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WO2004078975A1 - Nouvelle proteine et son utilisation - Google Patents

Nouvelle proteine et son utilisation Download PDF

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Publication number
WO2004078975A1
WO2004078975A1 PCT/JP2004/002902 JP2004002902W WO2004078975A1 WO 2004078975 A1 WO2004078975 A1 WO 2004078975A1 JP 2004002902 W JP2004002902 W JP 2004002902W WO 2004078975 A1 WO2004078975 A1 WO 2004078975A1
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WO
WIPO (PCT)
Prior art keywords
protein
antibody
disease
present
screening
Prior art date
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PCT/JP2004/002902
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English (en)
Japanese (ja)
Inventor
Nobuhiko Katunuma
Hiroshi Shiota
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Senju Pharmaceutical Co Ltd
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Senju Pharmaceutical Co Ltd
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Filing date
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Publication of WO2004078975A1 publication Critical patent/WO2004078975A1/fr
Anticipated expiration legal-status Critical
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Classifications

    • 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
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/81—Protease inhibitors
    • C07K14/8107—Endopeptidase (E.C. 3.4.21-99) inhibitors
    • C07K14/8139—Cysteine protease (E.C. 3.4.22) inhibitors, e.g. cystatin
    • 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
    • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
    • C12Q1/37—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving peptidase or proteinase
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value

Definitions

  • a protein characterized by having an amino acid sequence represented by SEQ ID NO: 1 in the sequence at the N-terminal and having a molecular weight of 31 kDa.
  • the present invention relates to a screening method for a disease such as Behcet's disease, which is characterized by the following. Background art
  • Examples of the protein containing the amino acid sequence represented by SEQ ID NO: 1 include human salivary gland proline-rititanium protein known as acidic PRP-1 and pHL E 1 F 1 encoded from mRNA in human lacrimal gland. Proline-rich proteins (molecules 4: 15, 097) are known (see, for example, Douglas P. Dickinson et al., (1995) Investigative Ophthalmology & Visual Science, 36 (10), 2020-203). Among the above prolinritsu titanium proteins, human salivary gland prolinritz titanium protein acts at the point of contact between teeth and saliva, regulates calcium phosphate precipitation and crystal formation, and acts as a protein that binds oral bacteria such as Streptococcus mutans. (See, for example, Japanese Patent Publication No. 2002-5 16997).
  • Behcet's disease is a multi-organ invasive inflammatory disease, the cause of which is currently unknown. The main symptoms are
  • Behcet's disease diseases are classified into complete and incomplete types according to the appearance of the above symptoms. As described above, diagnosis of Behcet's disease can be difficult because it is diagnosed only according to the type of the symptoms.Particularly, diagnosis is made in cases where the disease is suspected as Behcet's disease or a disease which has a finding that is confusingly similar to Behcet's disease. It is difficult at present.
  • Zymography is a method that uses electrophoresis.It uses a gel in which a protein that serves as an enzyme substrate such as gelatin or zein is encapsulated.After electrophoresis of a sample, the gel is placed in an appropriate solution in which the enzyme and enzyme substrate react. After incubating, the gel is stained in an appropriate protein staining solution, and the portion where the substrate has been degraded by the protease is detected as a transparent band. The approximate activity of the protease is determined by its position and the degree of omission. That's the way you can. If SDS-polyacrylamide gel is used, its activity and molecular weight can be measured simultaneously.
  • lipase zymography similarly, after electrophoresis on a substrate-containing gel, a protease is added to an enzyme reaction solution to digest the substrate in the gel. If the protease inhibitor is present in the separated protein, the whole gel after staining will have substrate degradation The part where the enzyme activity inhibitor is present is detected as a stained band because the substrate is not degraded, and is used for the analysis of protease inhibitors.
  • this lipase zymography is used for the study of cancer metastasis and the screening method of matrix meta-oral protease inhibitors (eg, Kaori Takasaki et al., (1998) Biophysical Chemistry, 42, 87- See 92.).
  • the present invention is expressed in diseases such as Behcet's disease, belongs to cysteine protease inhibitory protein due to its characteristic of lipase zymography, and is used for screening an excellent anti-Behcet's disease agent and the like. Cysteine proteinase inhibiting protein and its DNA.
  • the present inventors have conducted repeated investigations and studies on samples such as tears, saliva, and blood collected from patients, and found that proteins specifically detected when Behcet's disease or Harada's disease developed, and (4) The inventors discovered that there was a protein whose concentration increased in the sample, and conducted further research to complete the present invention. Therefore, by detecting and identifying such substances, that is, these specific proteins, from the sample, the function of diseases such as Behcet's disease can be examined, and furthermore, diagnosis of Behcet's disease and the like can be performed. It can be used for the objective decision-making, and for the screening and treatment of prophylactic and therapeutic agents such as Behcet's disease.
  • an antisense D having a nucleotide sequence complementary to or substantially complementary to the DNA encoding the protein of the above [1] or a part thereof, and having an action of suppressing the expression of the protein; NA,
  • a protein having a cysteine protease inhibitory activity wherein the N-terminal sequence has the amino acid sequence represented by SEQ ID NO: 5, and the molecular weight is 65 kDa.
  • [[26] A function of the protein, characterized by using a protein having an amino acid sequence represented by SEQ ID NO: 5 in the N-terminal sequence and having a molecular weight of 65 kDa. (27) The screening method according to the above (25), wherein the function is a cystine protease inhibitory activity or a factor associated with Behcet's disease, wherein the compound has a promoting or inhibiting activity or a salt thereof.
  • Cells having the ability to express the protein gene according to the above [1] are cultured in the presence or absence of the test compound, and in each case, the expression level of the mRNA of the protein is determined. Measuring and comparing the screening method according to the above (25), (31) the promoter region and the enhancer region of the protein according to the above (1), or the protein according to the above (1). Promoter region upstream of reporter gene.
  • the cells described above which are characterized by culturing the cells transformed with the DNA ligated to, in the presence and absence of the test compound, and measuring the expression level of the reporter gene in each case. Screening method,
  • (33) having the activity of promoting or inhibiting the function of the protein of (1), which can be obtained using the screening method of (29) or the screening kit of (32) A compound or a salt thereof,
  • Prevention and treatment of Behcet's disease containing salt
  • FIG. 1 shows a lipase zymography of tears from patients with Behcet's disease and Harada's disease.
  • FIG. 2 shows Western plotting using three kinds of egret polyclonal antibodies.
  • Lane 1 marker
  • lane 2 polyclonal antibody after ammonium sulfate treatment
  • lane 3 polyclonal antibody after DEAE-Affi-Ge1B1ue column treatment
  • lane 4 polyclonal after treatment with Mono Q column antibody.
  • the 31 kDa protein of the present invention is a substance that is detected in association with, for example, Behcet's disease or the like, or whose concentration is confirmed to increase.
  • the 31 kDa protein of the present invention is a PRP-1 containing the same amino acid sequence as SEQ ID NO: 1, since the 15-mer amino acid at the N-terminal shows the sequence represented by SEQ ID NO: 1. Or at least about 50% or more of the amino acid sequence having homology with the amino acid sequence of aspartic acid 76 to as amino acid 134 of tryptophan in the amino acid sequence of pHL E 1 F 1 proline litupitamin protein, and further about 60, 70, 75, 80, 85, 90, 95 ° / 0 or more.
  • the 31 kDa protein of the present invention can be produced from tears, blood, cells or tissues of patients with Behcet's disease or the like by a known protein separation / purification method, and the protein of the present invention can be coded. It can also be produced by culturing a transformant containing the DNA of interest.
  • the tears When manufactured from patient's tears, blood, cells or tissues, for example, the tears may be used as-is, such as chromatographic methods such as reverse-phase chromatography,
  • the blood can be purified and isolated by combining the fibrils, and the blood can be purified and isolated by centrifugation to separate the serum or plasma and then to the above-mentioned mouth chromatography.
  • the cells or tissues After homogenizing the cells or tissues, the cells or tissues are extracted or fractionated with salting out acid and the like, and the extracts or fractions are purified and purified by a combination of chromatography such as reverse phase chromatography and ion exchange chromatography. Can be released.
  • the 31 kDa protein of the present invention is produced by recombinant DNA technology, for example, a DNA encoding the above protein is prepared, and inserted into an expression vector to produce Escherichia coli, Bacillus subtilis, and actinic radiation.
  • a transformant may be introduced into a host such as a fungus or yeast to obtain a transformant, and the protein of the present invention may be collected from the culture.
  • the present invention also includes providing a DNA comprising the nucleotide sequence encoding the protein of the present invention.
  • a DNA comprising the nucleotide sequence encoding the protein of the present invention.
  • those incorporated in a plasmid vector to be maintained in transformed Escherichia coli described below can be suitably used.
  • the present invention provides a recombinant vector containing the above DNA.
  • a recombinant vector that is incorporated into a vector that enables the expression of a peptide consisting of an amino acid sequence encoded by the above-mentioned DNA can be suitably used.
  • a method for preparing a DNA having a desired nucleotide sequence for example, chemically synthesizing sense and antisense nucleotides, which are partial sequence nucleotides of the desired DNA and having both ends overlapping, are then subjected to a polymerase chain reaction.
  • a polymerase chain reaction for example, chemically synthesizing sense and antisense nucleotides, which are partial sequence nucleotides of the desired DNA and having both ends overlapping.
  • Prokaryotic or eukaryotic host cells can be transformed by incorporating a DNA encoding the amino acid sequence of the protein of the present invention into a suitable vector. Involved in various promoters and transformations By introducing such a sequence, the DNA can be expressed in each host cell. That is, the present invention also relates to a host cell having a recombinant vector having the DNA of the present invention incorporated in a vector enabling the expression of the protein of the present invention.
  • Prokaryotic host cells include, for example, Escherichia coli and Bacillus subtilis.
  • the host cells can be transformed with a replicon from a species compatible with the host, i.e., a plasmid vector containing an origin of replication and regulatory sequences. Just fine. It is also desirable that the vector has a sequence capable of imparting phenotypic (phenotypic) selectivity to the transformed cells.
  • E. coli K12 strain, JM109 strain and the like are often used, and as a vector, pBR322 and pUC-type plasmids are generally used, but not limited thereto, and various known strains and vectors may be used. Both can be used.
  • promoters include E. coli tryptophan (trp) promoter, ratatose (lac) promoter, tryptophan lactose (tac) promoter, V-poplin (1p) promoter, lambda (X) PL promoter derived from pacteriophage, and poly (E. coli).
  • Peptide chain elongation factor Tu (tuf B) promoter, 1 ac UV5 promoter and the like, and any promoter can be used for production of the protein of the present invention.
  • Bacillus subtilis for example, strain 207-25 is preferable, and as a vector, pTUB228 [see Ohmura, K., et al. (1984) J. Biochem. 95, 87-93] and the like are used. It is not limited.
  • pTUB228 As a promoter for Bacillus subtilis, the regulatory sequence of the ⁇ -amylase gene of Bacillus subtilis is often used, and if necessary, the bacterial sequence is ligated by linking a D D sequence encoding the signal peptide sequence of ⁇ -amylase. It also enables extracellular secretory expression.
  • a plasmid having a pBR322 replication origin, capable of autonomous growth in Escherichia coli, and further having a transcription promoter and a translation initiation signal can be used as an expression vector.
  • the expression vector was prepared by the calcium monochloride method [Mandel, M. and Higa, A. (1970) J. Mol.
  • Eukaryotic host cells include cells such as vertebrates, insects, and yeast.
  • vertebrate cells include COS cells derived from monkey kidney cells [Gluzman, Y. (1981) Cell23, 175- 182], and hamster ovary cells (CHO), human malva cells, and muster BHK cells are often used, but are not limited thereto.
  • a vertebrate cell expression vector a vector having a promoter, an RNA splice site, a polyadenylation site, a transcription termination sequence, and the like located upstream of a gene to be normally expressed can be used. May be included. Examples of such expression vectors include the early promoter of SV40.
  • yeasts are commonly used. Among them, yeasts belonging to the genus Saccharomyces and Saccharomyces' Saccharorayces cerevisiae are preferred. Examples of eukaryotic expression vectors such as the yeast include, for example, a promoter for an alcohol dehydrogenase gene [Bennetzen, J.L. and Hall, BD (1982) J. Biol.
  • the expression vector has an SV40 replication origin and is capable of autonomous growth in COS cells. Furthermore, those having a transcription promoter, a transcription assembly signal, and an RNA splice site can be used.
  • the expression vector is a DEAE-dextran method
  • the transformed cells may be any cells as long as they have been transformed to produce the protein of the present invention, and are not particularly limited.
  • the desired transformant obtained above can be cultured according to a conventional method, and the protein of the present invention is produced intracellularly or extracellularly by the culture.
  • the medium used for the culture can be appropriately selected from those commonly used depending on the host cell used. For example, in the case of Escherichia coli, tryptone-yeast medium (batato tryptone 1.6%, yeast extra Tat 1.0%, sodium chloride 0.5% (pH 7.0)) and peptone medium (manufactured by Difco) can be used.
  • a culture medium such as RPMI 1640 medium or Dulbecco's modified Eagle medium (DMEM) to which serum components such as fetal bovine serum (FBS) are added as necessary
  • DMEM Dulbecco's modified Eagle medium
  • FBS fetal bovine serum
  • Examples of such a method include, for example, treatment with a normal protein precipitant, ultrafiltration, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, abinity chromatography, partition chromatography, Examples include various types of chromatography such as high performance liquid chromatography (HPLC), dialysis methods, and combinations thereof.
  • HPLC high performance liquid chromatography
  • the peptide can be solubilized by denaturing the peptide with a strong denaturing agent such as guanidine isothiosinate.
  • the protein of the present invention may be in the form of a complex obtained by adding a saccharide-polyethylene recall to the thus obtained polypeptide, and further, the polypeptide may be acetylated, amidated, and / or It may be in the form of a derivative or a polymer obtained by crosslinking polymerization with a polyfunctional reagent.
  • the protein of the present invention can be separated and purified from the culture by, for example, the following method.
  • the cells or cells are collected by a known method after culturing, suspended in an appropriate buffer, and subjected to ultrasonication, lysozyme and Z or freeze-thawing. After the cells or cells are destroyed by such methods as mentioned above, a method of obtaining a crude extract of polypeptide by centrifugation or filtration is used as appropriate.
  • the buffer may contain a protein denaturing agent such as urea or guanidine hydrochloride, or a surfactant such as ketoxynol (for example, Triton X-100).
  • the polypeptide When the polypeptide is secreted into the culture solution, after completion of the culture, the supernatant is separated from the cells or cells by a known method, and the supernatant is collected. Purification of the polypeptide contained in the culture supernatant or extract obtained in this manner can be performed by appropriately combining known separation and purification methods. These known separation and purification methods include methods using solubility such as salting out and solvent precipitation, Methods that mainly use the difference in molecular weight, such as dialysis, ultrafiltration, gel filtration, and SDS-polyacrylamide gel electrophoresis, methods that use the difference in charge, such as ion exchange chromatography, and abundance chromatography.
  • solubility such as salting out and solvent precipitation
  • Methods that mainly use the difference in molecular weight such as dialysis, ultrafiltration, gel filtration, and SDS-polyacrylamide gel electrophoresis
  • methods that use the difference in charge such as ion exchange chromatography, and abundance chromat
  • Methods that use specific affinity such as chromatography, methods that use differences in solubility such as partition chromatography, methods that use differences in hydrophobicity such as reverse-phase high-performance liquid chromatography, and isoelectric focusing. For example, a method utilizing the difference between isoelectric points is used.
  • the presence or activity of the protein of the present invention thus produced can be measured by, for example, enzyme immunoassay using a specific antibody.
  • the protein of the present invention has an N-terminus at the left end (amino terminus) and a right end according to the convention of peptide notation. Terminal (carboxyl terminal). Protein of the present invention, C-terminal, usually force Rupokishiru group (- COOH) s-carboxylate (one COO-), amide (one CO NH 2) or an ester (- COOR) or may be any of the salts thereof .
  • alkyl group e.g. methyl, E Ji Le, propyl, isopropyl, etc. heptyl
  • C 6 - 1 2 Ariru group e.g. Hue - le, 1-naphthyl, etc.
  • the protein of the present invention includes a carboxyl group amidated or esterified.
  • ester for example, the above-mentioned c-terminal ester and the like are used.
  • the amino group of the N-terminal amino acid residue (for example, methionine residue) is protected by a protecting group (for example, Ci-eacyl group such as formyl group, acetyl group, etc.).
  • a protecting group for example, Ci-eacyl group such as formyl group, acetyl group, etc.
  • N-terminal glutamine residue generated by cleavage in vivo, pyroglutamic acid residue, substituent on the side chain of amino acid in the molecule (eg, hydroxy group, sulfanyl group)
  • An amino group an imidazolyl group, an indolyl group, a guanidino group, etc.
  • protecting groups for example, an acyl group such as an alkanol group such as a formyl group or an acetyl group.
  • Protected or complex polypeptides such as so-called sugar polypeptides having sugar chains bonded thereto are also included.
  • a salt with a physiologically acceptable acid for example, an inorganic acid or an organic acid
  • a base for example, an alkali metal salt
  • a physiologically acceptable salt is used, and particularly, a physiologically acceptable salt is used.
  • Acid addition salts are preferred.
  • salts include salts with inorganic acids (eg, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid) or organic acids (eg, acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, succinic acid) Acid, tartaric acid, citric acid, malic acid, oxalic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid) and the like.
  • inorganic acids eg, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid
  • organic acids eg, acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, succinic acid
  • Acid tartaric acid, citric acid, malic acid, oxalic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid
  • the antisense DNA of the present invention may be a modified DNA.
  • the modified DNA include a sulfur derivative and a thiophospho derivative of DNA, and those which are resistant to the decomposition of polynucleoside amide polynucleonucleoside amide, but are not limited thereto. Absent.
  • the antisense DNA of the present invention is intended to be more stable in cells, to enhance cell permeability, to increase the affinity for a target sense strand, or to reduce toxicity. It can be preferably designed and modified.
  • the antisense DNA of the present invention may contain altered or modified sugars, bases, or bonds, and may be provided in a special form such as ribosome or microsphere, applied by gene therapy, It could be provided in an added form.
  • antisense DNA examples include polycations such as polylysine, which act to neutralize the charge on the phosphate backbone, to enhance interaction with cell membranes, and to increase nucleic acid uptake. Lipids (eg, phospholipids, cholesterol, etc.). Such additions can be attached to the nucleic acid at the 3, 5 or 5 ends, and can be attached through bases, sugars, or intramolecular nucleoside linkages.
  • the inhibitory activity of antisense DNA can be examined using the transformant of the present invention or the in vivo or in vitro translation system of the protein of the present invention.
  • the DNA can be applied to cells by various known methods.
  • the antibody against the protein of the present invention may be any of a polyclonal antibody and a monoclonal antibody as long as it can recognize the protein of the present invention, but a monoclonal antibody is more preferable.
  • An antibody against the protein of the present invention may be a known antibody or antibody using the protein of the present invention or a part of the amino acid sequence of the protein of the present invention or a polypeptide having antigenicity having a part of the amino acid sequence as an antigen. It can be produced according to the method for producing serum.
  • an antibody against the 31 kDa protein a polypeptide consisting of the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 4, or a partial sequence thereof is specifically used.
  • An antibody that recognizes is preferred, and an antibody against the 61 kDa protein is preferably an antibody that specifically recognizes a polypeptide consisting of the amino acid sequence of SEQ ID NO: 5 or a partial sequence thereof.
  • Such an antibody can be prepared by using, as an antigen, a polypeptide consisting of the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 5, or a partial sequence thereof.
  • the polypeptide comprising the partial distribution sequence is not particularly limited as long as it has antigenicity.
  • the amino acid of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 5 It may be a polypeptide consisting of at least 6, preferably at least 8, more preferably at least one or more contiguous amino acids selected from the sequence.
  • the polypeptide having the antigenicity can be prepared, for example, by a conventional peptide synthesis method known as a “solid phase method” or “liquid phase method”. For example, details of peptide synthesis can be found in “The New Chemistry Laboratory Course”, edited by The Biochemical Society of Japan, Vol. 1, “Protein VI”, pp. 3-44, 1992, published by Tokyo Chemical Dojin. Has been described.
  • a part of the amino acid sequence of the protein described in the present invention or a polypeptide having a part of the amino acid sequence can be synthesized by a solid-phase synthesis method using Fmoc (9-fluorenyl methyloxycarbonyl) using a peptide synthesizer (Shimadzu Corporation).
  • Fmoc 9-fluorenyl methyloxycarbonyl
  • a peptide synthesizer Shiadzu Corporation
  • DMF piperidinenodimethylformamide
  • the above-mentioned polypeptide having antigenicity is administered to a warm-blooded animal at a site where the antibody can be produced by administration to itself or together with a carrier or a diluent.
  • • Complete Freund's adjuvant / incomplete Freund's adjuvant may be administered to enhance antibody production.
  • the administration is usually performed once every 2 to 6 weeks, preferably about 2 to 10 times in total.
  • Examples of the warm-blooded animal to be used include monkeys, puppies, dogs, guinea pigs, mice, rats, sheep, goats, and chickens, and preferably mice, rats, and puppies.
  • a warm-blooded animal immunized with the antigen for example, an individual with an antibody titer from a heron is selected, and the spleen or lymph node is collected 2 to 5 days after the final immunization.
  • a monoclonal antibody-producing hybridoma can be prepared.
  • the measurement of the antibody titer in the antiserum is performed, for example, by reacting a labeled polypeptide labeled with a radioactive substance or an enzyme with the antiserum, and then measuring the activity of the labeling agent bound to the antibody. be able to.
  • the fusion operation can be carried out according to a known method, for example, the method of Koehler and Milstein [Nature, 256, 495 (1975)].
  • the fusion promoter include polyethylene glycol (PEG) and Sendai virus, but PEG is preferably used.
  • myeloma cells examples include NS-1, P3U1, SP2 / 0, and AP-1.
  • P3U1 is preferably used, although myeloma cells from any warm-blooded animal can be used.
  • the preferred ratio between the number of antibody-producing cells (spleen cells) and the number of myeloma cells used is about 1: 1 to 20: 1, and PEG (preferably PEG100 to PEG600) is used.
  • Cell fusion can be carried out efficiently by adding at a concentration of about 10 to 80% and incubating at 20 to 40 ° C, preferably 30 to 37 ° C for 1 to 10 minutes.
  • Various methods can be used for screening the monoclonal antibody-producing hybridoma.
  • the hybridoma culture supernatant is added to a solid phase (for example, a microplate) on which the polypeptide antigen is adsorbed directly or together with a carrier.
  • a solid phase for example, a microplate
  • Anti-immunoglobulin antibody anti-mouse immunoglobulin antibody is used if the cells used for cell fusion are mice
  • the monoclonal antibody bound to the solid phase is added to the antibody.
  • Detection method Monoclonal antibody bound to solid phase by adding hybridoma culture supernatant to solid phase to which anti-immunoglobulin antibody or protein A is adsorbed, adding polypeptide labeled with radioactive substances, enzymes, etc. And the like.
  • Selection of the monoclonal antibody can be performed according to a known method or a method analogous thereto. Usually, it can be performed in a medium for animal cells supplemented with HAT (hippoxanthin, aminopterin, thymidine).
  • HAT hippoxanthin, aminopterin, thymidine
  • any medium can be used as long as it can grow a hybridoma.
  • RPMI 1640 medium containing 1 to 20%, preferably 10 to 20% fetal bovine serum, GIT medium containing 1 to 10% fetal bovine serum (Wako Pure Chemical Industries, Ltd.)
  • a serum-free medium for hybridoma culturing SFM-101, Nissui Pharmaceutical Co., Ltd.
  • the culturing temperature is usually 20 to 40 ° C, preferably about 37 ° C.
  • the culture time is usually 5 days to 3 weeks, preferably 1 week to 2 weeks.
  • the cultivation can usually be performed under 5% carbon dioxide.
  • the antibody titer of the hybridoma culture supernatant can be measured in the same manner as the measurement of the antibody titer in the antiserum described above.
  • Monoclonal antibodies can be separated and purified by known methods, for example, immunoglobulin analysis. Separation / purification method [eg, salting out method, alcohol precipitation method, isoelectric point precipitation method, electrophoresis method, adsorption / desorption method with ion exchanger (eg, DEAE), ultracentrifugation method, gel filtration method, antigen-binding solid phase Alternatively, a specific purification method in which only the antibody is collected using an active adsorbent such as protein A or protein G and the bond is dissociated to obtain an antibody].
  • separation / purification method eg, salting out method, alcohol precipitation method, isoelectric point precipitation method, electrophoresis method, adsorption / desorption method with ion exchanger (eg, DEAE), ultracentrifugation method, gel filtration method, antigen-binding solid phase
  • ion exchanger eg, DEAE
  • ultracentrifugation method eg, ultracentr
  • scFv single-chain antibody
  • a monoclonal antibody eg, Mol. Biol. 296, 55 (2000); http: //. Morphosys. Com / start, see php ".
  • Hu CAL registered trademark
  • Hu CAL which is a single-chain FV-based phage display library
  • Conversion of the scFv into a monoclonal antibody is performed by cloning the heavy chain and light chain variable regions from the scFV obtained above and linking them to the immunoglobulin heavy chain and light chain constant regions, respectively.
  • nucleotide mutations are introduced as necessary to add a leader sequence, modify the Kozak sequence, remove or add a restriction site, and the like.
  • the ligated product thus obtained is introduced into the Clawing site of the expression vector, and the host cell is transformed to obtain a transformant.
  • those expressing an immunoglobulin capable of binding to an anti-immune glopurin antibody are selected by a method known per se such as ELISA.
  • a monoclonal antibody is expressed in the selected transformant, and the target monoclonal antibody can be obtained by separation and purification.
  • the expression vector, host cell The same method as described above can be used, and the transformation method, separation and purification method, etc. can be performed in the same manner as described above.
  • the polyclonal antibody of the present invention can be produced according to a known method or a method analogous thereto.
  • an immunizing antigen having antigenicity against the protein of the present invention
  • a carrier protein is prepared, and immunization is performed on a warm-blooded animal in the same manner as in the above-described method for producing a monoclonal antibody.
  • the antibody can be produced by collecting an antibody-containing substance against the protein of the present invention from an immunized animal and separating and purifying the antibody. Complete Freund's adjuvant ⁇ Incomplete Freund's adjuvant may be administered in order to enhance the antibody-producing ability upon administration.
  • the administration is usually performed once every about 2 to 6 weeks, for a total of about 3 to 10 times.
  • the polyclonal antibody can be collected from the blood, ascites, or the like of a warm-blooded animal immunized by the above method, preferably from the blood.
  • the polyclonal antibody titer in the antiserum can be measured in the same manner as the measurement of the antibody titer in the antiserum described above. Separation and purification of the polyclonal antibody can be performed according to the same immunoglobulin separation and purification method as in the above-described separation and purification of the monoclonal antibody.
  • serum was subjected to ammonium sulfate precipitation and then treated with a DEAE-Affi-Ge1B1ue column and / or a Mono Q column to achieve a higher purity.
  • Excellent polyclonal antibodies can also be isolated and purified.
  • the antibody against the protein of the present invention include, for example, a partial amino acid sequence of human salivary gland proline rititanium protein (PP-1).
  • a polypeptide consisting of Gln-Gln-Arg-Pro-Pro-Arg-Arg-Gly-His-Arg-Gln-Leu-Ser-Leu-Pro-Arg-Phe-Pro-Ser-Val (SEQ ID NO: 3)
  • Antibodies used as antigens can be mentioned.
  • the protein of the present invention can be suitably detected when the peptide is separated and visualized by reverse zymography using electrophoresis.
  • the gel used for reverse zymography contains polyacrylamide.
  • the content of the polyacrylamide is preferably about 2 to 30 w / V%, and more preferably about 5 to 15 w / v%.
  • the gel used in the Lipase Zymodarafi method preferably contains a surfactant such as SDS, and the concentration of the SDS is about 0.0001 to 15 wZv%. More preferably, it is about 0.01-5 wZv%.
  • the gel used in the reverse zymography method preferably contains a substrate such as gelatin, casein, elastin and fipurin, and gelatin is particularly preferred.
  • the substrate concentration is preferably about 0.01 to lw / v%, more preferably about 0.05 to 0.5 w / v%.
  • cysteine protease for example, papain, fusin, promelain, cathepsin B, H, L, calpain and the like are preferable, and papain is particularly preferable.
  • the protein of the present invention is detected or its concentration is increased at least at the onset of Behcet's disease or Harada disease. According to the reverse zymography method, if the protein of the present invention is detected in the organism from which the specimen was collected, it may be a polypeptide that is specifically expressed in diseases such as Behcet's disease and Harada's disease. The protein of the present invention can be used as an indicator of Behcet's disease ⁇ Harada's disease.
  • the 31 kDa protein of the present invention As an indicator substance which is detected at least at the onset of Behcet's disease or whose concentration is confirmed to be increased, for example, the 31 kDa protein of the present invention can be mentioned.
  • the “31 kDa protein” refers to, for example, a substance that is detected as a band near 31 kDa when an indicator substance is detected by the reverse zymography method.
  • an indicator substance that is detected or whose concentration is confirmed to be increased is, for example, the amino acid sequence at the N-terminal side is SEQ ID NO: 5 and has a molecular weight of about 65 kDa.
  • Cysteine protease inhibitors and the like are, for example, 65% when an indicator substance is detected by the reverse zymography method described above. A substance detected as a band near kDa.
  • the protein of the present invention has a protein inhibitory activity due to one of the characteristics of reverse zymography. Therefore, the protein of the present invention, or an amide or ester thereof, or a salt thereof can be used as a composition for a therapeutic or prophylactic agent for diseases involving cysteine protease, for example, osteoporosis.
  • the DNA encoding the protein of the present invention When used as the above-mentioned therapeutic or prophylactic agent, the DNA may be used alone or in a suitable vector such as a retrovirus vector, an adenovirus vector, or an adenovirus associated virus vector. After insertion into a human, it can be administered to humans or warm-blooded animals according to conventional means.
  • the DNA of the present invention can be administered as it is or in the form of a formulation together with a physiologically acceptable carrier such as an adjuvant for promoting uptake, and administered by a gene gun or a catheter such as a hydrogel catheter.
  • the protein of the present invention When the protein of the present invention is used as the above-mentioned therapeutic / prophylactic agent, it is purified to at least 90%, preferably 95% or more, more preferably 98% or more, and still more preferably 99% or more. It is preferable to use one.
  • the protein of the present invention or an amide or estenole thereof or a salt thereof can be used, for example, orally as tablets, capsules, elixirs, microcapsules, etc., if necessary coated with sugar or water or other water.
  • Pharmaceutically acceptable It can be used parenterally in the form of an injectable preparation, such as a sterile solution with an acceptable liquid, or a suspension.
  • an injectable preparation such as a sterile solution with an acceptable liquid, or a suspension.
  • physiologically acceptable carriers, excipients, preservatives, stabilizers, binders, sweeteners, and the like in the unit dosage form required for generally accepted pharmaceutical practice. can do.
  • the amount of active ingredient in these preparations is such that a suitable dosage in the specified range can be obtained.
  • Additives that can be incorporated into tablets, capsules, etc. include, for example, binders such as gelatin, corn starch, tragacanth, gum arabic, excipients such as crystalline cellulose, corn starch, gelatin, Swelling agents such as alginic acid, lubricants such as magnesium stearate, sweeteners such as sucrose, lactose or saccharin are used.
  • suitable coating agents eg, cellulose acetate phthalenolate, methacrylic acid copolymer, hydroxypropylcellulose phthalate, carboxymethylethyl cellulose, etc.
  • enteric coating agents eg, cellulose acetate phthalenolate, methacrylic acid copolymer, hydroxypropylcellulose phthalate, carboxymethylethyl cellulose, etc.
  • the skin may be applied with such as.
  • a liquid carrier such as oil and fat can be further contained in the above-mentioned type of material.
  • the capsule can be an enteric-coated capsule, a gastric resistant capsule, or a controlled-release capsule other than ordinary capsules.
  • Sterile compositions for injection can be formulated according to normal pharmaceutical practice of dissolving or suspending the active substance in vehicles such as water for injection, and naturally occurring vegetable oils such as sesame oil, coconut oil and the like. .
  • aqueous liquids for injection include physiological saline, isotonic solutions containing butudose and other adjuvants (eg, D-sorbitol, D-mantol, sodium chloride, etc.).
  • Solubilizers for example, alcohols (eg, ethanol, etc.), polyalcohols (eg, propylene glycol, polyethylene glycol, etc.), nonionic surfactants (eg, polysorbate 80, polyoxyethylene hydrogenated castor oil 50) Etc.).
  • examples of the oily liquid include sesame oil and soybean oil, and may be used in combination with benzyl benzoate, benzyl alcohol and the like as a solubilizing agent.
  • buffers eg, phosphate buffer, sodium acetate buffer, etc.
  • soothing agents eg, proforce hydrochloride, etc.
  • stabilizers eg, human serum albumin, polyethylene glycol, etc.
  • storage Agents e.g., penzino oleanol, phenol, etc.
  • antioxidants e.g., antioxidants and the like.
  • the prepared injection solution is usually filled in a suitable ampoule.
  • the vector into which the DNA of the present invention has been inserted is also formulated in the same manner as described above, and is usually used parenterally.
  • the preparations obtained in this way are safe and have low toxicity, for example, warm-blooded animals (e.g., humans, rats, mice, guinea pigs, egrets, birds, higgies, stags, pests, pomas, cats, Dogs, monkeys, chimpanzees, etc.).
  • the dosage of the protein of the present invention varies depending on the target disease, the subject of administration, the administration route, and the like.
  • the protein of the present invention is orally administered for the purpose of treating osteoporosis, generally the adult (60 kg) is used. ).
  • the administration is about 0.1 mg to 100 mg, preferably about 1.0 to 500 mg per day.
  • the protein of the present invention or DNA encoding the protein of the present invention is also useful as a probe for screening a compound or a salt thereof that promotes or inhibits the expression of the protein of the present invention. That is, the present invention provides a method for screening a compound having an activity of promoting or inhibiting the expression of the protein of the present invention, which comprises using the protein of the present invention. Specifically, for example, cells having the ability to express the gene of the protein of the present invention are cultured in the presence of a test compound, and the DNA encoding the protein of the present invention or its complementary DNA or a partial DNA thereof is used. And a method for screening a compound having an activity of promoting or inhibiting the expression of the protein of the present invention or a salt thereof, which comprises measuring the amount of mRNA encoding the protein of the present invention.
  • Examples of the cells capable of expressing the protein gene of the present invention include animal cells into which the protein gene of the present invention has been introduced and transformed.
  • Animal cells into which the protein gene of the present invention has been introduced and transformed can be produced by the method described above. Wear.
  • the cells having the ability to express the protein gene of the present invention are cultured in the same manner as in a known animal cell culture method.
  • a MEM medium containing about 5 to 20% fetal bovine serum [Science, 122, 501 (1952)], a DMME medium [Virology, 8 volumes, 396 (1959)], RPMI 1640 medium [The Journal of the American Medical Association, 199, 5 19 (1967)], 199 medium [Proceeding of the Society for the Biological Medicine, 73, 1 (1950)] and the like are used.
  • the pH is about 6-8.
  • the cultivation may be carried out usually at about 30 to 40 ° C. for about 15 to 60 hours, and subculturing may be carried out if necessary. C The culturing may be conducted with aeration or stirring as necessary.
  • the protein of the present invention is encoded!
  • the amount of nRNA is measured by contacting RNA extracted from a cell according to a known method with DNA complementary to the DNA encoding the protein gene of the present invention or a partial DNA thereof, and It is performed by measuring the amount of niRNA bound to the complementary DNA of the gene DNA of the protein.
  • the amount of mRNA bound to the complementary DNA of the gene DNA of the protein of the present invention by labeling the DNA complementary to the gene DNA of the protein of the present invention or a partial DNA thereof with, for example, a radioisotope, a dye, or the like. Can be easily measured.
  • the radioisotope for example, [ 32 P], [ 3 H] or the like is used, and as the dye, for example, a fluorescent dye such as fluorescein is used.
  • the amount of the mRNA of the protein of the present invention is determined by converting RNA extracted from cells into complementary DNA by reverse transcriptase, and then transforming the DNA encoding the protein gene of the present invention or its complementary DNA. It can be performed by measuring the amount of complementary DNA to be amplified by PCR using A or its partial DNA as a primer. Measurement of the amount of mRNA of the protein of the present invention
  • Examples of the DNA complementary to the DNA of the protein of the present invention used for determination include DNA (lower chain) having a sequence complementary to the gene DNA (upper chain) of the protein of the present invention.
  • the present invention relates to a cell (eg, an adipocyte, a macrophage, a skeleton) transformed with a known promoter-enhancer region of the protein of the present invention, which is cloned from genomic DNA and ligated upstream of a suitable reporter gene.
  • Stimulating or inhibiting the expression of the protein of the present invention which comprises culturing a muscle cell or the like in the presence of a test compound and detecting the expression of a reporter gene in place of the expression of the protein of the present invention.
  • a method for screening a compound having the activity of As a reporter gene for example, a staining marker gene such as 1 & cZ galactosidase gene) or the like is used.
  • a test compound that increases the amount of the reporter gene product promotes the expression of the protein gene of the present invention.
  • a test compound that reduces the amount of a reporter gene product can be selected as a compound that inhibits the expression of the protein gene of the present invention.
  • the cells can be cultured in the same manner as in the known animal cell culture described above.
  • the present invention provides (i) expressing the protein of the present invention in, for example, Escherichia coli, purifying the protein, and adding a test compound together with the cysteine protease inhibitory activity of the protein of the present invention and (ii) the protein of the present invention.
  • a compound having the activity of promoting or inhibiting the function of the protein of the present invention which is characterized in that the cysteine protease inhibitory activity when cultivated is measured using lipase zymography and the like, and comparison is performed. And a method for screening for a salt thereof.
  • the present invention provides a method for culturing cells capable of expressing the gene of the protein of the present invention in the presence of a test compound, and measuring the expression level of the protein of the present invention using the antibody of the protein of the present invention.
  • Promotes or inhibits the expression of the protein of the present invention which is characterized by A method for screening a compound having a harmful activity or a salt thereof, more specifically, (i) the expression level of the protein of the present invention when cells having the ability to express the gene of the protein of the present invention are cultured; ) Measuring the expression level of the protein of the present invention when cells having the ability to express the gene of the protein of the present invention in the presence of the test compound are measured using the antibody of the protein of the present invention, and compared.
  • the antibody of the protein of the present invention can be produced by the method described above.
  • the cells can be cultured in the same manner as in the known animal cell culture described above.
  • a cell having the above is cultured in the presence of a test compound, and the culture solution (test solution) and the antibody of the protein of the present invention insolubilized on the carrier and another labeled antibody of the present invention are simultaneously or continuously treated.
  • Measuring the activity of a labeling agent on an insolubilized carrier in the case of reacting with a protein, or a method for screening a compound or a salt thereof having an activity of promoting or inhibiting the expression or secretion of a protein according to the present invention. provide.
  • one of the antibodies is the N-terminal of the protein of the present invention. It is desirable that the antibody recognize the end and the other antibody react with the C-terminal of the protein of the present invention.
  • examples of the test compound include peptides, proteins, non-peptide compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts, and the like.
  • the product may be a novel compound or a known compound.
  • the screening kit of the present invention includes a cell capable of expressing the gene of the protein of the present invention, a labeled protein of the present invention, an antibody of the protein of the present invention, a DNA encoding the protein of the present invention, Alternatively, it contains a DNA complementary to the DNA encoding the protein of the present invention.
  • Compounds or salts thereof obtained using the screening method or screening kit of the present invention may be any of the test compounds described above, for example, peptides, proteins, non-peptidic compounds, synthetic compounds, fermentation products, cell extracts, and plants. It is a compound selected from extracts, animal tissue extracts, plasma and the like, and is a compound having an activity of promoting or inhibiting the function of the protein of the present invention.
  • the salt of the compound those similar to the aforementioned salts of the protein of the present invention are used.
  • a compound obtained by using the screening method or the screening kit of the present invention is used as a therapeutic / prophylactic agent for diseases such as Behcet's disease, it can be carried out according to a conventional method.
  • a conventional method for example, in the same manner as the above-mentioned composition containing the protein of the present invention, orally or parenterally as tablets, forcepsels, elixirs, microforces, sterile solutions, suspensions, etc. Can be administered.
  • the preparations obtained in this way are safe and have low toxicity, for example warm-blooded animals
  • the dose of the compound or a salt thereof varies depending on its action, target disease, subject to be administered, administration route, and the like.
  • a compound that inhibits the function of the protein of the present invention for the purpose of treating Behcet's disease may be used.
  • oral administration generally adult (assuming a body weight of 6 O kg) , The compound is administered in an amount of about 0.1 to 100 mg, preferably about 1.0 to 50 mg, more preferably about 1.0 to 20 mg per day.
  • an antibody against the protein of the present invention can specifically recognize the protein of the present invention, and therefore, the quantification of the protein of the present invention in a test solution, particularly It can be used for quantification by sandwich immunoassay. That is, the present invention provides: (i) a step of competitively reacting an antibody of the present invention with a test solution and a labeled protein of the present invention to form a labeled protein of the present invention bound to the antibody.
  • a method for quantifying the protein of the present invention in a test solution which comprises measuring the ratio, and (ii) the antibody of the present invention insolubilized on the test solution and a carrier and another method of the labeled present invention.
  • the present invention provides a method for quantifying the protein of the present invention in a test solution, which comprises reacting an antibody simultaneously or successively and then measuring the activity of a labeling agent on an insolubilized carrier.
  • a quantitative method for quantifying the protein of the present invention comprises reacting an antibody simultaneously or successively and then measuring the activity of a labeling agent on an insolubilized carrier.
  • one of the antibodies is an antibody that recognizes the N-terminal portion of the protein of the present invention, and the other antibody is an antibody that reacts with the C-terminal portion of the protein of the present invention.
  • the protein of the present invention can be quantified using a monoclonal antibody against the protein of the present invention (hereinafter sometimes referred to as the monoclonal antibody of the present invention), and can also be detected by tissue staining or the like.
  • the antibody molecule itself may be used, or F (ab ') 2 , Fab, or Fab fraction of the antibody molecule may be used.
  • the method for quantifying the protein of the present invention using the antibody of the present invention is not particularly limited, and may be an antibody, an antigen, or an antibody-antigen complex corresponding to the amount of an antigen (eg, the amount of a polypeptide) in a test solution.
  • any method can be used, as long as the amount is determined by chemical or physical means, and this is calculated from a standard curve prepared using a standard solution containing a known amount of antigen.
  • the sandwich method described below in terms of the sensitivity and specificity in which the nef mouth method, the competitive method, the immunometric method and the sandwich method are suitably used.
  • a labeling agent used in a measurement method using a labeling substance for example, a radioisotope, Enzymes, fluorescent substances, luminescent substances and the like are used.
  • a radioisotope for example,
  • a stable enzyme having a high specific activity is preferable.
  • 3-galactosidase, ⁇ -dalcosidase, alkaline phosphatase, peroxidase, malate dehydrogenase and the like are used.
  • the fluorescent substance for example, fluorescamine, fluorescein isothiosinate and the like are used.
  • the luminescent substance for example, luminol, luminol derivative, / reciferin, lucigenin and the like are used.
  • a biotin-avidin system can be used for binding the antibody or antigen to the labeling agent.
  • the insolubility of an antigen or an antibody physical adsorption may be used, or a chemical bond usually used to insolubilize and immobilize polypeptides or enzymes may be used.
  • the carrier include insoluble polysaccharides such as agarose, dextran, and cellulose; synthetic resins such as polystyrene, polyacrylamide, and silicon; and glass.
  • the test solution is reacted with the insolubilized monoclonal antibody of the present invention (primary reaction), and further reacted with another labeled monoclonal antibody of the present invention (secondary reaction). By measuring the activity of the labeling agent, the amount of the protein of the present invention in the test solution can be determined.
  • the primary reaction and the secondary reaction may be performed in the reverse order, may be performed simultaneously, or may be performed at different times.
  • the labeling agent and the method of insolubilization can be the same as those described above.
  • the antibody used for the solid phase antibody or the labeling antibody does not necessarily need to be one kind, and a mixture of two or more kinds of antibodies is used for the purpose of improving measurement sensitivity and the like. You can.
  • the monoclonal antibody of the present invention used in the primary reaction and the secondary reaction is preferably an antibody having a different site to which the protein of the present invention binds.
  • the antibody used in the primary reaction and the secondary reaction is, for example, the antibody used in the secondary reaction
  • the antibody used in the primary reaction is preferably an antibody which recognizes other than the C-terminal part, for example, the N-terminal part.
  • the monoclonal antibody of the present invention can be used in a measurement system other than the sandwich method, for example, a competition method, an immunometric method, or a nephrometry.
  • a competition method the antigen in the test solution and the labeled antigen are allowed to react competitively with the antibody, and then the unreacted labeled antigen (F) is separated from the labeled antigen (B) bound to the antibody. (B / F separation), and the amount of B or F label is measured, and the amount of antigen in the test solution is quantified.
  • a soluble antibody is used as the antibody
  • BZF separation is performed using polyethylene glycol
  • a liquid phase method using a second antibody to the antibody a solid phase antibody is used as the first antibody
  • An immobilization method using an immobilized antibody as the second antibody and an immobilized antibody as the second antibody is used.
  • the immunometric method the antigen in the test solution and the immobilized antigen are subjected to a competitive reaction with a certain amount of labeled antibody, and then the solid phase and the liquid phase are separated.
  • the antigen is allowed to react with an excessive amount of the labeled antibody, then the immobilized antigen is added, and the unreacted labeled antibody is bound to the solid phase, and then the solid phase and the liquid phase are separated.
  • the amount of the label in the phase is measured to determine the amount of the antigen in the test solution.
  • the amount of insoluble sediment resulting from an antigen-antibody reaction in a gel or in a solution is measured. Even when the amount of antigen in the test solution is small and only a small amount of sediment is obtained, laser nephrometry utilizing laser scattering is preferably used.
  • the protein measurement system of the present invention may be constructed by adding ordinary technical considerations of those skilled in the art to ordinary conditions and operation methods in each method.
  • the protein measurement system of the present invention may be constructed by adding ordinary technical considerations of those skilled in the art to ordinary conditions and operation methods in each method.
  • the protein of the present invention can be quantified with high sensitivity by using the antibody of the present invention.
  • the protein of the present invention when the protein of the present invention is detected by quantifying the concentration of the protein of the present invention using the antibody of the present invention, for example, there is a possibility of Behcet's disease or future disease. It can be diagnosed as high.
  • the antibody of the present invention can be used for detecting the protein of the present invention present in a subject such as a body fluid or a tissue.
  • detecting the protein of the present invention in each fraction during purification analyzing the behavior of the protein of the present invention in test cells, and the like. Can be used for
  • a composition containing the antisense DNA of the present invention for the DNA encoding the protein of the present invention can complementarily bind to the polynucleotide of the present invention (for example, DNA), and enhance the expression of the polypeptide. Can be suppressed. Since the antisense DNA of the present invention has low toxicity and can suppress the expression of the protein of the present invention or the polynucleotide of the present invention in vivo, for example, diseases caused by overexpression of the protein of the present invention, Behcet, It can be used as a preventive and therapeutic agent for diseases such as Harada disease.
  • the above-mentioned antisense polynucleotide is replaced with the above-mentioned polynucleotide of the present invention. It can be formulated in the same manner as in the case of the peptide.
  • the preparations thus obtained have low toxicity and may be orally or non-toxic to humans or non-human mammals (eg, rats, puppies, sheep, sheep, puppies, cats, cats, dogs, monkeys, etc.). It can be administered orally.
  • the antisense 'polynucleotide can be administered as it is or together with a physiologically acceptable carrier such as an auxiliary for promoting uptake, using a gene gun or a catheter such as a hydrogel catheter.
  • a physiologically acceptable carrier such as an auxiliary for promoting uptake
  • a catheter such as a hydrogel catheter.
  • the dose of the antisense polynucleotide varies depending on the target disease, the subject of administration, the route of administration, and the like.For example, an antisense nucleotide against DNA encoding the protein of the present invention for the purpose of treating Behcet's disease may be used. When administered topically to the eyes and the like, the dose is preferably about 0.1 to 100 mg per day for an adult (body weight 60 kg).
  • the antisense polynucleotide can also be used as a diagnostic oligonucleotide probe for examining the presence of the DNA of the present invention in tissues or cells and the state of expression thereof.
  • the antibody of the present invention which has the activity of neutralizing the activity of the protein of the present invention, is useful for, for example, preventing diseases caused by overexpression of the protein of the present invention, such as Behcet's disease and Harada's disease. It can be used as a composition such as a therapeutic agent.
  • the therapeutic or prophylactic agent for the above-mentioned diseases containing the antibody of the present invention can be used as it is as a liquid or as a composition of an appropriate dosage form in a human or non-human mammal (for example, rat, porch egret, sheep, pig, porcine, It can be administered orally or parenterally to cats, dogs, monkeys, etc.).
  • the dosage varies depending on the administration subject, target disease, symptoms, administration route, etc.
  • the antibody of the present invention when used in adults, is usually administered in a single dose of 0.01 to 2 Omg / kg body weight.
  • Intravenous injection preferably about 0.1 to 10 mg / kg body weight, more preferably about 0.1 to 5 mg / kg body weight, about 1 to 5 times a day, preferably about 1 to 3 times a day
  • an equivalent dose can be administered, but it may be increased or decreased depending on the symptoms.
  • the antibody of the present invention may be administered by itself or as a suitable composition.
  • composition used for the above administration contains the above or a salt thereof and a pharmacologically acceptable carrier, diluent or excipient.
  • Such compositions are provided in dosage forms suitable for oral or parenteral administration. That is, for example, compositions for oral administration include solid or liquid dosage forms, specifically tablets (including sugar-coated tablets and film-coated tablets), pills, granules, powders, capsules ( Soft capsules), syrups, emulsions, suspensions and the like.
  • Such a yarn composition is produced by a known method and contains a carrier, a diluent or an excipient commonly used in the pharmaceutical field.
  • compositions for parenteral administration for example, injections, suppositories, etc. are used. Injections are in the form of intravenous injections, subcutaneous injections, intradermal injections, intramuscular injections, drip injections, etc. Is included. Such injections are prepared according to known methods, for example, by dissolving, suspending or emulsifying the antibody or a salt thereof in a sterile aqueous or oily liquid commonly used for injections.
  • aqueous liquid for injection for example, physiological saline, isotonic solution containing pudose and other adjuvants and the like are used, and suitable solubilizing agents, for example, alcohol (eg, ethanol), polyalcohol (eg, , Propylene glycol, polyethylene dalicol), nonionic surfactants (eg, polysorbate 80, polyoxyethylene hydrogenated castor oil 50) and the like.
  • suitable solubilizing agents for example, alcohol (eg, ethanol), polyalcohol (eg, , Propylene glycol, polyethylene dalicol), nonionic surfactants (eg, polysorbate 80, polyoxyethylene hydrogenated castor oil 50) and the like.
  • oily liquid for example, sesame oil, soybean oil, and the like are used, and benzyl benzoate, benzyl alcohol, and the like may be used in combination as a solubilizing agent.
  • the prepared injection solution is usually filled in a suitable ampoule.
  • Suppositories used for rectal administration are prepared by mixing the above-mentioned antibody or a salt thereof with a usual base for suppositories.
  • the above-mentioned oral or parenteral compositions are conveniently prepared in dosage unit form so as to be compatible with the dosage of the active ingredient.
  • dosage unit dosage forms include tablets, pills, capsules, injections (ampoules), suppositories, etc., and usually 5 to 500 mg per dosage unit form, especially for injections. 5 to 100 mg, other It is preferable that the above dosage form contains 10 to 250 mg of the above antibody.
  • Each of the above-mentioned compositions may contain another active ingredient as long as the composition does not cause an undesirable interaction with the above-mentioned antibody.
  • DNA Deoxyribonucleic acid
  • RNA Liponucleic acid
  • mRNA messenger ribonucleic acid
  • PVDF Polyvinylidene fluoride
  • EDTA ethylenediaminetetraacetic acid
  • Ser Serine G 1 u: gnoretamic acid
  • H histidine
  • Trt trityl group
  • sequence numbers in the sequence listing in the present specification indicate the following sequences.
  • Example 1 Separation of proteins by reparse zymography
  • the tears are diluted 2-fold with a normal temperature (20 ° C) sample buffer (0.125 M Tris-HCl (pH 6.8) / 4% SDS / 20% glycerol / 0.02% bromphenol blue) did.
  • a normal temperature (20 ° C) sample buffer (0.125 M Tris-HCl (pH 6.8) / 4% SDS / 20% glycerol / 0.02% bromphenol blue) did.
  • 10-15 L of tears diluted with this buffer for electrophoresis was applied to a polyacrylamide gel (0.1% gelatin 0.1% SDS / 10-12% polyacrylamide) containing gelatin as a substrate.
  • Electrophoresis was performed using running buffer (0.025 M Tris-HCl Z0.12 M glycine 0.1% SDS). After the electrophoresis, the gel was incubated with papain solution (0.0075 U papain).
  • the gel was washed with 2.5% Triton X-100 and purified water, and incubated with 50 mM acetic acid-10 IBM cysteine buffer (pH 6.0) for 10 hours. After immersion in 20% TCA for 1 minute, the cells were immersed in a staining solution (Coomassie Brilliant Blue 0.025 ° /./ methanol 40 ° /./ acetic acid 10% / water 49.975%) for 2 hours or more for staining. After staining, wash the gel with the first wash solution (methanol 40 ° / acetic acid 10% / water 50%), then wash the gel with the second wash solution (methanol 5% Z acetic acid 7% Z water 88%). Was.
  • Fig. 1 shows the results of reverse zymography of tears from patients with Behcet's disease and Harada's disease.
  • the amino acid sequence at the N-terminal of the protein was determined using the HPG1005A protein sequencing system (Hewlett-Packard, Palo Alto, CA). That is, after the electrophoresis in Example 1, the stained band was transferred to a PVDF membrane, and the method of Majima et al. [E. Majiraa, et al., J. Biol. Chem. 276 (13), 9792-9799 (1991) ] was used for an amino acid sequence analyzer.
  • the amino acid sequence on the N-terminal side of the 31 kDa protein was as shown in SEQ ID NO: 1. This sequence showed 100% homology with the amino acid sequence at positions 76 to 90 of the known PRP-1 and pHLE1F1 proline-rich proteins. This means that the amino acid sequences of PRP-1 and pHL E1F1 prolin litita titan protein from position 91 onwards are at least 50 ° /. It is considered that the above homology is shown.
  • Example 4 Synthesis of polypeptide of SEQ ID NO: 3
  • Fmoc-Val-Wang resin After swelling Fmoc-Val-Wang resin into which amino acid (Val) corresponding to the C-terminal residue of the polypeptide has been introduced with dimethylformamide (DMF), it is transferred to a reactor of a peptide synthesizer (manufactured by Shimadzu Corporation). The resin was treated with piperidine / DMF to remove the Fmoc group to release amino acids, and washed with DMF. This amino group has the following Fmoc-Ser (tBu) corresponding to the amino acid was condensed by the HOBt / PyBop method.
  • DMF dimethylformamide
  • Fmoc-Pro, Fmoc-Phe, Fmoc-Arg (Pbino, Fmoc-Pro, Fmoc Leu, Fmoc-Ser (tBu), Fmoc-Leu, Fmoc-Gin (Trt), Fmoc-Arg (Pbf) , Fmoc-His (Trt), Fmoc-Gly, 'Fmoc-Arg (Pbf), Fmoc-Arg (Pbf), Fmoc-Pro, Fmoc-Pro, Fmoc-Arg (Pbf),
  • Fmoc-Trp-Wang resin After the Fmoc-Trp-Wang resin was swollen with DMF, it was put into a reactor of a peptide synthesizer (manufactured by Shimadzu Corporation). The resin was treated with piperidine and DMF to remove the Fmoc group to release the amino acid, and washed with DMF. Fmoc-Leu corresponding to the next amino acid was condensed to this amino group by the HOBt / PyBop method. Thereafter, similarly, Fmoc- Pro, Fmoc- Gin (Trt),
  • Fmoc-Glu OtBu N Fmoc-Gin (Trt), Fmoc-Pro, Fmoc-His (Trt), Fmoc-Arg (Pbf), Fmoc-Ala, Fmoc-Pro, Fmoc-Arg (Pbf), Fmoc-Asp (OtBu), Fmoc-Arg (Pbf),
  • Fmoc-Gln (Trt) was sequentially condensed to complete the reaction. After the resin was dried, it was treated with a solution containing TFA for 5 hours according to a conventional method to cut off the peptide from the resin and remove the peptide protecting group. The deprotected peptide was washed with getyl ether and dried. This was dissolved in a solution containing acetonitrile / TFA according to a conventional method, and was eluted with a gradient using an HPLC apparatus, and purified. The purified peptide fraction was collected and freeze-dried to obtain a white powder.
  • Example 6 Preparation of Egret Polyclonal Antibody
  • KLH Keyhole Limbet Hemosian
  • a rabbit egret polyclonal antibody was prepared using the prepared polypeptide-KLH complex as an antigen.
  • the immunized animals were Japanese white egrets (male, 2.5-3 kg)-feathers, and the sensitization was with an adjuvant (first time: complete, second and third time: incomplete) suspensions, intradermally on the back This was done by injection and repeated four times every 14 B. Seven days after the last sensitization, blood was collected from the carotid artery under anesthesia to obtain serum. The serum thus obtained was used as a polyclonal antibody. The increase in the antibody titer in the antiserum was confirmed by Enzymnoassy.
  • Example 7 Western plotting
  • the samples were subjected to SDS-electrophoresis using a 15-25 w / v% gradient polyacrylamide gel in the presence of 5 w / v% 2-mercaptoethanol.
  • SDS-electrophoresis was based on a modified method of Lae orchid li [K. U. Laemmli, Nature, 227, 680-685 (1920)].
  • the protein was electrically transferred to a PVDF transfer membrane (Millipore, Bedford, MA).
  • the perforated polyclonal antibody prepared in Example 6 was used as the primary antibody.
  • Goat anti-Peacock IgG conjugate was used as a secondary antibody.
  • the alkaline phosphatase reaction was carried out on the membrane using nitro blue tetrazolium and 5-bromo-4-oct-3-indolyl phosphat.
  • cysteine protease inhibitory activity was based on the method of Barrett [JA Barrett et al., Methods in Enzymology, 80, 535-561 (1981)]. That is, after preincubating a solution containing 85 mM acetate buffer (H5.5), 2 mM dithiolate, 1 mM EDTA, papain (0.0075 U) and test substance for 15 minutes, Z- Phe-Arg-MCA (80 nM) was added to start the reaction. After reacting at 37 ° C for 10 minutes, acetate buffer (pH 4.0) was added to stop the reaction.
  • acetate buffer pH 4.0
  • the amount of released 4-methyl-7-aminocoumarin was measured using a fluorometer at an excitation wavelength of 360 nm and an emission wavelength of 440 nm. Control values those treated in the same manner with the test substance without addition, those papain no added pressure as the blank value, IC 5. I asked.
  • KLH was bound to the polypeptide synthesized in Example 5.
  • a heron polyclonal antibody was prepared using the prepared polypeptide-KLH complex as an antigen.
  • the immunized animal used two Japanese white egrets (male, 2.5-3 kg), and the sensitization was a suspension of adjuvant (first time: complete, second to sixth: incomplete), and the skin on the back It was performed by intravenous injection and repeated 6 times every 14 days. Seven days after the final sensitization, blood was collected from the carotid artery under anesthesia to obtain serum. The serum thus obtained was used as polyclonal-nanore antibody.
  • Example 10 Purification of Escherichia coli Polynal Antibody
  • the DEAE-Affi-Gel Blue column was equilibrated with 5 volumes of binding buffer (20 mM Tris-HCl, pH 7.5).
  • the sample obtained in (1) was applied to a column, the flow fraction was collected (flow rate 1-3 ml / min), and the unbound protein was eluted with 3 to 15 volumes of the binding buffer.
  • the bound IgG fraction was eluted with an elution buffer (20 mM Tris-HC1, 1 M NaCl, pH 7.5), and the eluted fraction was analyzed by 12% SDS-PAGE under reducing conditions. ⁇ 1 was analyzed to identify an IgG-containing fraction.
  • the IgG-containing fractions were pooled and desalted on a PD-10 column.
  • the sample is 5 ° /.
  • SDS-electrophoresis was performed using a 15-25% (w / v) gradient polyacrylamide gel in the presence of (w / v) 2 -mercaptoethanol.
  • SDS-electrophoresis was based on a modified method of Lae bandit li [KU Laemmli, Nature, 227, 680-685 (1920)].
  • the protein was electrically transferred to a PVDF transfer membrane (Millipore, Bedford, MA).
  • the egret polyclonal antibody obtained in Example 10 was used as a primary antibody.
  • Goat anti-Peacock IgG conjugate was used as a secondary antibody.
  • Alkaline phosphatase reaction was performed on the membrane using nitro blue tetrazolium and 5-bromo-4 -.- coupe raw 3-indolinolephosphate. SDS-PAGE low-range standard as molecular weight marker (Bio-Rad Laboratories, CA) was used.
  • FIG. 2 shows the results of estan printing using three kinds of egret polyclonal antibodies obtained in Example 10 (after treatment with ammonium sulfate, DEAE-Affi-Gel Blue column, and MonoQ column, respectively).
  • Example 10 recognized the 31 kDa protein. Since this polyclonal antibody is an antibody against a protein corresponding to the known amino acid sequence at positions 119 to 134 of PRP-1, the results were as follows: PRP-1 and pHL as partial sequences of the 31 kDa protein. This is considered to indicate that the sequence has a higher homology with the amino acid sequence of the ElFl proline litz titanium pack than the amino acid number 119. Further, it was confirmed that the purified polyclonal antibody obtained in Example 10 had high specificity.
  • Example 1 2 Synthesis of polypeptide of SEQ ID NO: 1
  • Fmoc-His-Wang resin After swelling Fmoc-His-Wang resin with dimethylformamide (DMF), put it into a reactor of a peptide synthesizer (manufactured by Shimadzu Corporation). The above resin is treated with piperidine / DMF to remove the Fmoc group to release the amino acid, and washed with DMF. Fmoc-His (Trt) corresponding to the following amino acid is condensed to this amino group by the HOBt / PyBop method. Thereafter, similarly,
  • Fmoc-Asp (OtBu) is sequentially condensed to complete the reaction.
  • the resin After the resin is dried, it is treated with a solution containing trifluoroacetic acid (TFA) for 5 hours according to a conventional method to separate the peptide from the resin and to remove the peptide protecting group.
  • TFA trifluoroacetic acid
  • the deprotected peptide is washed with getyl ether and dried.
  • Dissolve the solution containing acetonitrile / TFA according to the standard method.
  • gradient elution is performed using an HPLC apparatus, and purification is performed.
  • the purified peptide fraction is collected and lyophilized to give a white powder.
  • HuCAL registered trademark
  • HuCAL-scFv in E. coli TG-1 is amplified with 2XTY medium (hereinafter abbreviated as 2XTY-CG) containing Kucamu ramphenicol (appropriate amount) and 1% glucose. 0D 6 . .
  • 2XTY-CG 2XTY medium
  • Kucamu ramphenicol appropriate amount
  • 1% glucose 0D 6 .
  • the phage is recovered by PEG precipitation, resuspended in PBS / 20% glycerol, and stored at -80 ° C.
  • TG-1 cells infected with the phage are grown in liquid culture, and those obtained after helper virus infection are directly used. Change the carrier (transferrin, BSA) used in each round of Banning. After two paungings, prepare polyclonal phagemid DNA (BioRobot, Qiagen). Next, the polyclonal phage mid DNA is digested with a restriction enzyme, the resulting insert is subcloned into an expression vector, and then transformed into JM83 cells (Gene 33, 103 (1985)). The transformant is subjected to screening.
  • HuCAL registered trademark
  • BSA carrier
  • the resulting transformant is plated on an agar plate supplemented with chloramphenicol (appropriate amount) and 1% dalcose.
  • the obtained coloeie is transferred to 2XTY-CG medium, grown, and then transferred to an expression plate containing 2XTY / chloramphenicol medium. Add IPTG (final concentration 1 mM) and allow to grow.
  • the periplasmic E. coli extract [Current Protocols in Molecular Biology. Wiley, New York, USA] is transferred to an antigen-coated MaxiSorp TM plate (Nunc) and ELISA is performed.
  • the scFv is detected using an anti-FLAG Ml and M2 antibody (manufactured by Kodak), and an anti-mouse IgG antibody-specific phosphatase conjugate (manufactured by Sigma) and an AttoPhos substrate (manufactured by Roche Diagnostics). Then, after growing positive clones, DNA is isolated and the nucleotide sequence is determined.
  • a stuffer matching the restriction site used for HuGAL® design was used as a leader sequence, the VH domain obtained in (3) above, Ligation for ligation of immunoglobulin constant region. Subjecting the Kozak sequence leader sequence (EMBL M 83 133) [:... ⁇ Mol Biol 196 , 947 (1987) ].
  • the constant region of human IgGl (PIRJ00228) is synthesized by cleaving this sequence into overlapping oligonucleotides having a length of about 70 bases. Introduce silent mutations to eliminate restriction sites that are incompatible with HuCAL® design. These oligonucleotides are linked by overlap extension PCR.
  • Cotransfect CH0-K1 cells with an equimolar mixture of an IgG heavy chain expression vector and an IgG light chain expression vector are selected using G418 and Zeomycin (Invitrogen). Next, the obtained culture supernatant of the CH0-K1 cell clone is evaluated by IgG capture-ELISA.
  • the microtiter wells are coated with a heron anti-human IgG (Fcy specific, Dako).
  • Proskin with Tris buffered physiological saline / 5% non-fat dry milk After that, the culture supernatant of the obtained CH0-K1 cell clone is added. After washing several times, goat anti-human kappa or e-chain lipophosphatase conjugate, and p-tropophyl phosphate (manufactured by Sigma) are added to determine the presence or absence of IgG.
  • the obtained CHO-K1 cell clone is grown in RPMI-1640 medium supplemented with 10% ultra-low IgG-FCS (manufactured by Life Technologie).
  • the P H of the culture supernatant was adjusted to 8.0, after subjected to sterilization filtration, and subjected solution Protein A column chromatography (Poros 20A, PE manufactured Biosystems) to purified human IgG.
  • the protein of the present invention is a unique protein that is expressed in diseases such as Behcet's disease and Harada's disease, it is possible to accurately and efficiently diagnose, for example, Behcet's disease and Harada's disease.

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Abstract

L'invention concerne une protéine exprimée spécifiquement, entre autres, dans la maladie de Bechet et l'uvéo-encéphalite de Harada ; un polynucléotide codant pour cette protéine ; un procédé de criblage d'une protéine possédant une activité inhibitrice de la cystéine protéase, caractérisé par l'utilisation de la zymographie inverse ; un procédé de criblage d'un composé ou de son sel, favorisant ou inhibant la fonction de ladite protéine. Selon le procédé de criblage, un polypeptide spécifique d'une maladie, telle que la maladie de Bechet, peut être détecté et identifié. Ainsi, la fonction de la maladie, telle que la maladie de Bechet, peut être examinée. Par ailleurs, le polypeptide peut être utilisé en tant que matériel d'évaluation objective de la maladie de Bechet et le criblage d'un remède contre, entre autres, ladite maladie.
PCT/JP2004/002902 2003-03-05 2004-03-05 Nouvelle proteine et son utilisation Ceased WO2004078975A1 (fr)

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Title
DICKINSON D.P. ET AL: "A major human lacrimal gland mRNA encodes a new proline-rich protein family member", INVEST. OPHTHALMOL. VIS. SCI., vol. 36, no. 10, 1995, pages 2020 - 2031, XP002980398 *

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