[go: up one dir, main page]

WO1999006555A1 - Nouvelle proteine humaine mmp-20 et son utilisation - Google Patents

Nouvelle proteine humaine mmp-20 et son utilisation Download PDF

Info

Publication number
WO1999006555A1
WO1999006555A1 PCT/JP1998/003381 JP9803381W WO9906555A1 WO 1999006555 A1 WO1999006555 A1 WO 1999006555A1 JP 9803381 W JP9803381 W JP 9803381W WO 9906555 A1 WO9906555 A1 WO 9906555A1
Authority
WO
WIPO (PCT)
Prior art keywords
protein
human
salt
mmp
dna
Prior art date
Application number
PCT/JP1998/003381
Other languages
English (en)
Japanese (ja)
Inventor
Alberto M. PENDÁS
Elena Llano Cuadra
Carlos LÓPEZ-OTÍN
Takanori Aoki
Kazushi Iwata
Original Assignee
Fuji Yakuhin Kogyo Kabushiki Kaisha
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Fuji Yakuhin Kogyo Kabushiki Kaisha filed Critical Fuji Yakuhin Kogyo Kabushiki Kaisha
Publication of WO1999006555A1 publication Critical patent/WO1999006555A1/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
    • C12N9/6489—Metalloendopeptidases (3.4.24)
    • C12N9/6491—Matrix metalloproteases [MMP's], e.g. interstitial collagenase (3.4.24.7); Stromelysins (3.4.24.17; 3.2.1.22); Matrilysin (3.4.24.23)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to a novel protein and a novel protein useful in studies relating to tooth development and the process of maturation of enamel, in particular, enamel formation and mineralization of tooth matrix.
  • the present invention relates to a novel human-derived protein, which is a kind of matrix metalloprotease (MMP) having enamelysin activity, and a gene encoding the same. More specifically, the present invention relates to a novel matrix meta-oral protease cloned from a human odontoblast cDNA library [a novel matrix meta-oral protease disclosed in the present invention is expressed by human MP-20 (Matrix metal loprotenase-20)), a DNA containing a nucleotide sequence encoding the DNA, a host cell transformed with the DA, a method for producing the matrix meta-oral protease using the host cell, The present invention relates to an antibody (eg, a monoclonal antibody) that binds to a matrix meta-oral protease protein, and further relates to the use of the protein, some of its peptides and antibodies, and nucleic acids or nucleic acids.
  • MMP matrix metalloprotease
  • Tooth enamel is formed by matrix-mediated biominera lization. In secretion, an extracellular organic matrix is formed by the enamela tissue surrounding the developing tooth bud. Apatite crystals (apatitic crystal li tes) When formed strongly, the enamel protein power of the fine organic matrix 3 ⁇ 4S is secreted continuously, but is degraded by several different proteases to form the original enamel Protein is reduced quantitatively with respect to degradation products. Substitution and subsequent removal of the organic matrix component from the enamel in subsequent development results in an inorganic tissue. Several research groups have detected serine proteinase and meta-oral protease in bushes and eggs during enamel matrix formation. These proteases have been suggested to be involved in the degradation of fine matrix proteins, especially enamel, in the transformation of enamele.
  • the extracircular matrix is composed of complex components such as type IV collagen, floteoglycan, elastin, fibronectin, laminin, and heparan sulfate.
  • a group of enzymes collectively referred to as matrix meta-oral proteases of different specificities (hereinafter abbreviated as TMMP), are involved.
  • MMP-1 interstitial collagenase
  • MMP-2 gelatinase A
  • gelatinase B gelatinase B
  • MP-7 matrilysin
  • MP-7 matrilysin
  • MP-7 Neutrophil Collagenase
  • MMP-10 Stromlysin 2
  • MMP-11 Stromlysin 3
  • P-12 Collagenase Ze 3
  • Amino acid sequences deduced from the cDNA sequences of these MPs show homology, and are known to form MMP families.
  • These rigid Ps are basically N-terminal signal peptides that are removed during secretory production, followed by propeptide domain, Zn-bound horn butterfly domain, proline-rich hinge domain consisting of 5 to 50 amino acids, and C Moxin II is composed of an enzyme-like domain.
  • the present inventors have thought that there exists MP that acts as a factor having enamelysin activity in humans as well as pig, and as a result of various studies using genetic engineering techniques, a new enamel
  • the inventors succeeded in isolating a human gene encoding an active factor, elucidated the entire gene base sequence and amino acid sequence, and led to the present invention. Therefore, the present invention provides a novel human-derived protein which is a kind of MMP having enemalysin activity and is a factor having enamelysin activity other than porcine enamelysin, a method for producing the same, its use, and encoding the protein.
  • the purpose of the present invention is to provide a description of the family and its use, an antibody against the protein, a method for producing the antibody, and a use of the antibody.
  • the human-derived protein cloned according to the present invention which is a factor having enamelysin activity, was named human MMP-20, and the gene encoding the human MMP-20 was named human P-20 gene.
  • the present invention relates to a novel human MMP-20 family, a human-derived protein human MMP-20, and analogs thereof. Further, the present invention relates to a DNA sequence encoding the whole or a part of the novel human MMP-20, a vector having such a DNA sequence, and a host transformed or transfected with such a vector. It also relates to cells. Furthermore, the present invention also encompasses a method for producing recombinant human MMP-20 and its use. It also relates to antibodies that bind to human MMP-20, particularly monoclonal antibodies and hybridoma cells that carry the antibodies.
  • the present invention also relates to a nucleic acid or a nucleic acid oligomer that hybridizes to the human MMP-20 gene, for example, a DNA or RNA probe.
  • detection and measurement reagents for human MMP-20 using the above products, and detection and measurement using the reagents are performed.
  • the present invention also relates to a neutralizing antibody that inhibits enamelysin activity or extracellular matrix activity, in particular, a monoclonal antibody that binds to human P-20.
  • the present invention also relates to a partial peptide of human garden P-20 having no enamelysin activity and a derivative thereof or a salt thereof.
  • the present invention relates to antisense oligonucleotides and derivatives thereof that Pi the transcription or translation of human MP-20, and drugs containing the same as an active ingredient.
  • a human-derived protein or a salt thereof which is characterized by having or not having a property to enamel protein and being a kind of matrix meta-oral protease having substantially the same activity. Or parts thereof;
  • the protein is natural human P-20 or a salt thereof, has substantially the same activity, or has a primary structure conformation substantially the same.
  • amino acid sequence represented by SEQ ID NO: 1 has the Tyr force at position 108, the Cys sequence at position 483, or an amino acid sequence substantially equivalent thereto Any one of the above-mentioned [1] to [3], wherein the human-derived tamper is Substance or its salt or a part thereof;
  • the present invention provides: [22] a method for immunologically measuring the protein or salt thereof according to [1], wherein a monoclonal antibody specifically immunoreactive with the protein or salt thereof of [1] is used as a fita;
  • an immunological assay drug for the protein or salt thereof according to [1] which comprises a monoclonal antibody that specifically immunoreacts with the protein or salt thereof of [1];
  • amino acid sequence represented by SEQ ID NO: 1 having a homology higher than 89 ° and higher than 89 °, preferably 90 ° ′ or more, to the amino acid sequence represented by SEQ ID NO: 1.
  • a human-derived protein having the amino acid sequence represented by the sequence number: 1 in the sequence list, or one or more amino acid residues unique to the protein (for example, 1 to 80, preferably 1 to 60, more preferably 1 to 40, more preferably 1 to 20 and especially 1 to 10) missing ⁇ I analog, specific to the human MMP20
  • One or more amino acid residues e.g., 1 to 80, preferably 1 to 60, more preferably 1 to 40, more preferably 1 to 20, especially 1 to i 0 Pseudo-page liposome substituted with the residue of force M, and one or more (eg, 1 to 80, preferably 1 to 60, more preferably 1 to 40, (Preferably 1 to 20, especially 1 to 10) amino acid residues are selected from the group consisting of ⁇ J-opened addition analogs.
  • nucleic acid characterized by having a base sequence encoding the protein according to any one of [24] to [27];
  • a transformant comprising the vector of the above-mentioned [29]; [31] that the host cell is selected from the group consisting of Escherichia coli, yeast, CH0 cells and COS cells
  • the transformant according to the above [30] which is characterized by:
  • the present invention provides:
  • the present invention provides:
  • nucleic acid having a nucleotide sequence that hybridizes with a nucleotide sequence represented by SEQ ID NO: 2 under stringent conditions;
  • [66] Evaluating the test, the antibody of [11] insolubilized on the carrier and the labeled antibody of [11] above, and binding to the label on the insolubilized carrier or the insoluble carrier.
  • the method according to any one of [1] to [4] above, in which the determination of unlabeled protein is carried out, is performed using the method described in [1] to [4] above.
  • the term “and z or” means that there is both an ⁇ -merging connection and (2) a selective connection, such as “treatment and ⁇ or prevention.
  • FIG. 1 shows an SDS-PAGE showing the enzyme activity of recombinant human P-20.
  • porcine melogenin 200 ng / ⁇ l
  • porcine melogenin 200 ng / ⁇ l
  • the degradation products of amelodidinin (23,000, 22,000 and 20,000,000) are indicated by arrows (lane 1).
  • No degradation products were observed in the presence of T1MP-2 (lane 2).
  • O The molecular weight (kDa) was added to the left side of the figure.
  • FIG. 2 shows chromosome mapping (PCR) electrophoresis of the human MMP-20 gene.
  • FIG. 3 shows chromosome mapping (FISH) of the human MMP-20 gene.
  • FISH chromosome mapping
  • FIG. 4 shows western blotting using an anti-MP-20 monoclonal antibody.
  • the purified recombinant P-20 (21 / lane) was developed by SDS-PAGE (under reducing conditions), transferred to a nitrocellulose membrane, and stained with an anti-MP-20 monoclonal antibody.
  • Lane 1 used monoclonal antibody 200-10B2
  • lane 2 used monoclonal antibody 203-1C7.
  • a natural MMP-20 (e.g., human MMP-20) which is a type of MMP having enamelin activity but has an enamelysin activity other than porcine enamelin, or a protein having an activity substantially equivalent thereto.
  • Transgenic animals such as transgenic mice containing the gene, knockout animals such as knockout mice that specifically delete (delete) the gene, and host cells containing the gene,
  • transgenic animals such as transgenic mice containing the gene, knockout animals such as knockout mice that specifically delete (delete) the gene, and host cells containing the gene,
  • the present invention utilizes the activity disclosed and described herein, and for example, a drug or ⁇ ⁇ containing the activity is provided, and a disease or a disease using the activity tti * is used. Alternatively, treatment of abnormal conditions and Z or prophylaxis, and screening methods are also used.
  • the present invention relates to human P20 or a salt thereof, which has the amino acid sequence represented by SEQ ID NO: 1 in the sequence listing.
  • the human fraction -20 of the present invention is a kind of ⁇ ⁇ ⁇ having enamel protein content and characterized by having an enamel protein content performance other than porcine enamelinsin, and is a factor having enamelase activity and a novel factor. What is necessary is just to have an amino acid sequence.
  • the human -20 of the present invention includes all those having an amino acid sequence represented by SEQ ID NO: 1 in the sequence listing or an amino acid sequence substantially equivalent thereto.
  • the human rigid-20 of the present invention may have a part or ⁇ of the amino acid sequence starting with the Tyr force at position 108 of the amino acid sequence represented by SEQ ID NO: 1 in the sequence listing, or Good. Any having such an arrangement may be included.
  • the human MMP-20 of the present invention is composed of the ATG at positions 14 to 16 and the TAA at positions 1463 to 1446 in the nucleotide sequence represented by the sequence number: 2 in the sequence listing.
  • ⁇ 3 ⁇ 4 sequence (the stop codon TAA from position 1463 to position 1465 may be TGA or TAG). It has a sequence other than porcine enamelisin, and is encoded by a DNA sequence containing a nucleotide sequence having the same effect as that of having enamel content.
  • the nucleotide sequence of the human secreted-20 may be modified (for example, And such modifications may be included.
  • the nucleic acid of the present invention may encode the protein of the present invention or a part thereof, and is preferably a DNA molecule.
  • equivalent nucleotide sequence is, for example, one which hybridizes under stringent conditions with the nucleotide sequence represented by SEQ ID NO: 2 in the sequence listing, or an amino acid substantially equivalent to human MP-20
  • SEQ ID NO: 2 in the sequence listing
  • amino acid substantially equivalent to human MP-20 The one that codes the rooster sequence is a powerful example.
  • the DNA of the present invention containing the nucleotide sequence represented by SEQ ID NO: 2 in the rooster sequence table or a nucleotide sequence equivalent thereto can be obtained, for example, by the following method.
  • a human DNA constructed from various human filaments or cultured cells, etc. was probed with a DNA fragment obtained from porcine ⁇ ⁇ famili (preferably porcine enamelysin) in which the entire sequence or a partial sequence was cloned. Screening of the nomic DNA library, selecting a clone that hybridizes to the probe, determining the base sequence of the inserted sequence of the DNA in the clone, and determining the DNA fragment having a novel P sequence get. If necessary, import sequence of DNA in the clone Can be subcloned.
  • porcine ⁇ ⁇ famili preferably porcine enamelysin
  • a powerful and commercially available sequencing kit such as the Taq Dye Primer One-Cycle Sequencing, which can be determined using the Maxam-Gilbert method, such as the Didoxy method, for example, the M13 Didoxy method, etc. It can be performed using a kit, Sequenase v2.0 kit, etc., or a self-sequence determination device such as a fluorescent DNA sequencer.
  • the polymerase used in the dideoxy method include Klenow 'fragment of DNA polymerase 1, continuous inversion, Taq DNA polymerase, T7 DNA polymerase, and modified T7 DNA polymerase. .
  • the cDNA encoded by the MMP gene is obtained based on the DNA sequence of the new gene P thus obtained by a novel method.
  • a preferable method is reverse transcription PCR (polymerase chain reacti). on coupled reverse transcrption; RT-PCR).
  • a sense primer and an antisense primer are synthesized based on the DNA sequence of the novel P gene.
  • the primer can be prepared by a method known in the art.
  • the primer can be synthesized using an automatic DNA synthesizer by a phosphogester method, a phosphotriester method, a phosphoramidite method, or the like.
  • mRNA is isolated from various human tooth tissues or cultured cells (particularly, human odontoblasts, dental tissues such as dental pulp, etc.). Particularly preferably, mRNA can be isolated from human dental pulp.
  • the isolation of mRNA can be performed by a method known in the art or a method substantially similar to or a modification thereof.
  • T. Maniat is et al., "Molecular Cloning, 2nd ed., Chapter 7, Cold Spring Harbor Laboratory, Cold Spring Harbor, NT (1989); DM Gl over et al. Ed., "DNA Cloning", 2nd ed., Vol. 1, (The Practical Approach Series), 1R Press, Oxford University Press (1995); L. Grossman et al. Ed., "Methods in
  • Oligo (dT) primer can be used in the reverse transcription reaction. Oligo (dT) primers, preferably having 12 to 18 T residues, can be used. For directional cloning, it is also preferable to use a synthetic oligonucleotide primer having a restriction enzyme site linked to the 5 'side of 12 to 18 T residues.
  • a primer is an XbaI oligo (dT) primer adapter.
  • a random hexamer primer increases the possibility of obtaining the 5 'end of mRNA, and this random hexamer primer can be used alone or in combination with an oligo (dT) primer. it can.
  • RNaseP and a harmful agent for example, RNasin (Boehringer) can be added as necessary.
  • 1st strand DM synthesis using mRNA and inversion can be performed by a method known in the art or a method substantially similar thereto or a modified method, but H. Land et al., Nucleic Acids Res. , 9: 2251, 1981; U. Gubler et a. Gene, 25: 263-269, 1983; S. shi.
  • PCR Polymerase 'chain' reaction
  • the PCR reaction is carried out by a method known in the art or a method substantially similar to or a modification thereof, for example, R. Saiki, et al., Science, 230: 1350, 1985. R.
  • RACE rapid amplification of cDNA ends
  • RACE is described in, for example, MA Innis et al. Ed., "PCR Protocols” (MA. Frohman, "a guide to methods and applicarions"), pp. 28-38, Academic Press, New York (1990). It can be performed according to the method described.
  • RT-PCR products can be cloned into plasmid vectors, which can be introduced into highly efficient, competent cells.
  • plasmid vectors for example, commercially available p-Direct (Clontech), pCR-Script T i SK (+) (Stratagene), pGEM-T (Promega), pAmp '''' (Gibco-BR can be used plasmid vector further method that can isolate and purify RNA from it * of cells or tissues, e.g., REX kit, United States Biochmeical; .!
  • the DNA fragment constituting the novel MMP gene obtained as described above may be used, if necessary.
  • a probe as a probe, a cDNA library constructed from various human weaves or cultured cells as described above is screened, and a clone that hybridizes to the probe is selected. The base sequence of the inserted sequence is determined, and a novel DNA fragment constituting the novel MMP gene is identified and obtained.
  • the input sequence of the cDNA in the clone can be subcloned if necessary. Isolate the target DNA based on the determined 3 ⁇ 4S sequence. Can be powerful.
  • a human dental pulp-derived cDNA library is screened, the nucleotide sequence is determined, and the desired DNA is isolated.
  • the labeling of a probe or the like with a radioisotope can be performed using a commercially available labeling kit, for example, a random primed DNA labeling kit (Boehringer Mannhaim).
  • a cDNA library it is necessary to obtain cDNA, which can be obtained, for example, as follows. First, mRNA is isolated from various human tooth tissues or cultured cells (particularly, human odontoblasts, tooth tissues such as dental pulp, etc.) as described above.
  • the mRNA can be isolated by a method known in the art as described above, or a method substantially similar to or a modification thereof, and further according to the method described in the above-mentioned literature. it can.
  • CDNA is prepared using this mRNA and reverse transcriptase.
  • cDNA synthesis using mRNA and reverse transcriptase can be performed by a method known in the art or a method substantially similar thereto or a modification thereof.
  • a cDNA library can be constructed using a phage vector and a plasmid vector.
  • transformation of host cells such as bacteria can be performed, for example, by the calcium method, the rubidium-calcium method, the calcium / manganese method, the TFB high-efficiency method, the FSB
  • the method can be carried out by a method known in the art such as a colony method or an electroborane-on method or a method substantially similar thereto (D.
  • a commercially available cDNA library derived from various tissues can also be used directly.
  • a cDNA library commercially available from gene, Invitrogen, C 1 tech, etc. can be used.
  • PCR it is also possible to carry out PCR using the cDNA prepared in this manner as type III.
  • highly conserved amino acid sequences eg, precursors selected from amino acid sequences obtained from the abundant MMP family (particularly porcine enamelysin), whose entire or partial sequence has been cloned. (Pro-body) and the sequence near the cut part of the active ft) and the region specific to MMP Amino acid sequence (for example, the amino acid sequence in the transmembrane domain region at the C-terminal side, the amino acid sequence in the region between the probe peptide domain and the «domain, the active site of the « domain Based on the amino acid sequence of the region), create a digieneated * primer.
  • PCR is performed. PCR can be performed as described above.
  • the obtained PCR product can be cloned in the same manner as described above, the nucleotide sequence of the obtained PCR product can be determined, and a DNA fragment having a novel MMP gene sequence can be obtained.
  • the target DNA can be isolated by screening various cDNA libraries in the same manner using this DNA fragment as a probe.
  • probe DNA identify the new 20 ⁇ -20 gene from the gene library by screening, and code for the new ⁇ -20 gene obtained from the cloned DNA insert.
  • porcine enamelysin (Gene, 183: 123-128, 1996) is used, and it can be labeled and used as a probe.
  • the label can be a DNA labeling kit, for example, a random primed DNA labeling kit Bok (Boehri nger Mannhaim), etc.
  • the DNA probe was ⁇ ®3 ⁇ 4 the [a - j 2 P] dCTP (Amersham ) Can be used to obtain a radioactive probe.
  • Hybridization using the labeled DNA fragment and a gene library prepared from human tissues and cells for example, a human PI artificial chromosome dienomic library (Human Genome Mapping Resource Center).
  • a human PI artificial chromosome dienomic library Human Genome Mapping Resource Center.
  • the human dental pulp cDNA library can be constructed, for example, in a phage such as: igtl0, and then infecting it with a host bacterium such as a bacterium C600hfl strain to form a plaque. it can.
  • the black formed as described above is transferred to a membrane such as a nylon filter, and then, if necessary, subjected to a treatment, an immobilization treatment, a washing treatment, and then transferred to the membrane.
  • the resulting DNA is reacted with a denatured labeled probe DNA fragment, if necessary, in a hybridization buffer.
  • the hybridization process is usually about 40. C to about 80 ° (:, more preferably about 55. (: to about 65 ° C, about 15 minutes to about 24 hours, more preferably about 1 hour to about 4 hours)
  • the hybridization treatment is performed at about 60 ° C. for about 2 hours, and the hybridization for hybridization is not limited to those commonly used in the art.
  • the denaturation treatment of the transferred membrane includes a method using an alkali denaturing solution. After the treatment, it is preferable to carry out the treatment with a neutralizing solution or a buffer solution, and the membrane is usually immobilized at about 40 ° C to about 100 ° C, more preferably at about 70 ° C to about 90 ° C. By baking for about 15 minutes to about 24 hours, more preferably for about 1 hour to about 4 hours For example, baking of the filter at about 80 ° C for about 2 hours is used to immobilize the transferred membrane.
  • washing solution for example, 50m Tris-HCl buffer containing 1M NaCl, Im EDTA and 0. ⁇ % Sodium Dodecyl sul fate (SDS), pH 8.0, etc.
  • the membrane of the nylon filter or the like can be selected from those commonly used in the field, for example, a nylon filter [Hybond-N, Amsham], etc. Can be mentioned.
  • alkali denaturing solution can be selected from those commonly used in the art, and examples of the alkali denaturing solution include 0.5M NaOH and 1.5M NaCl.
  • examples of the neutralizing solution include a 1.5 M NaCl-containing 0.5 M Tris-HC1 buffer, pH 8.0, and the like.
  • examples of the buffer include: 2 XSSPE (0.36 M NaCl, 20 mM NaH 2 P0., And 2 mM EDTA).
  • the pre-hybridization treatment may be performed, for example, using a pre-hybridization solution [50% formamide, 5 x Denhardt's solution (0.2% ⁇ serum albumin, 0.2% polyvinylpyrold one), 5 x SSPE, 0. 1% SDS.100 zg / ml heat-denatured salmon sperm DNA] etc. at about 35 ° C to 50 ° C, preferably about 42 ° C, for about 4 to 24 hours, preferably for about 6 to 8 hours. Force that can be Performed by Reaction Under these conditions, those skilled in the art can determine more preferable conditions by repeating experiments as appropriate. Denaturation of the labeled probe DNA fragment used for hybridization is, for example, about 70 ° C i00.
  • the hybridization can be carried out by a method known per se or a method analogous thereto.
  • the stringent conditions are, for example, about 1550 m, preferably about 150 m in sodium concentration.
  • the filter is thoroughly washed, and the labeled probe other than the DNA fragment that has undergone the specific hybridization reaction is removed.
  • the filter can be washed and selected from those commonly used in the art. For example, 0.5 x SSC containing 0.1% SDS (0.15M NaCl 15m citrate) It can be carried out by washing with a pad.
  • the hybridized black can be typically detected by autoradiography, but can also be appropriately selected from methods used in the art and used for the black detection.
  • the plaque corresponding to the detected signal is suspended in an appropriate buffer, for example, an SM solution (50 mM In-Tris-HCl buffer containing 100 mM NaCl and 10 M MgSO: pH 7.5), and then the phage suspension is suspended.
  • the solution is diluted appropriately to infect E. coli and is obtained; the bacteria are cultured to obtain the desired recombinant phage from the cultured: ⁇ bacteria.
  • the above-described probe DNA can be used to repeatedly screen the target recombinant phage, such as the genetic library and cDNA library, by hybridization. .
  • the target recombinant phage can be obtained by subjecting the cultured phage to extraction, centrifugation and the like from the bacteria.
  • the obtained phage particles can be purified and separated by a method commonly used in the art.For example, Glyce Mole Gradient® 1-core separation method (Molecular clonin g, a laboratory manual, ed. Maniatis, Cold Spring Harbor Laboratory, 2nd ed. 78, 1989). From the phage particles, DNA can be purified and separated by a method commonly used in this field.For example, the obtained phage is suspended in a TM solution (10 mM MgS (L containing 50 mM Tris-HCl buffer, pH 7.8)).
  • TM solution 10 mM MgS (L containing 50 mM Tris-HCl buffer, pH 7.8)
  • the target DNA can also be obtained by subcloning, etc. For example, subcloning is performed using Escherichia coli as a host and a plasmid vector. For such subcloning Ri resulting DNA also centrifuged as above, Fueno Ichiru extraction, purified fraction separated by methods such as E evening Nord precipitation.
  • the nucleotide sequence of the DNA thus obtained for example, the single-stranded D ⁇
  • a nucleotide sequence can be sequenced in the same manner as described above.
  • a fluorescent DNA sequencer Model 373A (Applied Biosystems)
  • the sequence can be determined using a Taq dye primer cycle sequencing kit (Applied Biosystems) or the like.
  • Applied Biosystems Applied Biosystems
  • a primer is designed for the cDNA of the novel putative MP gene.
  • the sense primer is preferably selected and synthesized from the exon site at the 5 ′ end of the analyzed putative novel MMP gene, and the antisense primer is preferably the analyzed novel novel MMP. It can be synthesized by selecting from the exon site at the 3 'end of the gene putative for MMP, and more preferably from the exon region used for the synthesis of the sense primer.
  • the remaining cDNA may aim to obtain the full length at one time, but it is possible to design and synthesize multiple primers using the exon site (multiple exon sites) that have been
  • the PCR is designed and performed, and the DNA fragment whose nucleotide sequence is determined in this manner is raised, the entire nucleotide sequence of the cDNA of the gene is determined, and the cDNA of the fragment is obtained from the DNA fragment cloned based on it.
  • ply Is preferably an oligonucleotide consisting of 5 or more bases, more preferably an oligonucleotide consisting of 1825 bases.
  • the primer is synthesized using an automatic DNA synthesizer, for example, a model 381A DNA synthesizer Applied Biosystems.
  • a 1st strand cDNA is prepared by inversion using mRNA isolated from human teeth, especially human odontoblasts, dental pulp and the like.
  • the pulp of the tooth obtained by Si can be obtained by using a commercially available mRNA extraction / separation kit such as Trizol Reagent, Life Technologies, or a cDNA synthesis kit (mRNA extraction kit
  • poly (A) -RNA is added to 5X RT buffer (containing 250 mM Tris-HCl (pH 8.3), 50 mM MgCl, 375 mM KC1, 50 mM ⁇ ), dNTPs (Doxy Nucleoside triphosphate dATP, dGTP, dCTP, (mixture of ⁇ ), oligo dT, primer, RNase inhibitor (Boehringer) MLV RT (Gibco-BRL) or Superscript RT plus (Li fe Technologies) and deionized distillation This can be achieved by mixing with water and incubating for about 1 hour at about 37 ° C.
  • 5X RT buffer containing 250 mM Tris-HCl (pH 8.3), 50 mM MgCl, 375 mM KC1, 50 mM ⁇ ), dNTPs (Doxy Nucleoside triphosphate dATP, dGTP, dCTP, (mixture of ⁇ ), oligo
  • the obtained 1st strand DNA is combined with a primer designed based on the exon of the analyzed gene in a 10 ⁇ reaction buffer.
  • dNTPs mixture of deoxynucleoside triphosphates dATP, dGTP, dCTP, ( ⁇ )
  • dNTPs mixture of deoxynucleoside triphosphates dATP, dGTP, dCTP, ( ⁇ )
  • dNTPs mixture of deoxynucleoside triphosphates dATP, dGTP, dCTP, ( ⁇ )
  • Mix with Taq DNA polymerase and deionized distilled water , Gene neAmp 2400 PCR system, Perkin-Elmer / Cetus, etc.
  • the number of cycles for amplification can be set to an appropriate number according to the purpose.
  • PCR cycle conditions include, for example, Denaturation 90 95 ° C 5 100 seconds, annealing 40 60 ° C 5 150 seconds, extension 65 75 ° C 30 300 seconds cycle, preferably denaturation 94 ° C 15 seconds, annealing 58 ° C 15 seconds, extension 72 C
  • the time and the time of the cleaning are il: Appropriate values can be selected by experiment, and the denaturation and extension Sit time also depends on the expected chain length of the PCR product.
  • the annealing reaction is usually performed by hybridizing the primer with type I DNA. It is strongly preferable to change according to the Tm value of the lid.
  • the extension time is generally about 1 minute per lOOOObp of chain length, but it is possible to select a shorter time.
  • the resulting PCR product is usually subjected to 1-agarose gel electrophoresis, cut out from the gel as a specific band, and DNA is extracted using a commercially available extraction kit such as gene clean kit or Stratagene.
  • the extracted DNA is cleaved with an appropriate restriction enzyme and, if necessary, purified, or, if necessary, phosphorylated at the 5 'end with T4 polynucleotide kinase or the like, and then a pUC vector such as pUC18.
  • the ligation is performed on an appropriate plasmid vector, and an appropriate competent cell is transformed.
  • the nucleotide sequence of the cloned PCR product is analyzed.
  • a 5' end cDNA of the gene of interest is obtained using a designed primer.
  • a primer designed based on the base sequence of the exon site is used to construct the 3' end of the cDNA of the gene. Then, if necessary, primers designed using these primers and the base sequence of the 5'-end cDNA and the 3'-end cDNA of the obtained cDNA of the gene are obtained.
  • human tooth tissue especially human odontoblasts
  • 1st strand cDNA produced by reverse transcriptase from mRNA isolated from dental pulp, type III, amplify by PCR, and cDNA of the gene concerned Can be obtained.
  • the primer on the 5 'end side is selected so as to have at least an initiation codon or amplifying the initiation codon, and the primer on the 3' end side contains at least a stop codon. Or it is preferable to select so as to enable amplification including the stop codon.
  • PCR cycle conditions include, for example, denaturation at 92 to 95 ° C for 10 to 20 seconds, annealing at 55 to 60 ° C 10 to 30 seconds, extension 65 to end 5.
  • the obtained PCR product is cloned in the same manner as described above, and its nucleotide sequence is determined.
  • primers are designed based on the determined DNA base sequence, and screening is performed using these primers and cDNA libraries derived from various animal cells (for example, cDNA libraries derived from various human cells). Thus, a desired clone can be obtained in the same manner.
  • PCR amplification can be performed using these primers to obtain a target gene, a novel gene, a fragment thereof, and the like. Thus, it is possible to search for a PCR product that has homology to human MMP-20 but has a novel sequence.
  • a clone containing the target DNA (for example, as a recombinant phage) can be obtained.
  • the full length of the determined nucleotide sequence of the DNA of the gene isolated from this cloned recombinant phage is 1674 bp, and the sequence shown in SEQ ID NO: 2 in the rooster sequence table is obtained. Is recognized. When the base sequence described in SEQ ID NO: 2 in the sequence listing was searched using GBNBANK / EMBL DNA Data Base, no identical sequence was found.
  • the obtained DNA fragment is used in a suitable vector as described in detail below, for example, a vector such as plasmid pEX, pMAMneo, pKG5, etc., and a suitable vector as described in detail below, for example, It can be expressed in E. coli, yeast, CH0 cells, COS cells, etc.
  • the DNA fragment may be used as it is or as an appropriate control sequence.
  • mice examples include sill animals, for example, mice, rats, egrets, guinea pigs, and horses.
  • a transgenic animal can be prepared by introducing the DNA fragment into a fertilized egg of an animal such as a mouse.
  • Confirmation of the human MMP-20 gene product can be performed using animal cells suitable for it, such as C0S-1 cells transfected with human MP-20 gene.
  • the method of introducing this gene into animal cells such as mammals can be carried out by a method known in the art or a method substantially similar thereto.
  • a calcium phosphate method for example, FL Graham et al., Virology, 52: 456, 1973, etc.
  • DEAE-dextran method eg, D. Warden et al.. J. Gen. Virol., 3: 371, 1968, etc.
  • electro-volatilization Methods eg, E.
  • the gene products produced by animal cells transfected with the human MP20 gene can be analyzed by immunoprecipitation experiments using anti-human MP-20 monoclonal antibody.
  • - Human MMP-20 Plasmids containing genes include host cells commonly used in genetic engineering (eg, prokaryotic host cells such as easy bacteria and Bacillus subtilis, eukaryotic host cells such as yeast, CH0 cells, COS cells, and insects such as Sf21). Any plasmid can be used as long as it can express the DNA in a cell host.
  • a codon suitable for expression in the selected host cell ⁇ may be introduced, a restriction enzyme site may be provided, and Selection of linkers, adapters, etc. that are useful for binding the target gene, such as control sequences for promoting expression, promoter sequences, etc., as well as control of substance resistance, metabolism, etc. And the like.
  • an appropriate promoter for example, a plasmid using ⁇ bacterium as a host, includes tryptophan (trp) promoter, lactose (lac) promoter, tryptophan 'lactose (tac) promoter, and lipoprotein (lpp) promoter.
  • trp tryptophan
  • lac lactose
  • tac tryptophan 'lactose
  • lpp lipoprotein
  • a 40-rate promoter, an MMTV LTR promoter, an RSV LTR promoter, a CMV promoter, an SR promoter, and the like can be used.
  • a plasmid using yeast as a host can use the GAL1, GAL10 promoter, and the like.
  • Plasmid vectors suitable for expression in E. coli include pAS, KK223 (Pharmacia), pMC1403, pMC931, pKC30, and pRSET-B (Invitrogen).
  • Plasmids that use animal cells as hosts include SV40 vector, polyoma virus vector, vaccinia virus vector, retrovirus vector, etc., for example, pcD, pcD-SRa, ⁇ , Strong mention is made of pCEV4, pME18S, pBC12B I, pSG5 (Stratagene) and the like.
  • Examples of a plasmid using yeast as a host include a Yip-type vector, a YEp-type vector, a YRp-type vector, a YCp-type vector, and, for example, pGPD-2.
  • host cells in the case of a host cell bacterium, for example, those derived from the ⁇ !
  • Bacterium K12 strain may be mentioned.
  • DH10B HB101, C106K JM109, STBL2, BL21 (DE3) pLysS and the like.
  • the host cell is an animal cell, such as African green monkey fibroblast-derived C0S-7 cell, COS-1 cell, CV-1 cell, mouse fibroblast-derived COP cell, MOP cell, W0P cell, Chinese hamster Cell-derived CH0 cells, CHO DHFR "cells, human HeLa cells, mouse cell-derived C127 cells, mouse cells derived from NIH 3T3 cells, and the like.
  • Insect cells include the dystrophic virus (Bombyx mor i nucl ear pol yhedros isvi rus) or a substance derived therefrom is used as a vector, for example, using silkworm larvae or silkworm culture cells, for example, BM-N cells.
  • Dytrophic virus Bostox mor i nucl ear pol yhedros isvi rus
  • silkworm larvae or silkworm culture cells for example, BM-N cells.
  • the genetic method of the present invention involves modifying or converting restriction enzymes, inversions, or DNA fragments known or commonly used in the art to structures suitable for cloning.
  • DNA modification / digestion, DNA polymerase, terminal nucleotidyl transferase, DNA ligase, etc. I can do it.
  • restriction enzyme include RJ Roberts, Nucleic Acids Res., 13: rl65, 1985; S. Linn et al. Ed. Nucleases, p. 109, Cold Spring Harbor Lab., Cold Spring Harbor, New York, 1982; RJ Roberts, D. Macelis, Nucleic Acids Res., 19: Suppl. 2077, 1991 and the like.
  • the inverting element examples include a reverse transcriptase derived from mouse Moloney leukemia virus (MMLV) and an inverted avian myeloblastosis virus (AV) derived from avian myeloblastosis virus (AV).
  • MMLV mouse Moloney leukemia virus
  • AV avian myeloblastosis virus
  • No. RNase H-deficients and the like can be preferably used as the invertor, and in particular, modified MLV RTs lacking RNase H activity can be preferably used, and more preferably those having high heat stability.
  • Suitable reverse transcriptases include MMLV RT (Gibco-BRL), Superscript RT plus (Life Technologies) and the like.
  • DNA polymerases examples include ⁇ I bacterium DNA polymerase, Klenow fragment, which is an inducer thereof, phage T4 DNA polymerase, phage T7 DNA polymerase, and thermostable DNA polymerase.
  • terminal nucleotidyl transferase examples include the 3'-OH terminal described in R. Wu et al. Ed., "Methods in Enzymology", Vol. 100, p. 96, Academic Press, New York (1983). TdTase to add deoxynucleotides (dMP).
  • DNA modification ⁇ examples include exonucleases and endonucleases, such as snake venom phosphodiesterase, spleen phosphodiesterase, fungal DNA exonuclease, and fungal DNA exonuclease 111, and ⁇ fungal DNA exonuclease. Examples include VII, exonuclease, DNase I, nuclease Sl, and Micrococcus nuclease. Examples of the DNA ligase include: ⁇ i bacteria DNA ligase, T4 DNA ligase and the like.
  • Vectors suitable for constructing a DNA library by cloning the DNA fragment iS ⁇ include plasmid, sphage, cosmid, P1 phage, factor F, YAC and the like.
  • Charon 4A, Charon 21A, AtlO, gtll, ADASHIK F1X1I, AEMBL3 ⁇ TM (Stratagene) and the like can be mentioned.
  • 111 the amino acid sequence of human MMP-20; the substitution of one or more amino acids, deletion, insertion, transposition, Protein can be produced.
  • Transformants transformed with an expression vector containing a nucleic acid encoding the protein of the present invention can be expressed at a high level by repeatedly performing clones using an appropriate selection marker as necessary. It is possible to obtain a cell line having stable performance. For example, in a transformant using animal cells as the inn ⁇ spores, the dhfr residue was used as a selection marker ⁇ , the MTX concentration was gradually increased, and culture was performed to select resistant strains. It can amplify the DNA that encodes the protein and obtain a cell line that can achieve higher expression.
  • the transformant of the present invention can be cultured under conditions in which the nucleic acid encoding the protein of the present invention can be expressed, and the target substance can be accumulated.
  • the transformant can be cultured in a medium commonly used in the art.
  • a transformant using a prokaryotic host such as a bacterium or Bacillus subtilis, or a yeast as a host can preferably use a liquid medium.
  • the medium contains a carbon source, a nitrogen source, inorganic substances and the like necessary for the growth of the transformant. .
  • Examples of carbon sources include glucose, dextrin, soluble starch, sucrose, etc.
  • Examples of nitrogen sources include ammonium page, nitric acid page, corn chip liqueur, peptone, casein, meat extract, malt extract, soybean meal
  • Inorganic or organic substances such as potato extract, and inorganic substances include, for example, calcium chloride, sodium dihydrogen phosphate, magnesium chloride, calcium carbonate and the like.
  • yeast, vitamins, casamino acids, growth promoting factors and the like may be added. If necessary, a drug such as 3 ⁇ -indolylacrylic acid can be added to make the promoter work efficiently.
  • the ⁇ ⁇ of the medium is preferably about 5 to 8 force.
  • cultivation of Escherichia coli is usually performed at about 15 to 45 ° C. for about 3 to 75 hours, and if necessary, aeration and stirring may be added.
  • a transformant in which the host is an animal cell
  • a MEM medium containing about 5 to 20% fetal bovine serum, PRMI 1640 ⁇ ground, or a DMEM medium is used as a medium.
  • the pH is about 6-8.
  • Cultivation is usually carried out at about 30 ° C to 40 ° C for about 15 to 72 hours, and aeration and stirring are added as necessary.
  • the cells or cells are collected by culture, by a known method, suspended in an appropriate buffer, and lysed by ultrasonication, lysozyme and / or freeze-thaw.
  • a method of disrupting cells and then obtaining a crude extract by centrifugation or filtration can be used as appropriate.
  • the buffer may contain a protein denaturant such as urea or guanidine hydrochloride, or a surfactant such as Triton X-100 (trade name) or Wien 80 (trade name).
  • the supernatant is separated from the cells or cells by a method known per se, and the supernatant is collected.
  • the target product contained in the culture supernatant or extract thus obtained can be purified by appropriately combining known separation and purification methods, for example, ammonium sulfate precipitation method.
  • Gel filtration by Sephadex, etc. for example, ion-exchange chromatography using a carrier having getylaminoethyl group or carboxymethyl group, etc., hydrophobicity such as butyl group, octyl group, phenyl group, etc.
  • the antibody can be purified and separated by treating it with affinity chromatography, such as monoclonal antibodies, which are antibodies that specifically react with the antibody.
  • affinity chromatography such as monoclonal antibodies, which are antibodies that specifically react with the antibody. Examples thereof include gelatin-agarose-affinity-1 chromatography-1 and heparin-agarose-chromatography-1.
  • the obtained protein of the present invention can modify the amino acid residue contained therein by a lignological method, and can also be used for peptidases such as pepsin, chymotrypsin, papain, and bromelain. It can be modified using enzymes such as endopeptidase and exopeptidase, or can be partially degraded to give its name.
  • Proteins of the present invention C-terminal, usually a carboxyl group (-C00H) or Cal Bokishireto be - - (C0NH 2) or ester (-CO OR) (C00-) in which Chikaraku, C-terminal, the amino de Good.
  • R in the ester e.g., methyl, E chill, n- propyl, C WINCH ⁇ alkyl groups isopropyl or n- butyl
  • C 3 such as cyclohexyl cyclopentyl, cyclohexylene - 8 cycloalkyl Groups, for example, C t) ,, and aryl groups such as phenyl and mono-naphthyl; and C such as phenyl-alkyl groups such as benzyl and phenyl or ⁇ -naphthyl-alkyl groups such as mono-naphthylmethyl; i;
  • pinocyloxymethyl groups widely used as oral esters are used.
  • Tano of the present invention A protein having a carboxyl group (or carboxylate) other than the C-terminus and having a hydroxyl group amide or esterified is also included in the protein of the present invention.
  • ester of ⁇ for example, the above-mentioned ester of C3 ⁇ 4 is used.
  • the amino group of the methionine residue of N is protected by a protecting group (for example, C 1, such as a formyl group or acetyl, C 1-5 alkyl mono-lponyl group or the like).
  • 6- protected group N-terminal force ⁇ Pyroglutaminolization of ripped glutamyl group cleaved in vivo, substitution of amino acid side IS ⁇ (eg, -OH, -C00H , Amino, imidazole, indole, guanidino, etc.) Strongly protected with a suitable protecting group (for example, formyl, acetyl, etc., 6- acyl, etc.) It also includes complex protein such as so-called sugar protein combined. In addition, it is expressed as a fusion protein when it is produced by the recombinant method, It may be converted and processed into a substance having substantially the same biological activity as natural human MP-20 in vivo or in vitro.
  • the biological activity may include having an immunological activity, for example, having an objectivity.
  • the human-derived protein of the present invention is different from a natural protein in that one or more amino acid residues are different from a natural protein in that the amino acid residues are the same, and a position of one or more amino acid residues is a natural protein. It may be different.
  • the human-derived protein of the present invention has one or more amino acid residues specific to human MP-20 (for example, 1 to 80, preferably 1 to 60, more preferably 1 to 40, Preferably 1 to 20, especially 1 to 10, etc. missing missing analog, one or more unique amino acid residues (eg, 1 to 80, preferably 1 to 60, more preferably Is 1 to 40, more preferably 1 to 20, particularly 1 to 10, etc.
  • the human MMP-20 of the present invention may include those having a primary structure conformation substantially equivalent to natural human MMP-20 or a part thereof. It is considered that those having substantially the same biological activity as natural human MP-20 may be included. It can also be one of the naturally occurring variants.
  • the human-derived protein of the present invention includes, for example, those having a homology higher than 89 ° to the amino acid sequence represented by SEQ ID NO: 1 in the sequence listing, and more preferably 90%. Those having a homologous amino acid sequence of! 3 ⁇ 4 or more are strongly cited.
  • a part of the human-derived protein of the present invention is a part of the human-derived protein. That is, a partial peptide of the protein) which is substantially the same as the human MMP-20 of the present invention may be shifted.
  • the partial peptide of the protein of the present invention has at least 5 or more, preferably 20 or more, more preferably 50 or more preferably 70 or more of the constituent amino acid sequences of the human MMP-20 of the present invention.
  • Preferable examples include a peptide having an amino acid sequence of 100 or more, more preferably 200 or more.
  • a peptide having an amino acid sequence of 100 or more more preferably 200 or more.
  • SEQ ID NO: 1 amino acid sequence represented by SEQ ID NO: 1 in the sequence listing.
  • substantially equivalent means that the activities of the proteins, for example, catalytic activity, physiological activity, and biological activity are substantially the same. Furthermore, the meaning of the term may include the case where the substance has substantially the same activity. Examples of the substantially same activity include a matrix meta-oral protease activity and an enamel. It can be mentioned that it has a property for protein, and also a property for amelodidinin. The term “substantially the same activity” means that they have the same active and qualitative properties, such as being physiologically, pharmacologically or biologically the same.
  • the activity such as matrix metalloprotease activity is equivalent (for example, about 0.001 to 1000 times, preferably about 0.01 to 100 times, more preferably about 0.1 to 20 times, more preferably about 0.1 to 20 times. It is preferable that the activity is about 0.5 to '2 times), but the quantitative factors such as the activity of these activities and the molecular weight of the protein may be different.
  • amino acid substitutions, deletions, or insertions often do not significantly alter the physiological growth or chemical properties of polypeptides, and in such cases, the substitutions, deletions, etc. Or the inserted polypeptide will be substantially identical to those without such substitutions, deletions or insertions. O Substantially the amino acid in the amino acid sequence.
  • ffi (hydrophobic) amino acids include alanine, phenylalanine, oral isin, isoleucine, 'phosphorus, proline, tributofan, methionine, and the like.
  • the basic amino acid include arginine, lysine and histidine, and examples of the negatively charged amino acid (acidic amino acid) include aspartic acid and glutamic acid.
  • the protein of the present invention and some of its peptides can be converted into salts by a method known per se or a method analogous thereto when they are obtained as an awake form.
  • the if ⁇ obtained as a salt can be converted into a free form or another salt by a method known per se or a method analogous thereto.
  • the salt of the protein of the present invention and some of its peptides are preferably physiologically acceptable or pharmaceutically acceptable, but are not limited thereto.
  • Such salts include, for example, salts with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like, for example, sulfuric acid, formic acid, maleic acid, fumaric acid, succinic acid, citric acid, tartaric acid.
  • salts with organic acids such as linco, benzoic acid, methanesulfonic acid, ⁇ -toluenesulfonic acid, and benzenesulfonic acid.
  • examples of the salt include an ammonium salt, for example, a salt with an organic base such as ethylamine, dimethylamine, trimethylamine, and hydroxyshetylamine.
  • Natural ⁇ (particularly human 20-20) or a substance substantially equivalent thereto, which is a kind of ⁇ which has an affinity for the enamel protein of the present invention thus obtained and is an enamelin activator other than porcine enamelisin, or Proteins or salts thereof, or their partial peptides, whose remarkable activities are to have substantially the same activity, are involved in research on the development and search of enzyme inhibitors, drug development research, and human 20-20 involvement It can be used to study biological phenomena and reactions that can be considered, can be used to generate antibodies against it, and can be used to research specific analytes or analytes You can also.
  • the present invention provides a DNA sequence encoding the above-described polypeptide, a human MMP-20 polypeptide having ⁇ or a part of natural characteristics, and an analog or derivative thereof. Includes coding DNA roosters.
  • the DNA sequence of the present invention has been known so far and relates to the amino acid sequence of a mammalian protein, the use of such information is also encompassed by the present invention.
  • Such uses include, for example, the design of probes for the detection of genomic DNA and cD ⁇ A in humans, particularly for mammals encoding human -20 and related proteins, particularly humans.
  • the DNA sequence of the present invention may be, for example, the genomic DM and cDNA of a mammal, particularly preferably a mouse, encoding human P-20 and related proteins. It is useful as a probe for detecting shrinkage.
  • the PCR method and, furthermore, the reverse method: PCR method using nitrogen (RT) (RT-PCR) can be used.
  • the human P-20 cDNA and its related DNA were cloned, and the number of glue sequences was determined based on the amino acid sequence deduced from the human MMP-20 cDNA sequence determined by rooster sequence.
  • DNA obtained by the present invention (for example, DNA encoding human MMP-20) is used as a pair In transferring, it is generally advantageous to use it as a DNA fragment or by binding the DNA downstream of a promoter capable of expressing the DNA in animal cells.
  • a promoter capable of expressing the DNA for example, if ⁇ , which introduces human MP-20 DNA into a mouse, a gene that binds human MMP-20 DNA derived from an animal with high homology to it, downstream of various promoters capable of expressing it in animal cells.
  • a fertilized egg such as a mouse fertilized egg, for example, a mouse fertilized egg
  • transgenic mice that produce high levels of human MMP-20 can be produced.
  • the mouse is not particularly limited to a pure mouse, but includes, for example, C57BL / 6, Balb / C. C3H, (C57B / SxDBA / Z, (BDF,)).
  • a ubiquitous expression promoter such as a virus-derived promoter or metallothionein can be used preferably, and the human MP-20 DNA is used as a guide, and it is used in a recombinant retrovirus.
  • a mouse fertilized egg into which the target DNA has been introduced can be grown in a foster parent mouse such as, for example, an ICR.
  • DNA obtained by the present invention eg, DNA encoding human MP-20
  • DNA encoding human MP-20 DNA encoding human MP-20
  • the presence of DNA that encodes human P-20 in the germ cells of the animal after DNA transfer indicates that the offspring of the animal produce the human MP-20 in all of its germ cells and somatic cells
  • the offspring of this type of animal that has inherited the gene have the potential to express the human P-20 in all of its germinal and somatic cells.
  • the animal After confirming that the human MMP-20 DNA-transferred animal stably retains the gene by breeding, the animal can be bred and passaged in a normal environment as the DNA-bearing animal. In addition, by crossing the animals having the target DNA, a homozygous animal having both the homologous chromosome and the introduced gene is obtained. Passage can be increased to have the DNA. In the animal into which the human MMP-20 DNA has been introduced, since the human MMP-20 protein is highly expressed, screening for an inhibitor (inhibitor) for the human MMP-20 protein is performed. Useful as animals. Antisense oligonucleotides that can inhibit the replication of the human marauder P-20 gene, such as antisense DNA It is useful as an animal for any screening.
  • This transgenic animal can also be used as a cell source for tissue culture
  • tissue of transgenic mice directly analyze DNA or RNA in the tissue of transgenic mice, or analyze protein proteins associated with MMPs by analyzing the protein filaments expressed by the transgenic mice.
  • the cells of the tissue having the P-20 are cultured by standard tissue culture techniques and used to study the function of cells from tissues difficult to cultivate, for example, from teeth or other tissues. it can.
  • the use of the cells can contribute to the development of medicines that enhance the functions of various types, for example.
  • MP-20 can be isolated and purified. Technologies related to transgenic mice and the like are described, for example, in Blinster, R. Shi, et al., Pro at l. Acad. Sci.
  • Mutant mice that have mutations in the gene obtained in the present invention (eg, DNA encoding mouse awake P-20 corresponding to human MMP-20) and do not express mouse MMP-20 at all are Can be produced.
  • a mutant gene obtained by inserting a gene cassette comprising a neo- resistant poly yA addition signal into an exon located near the center of a genomic DNA of about 8 kb including 4 kb before and after the translation initiation codon of the gene and near the translation initiation codon. It is possible to construct a targeting vector.
  • Examples of the gene cassette to be inserted include a DTA cassette, a tk cassette, and a lacZ cassette in addition to the neo resistance gene cassette.
  • the targeting vector is opened linearly, electroporated into established mouse embryonic stem cells (embryonic stem cells: ES cells), and cultured to select ES cells that have acquired neo resistance. I do.
  • ES cells can be prepared by selecting from mouse strains such as 129, C57BL / 6 and F1 CC57BL / 6 XCBA) mice. It is assumed that the ES cells that have acquired neo resistance have undergone homologous recombination with the targeting vector into which the genetic force set has been inserted in the mouse MMP-20 genetic page, and at least the mouse MMP-20 gene allele. One of them is destroyed mouse MMP-20 cannot be expressed normally. For the selection, an appropriate method is selected depending on the inserted gene cassette, and the introduction of the mutation is confirmed by a method such as PCR, Southern hybridization or Northern hybridization. You can force.
  • ES cells that have induced the mutation are injected into 8-cell stage embryos taken from C57BL / 6, BALB / c, ICR mice, etc., cultured for one day, and those that develop in E are transplanted to a foster parent such as ICR.
  • Individuals can be grown.
  • Born offspring mice are chimeric mice derived from ES cells with mutations and normal cells ⁇ ⁇ , and the amount of cells derived from ES cells is determined by the coat color of the individual. Therefore, it is desirable to use a combination of strains with different hair colors for ES cells and lodgings :)
  • Mutations in the obtained chimeric mice are heterozygous, and homozygous mice can be obtained by crossing them appropriately. be able to.
  • the homozygous mutant mouse thus obtained has only the mouse ⁇ -20 gene in all viable and somatic cells (disrupted and does not express mouse ⁇ 20-20 at all). It also has a similar expression system.
  • This knockout mouse analyzes the role of ⁇ -20 in the life cycle of an individual, such as development, growth, reproduction, aging, and death, and the function of ⁇ -20 in various organs and tissues, compared to normal mice. Useful for It can also be applied to tooth-related drug development. Knock-out mice can be used not only as model animals but also as a cell source for tissue culture, and can be used as a function of -20 at the cell level.
  • an antisense 'oligonucleotide (nucleic acid) capable of inhibiting the replication or expression of the MP-20 gene has been cloned or determined.
  • Such oligonucleotides (nucleic acids) are hybridized with RNA (or DNA) of the MMP-20 gene.
  • ⁇ 3 ⁇ 4 ⁇ is the ability to inhibit the function ⁇ , or regulates and regulates the expression of the ⁇ -20 gene through interaction with ⁇ 20-20-related RNA, etc. can do.
  • Oligonucleotides that are complementary to the selected rooster sequence of the ⁇ -20 gene and oligonucleotides that can specifically hybridize with the ⁇ -20 gene are in vivo and in vivo. It is useful for regulating and controlling the expression of the MP-20 gene outside, and is also useful for treating or diagnosing diseases related thereto.
  • Corresponding means having homology or being complementary to a specific sequence of nucleotides, base sequences or nucleic acids including genes.
  • the “correspondence” between a nucleotide, base sequence or nucleic acid and a peptide (protein) is defined as the amino acid of the peptide (protein) specified by the sequence derived from the sequence of the nucleotide (nucleic acid) or its complement. Usually pointing.
  • 5 'end hairpin loop 5' end 6-bead of the gene-spare 'repeat, 5' end untranslated region, polypeptide translation start codon, protein code region, 0RF translation start codon, 3 'end
  • the untranslated region, the 3'-end palindrome region, and the 3'-end hairpin loop are preferable, and any region within the gene can be selected as a target.
  • the relationship between the target nucleic acid and an oligonucleotide complementary to at least a part of the target region means a relationship between the target nucleotide and an oligonucleotide capable of hybridizing with the target, which is referred to as "antisense". be able to.
  • Antisense 'oligonucleotides are polydeoxynucleotides containing 2-dexoxy-D-ribose, polydeoxynucleotides containing D-ribose, N-glycosides of purine or pyrimidine bases and other types.
  • Polynucleotides or other polymers with non-nucleotide backbones eg, commercially available protein nucleic acids and synthetic sequence-specific nucleic acid polymers
  • polymers containing special bonds where the polymer is a DNA And nucleotides having a configuration permitting the attachment of ⁇ S, as found in RNA and RNA).
  • RNA hybrids can be unmodified polynucleotides or unmodified oligonucleotides, or even known Modified, e.g., with labels known in the art, capped, methylated, one or more Above natural nucleotides substituted with analogs, modified intramolecular nucleotides, such as those with uncharged bonds (eg, methylphosphonate, phosphotriester, phosphoramidate, olebamate, etc.) Having a charged bond or an E-yellow-containing bond (eg, phosphorothioate, phosphorodithioate, etc.), such as proteins (nucleases, nucleases' inhibitors, toxins, antibodies, signal peptides, poly- Those with side groups such as lysine and sugars (eg, monosaccharides), those
  • nucleoside., Nucleotide and nucleic acid include not only those containing known purine and pyrimidine bases but also those containing other modified heterocyclic bases. Good, such modifications may include methylated purines and pyrimidines, acylated purines and pyrimidines, or other heterocycles: modified nucleosides and modified nucleotides may also have a sugar moiety. May be modified (for example, one or more hydroxyl groups may be substituted with a halogen and a force, a fatty group, or the like, or may be converted to a functional group such as an ether or a olefin).
  • the antisense nucleic acid of the present invention is RNA, DNA, or a modified nucleic acid.
  • modified nucleic acids include, but are not limited to, sulfur derivatives of nucleic acids, thiophosphoate derivatives, and resistance to degradation of polynucleoside amide polynucleonucleoside amides.
  • the antisense nucleic acid of the present invention can be preferably designed according to the following policy. That is, it makes the antisense nucleic acid more stable in the cell, enhances the cell permeability of the antisense nucleic acid, increases the affinity for the target sense strand, and is toxic. Then, the toxicity of antisense nucleic acid is made smaller.
  • antisense nucleic acids of the invention may contain altered or modified sugars, bases, or linkages, may be in special forms such as ribosomes, microspheres, may be applied or added by gene therapy. could be given in a different form.
  • Such additional forms include polycations such as polylysine, which acts to neutralize the charge on the phosphate backbone, and lipids, which enhance the interaction with cell membranes or increase the uptake of nucleic acids (for example, hydrophobic substances such as phospholipids and cholesterol) can be mentioned.
  • Preferred lipids for addition include cholesterol and its derivatives (eg, cholesteryl chromate form, cholic acid, etc.). These can be attached to the 3 'end or 5' end of the nucleic acid, and can be attached via a base, sugar, or intramolecular nucleoside bond.
  • capping groups include, but are not limited to, hydroxyl-protecting groups known in the art, including glycols such as polyethylene glycol and tetraethylene glycol.
  • the inhibitory activity of the antisense nucleic acid can be examined using the transformant of the present invention, the in vivo or in vitro gene expression system of the present invention, or the MMP-20 in vivo or in vitro translation system. Can be.
  • the nucleic acid can be applied to cells by various methods known per se.
  • a method for transferring a human MMP-20 gene and a recombinant DNA molecule into a host, expressing human MMP-20, and obtaining a target human MMP-20 by the research of the present inventors You.
  • a recombinant or transfectant that substantially expresses the human ⁇ ⁇ -20 gene, a method for producing the same, and its use are also provided.
  • the present invention relates to a protein or a protein characterized in that it is a kind of ⁇ having enamelysin activity and has substantially the same activity as natural ⁇ -20, which is an enamelysin activator other than bushenamelysin.
  • the present invention can be applied to nucleic acids such as DNA and RNA which enable the expression of the polypeptide having all of them in prokaryotes such as Escherichia coli or in organisms such as mammalian cells.
  • nucleic acids are (a) a sequence capable of encoding the amino acid sequence represented by SEQ ID NO: 1 or a sequence complementary thereto, (b) the DNA sequence of (a) or a sequence thereof. It can be a sequence capable of hybridizing with the fragment, and (c) a sequence having a degenerate code capable of hybridizing to the sequence of (a) or (b).
  • Prokaryotes such as Escherichia coli, which can be transformed with such nucleic acids, and which can express the polypeptide of the present invention, and organisms such as mammalian cells also feature the present invention.
  • the antibody such as the monoclonal antibody according to the present invention immunizes animals by a known method using human MP-20 or a fragment thereof obtained according to the present invention as an immunogen, or is known or widely used in the relevant field. It can be produced by a method such as the method of Keller and Milstein et al. (Nature, 256: 495-97, 1975). In this method, the immunogen used was an amino acid selected from regions having low homology to other MP families in native human MP-20, recombinant human MP-20 and human MMP-20.
  • a synthetic peptide having an acid sequence or a fragment obtained from a protein for example, a synthetic peptide having a partial amino acid sequence of the human figure P-20 consisting of at least 8 contiguous amino acids, for example, 14 amino acids
  • Any synthetic peptide having an amino acid sequence having an acid residue, preferably a peptide having a characteristic sequence portion in human P-20, can be used.
  • Human MMP-20 can be obtained from in vivo and extracellular producer cells, for example, from cultured cells, excised tissues, culture tissues, etc., for example, from cells such as tooth odontoblasts, tooth II tissues, deciduous teeth, etc. Obtainable. Furthermore, human MMP-20 can be obtained as a recombinant human MMP-20. You can use it to gain. Human MMP-20 prepared according to the present invention or one derived therefrom can be suitably used as an immunogen. These human P-20s can be purified and separated from each material, for example, a production material such as a transformed cell such as a cultured cell or a cultured textile by a conventionally known method as described above. Purified recombinant human solid -20 is used for antibody production, Can be suitably used as immunity for producing monoclonal antibodies.
  • a peptide is synthesized based on the human MMP-20 gene obtained in the present invention, and the synthetic peptide is immunized to produce an antibody (eg, a monoclonal antibody)! ).
  • the antibodies can be appropriately labeled by a commonly used method.
  • an enzyme, an isotope (radioactive substance), a luminescent substance such as a luminescent compound, a fluorescent substance, a metal colloid, a prosthetic substance, a pigment substance, and biotin can be used. You. Hereinafter, production of the antibody will be described in detail.
  • the monoclonal antibody of the present invention may be a monoclonal antibody obtained by utilizing a cell fusion technique using myeloma cells.
  • the monoclonal antibody of the present invention can be produced, for example, by the following steps.
  • the recombinant mouse pro-MMP-20 prepared according to the above-described method can be used.
  • an appropriate oligopeptide is chemically synthesized and then synthesized.
  • human MMP-20 precursor human MMP-20 active human MMP-20 can be used.
  • a non-immunoconjugate may be used, but it can be used as it is for immunizing animals by mixing it with an appropriate adjuvant.
  • a fragment of human MMP-20 or a characteristic rooster sequence based on the amino acid sequence deduced from the cloned and sequenced cDNA sequence is selected.
  • the fragment may be a synthetic polypeptide fragment obtained by designing and chemically synthesizing the polypeptide.
  • the fragment is bound to various carrier proteins via an appropriate condensing agent to form an immunogenic conjugate such as a hapten protein, which is used to react only with a specific sequence. It can also be used to design a monoclonal antibody that can (or only recognizes a specific sequence).
  • a cysteine residue or the like can be added to the designed polypeptide in advance so that the immunogenic conjugate can be easily prepared.
  • the carrier proteins Upon binding to the carrier proteins, the carrier proteins can first be activated. The ability to introduce an activating binding group in such activation is mentioned.
  • activated bonding group examples include: (1) an activated ester or an activated carboxyl group, for example, a nitrophenyl ester group, a pentafluorophenyl ester group, a 1-benzotriazole ester group, or an N-succinimide ester group.
  • Active bioactive dithio group for example, 2-pyridyldithio group.
  • Carrier proteins include keyhole limpet hemosinin (KLH), bovine serum albumin (BSA), polypeptides such as ovalbumin, globulin, polylysine, and bacterial cell components such as BCG Is mentioned.
  • the dose of ⁇ 3 ⁇ 4 is, for example, about 1 to 400 g gZ for a mouse, and is generally measured intraperitoneally or subcutaneously in a host animal, and thereafter every 1 to 4 weeks, preferably every 1 to 2 weeks. Intra-, subcutaneous, intravenous or intramuscular boosts are repeated 2-10 times.
  • a mouse for immunization in addition to a BALB / c mouse, an F1 mouse of a BALB / c mouse and another mouse can be used. If necessary, an antibody titer measurement system can be prepared, and the antibody titer can be measured to confirm the model of animal immunity.
  • the antibody of the present invention may be obtained from the immunized animal thus obtained, for example, antiserum, polyclonal antibody, etc. Is included.
  • a cell line that does not produce immunoglobulin can be selected.
  • NS3-NS-to-Ag41 NS-1, Eur. J. Immunol., 6: 511-519, 1976
  • SP2 / 0-Agl4 SP2, Nature, 276: 269 -270, 1978
  • P3-X63-Ag8-Ul P3U1, Curr. Topi
  • M0PC-21 cell line cs Mi crobiol. Immunol., 81: 1-7, 1978
  • P3-X63-Ag8 X63, Nature, 256: 495-497, 1975
  • P3-X63-Ag8-653 653, J.
  • the azaguanine-resistant mouse myeloma cell line is prepared by adding substances such as penicillin and amikacin, fetal calf serum (FCS), etc. to cell culture medium such as Dulbecco's MEM medium (DMEM medium) and RPI-1640 medium.
  • cell culture medium such as Dulbecco's MEM medium (DMEM medium) and RPI-1640 medium.
  • DMEM medium Dulbecco's MEM medium
  • RPI-1640 medium Dulbecco's MEM medium
  • the cell strain used was prepared by completely thawing the cryopreserved strain at about 37 ° C, washing it three times or more with a normal medium such as RPM 1640 medium, and culturing it in a normal medium to prepare the required number of cell lines. It may be.
  • the immunized animal for example, a mouse
  • lymph node cells from various parts of the body can be obtained and used for cell fusion.
  • the thus obtained spleen cell suspension and the Mie cell line obtained in the above step 3 are placed in a cell medium such as a minimum essential medium (MEM medium), a DMEM medium, and an RPMI-1640 medium.
  • a cell fusion agent such as polyethylene glycol.
  • the cell fusion agent those known in the art can be used.
  • Examples of such a cell fusion agent include inactivated Sendai virus (HVJ: Hemagglutinating Virus of Japan).
  • HVJ Hemagglutinating Virus of Japan
  • polyethylene glycol having a molecular weight of 1,000 to 8,000 can be used, and further, a molecular weight of 1,000 to 8,000 can be used. Up to 4,000 polyethylene glycols can be used more preferably.
  • the concentration of polyethylene glycol in the fusion medium It is strongly preferable that the content be 30 to 60%. If necessary, for example, dimethyl sulfoxide may be used in a small amount to promote the fusion.
  • the ratio of the splenocytes (phosphospheres) Roman crescent cell line used for the fusion may be, for example, 1: 1 20: 1, and more preferably 4: 1 7: 1.
  • a cell culture medium such as RPMI 1640 medium.
  • the fusion reaction can be performed multiple times. After the fusion reaction, the cells are separated by centrifugation and transferred to a selection medium.
  • the selection medium examples include a FCS-containing MEM medium and a medium such as RPM1640 containing hypoxanthine, aminopterin and thymidine, and so-called HAT ⁇ ground.
  • the method of replacing the selective medium is generally the same as the volume dispensed to the culture plate, adding the same volume the next day, and then replacing the half volume with the HAT medium every 13 days. ⁇ Changes can be made to this.
  • the aminopterin is removed, and the medium can be replaced every 14 days in a so-called HT medium.
  • a feeder for example, mouse thymocytes can be used, which may be preferable in some cases.
  • the culture supernatant of culture cells with a high level of hybridoma growth can be analyzed using a measurement system such as radioimmunoassay (R1A), enzyme immunoassay (EL1SA), or fluorescence immunoassay (F1A), or a fluorescence-induced cell separation device ( Screening is performed by using human MMP20 or its fragment peptide as an antigen by FACS) or by measuring the target antibody using a labeled anti-mouse antibody.
  • R1A radioimmunoassay
  • EL1SA enzyme immunoassay
  • F1A fluorescence immunoassay
  • Screening is performed by using human MMP20 or its fragment peptide as an antigen by FACS) or by measuring the target antibody using a labeled anti-mouse antibody.
  • Cloning can be done by the ability to pick up colonies in an agar medium, or by limiting dilution. It can be performed more preferably by the limiting dilution method. Cloning is preferably performed multiple times.
  • the obtained hybridoma strain is cultured in an appropriate growth medium such as an FCS-containing MEM medium or RPM1-1640 medium, and a desired monoclonal antibody can be obtained from the culture iLJi culture.
  • an appropriate growth medium such as an FCS-containing MEM medium or RPM1-1640 medium
  • a desired monoclonal antibody can be obtained from the culture iLJi culture.
  • I can do it.
  • Each of the hybridomas is transplanted into ⁇ ⁇ of a histocompatible animal that is syngeneic to the animal derived from the myeoma cells, and is propagated, or each hybridoma is transplanted and grown in, for example, a nude mouse.
  • Monoclonal antibodies produced in the ascites of a substance can be recovered and obtained.
  • Animals can receive 5 ti of Hypridoma transplantation, and can be given ⁇ mineral oil such as pristane (2,6,10,14-tetramethylpentanedane).
  • ascites can be collected.
  • the ascites fluid may be used as it is or by a conventionally known method, for example, salting out such as ammonium sulfate precipitation, gel filtration using Sephadex, ion exchange chromatography, electrophoresis, dialysis, ultrafiltration, It can be used as a monoclonal antibody after purification by T-chromatography or high-performance liquid chromatography.
  • the ascites containing the monoclonal antibody can be subjected to ammonium sulfate fractionation, followed by treatment with an anion exchange gel such as DEAE-Sepharose and an affinity column such as a protein A column for purification and separation.
  • an anion exchange gel such as DEAE-Sepharose
  • an affinity column such as a protein A column for purification and separation.
  • affinity chromatography in which ⁇ 3 ⁇ 4 ⁇ or an antigen fragment (eg, a synthetic peptide, a recombinant thigh protein or peptide, a site specifically recognized by an antibody, etc.) is immobilized, and affinity chromatography in which protein A is immobilized is preferred. T Chromatography is one of them.
  • these antibodies may be treated with enzymes such as trypsin, papain, and pepsin, and used as antibody fragments such as Fab, Fab ', and F (ab') obtained by reduction with if ⁇ .
  • enzymes such as trypsin, papain, and pepsin
  • antibody fragments such as Fab, Fab ', and F (ab') obtained by reduction with if ⁇ .
  • an IgG fraction, or a specific binding Fab ′ obtained by digestion with pepsin can be used as the antibody to be labeled.
  • these labeled substances include enzymes (peroxidase, alkaline phosphatase or 5-D-galactosidase, etc.), diamine substances, fluorescent substances, and radioisotopes, as described below. There is.
  • Detection and measurement in the present invention are performed by immunostaining, for example, tissue staining, cell staining, and immunostaining.
  • immunostaining for example, tissue staining, cell staining, and immunostaining.
  • it can be performed with competitive immunoassay or non-competitive immunoassay, and radioimmunoassay, ELISA, etc. can be used, and BF separation may or may not be performed. It can be performed.
  • Preferable are a radioimmunoassay and an enzyme immunoassay, and further include San Diego Assay.
  • a sandwich type assay one of the antibodies against human MMP-20 is detectably labeled. Another antibody that can recognize the same fragment is immobilized on a solid phase.
  • the sample is subjected to an incubation treatment in order to sequentially react the labeled antibody and the immobilized antibody as necessary. After separating the unbound antibody, the labeled substance is measured. The amount of label measured is proportional to ⁇ ⁇ ⁇ ⁇ , the amount of human ⁇ -20.
  • a simultaneous sandwich type assay it is called a simultaneous sandwich type assay, a forward sandwich type assay or a reverse sandwich type assay according to the order of addition of an insolubilized antibody or a labeled antibody. For example, washing, stirring, shaking, filtration or pre-extraction of antigens are performed in the measurement process under specific circumstances;;: Concentration of specific reagents, buffers, etc.
  • measurement conditions such as the treatment time, can be varied according to factors such as the concentration of perturbations in the sample, the nature of the sample separator, and the like.
  • a person skilled in the art can carry out the measurement by appropriately selecting the optimum conditions effective for each measurement while using ordinary experimental methods.
  • carrier various carriers known to be used for antibody reaction and the like are known, and the carrier of the present invention can be selected from these known carriers.
  • Particularly preferably used are, for example, glass, for example, activated glass, porous glass, silica gel, silica-alumina, alumina, magnetized iron, magnetized alloys and other inorganic materials, polyethylene, polypropylene, polyvinyl chloride , Polyvinylidene fluoride, polyvinyl acetate, polymethacrylate, polystyrene, styrene-butadiene copolymer, polyacrylamide, cross-linked polyacrylamide, styrene-methacrylate copolymer, polyglycidyl methacrylate, acrolein-ethylene glycol Dimethacrylate copolymer, cross-linked albumin, collagen, gelatin, dextran, agarose, cross-linked albumin, collagen, gelatin, dextran, agarose, cross-linked albumin, collagen, ge
  • filter paper, beads, the inner wall of the test container for example, test tubes, cells made of synthetic materials such as tie plates, glass cells, synthetic resin cells, glass rods, rods made of synthetic materials, ends
  • a solid material such as a rod with a thicker or thinner, a rod with a rounded or flat protrusion at the end, or a thinned rod.
  • An antibody can be bound to these carriers, and preferably, a monoclonal antibody that specifically reacts with human P-20 obtained in the present invention can be bound thereto.
  • the binding between the carrier and those involved in the antibody reaction can be performed by a physical method such as adsorption, a chemical method using a condensing agent, or an activated material, or a mutual method. It can be performed by a method using the chemical bonding reaction of
  • Labels include enzymes, enzyme substrates, enzyme inhibitors, prosthesis pages, coenzymes, enzyme precursors, apoenzymes, fluorescent substances, coloring substances, chemiluminescent ⁇ !, luminescent substances, coloring substances, magnetic substances, metals Particles, for example, radioactive substances such as colloidal gold, can be mentioned.
  • enzymes include oxidases such as enzyme enzymes, reductases, and oxidases; transferases that catalyze the transfer of amino, carboxyl, methyl, acyl, and phosphate groups, such as esters
  • oxidases such as enzyme enzymes, reductases, and oxidases
  • transferases that catalyze the transfer of amino, carboxyl, methyl, acyl, and phosphate groups, such as esters
  • Examples include hydrolysates, lyases, lyases, isomerases, and ligases that hydrolyze bonds, glycosidic bonds, ether bonds, peptide bonds, and the like.
  • Enzymes can be used for detection by using multiple enzymes in combination. For example, elementary cycling can be used.
  • Representative enzyme labels include peroxidases such as horseradish biperoxidase, galactosidase such as y ⁇ fungal 3-D-galactosidase, maleate dehydrogenase, glucose-6-phosphate 'dehydrogenase, and glucose oxidase.
  • peroxidases such as horseradish biperoxidase, galactosidase such as y ⁇ fungal 3-D-galactosidase, maleate dehydrogenase, glucose-6-phosphate 'dehydrogenase, and glucose oxidase.
  • I glucoamylase
  • a Examples include cetylcholinesterase, lipase, alkaline phosphatase such as alkaline phosphatase, and small intestine alkaline phosphatase.
  • a substrate such as a dynamic cycling system, a luciferin derivative, a dioxetane derivative, or the like, it can be measured by the resulting fluorescence or luminescence. Luciferin and luciferase systems can also be used. Since it reacts with hydrogen peroxide using catalase to form oxygen, the oxygen can be detected by an electrode or the like.
  • the electrode may be a glass electrode, an ion electrode using poor solubility, a huge electrode, or a polymer membrane electrode.
  • the enzyme label can be replaced with a biotin label and an enzyme-labeled avidin (streptavidin).
  • the sign may be from several different Nada pages. It may be possible to make multiple measurements continuously, or discontinuously, and simultaneously or separately.
  • 4-hydroxyphenylacetic acid, 1,2-phenylenediamine, tetramethylbenzidine and the like, and horseradish beloxoxidase, umbelliferyl galactoside, nitrophenyl galactone and the like are used for signal formation.
  • Combinations of enzymes such as D-galactosidase and glucose 6-phosphate / dehydrogenase can also be used, and quinol compounds such as hydroquinone, hydroquinbenzoquinone, and hydroxyanthraquinone, and lipoic acid. And those capable of forming thiol compounds such as glucan thione, phenol derivatives, and phenol derivatives thereof by the action of enzymes and the like.
  • a fluorescent substance or a chemiluminescent compound may be fluorescein isothiosinate, for example, rhodamine inducers such as mouth-diamine diisothiosinate, tetramethylrhodamine disothiosinate, dansyl chloride, dansyl fluoride, and fluorescein.
  • rhodamine inducers such as mouth-diamine diisothiosinate, tetramethylrhodamine disothiosinate, dansyl chloride, dansyl fluoride, and fluorescein.
  • Examples include min, phycopyriprotein, acridinium salt, luminol such as noremiferin, noresiferase, and aequorin, imidazole, oxalate, rare ⁇ 11 chelate compounds, and coumarin derivatives.
  • Labeling can be carried out using a reaction between a thiol group and a maleimide group, a reaction between a pyridyldisulfate "group and a thiol group, or a reaction between an amino group and an aldehyde group.
  • the method can be appropriately selected from known methods or methods that can be easily performed by those skilled in the art, and further modified methods. Further, it is possible to use a condensing agent that can be used for producing the immunogenic complex, a condensing agent that can be used for binding to a carrier, and the like. Examples of the condensing agent include glutaraldehyde,
  • a labeled antibody such as a monoclonal antibody in which a substance to be measured is labeled with an enzyme or the like can be sequentially reacted with an antibody bound to a carrier, or simultaneously. Can also.
  • the order of addition depends on the type of carrier system chosen.
  • ⁇ using beads sensitized with plastic, etc. humans are first placed in an appropriate test tube together with a sample containing a substance to be measured, such as a monoclonal antibody labeled with an enzyme. ⁇ The measurement can then be made by adding beads such as the sensitized plastic.
  • an immunoassay method is used.
  • a solid support such as a polystyrene, polycarbonate, polypropylene or polyvinyl ball, a microsphere, or a microparticle that can adsorb proteins such as antibodies well is used.
  • Various materials and forms such as plates, sticks, microparticles or test tubes can be arbitrarily selected and used.
  • the measurement can be performed in an appropriate buffer system so as to maintain the optimum pH, for example, a pH of about 4 to 9.
  • Particularly suitable buffers include, for example, acetate buffer, citrate buffer, phosphate buffer, tris buffer, triethanolamine buffer, borate buffer, glycine buffer, carbonate buffer And Tris-HCl buffer. Buffering agents can be used by mixing them at an arbitrary ratio.
  • Antibody reactions should be performed at temperatures between about 0 and 60 ° C.
  • Incubation treatment with antibody reagents such as monoclonal antibodies labeled with enzymes, antibodies bound to carriers, and substances to be measured can be performed until W is reached. Shortly before ⁇ is achieved, the reaction can be stopped after a limited incubation by separating the solid and liquid phases at a point in time, such as the presence of enzymes in the liquid or solid phase. The presence of the sign can be measured.
  • the measurement operation can be performed using an automated measuring device. Using a noreluminescence 'detector' or 'photo' detector, etc., it is possible to detect the display signal generated by the conversion of the substrate by the action of the enzyme. Can also be measured.
  • EDTA ethylenediaminetetraacetate
  • Lumps commonly employed in the art may be subjected to a blocking treatment known to those skilled in the art to prevent heterologous binding reactions, for example, normal serum proteins such as mammals, albumin , Skim milk, fermented milk, collagen, gelatin, etc. These methods are not particularly limited and can be used as long as the purpose is to prevent the heterogeneous binding reaction.
  • the knives to be measured by the measuring method of the present invention include a force that can be used by any form of sickle colloid, a human body, and the like.
  • the purpose of the present invention is to use an antibody such as a monoclonal antibody against human MMP-20 and an antibody such as a monoclonal antibody against human MMP-20 for a solid phase carrier, or furthermore, if necessary, a human antibody. It is an object of the present invention to provide an excellent method for separating MM from human ⁇ -precursor tt human MMP-20 using an inhibitor against MP-20 and a 1 1 kit for the same.
  • the present invention is supposed to include all such aspects of the kit, which is capable of separating the precursor of ⁇ and the active form of human -20, into all aspects thereof.
  • an object of the present invention is to separate tooth precursors and active human MMP-20 using the above-mentioned method to form tooth woven fabric.
  • the purpose of this is to provide a method that can monitor damage and destruction, and / or a diagnostic agent. Therefore, the above-mentioned various uses in the medical and physiological fields, and the use of the above reagents for the purpose of studying, analyzing, and measuring cells and tissues of animals such as humans, including teeth, are all embodiments of the present invention. It is understood to be included in.
  • the human MMP-20 of the present invention or a salt thereof has an ft S-propidin content which is a major constituent protein of enamel matrix, and is, for example, an essential factor in the normal formation of enamel. It is believed that there is.
  • the protein is considered to be useful for treating enamel dysplasia, which is a pathological condition of enamel formation, enamel dysplasia, and enamel calcification dysfunction.
  • enamel dysplasia which is a pathological condition of enamel formation, enamel dysplasia, and enamel calcification dysfunction.
  • a drug containing a derivative of human P-20 or human MP-20 is used, the re-formation of enamel that has failed to form enamel due to insufficient degradation of the enamel matrix Alternatively, it is possible to make the teeth of a patient with the disease healthy by using Kyo-Dai.
  • human P-20 or human MP is used for the treatment of dental caries (caries), surgical treatment, promotion of regeneration and regeneration of enamel that has been lost due to other causes, or induction of enamel formation on exposed dentin.
  • Medications containing 20 derivatives are useful.
  • by enhancing enamel it is expected to have a preventive effect on dental caries (caries), and pharmaceuticals containing derivatives of human P20 or human MP-20 are also useful as agents for preventing dental caries.
  • the protein of the present invention is one of the median proteases, and is used as a therapeutic or Z or prophylactic agent for various diseases caused by a decrease in the activity of the immunoglobulin protease. It is useful as a medicine.
  • the protein of the present invention is useful as a medicament such as a therapeutic or Z or prophylactic agent for various diseases caused by a decrease in the activity of degrading enamel protein and resulting in i ⁇ .
  • a medicament such as a therapeutic or Z or prophylactic agent for various diseases caused by a decrease in the activity of degrading enamel protein and resulting in i ⁇ .
  • the biological activity in cells is not sufficiently obtained or the patient has abnormal symptoms due to a decrease or lack of human MMP-20 in vivo
  • C Improving the symptoms in the patient by transplanting cells into which the nucleic acid such as the DNA of the present invention can be expressed so as to replenish the protein of the present invention in the living body, etc.
  • Functions such as biological activity of human MP-20 of the present invention may cause human MP-20 dysfunction, enamel aplasia, enamel hypoplasia, enamel calcification pathology, enamel It can be used as a medicament for the treatment of various diseases such as dysplasia and dental caries (caries), as a Z or prophylactic agent, for promoting the regeneration and regeneration of defective enamel, or for inducing enamel formation in exposed dentin.
  • amelogenin degradation Activity eg., agonists or accelerators
  • salts thereof may cause human MP-20 dysfunction, enamel aplasia, enamel hypoplasia, enamel calcification pathology, enamel It can be used as a medicament for the treatment of various diseases such as dysplasia and dental caries (caries), as a Z or prophylactic agent, for promoting the regeneration and regeneration of defective enamel, or for inducing enamel formation in exposed dentin.
  • compounds (antagonists, or antagonists) that inhibit functions such as biological activity of human MP-20 of the present invention for example, protease activity, matrix meta-oral protease activity, affinity for enamel protein, amelogenin degradation activity, etc.
  • the inhibitor) or a salt thereof can be used as a medicament such as a therapeutic and / or prophylactic agent for various diseases such as hyperhuman MP-20 function and peramelodidinin dystrophy.
  • human MMP-20 is strongly active with amelodidinin, a major constituent protein of enamel matrix, and its activity is completely inhibited by TIMP-2.
  • T1MP-2 is useful as a medicament for treating enamel dysplasia caused by excessive degradation of the enamel matrix.
  • metalloproteinase inhibitors such as TIMP-1 are also useful as medicaments for such uses.
  • a substrate of the present invention, a part of the peptide or a salt thereof, etc. was brought into contact with a substrate, and (ii) a protein of the present invention, a part of the peptide, or a salt thereof. Compare with the substrate and the test tube for contact.
  • the biological activity eg, protease activity, affinity for enamel protein, amelogenin
  • Any substrate can be used as long as it can serve as a substrate for the protein or the like of the present invention.
  • it can be selected from those used for measuring protease activity, such as casein, collagen, and synthetic oligopeptides.
  • peptide substrates that can be used include, for example, (7 methoxycoumarin-4-yl) -acetyl-Pro- and eu-Gly-Leu L3- (2,4-dini torophenyl)-and 2,3 diaminopropionyl Ala-Arg-NH, (McaPLGLDpaARNH., McaPLANvaDpaARNH_, etc.
  • the substrate can be used as it is, and preferably a substrate labeled with a fluorescent, enzymatic or radioactive substance such as fluorescein can be used.
  • a fluorescent, enzymatic or radioactive substance such as fluorescein
  • Amerodidinin J. Dent. Res. 76: 641-647 (1997)
  • Test 1 includes, for example, proteins, peptides, non-peptidic compounds, synthetic compounds, fermentation products, plant extracts, tissue extracts of animals and the like, cell extracts, and the like.
  • test compounds used in the test container include preferably meta-oral proteinase inhibitors, compounds having inhibitory activity against MMP proteins, especially synthetic compounds, including hydroxamic acid, Carboxylic acids, phosphonic acids, thiol derivatives and the like may be included. These compounds may be new compounds or known conjugates.
  • the screening can be carried out according to the usual method for measuring protease activity, for example, by referring to the method described in Biochemistry, 32, pp. 4330-4337 (1993). Can be.
  • the protein or the like of the present invention can be treated with an activator such as mercury aminophenylacetate, or its precursor or latent form can be previously converted to active ⁇ form.
  • the measurement is usually performed in a buffer such as Tris-HCl buffer or phosphate buffer which does not adversely affect the reaction, for example, at ⁇ 4 to 10 (preferably ⁇ about 6 to 8). be able to.
  • the experimental system for the inhibition of human ⁇ -20 amelogenin is shown in Table 1. It can also be used to search for substances.
  • the human MMP-20 of the present invention has substantially the same activity as the human MMP-20 of the present invention, in addition to the usual conditions and procedures of each method, and the ordinary technical rooster of those skilled in the art.
  • a measurement system related to proteins or peptides may be constructed.
  • Methods in Enzymology, Vol. 1, 2, 5 and 6 Preparation and Assay of Enzymes); Ibid., Vol. 3 (Preparation and Assay of Substrates); ibid, Vol. 4 (Special Techniques for the Enzymologist); ibid, Vol. 19 (Proteolytic Enzymes); ibid, Vol. ); Ibid, Vol. 80 (Proteolytic Enzymes, Part C) (above, published by Academic Press (USA), etc.)).
  • Candidates for human MMP-20 medium oral genonin inhibitors include the specific inhibitory proteins TIMP-1, TIMP-2, TIMP-3, and T1MP-4, which are common to the matrix meta-proteinase family.
  • the synthesis inhibitor include synthetic compounds having meta-oral protease inhibitory activity such as inducing hydroxamic acid, carboxylic acid, phosphonic acid, and thiol.
  • a substance that inhibits the process of converting latent human ⁇ -20 having no enzymatic activity into active human ⁇ -20 is also effective in inhibiting amelogenin.
  • an antisense oligonucleotide (nucleic acid) capable of replicating or expressing the human MP-20 gene can be used.
  • the active ingredient of the present invention [for example, (a) the protein of the present invention, a partial peptide or a salt thereof, (b) a nucleic acid such as a DNA of the present invention, (c) a protein of the present invention, Antibodies (including monoclonal antibodies) described in some peptides or their induction, (c) antisense oligonucleotides to nucleic acids such as DNA of the present invention, (d) proteins of the present invention, and some peptides thereof Or a compound or a salt thereof that inhibits or inhibits the biological activity of a salt thereof), for example, a protein such as human MP-20 of the present invention, a partial peptide thereof or These salts can be mixed with ordinary insects or pharmacologically acceptable adjuvants, and administered as sterile or pharmaceutical preparations.
  • parenteral dosage forms can include topical, m., Intravenous, intramuscular, subcutaneous, intradermal or intravenous administration, but can also be delivered directly to the affected area. Is also suitable. It is preferably orally or parenterally to mammals including humans (eg, intracellular, paper, intravenous, intramuscular, subcutaneous, intradermal)
  • compositions include ⁇ preparations, bulking agents, semi-solid preparations, granular preparations, ⁇ preparations, leaching preparations, and the like.
  • examples include tablets, coated tablets, sugar-coated preparations, and pills.
  • Agents troches, hard capsules, soft capsules, microcapsules, powders, powders, powders, m ⁇ , fine granules, m, liquids, elixirs, emulsions, irrigants, syro-u, Solutions, emulsions, suspensions, liniments, lotions, aerosols, sprays, inhalants, orchids, ointments, plasters, patches, pastas, pastes and tablets. Preparations, creams, oils, suppositories (eg, skin suppositories), tinctures, skin solutions, eye drops, nose drops, ear drops, coatings, infusions, injections, etc. Powders, freeze-dried agents, gel preparations and the like.
  • the product can be formulated according to the usual method.
  • Physiologically acceptable carriers for example: ⁇ Physiologically acceptable carriers, pharmaceutically acceptable carriers, adjuvants, excipients, excipients, diluents, perfumes, flavors, sweeteners, m-vehicles, preservatives, as appropriate , Stabilizers, binders, pH regulators, buffers, surfactants, bases, solvents, bulking agents, solubility aids, solubilizers, isotonic agents, emulsifiers, suspending agents, Diversion, thickener, gelling agent, hardening agent, absorbent, adhesive, mu plasticizer, disintegration agent, propellant, shelf, antioxidant, shading agent, humectant, easing agent, antistatic agent
  • an analgesic agent or a combination thereof or the like and mixing the protein of the present invention with the agent, it is possible to produce in a simple form required for the preparation recognized in the above.
  • Formulations suitable for parenteral use include sterile reservoirs of the active ingredient and water or other pharmaceutically acceptable vehicle, or suspensions, such as liquors. I can do it.
  • water ⁇ water, dextrose water ⁇ and other related sugar rind, and glycols such as ethanol, propylene glycol and polyethylene glycol are preferred liquid carriers for sizing agents.
  • the preparation is carried out by a method known in the art using distilled water, Ringer's solution, a carrier such as menstrual fluid, a suitable dispersant or wetting agent and a suspending agent, and the like.
  • Suspension, Emulsion, etc. ⁇ Prepare in a form that can be measured.
  • aqueous liquids for ait include physiological fluids: ⁇ , isotonic liquids containing glucose and other adjuvants (eg, D-sorbitol, D-mannitol, ichinatrium chloride, etc.).
  • Suitable solubilizers that are acceptable in nature, such as alcohols (eg, ethanol), polyalcohols (eg, propylene glycol, polyethylene glycol), nonionic surfactants (eg, polysorbate)
  • the oily liquid includes sesame oil, soybean oil and the like, and may be used in combination with solubilizers such as benzyl benzoate and benzyl alcohol.
  • solubilizers such as benzyl benzoate and benzyl alcohol.
  • buffers eg, phosphate buffer, sodium acetate buffer, etc.
  • reagents for adjusting osmotic pressure e.g, phosphate buffer, sodium acetate buffer, etc.
  • soothing agents eg, benzalkonium chloride, proforce hydrochloride, etc.
  • stabilizers eg, , Human serum albumin, polyethylene glycol, etc.
  • retention: ⁇ ij eg, benzyl alcohol, phenol, etc.
  • An acid inhibitor such as ascorbic acid, an absorbent, and the like.
  • the adjusted is usually 5 mm to a suitable ampoule.
  • haze or water in a sterile pharmaceutically acceptable liquid such as water, ethanol or oil may be added, with or without surfactants and other pharmaceutically acceptable auxiliaries. It is formulated in the form of a suspension.
  • the oily vehicle or solvent to be converted into the drug product is natural or synthetic.
  • A is a semi-synthetic mono- or di- or triglyceride, natural, semi-synthetic or synthetic oil or fat or fatty acid;
  • vegetable oils such as peanut oil, corn oil, soybean oil, sesame oil and the like can be mentioned.
  • the sizing agent can be prepared so as to generally contain 0.1 to 10 S *% of the bacteria of the present invention.
  • Formulations suitable for topical, e.g., oral, or intractable use include, for example, mouthwashes, dentifrices, oral beta, inhalants, ointments, dental fibres, dental coatings, dental pastes, dentists Agents and the like. Mouthwashes and other dental agents are prepared by a conventional method using a pharmacologically acceptable carrier.
  • a pharmacologically acceptable carrier As oral orchids and inhalants, the compound of the present invention itself or a pharmacologically acceptable inert carrier may be dissolved in an aerosol or nebulizer at night, or as a fine powder for inhalation, such as teeth.
  • the ointment is prepared by adding a commonly used base, for example, an ointment base (white serine, paraffin, olive oil, macrogol 400, macrogol ointment, etc.) and the like, and then using a conventional method. It is usually prepared so as to contain the compound of the present invention in an amount of 0.001 to 30 fi *%.
  • a commonly used base for example, an ointment base (white serine, paraffin, olive oil, macrogol 400, macrogol ointment, etc.) and the like. It is usually prepared so as to contain the compound of the present invention in an amount of 0.001 to 30 fi *%.
  • Drugs for topical application to teeth and skin can be formulated as solutions or suspensions in suitably sterilized water or non-aqueous vehicles.
  • Additives include preservatives, including, for example, buffering agents such as sodium bisulfite or sodium sodium edetate; bactericidal and antifungal agents such as silver mercury acetate or phenyl nitrate, hyphenzalconium chloride or black hexidine. Thickening agents such as hypromelrose are mentioned.
  • Suppositories are carriers well known in the art, preferably non-irritating suitable excipients, for example, polyethylene glycols, lanolin, lactic acid, fatty acid triglycerides and the like, preferably solid at room temperature.
  • suitable excipients for example, polyethylene glycols, lanolin, lactic acid, fatty acid triglycerides and the like, preferably solid at room temperature.
  • the intestinal tract it is prepared by a conventional method using a substance that melts in a liquid and releases the drug in a convenient manner; it is usually prepared to contain about 0.1 to 95% by weight of the present compound. Is prepared.
  • the drug can be suspended or dissolved in the drug.
  • Adjuvants such as local anaesthetics, preservatives and buffering agents can be dissolved in the vehicle. These preparations are usually prepared so as to contain 0.001 to 95 ⁇ *% of the compound of the present invention.
  • Formulations suitable for oral classes include solid preparations such as tablets, pills, capsules, powders, granules and lozenges, and liquid threads such as liquids, syrups and suspensions. Objects and the like.
  • solid preparations such as tablets, pills, capsules, powders, granules and lozenges
  • liquid threads such as liquids, syrups and suspensions.
  • a formulation auxiliary known in the art and the like are used.
  • Locks and pills can also be manufactured with enteric coating. If the preparation is a capsule, the above type of material is required. Further, a liquid carrier such as an oil or fat may be contained.
  • the nucleic acid when used as a therapeutic and / or Z-prophylactic agent as described above, the nucleic acid can be used alone, or can be used in the above-mentioned genetic and recombinant techniques.
  • Any suitable vector for example, a vector derived from a virus, such as a vector derived from a retrovirus, can be used.
  • the nucleic acid such as the DNA of the present invention can be administered by a commonly known method, and until then, or a suitable auxiliary agent or a physiologically acceptable carrier, for example, so as to promote the intracellular activity.
  • it can be used in the form of a formulation, and can be administered as a pharmaceutical composition or a pharmaceutical preparation as described above.
  • a method known as gene therapy can be applied.
  • the activity of the present invention is the ability to select and administer the dosage over a wide range.
  • the administration * The number of administrations, etc., depends on the treatment patient's age, age, body weight, " ⁇ healthy condition, diet, administration The time, the mode of administration, the rate of administration, the combination of drugs, the type of medical condition being treated at the time of the patient, and other factors should be considered.
  • hybridoma producing the anti-human MMP-20 monoclonal antibody described in Example 5 (f): 203-1C7 (labeled as: Hypri-Doma 203-1C7) was obtained on July 22, 1998. (Original deposit date) Deposited with NIBH under the accession number FERM BP-6434. Male
  • the human PI art ifi ci al chromosome dienomic life rally was screened using the cDNA of porcine enamelisin (Gene, 183: 123-128, 1996) as a probe.
  • porcine enamelisin Gene, 183: 123-128, 1996)
  • double-stranded DNA of 780 bp corresponding to positions 300 to 1080 of the above-mentioned porcine enamelysin cDNA was labeled with 1 ⁇ 2ersham's random priming kit using ⁇ [ 3 ] (1 ( ⁇ ? (3000 ⁇ / citation 01)). of 50 ° C 13 ⁇ 4SDS, 0.
  • a sense primer (Ename 1: SEQ ID NO: 6 in the sequence listing) and an antisense primer to Exon 4 (Ename 4: Rooster column number: 7) in the column list was synthesized (model 381A DNA synthesizer, Applied Biosystems) and subjected to PCR.1st strand DNA was prepared from human odontoblast cells and dental pulp as follows.
  • RNA preparation should be performed according to the instructions in Trizol Reagent (CLife Technologies), and the concentration and! ⁇ was determined by the absorbance value.
  • PCR was performed using the obtained 1st strand DNA, primers Ename 1 (SEQ ID NO: 6), and Ename 1-4 (SEQ ID NO: 7). Denaturation was performed using the GeneAmp 2400 PCR system (Perkin-Elmer / Cetus). C 15 seconds, annealing 58. C 15 seconds, extension
  • the 610 bp cDNA fragment obtained by PCR was phosphorylated with T4 polynucleotide kinase, inserted into the pUC18 Smal site, and used for the base sequence.
  • the nucleotide sequence of the cDNA fragment was confirmed to consist of exon 1 to exon 4 of the dienomic clone obtained above.
  • the sequence of the obtained cDNA is shown in SEQ ID NO: 2 in the sequence listing.
  • the nucleotide sequence represented by SEQ ID NO: 2 in the sequence listing encodes a protein consisting of 483 amino acids, and its expected amount was 54.4 kDa. This amino acid sequence had a high homology of 89% with porcine enamelysin, strongly suggesting that this cDNA corresponds to human enamelysin.
  • This putative protein was named human MP-20 (human enamelisin).
  • Escherichia coli strain DH5a (pBlueHMMP20 / DH5a) harboring the vector pBluescript TM (Stratagene) into which the nucleotide sequence encoding the human MP-20 has been inserted, has been established since June 25, 1997 at Higashi 1 in Tsukuba, Ibaraki Prefecture. It has been deposited under the accession number FERM P-16284 at the Ministry of International Trade and Industry at IS-Technical Research Institute ⁇ (NIBH) at Chome 1-3 (zip code 305-8566). A request for transfer to the deposit based on the Budapest Treaty was made from this original deposit on July 22, 1998, and the deposit is stored at the NIBH under the accession number FERM BP-6438.
  • human MP-20 is composed of a predomain consisting of a hydrophobic sequence involved in the secretory process, a prodomain containing a conserved PRCGVPD motif (positions 98 to 104) involved in the latentization of protease activity, A conserved HEXGHXXGXXH containing three histidine residues that bind the «atoms of the active site (positions 226-236) ) With a corner butterfly domain.
  • the corner of human MMP-20; ⁇ domain also has a methionine at the 7-residue C-terminal side of HEXGHXXGXXH (positions 226 to 236) conserved in all MPs, and these sequences Is thought to play a common role in maintaining the active site structure of MMPs.
  • This human MMP-20 also had a hemopoxin II enzyme-like domain that is common to MP families other than MMP-7 for approximately 200 residues on the C-terminal side.
  • the cleavage site of the prodomain was inferred, and it was estimated that the active human P-20 was a protein with a molecular weight of 42.6 kDa starting from the 108th tyrosine.
  • Table 1 shows the known sites of activity conversion of collagenase, stromelysin, and gelatinase and the homologous regions of the human figure P20. Amino acid residues are indicated by conventional one-letter codes (hereinafter the same
  • MMP-20 Enamelysin 98 PRCGVPDVA YRLFPGEPKW 117
  • the MMP family 1 is classified into subfamilies such as collagenase, stromelysin, gelatinase, and membrane type ⁇ MMPs in terms of substrate specificity, form of existence, and homology of amino acid sequence.
  • the Tyr, Asp, and Gly 3-amino acids in the sequence near the zinc-binding site of collagenase-subsamammaly-1 (MMP-1, MMP-8, and MMP-13) are specifically conserved in the subfamily ( Table 2) is said to determine the substrate specificity.
  • MMP subfamily (Tl-MMP, MT2-MMP, T3-MMP and MT4 -Water-repellent water on the C-terminal side characteristic of '-hidden' 'penetration region, membrane type: / miP subfamily and the fibrin cleavage sequence found in "MP11" RXKR, not belonging to any subfamily It was shown vigorously.
  • human ⁇ P-20 includes MMP-1, MMP-8, GoP13, ⁇ P-12, Awake P-3, MP-10.
  • human MMP-20 is compatible with any existing ⁇ P subfamily. A new MMP that does not belong to This was consistent with the specific substrate specificity of human MMP-20 in the process of enamel formation.
  • Example 2 Expression of human MMP-20 in human tissue
  • the reaction was carried out for 20 hours under the same conditions as in the prehybridization, and washed with 0.1 x SSC containing 0.1% SDS at 50 ° C for 2 hours.
  • a actin probe was also added to estimate the state and amount of RNA.
  • RNA extracted from human odontoblasts, dental pulp and various human tissues liver, heart, muscle, placenta, brain, breast, breast cancer, ovary, testicle, lung, kidney, leukocyte
  • PCR was performed using 1st strand DNA prepared from (Clontech), primers Enamel-1 (SEQ ID NO: 6) and Enamel-4 (SEQ ID NO: 7). Denaturation was performed using the GeneAmp 2400 PCR system (Perkin-Elmer / Cetus) for the reaction. C 15 seconds, ani ring 58. Forty cycles of 15 seconds at C and 45 seconds at 72 ° C were performed. Only in odontoblasts and dental pulp, the band of human rigid P20 was amplified.
  • Example 3 Rice cake loaf with enzyme function of recombinant human MMP20 expressed in Escherichia coli
  • a gene fragment containing the human P20 cDNA excised by Pstl was inserted and ligated in frame with the expression vector PRSET-B (invitrogen), which was previously digested with Pstl. The direction of insertion was confirmed by the cutting pattern with EcoRI.
  • the expression vector was transformed into a competent cell of Escherichia coli BL21 (DE3) pLysS and cultured on an agar medium containing chloramphenicol and ampicillin.
  • the fraction containing recombinant human MMP-20 was dialyzed against a 20 mM Tris-HC1 buffer (pH 8.0) containing 150 mM NaCl, 5 mM CaCl ,, 50 / M ZnCl 2 and 0.01% NaN 3 and refolded. During this refolding process, human MMP-20 became active due to autonomous proteolysis. The precipitated protein was removed by centrifugation, and the night containing activated human MMP-20 was used for the measurement of enzyme activity.
  • the activity of activated human MMP-20 was measured using the Mps firefly ⁇ fci-synthetic peptide using the substrates McaPLGLD paARNH 2 and "McaPL fine vaDpaARNH 2 ".
  • the activity was measured at 37 ° C. respectively the k ca l / K m representing the. minutes ⁇ which was 0.5 / M, 1.5 ⁇ , sufficient cleavage activity indicates 4.55xlO '1 M_'s 5.88X1CTM-' s 1 was confirmed, respectively.
  • TI P- 3 an inducer containing TIMP-4 and a partial peptide thereof, an inducer containing a partial peptide of human MMP-20 having no amelodidinin-degrading activity, an antibody against human rigid P-20 (for example, Monoclonal antibodies described herein and derivatives containing partial peptides thereof; synthetic inhibitors (for example, synthetic compounds having meta-oral protease inhibitory activity such as hydroxamic acid, carboxylic acid, phosphonic acid, and thiol derivatives); Screening can be performed using. Chromosome mapping of human ⁇ -20 gene
  • the human-rodent hybrid cell panel (Human Genome Mapping Resource Center) was used to view the two primers STS-1 (SEQ ID NO: 11) and STS 2 ( It was subjected to PCR according to SEQ ID NO: 12) in the sequence listing, and screening of human figure P-20 was performed.
  • PCR was performed using the Expand Long PC Kit (Boehringer-Mannheim) described in Example 1 (b), denaturation at 94 ° C for 15 seconds, and annealing 58. A cycle of C 15 seconds and extension at 68 ° C. for 4 minutes was performed 35 times. As a result, PCR products were obtained from chromosome 11 and chromosome 13 panels (Fig. 2).
  • Two pig DNA fragments were labeled with biotin-16-dUTP by nick translation.
  • the probe hybridized to the metaphase chromosome of maglutinin-stimulated lymphocytes in human males. Further, c these labeled probes using Jigokishi genin probe specific for the centromere of chromosome 11 as an anchor one marker one can
  • the human P-20 probe was detected as yellow and the chromosome 11 centromeric probe was detected as red (Fig. 3).
  • human fighter P-20 was detected only on chromosome 11 and was not found on other chromosomes.
  • the chromosomes were banded with diamidinofenylindole dihydrochloride and subjected to CCD camera (P Observation with a firefly microscope (Zeiss) connected to a hotometri cs) revealed that the human MMP-20 probe hybridized to q22.3-23 of chromosome 11.
  • the conjugate was bound to serum albumin (BSA) via a cysteine residue. 19.6 mg of BSA was dissolved in 1 ml of 0.1 M phosphate buffer (pH 7.0), and 2.22 mg of EMCS (N- (6-maleimidecaproyloxy) -succinimi de) was added to 48.3 zl of The mixture was dissolved in dimethylformamide, reacted at 30 ° (:, 30 minutes), and then the above mixture was equilibrated with 0.1M phosphate buffer (pH 7.0).
  • BSA serum albumin
  • the resulting filtrate was gel-filtered using a Pharmacia method, and the concentration of the obtained maleamide-bound BSA was 6.22 mg / ml
  • concentration of the obtained maleamide-bound BSA was 6.22 mg / ml
  • Each of the polypeptides synthesized in (a) was mixed with a 0.1 M phosphate buffer (pH 7.0). 0) and mixed 50 times with maleimide-bound BSA: 35.8 nmol for 1788 nmol of polypeptide Enamel # 1 and 23.
  • each BSA polypeptide complex prepared in (b) above was administered together with complete Freund's adjuvant to 8-week-old Balb / c female mice) for the first immunization.
  • a 200 / g body was internally administered to mice immunized for the first time and boosted.
  • 200 // g of the BSA-polypeptide complex was intravenously administered for final immunization.
  • the spleen was excised and a spleen cell suspension was prepared.
  • 1640 medium RP 1-1640 (Flow Lab.) With sodium bicarbonate (2 machines), sodium pyruvate (lmM), potassium penicillin G (50U / ml), amikacin sulfate (100 / zg / ml) The mixture was adjusted to pH 7.2 with dry ice and sterilized and filtered through a 0.2 fim Toyo Membrane Filter.
  • NS-1 medium Fetal serum (FCS, A.A. Bioproducts) filtered and sterilized was added to the RPMI-1640 medium to a concentration of 15% (v / v).
  • PEG-4000 solution A serum-free medium was prepared by adding polyethylene glycol 4000 (PBG-4000, Merck & Co.) to RPMI-1640 medium to a concentration of 50% (w / w).
  • nucleated splenocytes (viable cell rate 100%) and myeloma cells (viable cell rate 100%) prepared in (c) were fused in the following procedure at a ratio of 5: 1.
  • Each of the polypeptide-immunized spleen cell suspensions and myeloma cells were washed with RPM11640 medium. Next, the cells were suspended in the same medium, and nucleated splenocytes and myeloma cells were mixed for fusion.
  • BSA- Poripe Puchito "Enamel # 1 Contact to the complex, Te is, 4.7 x 10 8 cells of the chromatic Kaku ⁇ cell pair were 9.3 ⁇ 10 7 cells of myeloma mouth Ichima cells, BSA- polypeptide Enamel # 2 complex
  • BSA-polypeptide Enamel # 3 complex In 5.3x10 8 nucleated spleen cells, 1.lxlO 8 myeloma cells, and in the BSA-polypeptide Enamel # 3 complex, the first 1.8X10 8 nucleated splenocytes (mouse 1 3. 6 X 10 7 cells of myeloma cells to animal), were mixed 7. 4 X 10 7 cells of myeloma cells to the second 3.
  • RPMI-1640 medium 7 times the volume of the added RPMI-1640 medium containing 50 ° PEG-4000, was added dropwise with stirring for 2 to 3 minutes to disperse the cells. This was centrifuged, and the supernatant was completely removed by suction. Next, add S-1 ⁇ ground, which has been heated to 37 ° C to 3 ⁇ 10 6 cells / ml, quickly to the precipitated cells, and carefully add large cell clumps. Dispersed by pipetting. Further, the same medium was added for dilution, and the cells were inoculated into a 96-well polystyrene microwell so that the number of myeloma cells per well became 6.0 ⁇ 10 5 . The microgel containing cells was cultured in 7% carbon dioxide / 93% air at a temperature of 37 ° C and a humidity of 100 ".
  • HAT medium NS1 medium described in (d) (1) above was supplemented with hiboxanthin (100 uM), aminopterin (0.4 ⁇ M) and thymidine (l (ii ⁇ 1)).
  • HTi base The same composition as the above HAT medium except that aminobuterin was removed.
  • HRP horseradish peroxidase
  • Cappel horseradish peroxidase-labeled goat anti-mouse immunoglobulin
  • the hybridomas in the positive wells for the peptide obtained in the above section (e) were monoclonally cloned using the limiting dilution method. That, NS-1 medium lml per Fi a black-learning medium containing 10 7 mouse thymocytes were prepared as an da one, 5 per Ueru the hybridoma to 96 Mai Kuroueru, one, 0.5 or , And added to 36, 36, and 24 wells, respectively. On days 5 and 12, about 0.1 ml of NS-1 medium was added to all the wells.
  • Hypridoma producing the above-mentioned anti-human MMP-20 monoclonal antibody 203-1C7 has been deposited with NIBH on July 22, 1998 under the accession number FERM BP-6434.
  • the gene fragment containing human MMP-20 cDNA cut out by Pstl in Example 3 was cut in advance with Pstl and inserted in frame into an Escherichia coli expression vector ( ⁇ 58 (11 ⁇ 611)) which had been treated with CIP.
  • Escherichia coli expression vector ⁇ 58 (11 ⁇ 611)
  • competent cells of E. coli strain DH5 were transformed and cultured on an agar medium containing 100 / g / ml ampicillin.A single colony was picked and 4 ml containing 100 g / ml ampicillin. The medium was inoculated into B medium and cultured at 37 ° C.
  • the obtained primary culture was re-inoculated into B medium containing 400 ml of 100 ⁇ g / mi ampicillin until the turbidity at A en reached 0.6.
  • the cells were cultured at 37 ° C.
  • IPTG was added to a final concentration of ImM to induce the expression of recombinant MMP 20.
  • the recombinant E. coli was collected by centrifugation and contained lmg / ml lysozyme.
  • the sample was applied to a Ni-cleaning Sepharose gel column (Pharmacia) equilibrated in step 1.
  • the molecule having a His tag at the N-terminus was adsorbed.
  • the recombinant MMP-20 was purified by eluting with a 50 mM phosphate buffer (pH 7.5) containing glue.
  • a main band of about 56 kDa was observed.
  • the expected molecular weight of the expression product is approximately 55 kDa. It was confirmed that the 56 kDa protein was recombinant rigid P20.
  • the recombinant MMP-20 purified in (1) was subjected to SDS-PAGE and transferred to a nitrocellulose membrane.
  • the membrane was blocked with 33 ⁇ 4 skim milk, 50 mM phosphate buffer (pH 7.5) containing 0.1 M NaCl for 1 hour at room temperature, and cut into 2.8 thigh strips.
  • the high prepared in (f) Pre-doma culture supernatant was added to each strip and reacted at room temperature for 16 hours.
  • FIG. 4 shows the results of Western blotting of the BSA-polypeptide Enamel # 1 complex and the BSA-polypeptide Enamel # 3 complex with the monoclonal antibody 200-10B2 and the monoclonal antibody 2031C7. These monoclonal antibodies reacted with recombinant MP-20 and showed a band of about 56 kDa.
  • the obtained hybridoma cells are cultured in MS 1 medium, and a monoclonal antibody having a concentration of 10 to 100 // g / ml can be obtained from the supernatant.
  • a combination of two suitable antibodies from the anti-human P-20 monoclonal antibody prepared in Example 5, for example, a combination of monoclonal antibody 200 10B2 and monoclonal antibody 203-1C7 This makes it possible to construct a sandwich EIA system that specifically detects and measures human MP-20.
  • the E1A system can use either the one-step method or the two-step method, and the labeled antibody is not limited to Fab'-HRP.
  • the composition and reaction conditions of each reaction buffer can be adjusted, such as shortened or extended, according to the purpose of measurement.
  • standard human MMP-20 can be purified from human odontoblasts, human dental pulp or tissue culture supernatant thereof, or a recombinant expressed by the method described in Examples 3 and 5 or other methods.
  • Purification includes ion exchange, gel filtration, and anti-human MMP-20 This is achieved by a combination of affinity chromatography using a nocturnal antibody and other various affinity chromatography.
  • the prepared antibody-bound microplate is washed three times with Tris-HCl buffer (pH 8.0) containing 0.05% Tween20, 0.1M NaCl, and 5mM CaC. Add each. 0. 053 ⁇ 4 Tween20 After reacting 1 hour at room temperature, 0. 1M NaCl, Tri s- HCl buffer containing 5mM CaCl 2 (pH8. 0) at you 3 times washed ⁇ . Next, 1.2 mg / ml o-phenylenediamine dissolved in 0.1 citrate monophosphate buffer ( ⁇ 4.9) containing 0.02% hydrogen peroxide was added at 100 ⁇ 1 per well, and the mixture was cooled to room temperature. After reaction for 20 minutes, add 100 n ⁇ of 2X sulfuric acid to stop the reaction. Measure the A492 of this reaction mixture using a microphone mouth plate reader to obtain a standard curve.
  • Samples to be measured include human serum, plasma, synovial fluid, urine and saliva, and other body fluid components derived from humans, extracts of various human tissues, cell extracts of various cultured cells such as human or recombinant, and culture. It is prepared from pure water.
  • Each measurement sample is supplied to the above-mentioned one-step sandwich E1A instead of the standard human P-20, and the reaction proceeds simultaneously with the standard human P-20.
  • Calculate the amount of human MMP-20 contained in the measurement sample by applying the A492 value obtained from the measurement sample to the standard curve obtained.
  • human P human enamelysin, human MMP20
  • human MMP20 human MMP20
  • odontoblasts The whole length of the gene encoding human P (human enamelysin, human MMP20), which is a powerful and novel protein with homology to the lysin gene sequence, is expressed in odontoblasts.
  • human MMP20 human MMP20
  • the expression of this gene in fungi can reveal its enzymological properties with recombinant proteins.
  • human MMP-20 The expression of human MMP-20 is limited to tooth tissue and the ability of human fraction P20 to have enamel resolution ⁇
  • human MMP-20 plays a specific role in tooth enamel formation, and the importance of its use can be recognized.
  • An antibody that specifically reacts with human MMP-20, especially a monoclonal antibody, is created and an immunological group! It can provide a means for detecting human MMP-20, such as color, as well as a URL therefor. Also, methods for detecting and measuring human MMP-20, such as the EIA system, are used.
  • amelodidinin-degrading action ij therapeutic agent for enamel dysplasia, agent for inducing and / or promoting regeneration of defective enamel, agent for treating and / or preventing dental caries (caries), exposed dentin
  • the present invention provides an agent for inducing and / or promoting enamel formation into Z, or an agent for enamel formation. Further, the present invention provides a drug having an activity of inhibiting amelodidinin degradation.
  • the protein and the like of the present invention are useful as screening reagents for compounds or salts thereof that promote or inhibit the biological activity of the protein or the like of the present invention.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Organic Chemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biomedical Technology (AREA)
  • Genetics & Genomics (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Medicinal Chemistry (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Molecular Biology (AREA)
  • Peptides Or Proteins (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

L'invention porte sur de nouvelles protéines utilisées dans l'étude du développement des dents et du processus de maturation de l'émail, notamment, la formation de l'émail ou la minéralisation de la matrice dentaire, ou sont utilisées à d'autres fins médicales et physiologiques. L'invention porte notamment sur la protéine humaine énamelysine (MMP-20) qui est une des protéines humaines dont l'activité décompose la protéine de l'émail. L'invention porte également sur des gènes codant ces protéines, et sur des anticorps contre ces protéines. L'invention porte encore sur de nouvelles métalloprotéases matricielles (notamment la MMP-20 humaine) clonées par une banque d'ADNc provenant de la pulpe dentaire humaine, sur des ADN contenant une séquence de base les codant; sur des cellules hôtes transformées par ces ADN, sur un procédé de production des protéines métalloprotéases matricielles au moyen des cellules hôtes, sur des anticorps (y compris les anticorps monoclonaux) capables de se lier aux protéines métalloprotéases matricielles et sur les utilisations de ces protéines, de leurs peptides, des anticorps, des acides nucléiques ou des oligomères d'acide nucléique.
PCT/JP1998/003381 1997-07-29 1998-07-29 Nouvelle proteine humaine mmp-20 et son utilisation WO1999006555A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP9/217038 1997-07-29
JP21703897 1997-07-29

Publications (1)

Publication Number Publication Date
WO1999006555A1 true WO1999006555A1 (fr) 1999-02-11

Family

ID=16697875

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP1998/003381 WO1999006555A1 (fr) 1997-07-29 1998-07-29 Nouvelle proteine humaine mmp-20 et son utilisation

Country Status (1)

Country Link
WO (1) WO1999006555A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999061655A1 (fr) * 1998-05-29 1999-12-02 Hawaii Biotechnology Group, Inc. Essai par fixation determinant un etat de transition

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
BARTLETT J.D. et al., "Molecular Cloning and mRNA Tissue Distribution of Novel Matrix Metalloproteinase Isolated from Porcine Enamel Organ", GENE, (1996), Vol. 183, p. 123-128. *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999061655A1 (fr) * 1998-05-29 1999-12-02 Hawaii Biotechnology Group, Inc. Essai par fixation determinant un etat de transition
US6569636B2 (en) 1998-05-29 2003-05-27 Hawaii Biotechnology Group, Inc. Assay for modulators of metallo-enzyme activity

Similar Documents

Publication Publication Date Title
JP3981416B2 (ja) Ｐｃａ３タンパク質、ｐｃａ３遺伝子、及びこれらの用途
JP3837494B2 (ja) 可溶型ｒａｇｅタンパク質
KR101363695B1 (ko) 항페리오스틴에 대한 항체, 및 페리오스틴이 관여하는 질환의 예방 또는 치료를 위해 그것을 함유하는 약제학적 조성물
US20090259019A1 (en) Masp-2, a complement fixing enzyme, and uses for it
JPH09509570A (ja) Ｍｔｓ−１遺伝子により転移性癌の診断
JP2003116587A (ja) ポリペプチド及び同じものをコードしている核酸
WO1997004080A1 (fr) Nouvelle proteine et anticorps monoclonal specifique a cette derniere
EP1225224A1 (fr) Adn reagissant a la contrainte de cisaillement
JPH10210982A (ja) 新規なタンパク質
WO2004064857A1 (fr) Medicaments contenant galectine 9
MXPA02009091A (es) Anticuerpos monoclonales para el receptor humano para lipoproteinas de baja densidad, su produccion y uso.
WO2000029441A1 (fr) Nouvelle proteine receptrice couplee a la proteine g, son adn et son ligand
JP4488140B2 (ja) セリンプロテアーゼであるコリン
JP2000050885A (ja) カテプシンに対する抗体およびそれらの利用
EA006874B1 (ru) Полипептид, обладающий активностью rap [белка, ассоциированного с rip (взаимодействующим с рецептором белком)], кодирующая его последовательность днк и способы их получения и использования
JPH0987299A (ja) Ｍｍｐ−２活性化因子に特異的なモノクローナル抗体
JP2001231578A (ja) Ｉｌ−１ファミリーに属する蛋白質
JP2003527091A (ja) 新規カルパインプロテアーゼ２６１７６およびその使用
JP4201712B2 (ja) Ｇｉｐ、グッドパスチャー抗原結合タンパク質と相互作用する転写因子活性を備えたポリペプチドのファミリー
JPH0984589A (ja) 新規なタンパク質
WO1999006555A1 (fr) Nouvelle proteine humaine mmp-20 et son utilisation
JP4171228B2 (ja) 可溶型ｒａｇｅ測定法
JPH09235300A (ja) ヒトｔｉｍｐ−３及び抗ヒトｔｉｍｐ−３モノクローナル抗体並びにその用途
JP2000270874A (ja) 新規な膜結合型メタロプロテアーゼ
JP2003321494A (ja) プロｍｍｐ−７の活性化調節方法

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): JP US

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE

DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
121 Ep: the epo has been informed by wipo that ep was designated in this application
122 Ep: pct application non-entry in european phase