WO2004101824A1 - Methode de detection d'une bronchopneumopathie chronique obstructive - Google Patents

Methode de detection d'une bronchopneumopathie chronique obstructive 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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Prior art keywords
nucleic acid
family
seq
protein belonging
gene
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PCT/JP2004/006905
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English (en)
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/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—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.

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Abstract

L'invention concerne une méthode destinée à détecter ou dépister facilement et rapidement un spécimen provenant d'individus susceptibles de présenter une BPCO infectieuse par mesure du niveau d'expression d'un gène codant pour une protéine appartenant à la famille SCGB, et notamment UGRP2, ainsi qu'une trousse permettant une mise en oeuvre efficace de cette méthode. Plus particulièrement, l'invention concerne une méthode destinée à détecter ou dépister un spécimen provenant d'individus susceptibles de présenter une bronchopneumopathie chronique obstructive infectieuse, et consistant à mesurer le niveau d'expression d'un gène codant pour une protéine appartenant à la famille des sécrétoglobines dans un échantillon de test provenant d'individus sujets de test et un échantillon de référence provenant d'individus normaux. Le fait que le niveau d'expression de l'échantillon de test soit supérieur à celui de l'échantillon de référence fournit une indication selon laquelle l'échantillon de test provient d'individus susceptibles de présenter une bronchopneumopathie chronique obstructive infectieuse. L'invention se rapporte en outre à une trousse de détection ou de dépistage destinée à la méthode susmentionnée et comprenant un acide nucléique permettant de détecter un acide nucléique codant pour une protéine appartenant à la famille des sécrétoglobines.
PCT/JP2004/006905 2003-05-14 2004-05-14 Methode de detection d'une bronchopneumopathie chronique obstructive WO2004101824A1 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014145357A3 (fr) * 2013-03-15 2014-12-31 Clarassance, Inc. Méthodes améliorées d'utilisation de sécrétoglobines humaines de recombinaison
US10294285B2 (en) 2013-03-15 2019-05-21 Therabron Therapeutics, Inc. Modification and compositions of human secretoglobin proteins

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2001522225A (ja) * 1997-01-31 2001-11-13 アボツト・ラボラトリーズ 肺の疾患の検出に有用な試薬及び方法

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Publication number Priority date Publication date Assignee Title
JP2001522225A (ja) * 1997-01-31 2001-11-13 アボツト・ラボラトリーズ 肺の疾患の検出に有用な試薬及び方法

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Title
GHAFOURI B, ET AL: "Newly identified proteins in human nasal lavage fluid from non-smokers and smokers using two-dimensional gel electrophoresis and peptide mass fingerprinting", PROTEOMICS, vol. 2, 2002, pages 112 - 120, XP002981714 *
NIIMI T, ET AL: "UGRP1, a uteroglobin/clara cell secretory protein-related protein, is a novel lung-enriched downstream target gene for the T/EBP/NKX2.1 homeodomain transcription factor", MOL. ENDROCRINOL., 2001, pages 2021 - 2036, XP002981713 *

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014145357A3 (fr) * 2013-03-15 2014-12-31 Clarassance, Inc. Méthodes améliorées d'utilisation de sécrétoglobines humaines de recombinaison
JP2016520531A (ja) * 2013-03-15 2016-07-14 セラブロン セラピューティクス，インコーポレイテッド 組換えヒトセクレトグロビンの改善された使用方法
US9765127B2 (en) 2013-03-15 2017-09-19 Therabron Therapeutics, Inc. Compositions and methods of use for recombinant human secretoglobins
US10294285B2 (en) 2013-03-15 2019-05-21 Therabron Therapeutics, Inc. Modification and compositions of human secretoglobin proteins
US10556938B2 (en) 2013-03-15 2020-02-11 Apc Research Assets, Llc Compositions and methods of use for recombinant human secretoglobins
US11512121B2 (en) 2013-03-15 2022-11-29 Apc Research Assets, Llc Compositions and methods of use for recombinant human secretoglobins

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