G2000SWXLカラムを用いた HPLCにて行う。 Of the two antibodies, the antibody that recognizes one of the epitopes is diluted to an appropriate concentration with 50 mM Tris-HCl buffered saline (TBS) and spread on a 96-well plate (Nunc) at 4 ° C. Physically adsorb by standing overnight. After washing, blocking is performed by adding a solution containing serum albumin, and an antibody solid phase plate is prepared. Antibodies that recognize the other epitope are labeled with HRP using the hinge method. The antibody is digested with pepsin to prepare F (ab ') 2, and then reduced with 2 mercaptoethylamine at a final concentration of 10 mM for 90 minutes to prepare Fal]'. HRP is reacted with 50-fold molar amount of Sulfo-H MCS (manufactured by Dojindo Laboratories) to produce maleimidated HRP. The above Fab ′ and maleimidated HRP are reacted in equimolar amounts to prepare an HRP-labeled Fab ′ (HRP-labeled antibody). The labeled antibody is purified by HPLC using a TSKgel G2000SWXL column.
ELISAは、 初めに標準 UGRP2あるいは UGRP2を含む検体を抗体固相プレートに 添加し、 反応させる。 反応後、 プレートを洗浄し、 HRP 標識抗体を添加する。 反 応後、 プレートを洗浄し Col orburs t Blue溶液 （テトラメチルベンチジン溶液、 ALerCHEK社製） を添加する。 呈色後、 等量の 0. 18N硫酸溶液を添加し酵素反応 を停止させる。 450nmにおける吸光度を Wal l ac ARV0 SX (パーキンエルマ一社製 ) にて測定し、 標準 UGRP2の吸光度から標準曲線を作成し、 検体の濃度を求める  In ELISA, first, standard UGRP2 or a sample containing UGRP2 is added to an antibody solid-phase plate and reacted. After the reaction, wash the plate and add HRP-labeled antibody. After the reaction, wash the plate and add Colorburst Blue solution (tetramethylbenzidine solution, ALerCHEK). After color development, add an equal volume of 0.18N sulfuric acid solution to stop the enzyme reaction. Measure absorbance at 450 nm with Walac ARV0 SX (PerkinElmer) and create a standard curve from the absorbance of standard UGRP2 to determine the sample concentration
配列表フリーテキスト Sequence listing free text
配列番号： 1 9は、 ヒト SCGB3A1 (UGRP2) RT-PCR フォワードプライマーの配 列を示す。  SEQ ID NO: 19 shows a sequence of a human SCGB3A1 (UGRP2) RT-PCR forward primer.
配列番号： 2 0は、 ヒト SCGB3A1 (UGRP2) RT-PCR リパースプライマ一の配列 を示す。 配列番号： 2 1は、 ヒト SCGB3A1 (UGRP2) RT-PCR フォワードプライマーの配 列を示す。 SEQ ID NO: 20 shows the sequence of human SCGB3A1 (UGRP2) RT-PCR lipase primer. SEQ ID NO: 21 shows a sequence of a human SCGB3A1 (UGRP2) RT-PCR forward primer.
配列番号： 2 2は、 ヒト SCGB3A1 (UGRP2) RT-PCR リバースプライマーの配列 を示す。  SEQ ID NO: 22 shows the sequence of a human SCGB3A1 (UGRP2) RT-PCR reverse primer.
配列番号： 2 3は、 ヒト SCGB3A1 (UGRP2)ノーザン解析用プローブの配列を示 す。 一  SEQ ID NO: 23 shows the sequence of a human SCGB3A1 (UGRP2) Northern analysis probe. One
配列番号： 2 4は、 マウス SCGB3A1 (UGRP2) RT-PCR フォワードプライマーの 配列を示す。  SEQ ID NO: 24 shows the sequence of mouse SCGB3A1 (UGRP2) RT-PCR forward primer.
配列番号： 2 5は、 マウス SCGB3A1 (UGRP2) RT-PCR リバースプライマーの配 列を示す。  SEQ ID NO: 25 shows a sequence of a mouse SCGB3A1 (UGRP2) RT-PCR reverse primer.

Claims

請求の範囲 The scope of the claims
1 . 被検対象の個体由来の被検試料及び正常個体由来の対照試料それぞれにおけ る Secretoglob inファミリーに属するタンパク質の遺伝子の発現レベルを測定す るステップを含み、 ここで、 被検試料における発現レベルが、 対照試料における 発現レベルよりも高くなる場合、 該被検試料が、 慢性閉塞性肺疾患に罹患してい る疑いがある個体由来の試料であることの指標となる、 慢性閉塞性肺疾患に罹患 している疑いがある個体由来の試料の検出又は選別方法。 1. Includes a step of measuring the expression level of a gene of a protein belonging to the Secretoglobin family in each of a test sample derived from an individual to be tested and a control sample derived from a normal individual, wherein the expression in the test sample is included. Chronic obstructive pulmonary disease, when the level is higher than the expression level in the control sample, indicates that the test sample is a sample derived from an individual suspected of having chronic obstructive pulmonary disease. A method for detecting or selecting a sample derived from an individual suspected of having a disease.
2 . 発現レベルが、 Secretoglobin ファミリーに属するタンパク質の遺伝子の mRNA転写物の量を指標として測定される、 請求項 1記載の方法。 2. The method according to claim 1, wherein the expression level is measured using an amount of an mRNA transcript of a gene of a protein belonging to the Secretoglobin family as an index.
3 . 発現レベルが、 Secretoglobin ファミリ一に属するタンパク質の遺伝子の遺 伝子産物の量を指標として測定される、 請求項 1記載の方法。 3. The method according to claim 1, wherein the expression level is measured using an amount of a gene product of a gene of a protein belonging to the Secretoglobin family as an index.
4 . 慢性閉塞性肺疾患が肺気腫である、 請求項 1〜 3いずれか記載の方法。 4. The method according to any one of claims 1 to 3, wherein the chronic obstructive pulmonary disease is emphysema.
5 . Secretoglobin ファミリーに属するタンパク質をコードする核酸を検出しう る核酸を用いて測定される、 請求項 2記載の方法。 5. The method according to claim 2, which is measured using a nucleic acid capable of detecting a nucleic acid encoding a protein belonging to the Secretoglobin family.
6 . Secretoglobin ファミリーに属するタンパク質をコードする核酸が、 配列番 号： 1、 3、 5、 7、 9、 1 1、 1 3及び 1 5からなる群より選ばれる塩基配列 からなる核酸である、 請求項 5記載の方法。 6. The nucleic acid encoding a protein belonging to the Secretoglobin family is a nucleic acid comprising a base sequence selected from the group consisting of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 11, 13 and 15. Item 5. The method according to item 5.
7 . 配列番号： 1 9、 2 0、 2 1、 2 2及び 2 3からなる群より選ばれる少なく とも 1つの核酸を用いて測定される、 請求項 6記載の方法。 7. The method according to claim 6, wherein the measurement is carried out using at least one nucleic acid selected from the group consisting of SEQ ID NO: 19, 20, 21, 22, and 23.
8. Secretoglobin ファミリーに属するタンパク質に対する抗体又はその断片を 用いて測定される、 請求項 3記載の方法。 8. The method according to claim 3, which is measured using an antibody against a protein belonging to the Secretoglobin family or a fragment thereof.
9. 前記抗体又はその断片が、 配列番号 26又は 27のアミノ酸配列を有するぺ プチド又は該ぺプチドを含むポリべプチドに対するモノクローナル抗体又はその 断片である、 請求項 8記載の方法。 9. The method according to claim 8, wherein the antibody or a fragment thereof is a monoclonal antibody against a peptide having the amino acid sequence of SEQ ID NO: 26 or 27 or a polypeptide containing the peptide or a fragment thereof.
10. 前記抗体が受領番号 FERM ABP- 10012又は FERM ABP- 10013であるハイブリ ドーマにより産生されるものである、 請求項 9記載の方法。 10. The method of claim 9, wherein the antibody is produced by a hybridoma having accession number FERM ABP-10012 or FERM ABP-10013.
11. Secretoglobin ファミリーに属するタンパク質をコードする核酸を検出し うる核酸を含有してなる、 請求項 5記載の方法に使用するための検出又は選別用 キッ卜。 11. A detection or selection kit for use in the method according to claim 5, comprising a nucleic acid capable of detecting a nucleic acid encoding a protein belonging to the Secretoglobin family.
12. Secretoglobin ファミリーに属するタンパク質をコードする核酸が、 配列 番号： 1、 3、 5、 6、 9、 1 1、 13及び 1 5からなる群より選ばれる配列か らなる核酸である、 請求項 11記載の検出又は選別用キット。 12. The nucleic acid encoding a protein belonging to the Secretoglobin family is a nucleic acid consisting of a sequence selected from the group consisting of SEQ ID NOs: 1, 3, 5, 6, 9, 11, 13, and 15. The detection or selection kit according to the above.
13. 配列番号： 19、 20、 21、 22及び 23からなる群より選ばれる少な くとも 1つの核酸を含有してなる、 請求項 12記載の検出又は選別用キット。 13. The detection or selection kit according to claim 12, comprising at least one nucleic acid selected from the group consisting of SEQ ID NOs: 19, 20, 21, 22, and 23.
14. Secretoglobin ファミリーに属するタンパク質に対する抗体又はその断片 を含有してなる、 請求項 8記載の方法に使用するための検出又は選別用キット。 14. A detection or selection kit for use in the method according to claim 8, comprising an antibody against a protein belonging to the Secretoglobin family or a fragment thereof.
15. 前記抗体又はその断片が、 配列番号 26又は 27のアミノ酸配列を有する ぺプチド又は該ぺプチドを含むポリぺプチドに対するモノクローナル抗体又はそ の断片である、 請求項 1 4記載の検出又は選別用キット。 15. The antibody or fragment thereof has the amino acid sequence of SEQ ID NO: 26 or 27. 15. The detection or selection kit according to claim 14, which is a monoclonal antibody against a peptide or a polypeptide containing the peptide, or a fragment thereof.
1 6 . 前記抗体が受領番号 FERM ABP- 1001 2又は FERM ABP- 10013であるハイブリ 'ドーマにより産生されるものである、 請求項 1 5記載の検出又は選別用キット。 16. The kit for detection or selection according to claim 15, wherein the antibody is produced by a hybridoma having accession number FERM ABP-10012 or FERM ABP-10013.
1 7 . 配列番号 2 6又は 2 7のアミノ酸配列を有するペプチド又は該ペプチドを 含むポリぺプチドに対するモノクローナル抗体。 17. A monoclonal antibody against a peptide having the amino acid sequence of SEQ ID NO: 26 or 27 or a polypeptide containing the peptide.
1 8 . 受領番号 FERM ABP- 10012又は FERM ABP-10013であるハイブリドーマによ り産生される抗体。 18. Antibodies produced by hybridomas having accession numbers FERM ABP-10012 or FERM ABP-10013.
1 9 . 受領番号 FERM ABP- 10012又は FERM ABP- 10013であるハイプリドーマ。 1 9. A hybridoma with accession number FERM ABP-10012 or FERM ABP-10013.
2 0 . 被検物質の存在下、 Secretoglob in ファミリーに属するタンパク質若しく は該タンパク質をコードする遺伝子の動態を検出又は測定することを特徴とする 、 慢性閉塞性肺疾患治療剤又は予防剤のスクリーニング方法。 20. Screening of a therapeutic or preventive agent for chronic obstructive pulmonary disease, characterized by detecting or measuring the dynamics of a protein belonging to the Secretoglob in family or a gene encoding the protein in the presence of a test substance. Method.
2 1 . 被検対象の個体由来の被検試料及び正常個体由来の対照試料それぞれにお ける Secre toglobinファミリ一に属するタンパク質の遺伝子の発現レベルを測定 するステップを含み、 ここで、 被検試料における発現レベルが、 対照試料におけ る発現レベルよりも高くなる場合、 該被検対象の個体が、 慢性閉塞性肺疾患に罹 •患している疑いがあることの指標となる、 慢性閉塞性肺疾患の診断方法。 21. measuring the expression level of the gene of a protein belonging to the Secre toglobin family in each of a test sample derived from an individual to be tested and a control sample derived from a normal individual, wherein: Chronic obstructive pulmonary disease, when the expression level is higher than that in the control sample, indicates that the subject is suspected of having chronic obstructive pulmonary disease. How to diagnose the disease.
2 2 . Secretoglob in ファミリ一に属するタンパク質をコードする核酸を検出し うる核酸を含有してなる、 請求項 2 1記載の方法に使用す ¾ための診断用キット 22. A diagnostic kit for use in the method of claim 21, comprising a nucleic acid capable of detecting a nucleic acid encoding a protein belonging to the Secretoglob in family 1
2 3 . Secretogl obin ファミリ一に属するタンパク質をコードする核酸が、 配列 番号： 1、 3、 5、 6、 9、 1 1、 1 3及び 1 5からなる群より選ばれる配列か らなる核酸である、 請求項 2 2記載の診断用キット。 23. The nucleic acid encoding a protein belonging to the Secretogl obin family 1 is a nucleic acid consisting of a sequence selected from the group consisting of SEQ ID NOs: 1, 3, 5, 6, 9, 11, 13, and 15. The diagnostic kit according to claim 22.
2 4 . 配列番号： 1 9、 2 0、 2 1、 2 2及び 2 3からなる群より選ばれる少な くとも 1つの核酸を含有してなる、 請求項 2 3記載の診断用キット。 24. The diagnostic kit according to claim 23, comprising at least one nucleic acid selected from the group consisting of SEQ ID NOs: 19, 20, 21, 22, and 23.
2 5 . Secretogl obin ファミリ一に属するタンパク質に対する抗体又はその断片 を含有してなる、 請求項 2 1記載の方法に使用するための診断用キット。 25. A diagnostic kit for use in the method according to claim 21, comprising an antibody against a protein belonging to the Secretogl obin family or a fragment thereof.
2 6 . 前記抗体又はその断片が、 配列番号 2 6又は 2 7のアミノ酸配列を有する ぺプチド又は該ぺプチドを含むポリぺプチドに対するモノクローナル抗体又はそ の断片である、 請求項 2 5記載の診断用キット。 26. The diagnosis according to claim 25, wherein the antibody or a fragment thereof is a monoclonal antibody against a peptide having the amino acid sequence of SEQ ID NO: 26 or 27 or a polypeptide containing the peptide, or a fragment thereof. Kit.
2 7 . 該抗体が受領番号 FERM ABP-10012又は FERM ABP-10013であるハイプリド 一マにより産生されるものである、 請求項 2 6記載の診断用キット。 27. The diagnostic kit according to claim 26, wherein said antibody is produced by a hybridoma having accession number FERM ABP-10012 or FERM ABP-10013.
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