WO2002057448A1 - Nouveaux peptides et leurs activites - Google Patents

Nouveaux peptides et leurs activites Download PDF

Info

Publication number
WO2002057448A1
WO2002057448A1 PCT/JP2001/011529 JP0111529W WO02057448A1 WO 2002057448 A1 WO2002057448 A1 WO 2002057448A1 JP 0111529 W JP0111529 W JP 0111529W WO 02057448 A1 WO02057448 A1 WO 02057448A1
Authority
WO
WIPO (PCT)
Prior art keywords
tes
polypeptide
amino acid
cells
salt
Prior art date
Application number
PCT/JP2001/011529
Other languages
English (en)
Japanese (ja)
Inventor
Hiroshi Sato
Takanori Aoki
Original Assignee
Daiichi Fine Chemical Co., Ltd.
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 Daiichi Fine Chemical Co., Ltd. filed Critical Daiichi Fine Chemical Co., Ltd.
Priority to JP2002558500A priority Critical patent/JPWO2002057448A1/ja
Publication of WO2002057448A1 publication Critical patent/WO2002057448A1/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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
    • A61P19/10—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease for osteoporosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P21/00—Drugs for disorders of the muscular or neuromuscular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00

Definitions

  • the present invention relates to a newly identified polynucleotide (or nucleic acid) and polypeptide (or protein) (X is D or a salt thereof; a variant of the polynucleotide (or nucleic acid) and polypeptide (or protein)) A method for producing the polynucleotide (or nucleic acid) and polypeptide (or protein), and variants and derivatives thereof; an antibody against the polypeptide (or protein) (or a part thereof); Immunological assay using antibodies and agonists and antagonists of the polypeptide (or protein), and the screening method and screening kit associated therewith; and the polynucleotide (or nucleic acid) , Polypeptide (or protein), mutant This is related to the use of invitation books, agonists, and angels.
  • Matrix metalloproteases are a family of Mlf-dependent endopeptidases that degrade the extracellular matrix and various constituents of the basement membrane component, the proteins. These enzymes are involved in the reconstitution of connective fibers, such as normal embryo development, bone growth or wound healing (Woessner, JF, FASEB J., 5, 2145-2154 (1991); Matrisian , L.., BioEssays, 14, 455-463 (1992); Birke (-Hansen, H., Moore WGI, Bodden, MK, Windsor, LJ, Birkedal-Hans en, B., DeCarlo, A., and Engler. Oral Biol.
  • connective fibers such as normal embryo development, bone growth or wound healing (Woessner, JF, FASEB J., 5, 2145-2154 (1991); Matrisian , L.., BioEssays, 14, 455-463 (1992); Birke (-Hansen, H., Moore WGI, Bo
  • MPs MMP-1 (collagenase); MMP-2 (gelatinase A); MMP-3 tromelysin-1); MP-7 ⁇ matrilysin); MMP-8 (neu trophil collagenase); MMP-9 (gelatinase) B); MMP- 10 (stromelysin-2); wallet-11 (stromelysin-3); MMP-12 (macrophage elastase); ⁇ P-13 (collagenase-3); MP-14 (MT1-MMP); MMP- MMP-16 (MT3-MMP); MMP-17 (MT4-MMP); MMP-18 (collagenase-4); MMP-19; MMP-20 (enamelysin); MMP-24 (T5 -MP); MMP-25 (MT6-MMP) etc.) Strongly known (Woessner, JF, FASEB J., 5, 2145-2154 (1991); Matrisian, LM, BioEssays, 14, 455-463 (1992); Birkedal-Hansen
  • banded Ps Based on primary neurons, substrate specificity and cell distribution, these banded Ps have been classified into at least four subfamilies: collagenase, gelatinase, stomata melysin, and membrane MMPs (MT-banded Ps).
  • the MT-MMPs subfamily is the most recently reported subclass of MMPs, and has so far been identified with more than five members by the use of digitized primers and RT-PCR for the region conserved in the PMPs.
  • -MMP, MT3-MP, T4-MMP, MT5-P, etc. Strongly isolated and identified (Sato, H., Takino, T., Okada, Y., Cao, J., Shinagawa, A. , Yamamoto, B., and Seiki, M., Nature, 370, 61-65 (1994); Will, H., and Hinzmann, B., Eur. J.
  • MT-MMPs are type I membrane proteins with a single transmembrane domain and a short intracellular tail behind the homopexin domain characteristic of many banded Ps. In addition, they insert a ⁇ S-amino acid between the propeptide and the active domain, and when activated by furin or a furin-like enzyme, activation of these membrane proteins can occur. Okiru (Pei, D ,, and Weiss, SJ, J. Biol.
  • Testican is the first to be discovered proteoglycans in the testes, from human testis plasma to chondroitin sulfate / as Roh N 0 run sulfate-flops opening Teogurikan isolated (B i ochem. J., 288 , 565-569 (1992) ).
  • the amino acid rooster J was inferred from human testis cDNA (Bur. J. Biochem, 214: 347-350 (1993)), ⁇ -40 / secreted protein, acidic and rich in cysteine (SPARO / osteonectin protein family). (Bio chem.
  • Testican has a multi-domain structure. Testican localization is a restricted area of the brain, located at the postsynapse. This suggests that Testican may be involved in receptor activity, neuromodulation, synapse formation, or mycelia transmission.
  • Testican and its related substances include Testican-1 (Bur. J. Biochem, 214: 347-350 (1993)) and Testican-2 (Journal of Neurochemistry, Vol. 73: 12-20 (1 999)).
  • Testican-3 such as Testican-3, ⁇ -40 / SPARC / osteonectin, QR1, SCl / hevin, tsc36 / Flik, etc.
  • Testican-3 is derived from a gene encoding 36 amino acids and has a multidomain structure. Its special domains are the Kazal-type domain (positions 148-183 of the amino acid rooster), a foll istatin-like domain that acts as a serine proteinase inhibitor, and the TY domain (Thyroglobul in type I repeats, amino acid rooster).
  • Testican-3 is recognized as having a Glycosaminoglycan attachment site.
  • HPTLG One having an amino acid sequence similar to this is HPTLG (W099 / 23110 A1).
  • This HPTLG is derived from the HPTLG gene from the lower part of the human liver and is thought to be involved in cell adhesion, migration, proliferation and the like. Because it is a testican-like protein, it is speculated that it acts as a growth factor receptor in the brain.
  • HPTLG has a multi-domain structure, and its prominent domain is the Kazato type domain (positions 136-182 of the amino acid sequence), which is known to have proteolytic inhibitory activities such as QR1, SC1, and foll istatin.
  • CWCV domain (332-381 of amino acid sequence), which is known to bind, for example, insul in-like growth factors.
  • HPTLG is also recognized as having Glycosaminoglycan attachment site.
  • the relationship between cancer and solid Ps and / or membrane-type Ps so far has been described, for example, in the activation of prozelatinase A (Okada et al., Bur. J. Biochem., 194, pp721-730, 1990). ), Activation of solid P-2 by MT1-MMP (Sato et al., Nature, 370, pp61-65, 1994) has been suggested, but the specific involvement is still unknown. It is.
  • the present inventors have constructed a screening system for genes involved in the regulation of marshal Ps activity, and conducted a search for genes involved in the regulation of cascade Ps activity in human genes. Novel genes that have inhibitory activity against mosquitoes T1-MMP and MT3- ⁇ P, which are very similar to a group of genes called I found a peptide.
  • polypeptide or the salt thereof according to the above (1) which is a polypeptide selected from the group consisting of those having an amino acid sequence substantially equivalent to any one of them. ;
  • MT- MMPs have the function of suppressing the ability to activate MMPs
  • nucleic acid of the above-mentioned [6] which has an open reading frame portion or a ⁇ S sequence substantially equivalent thereto in the nucleotide sequence represented by SEQ ID NO: 1 in the sequence listing;
  • the pharmaceutical according to the above [16] which is characterized by being a member of the group consisting of: [18] the polypeptide according to any one of the above [1] to [5], Drugs that promote or inhibit the biological activity of peptides or their salts, or that contain the salts thereof;
  • Expression plasmids selected from the group consisting of national Ps and sample genes are introduced into host cells, and the detection of the expressed P is used as an index. Screening of the secrets involved in the regulation of the activity of debris Methods and screening kits;
  • N-Tes gene or nucleic acid such as mRNA derived therefrom (c) N-Tes gene or nucleic acid such as mRNA derived therefrom, and
  • the method of detecting gloma is based on the fact that the index is selected from the group consisting of birds.
  • the invention relates to
  • polypeptide or a salt thereof according to any one of the above [1] to [5], which has an inhibitory activity on MT1-MMP and Z or MT3-MMP;
  • the term “and / or” means that there are both (1) merged connectivity and (2) selective connectivity, such as “treatment and Z or prevention”. : t is meant to encompass both (1) treatment and prevention and (2) treatment or prevention. Elsewhere, the term “and / or” is similarly used to encompass both (1) merged connections and (2) selective connections.
  • Figure 1 shows that the plasmids expressing MMP-9, MMP-2, and MT1-MMP and the test sample plasmid were introduced into 293T cells, and the latent, intermediate, and activated MP-9, The result of detecting MMP-2 is shown.
  • FIG. 2 shows the homology of Testican-1, Testican 2, Testican-3 and HPTLG, and N-Tes with respect to the amino acid rooster.
  • FIG. 3 shows the results of inhibition of activation of bandits P-2 through MT1-MP and MT3-MP by N-Tes-de FLAG analyzed by gelatin zymography.
  • FIG. 4 shows the results of an N-Tes expression search in glioma.
  • FIG. 5 shows the results of an investigation on the binding between MT-MMP and N-Tes.
  • FIG. 6 shows the results of square beveling of the expression levels of Testican 3 and N-Tes by semi-RT-PCR.
  • NB normal brain fiber
  • LGA melanoma
  • M undifferentiated astrocytoma
  • GB Glioblastoma
  • Meta Cancer of the brain metastasized to the brain
  • GCL Glioma cell line Best mode for carrying out the invention II
  • a human N-Tes polypeptide having a P-band inhibitory activity which is a kind of Testican family, has at least 60% homology with the amino acid rooster of the N-Tes polypeptide, and ⁇ Peptides or salts thereof having P-inhibitory activity or equivalent antigenicity, special partial peptides of the polypeptides or salts thereof, and genes encoding them, such as DNA, RNA, etc.
  • a transgenic animal such as a mouse, a knockout animal such as a knockout mouse in which the gene is specifically inactivated, and a transformed cell thereof are cultured.
  • the antibody-producing hybridoma cells, the isolated gene for example, DNA, RNA, etc., may be used as a probe, or may be used as a measurement / diagnosis means using the antibody, and may be used in conjunction with the present invention.
  • N-Tes is a peptide related to the proteoglycan Testican, and refers to a novel peptide disclosed in the present invention.
  • the N-Tes is a peptide of 313 amino acid residues, at its C-terminal has a tree ⁇ sequence Gly 3 "-Lys 3 1 2 -Ar 3 1 3, Testi can -Lack of TY domain and CWCV domain present in 3 and HPTLG, and also present in Testican-3 and HPTLG Lacking the Glycosaminoglycan attachment site.
  • the N-Tes has MT-MMPs P and a harmful activity, and particularly has a strong inhibitory activity against ⁇ -band P and MT3-sub-P, and more strongly against MT3-band P. Have activity.
  • the inhibitory activity includes, for example, inhibiting activation of P-2 in the MT-MMPs region.
  • This inhibitory activity against MT-solid Ps is predicted to be within the amino acid sequence at positions 22 to 122 of SEQ ID NO: 1 in the sequence listing, and these amino acid sequences or amino acid sequences substantially equivalent thereto Is meant to have an inhibitory activity on MT-MMPs.
  • HPTLG has inhibitory activity against MT-banded Ps.
  • testican-3 is considered to have an inhibitory activity against 1 ⁇ -figure 3 as well as the force of insertion of 3 amino acid residues into these roosters.
  • polypeptide may refer to any of the polypeptides described below. The basic structure of polypeptides is well known and is described in numerous references and other publications in the art. In view of these, the term “polypeptide” as used herein refers to any peptide containing two or more amino acids that are linked to each other by a peptide bond or a modified peptide bond. Or any protein. As used herein, the term “polypeptide” is generally referred to in the art as, for example, a peptide, an oligopeptide or a peptide chain, a short chain, and in many forms, generally referred to as a protein. It may mean both long and long chains.
  • Polypeptides often contain amino acids other than the 20 naturally occurring amino acids (the naturally occurring amino acids: or amino acids encoded by the gene) other than the 20 amino acids. May be contained. Polypeptides may also be processed and otherwise altered (or modified) after many of their amino acid residues have been translated, including the terminal amino acid residues, as well as by natural processes. It will be understood that the above polypeptides can also be modified (modified) by chemical modification techniques well known in US Pat. The alterations (modifications) made to the polypeptide are well known in many forms, and are described in basic reference books and more detailed articles in the art, as well as in a number of studies. They are well described in the literature and are well known to those skilled in the art.
  • Some particularly conventional modifications and modifications include, for example, glycosylation, lipid binding, sulfation, glutamation, 7-carboxylation of acid residues, hydroxylation and ADP-ribosylation, and the like.
  • polypeptide of the present invention particularly includes N-Tes and related polypeptides.
  • N-Tes and related polypeptides include those derived from humans, and those having an inhibitory activity on marshal Ps, for example, Ml-MMP, MT3-maraudal P, etc. Specific examples include those lacking the Glycosaminoglycan attachment site and / or lacking the TY domain or the CWCV domain.
  • amino acid sequences represented by SEQ ID NO: 2 in the sequence listing Having at least the amino acid sequence at positions 311 to 313, having the amino acid sequence at positions 22 to 122, and having the amino acid sequence B ⁇ ij at positions 22 to 313 Having an amino acid sequence of positions 1 to 313, and a homology of at least 60%, preferably 70% or more, more preferably 80% or more with any one of them. And preferably at least 85% homology, preferably 90% or more homology, more preferably 95% or more homology, particularly preferably 97% or more homology and PP and harmful activity, or substantially equivalent antigenicity. All those having an amino acid sequence having a biological activity equivalent to that described above are listed.
  • the human N-Tes of the present invention has inhibitory activities such as MT1-bandwidth P, MT3-bandwidth P, A variant of the Testican family, such as ican-3, which lacks the Glycosaminoglycan attachment site and has a novel amino acid sequence. More preferably, the peptides of the present invention include those having an amino acid sequence having homology at least higher than 60% with the Testican family, but lacking the glycosam inoglycan attachment site.
  • Ku is 90 or more, most preferably 100 or more, and preferably include rather force those having more than 110 pieces).
  • the peptide of the present invention includes, among the amino acid sequences represented by SEQ ID NO: 2 in the sequence listing, those having at least the amino acid sequence at positions 22 to 313, and any one of them. Are selected from the group consisting of those having substantially the same amino acid sequence. Further, the peptide of the present invention may have a part of the amino acid sequence represented by SEQ ID NO: 2 or SEQ ID NO: 2 in the sequence listing. Any having such an arrangement may be included.
  • the term “homology” refers to the residue of each amino acid constituting two strands in a polypeptide sequence (or amino acid sequence) or polynucleotide sequence (or base sequence).
  • Preferred methods for determining homology include those designed to obtain the largest match between the two sequences tested. Such methods are those that are assembled as computer programs.
  • Preferred computer programming methods for determining homology between two sequences include the GCG program package (Devereux, J. et al., Nucleic Acids Research, 12 (1): 387 (1984)), BLASTP ⁇ Forces such as BLASTN and FASTA (Atschul, 'SF et al., J. Molec.
  • the gene encoding N-Tes of the present invention typically has a nucleotide sequence encoding the peptide represented by SEQ ID NO: 2 in the sequence listing and a partial continuous amino acid sequence thereof.
  • the start codon in the base sequence eg Met Code codons (and stop codons) which has at least 80% homology with the protein whose nucleotide sequence encodes, and at least one of Ps such as MT1- ⁇ P, MT3-MMP, etc.
  • Ps such as MT1- ⁇ P, MT3-MMP, etc.
  • the gene encoding the N-Tes is a nucleic acid such as a single-stranded DNA, a double-stranded DNA, an RNA, a DNA: RNA hybrid, or a synthetic DNA, and a human genomic DNA, a human dinoomic DNA library, and a human paper fabric. It may be either cell-derived cDNA or synthetic DNA.
  • the remaining nucleotide sequence encoding the N-Tes can be modified (eg, ira, removed, substituted, etc.), and such modified ones can be included.
  • the nucleic acid of the present invention may encode the peptide of the present invention or a part thereof, and preferably includes DNA.
  • the “equivalent nucleotide sequence” refers to, for example, 5 or more consecutive nucleotide sequences, preferably 10 or more nucleotide sequences of the nucleotide sequence of SEQ ID NO: 1 in the sequence listing under stringent conditions, more preferably Is one that hybridizes with 15 or more base sequences, more preferably 20 or more sequences, and encodes an amino acid sequence substantially equivalent to N-Tes.
  • the DNA of the present invention containing the nucleotide sequence represented by SEQ ID NO: 1 in the sequence listing or a nucleotide sequence equivalent thereto can be obtained, for example, by the following method.
  • Plasmids with cDNA inserts obtained from cDNA libraries can be converted into at least one of MMPs by an appropriate detection system. Sorting is performed using the index that suppresses one activity as an index. Specifically, for example, a plasmid having a cDNA insert sequence obtained from the cDNA library to be tested, together with a vector that expresses the full length cDNA of IMP-9, MP-2, and Ml band P. Is transfected into a suitable host cell, for example, to detect latent, intermediate, and activated MMMP-9 and MMP-2 to produce activated MMP-9 or activated solid P-2. Search for suppressive gene plasmid populations.
  • the sequence of the gene thus identified is determined. Based on the sequence identified in this manner, appropriate primers are designed and synthesized.In some cases, a cDNA library derived from an animal, which is the origin of the identified sequence, is used. Amplification by chain reaction (polymerase chain reaction: PCR). The obtained DNA fragment is used as a probe to screen a human genetic DNA library or a human-derived cDNA library constructed from various human threads or cultured cells, and to select a clone that hybridizes to the probe. The nucleotide sequence of the human sequence of DNA in the clone can be determined to obtain and obtain a novel N-Tes and a DNA fragment having a related 5′-sequence.
  • the sense primer and antisense primer can be designed and synthesized.
  • Sense primer is preferably a corner ?
  • the exon site at the 5 'end of the putative gene of the putative desired peptide can be selected and synthesized, and the nucleic acid primer is preferably analyzed.
  • the cDNA of the gene may aim to obtain the full length at one time, but using the analyzed exon site (the exon site of the exon site), the primers are designed and synthesized, and the PCR of the fiber is performed.
  • the resulting DNA is designed and designed (if necessary, the DNA fragment whose base sequence has been determined is squared to determine the entire base sequence of the cDNA of the gene, and cloned based on that). It is possible to obtain cDNA of the gene from the fragment.
  • the primer preferably includes an oligonucleotide consisting of 5 or more bases, more preferably an oligonucleotide consisting of 18 to 25 bases.
  • the preparation of the primer can be carried out by a method known in the art.
  • the primer can be synthesized by a phosphodiester method, a phosphotriester method, a phosphoramidite method, and the like.
  • PCR can be carried out using the cDNA library and the above-mentioned sense primer and antisense primer to amplify the cDNA.
  • a specific hybridization probe is prepared from the mouse identified as described above, a human-derived DNA library is screened, and the probe is hybridized. This can be done by selecting a clone.
  • a commercially available labeling kit such as a random primed DM labeling kit (Boehdnger Mannheim) can be used.
  • cDNA library used as ⁇ can be prepared from various commercially available cDNA libraries.
  • a cDNA library commercially available from Stratagene, Invitrogen, Clontech, etc. can be used.
  • a typical example is a gene library prepared from the labeled DNA fragment and human paper tissue and cells, such as a human PI articial chromosome genomic library (Human Genome Mapping Resource Center), a human brain cDNA library (for example, , Available from Clontech, etc.).
  • the human brain cDNA library can be constructed, for example, in a phage such as igU0, and it can be obtained by infecting host E. coli such as E. coli C600hil strain to form a black. If necessary, the inserted sequence of the cDNA in the fragment can be sub-ligated.
  • the target DNA can also be isolated based on the determined base sequence.
  • the determination of the base sequence can be carried out using the Maxam-Gilbert method, such as the Didoxy method, for example, the M13 Didoxy method. Applied Biosystems), Sequenase v2.0 kit, etc., or by using an automatic base rooster ij determination, for example, a fluorescent DNA sequencer (eg, odel 373A, Applied Biosystems). I can do it.
  • the polymerase used in the dideoxy method include Klenow fragment of DNA polymerase I, layer reverse transcriptase, Taq DNA polymerase, T7 DNA polymerase, and modified T7 DNA polymerase. It is.
  • PCR Polymerase Chain Reaction
  • U.S. Pat refers to a method for enzymatically amplifying a desired nucleotide sequence in vitro.
  • the PCR method prefers type I nucleic acids The method involves ⁇ ffl of two oligonucleotide primers that can be hybridized to the same, and repeating a cycle for performing primer extension synthesis.
  • the primers used in the PCR method can use a primer that is complementary to the internal nucleotide sequence to be amplified, e.g., the nucleotide sequence to be amplified and its Preferably, a force that is complementary at both ends, or one that matches the nucleotide sequence to be amplified, may be used.
  • the PGR reaction can be 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. Saiki, et al., Science, 239: 487, 1988; HA Erliched., PCR Technology, Stockton Press, 1989; DM Glover et al. ed., "DNA Cloning", 2nd ed., Vol. J. Practical Approach Series), IRL Press, Oxford University Press (1995); MA Innis et al. Ed., "PCR Protocols: a guide to methods and applications", Academic Press, New York (1990));. McPherson, P.
  • PCR method can be performed using a commercially available kit suitable for the PCR method, and can also be performed according to a protocol specified by the kit manufacturer or kit distributor.
  • Representative i ⁇ include, for example, 1st strand DNA and primers, 10X reaction buffer (attached to DNA polymerase such as Taq DNA polymerase), dNTPs (doxynucleoside triphosphate dATP , dGTP, dCTP, (mixture of ⁇ ), Taq DNA polymerase and deionized distilled water
  • DNA polymerase such as Taq DNA polymerase
  • dNTPs doxynucleoside triphosphate dATP , dGTP, dCTP, (mixture of ⁇ )
  • Taq DNA polymerase and deionized distilled water
  • the mixture is mixed using, for example, an automatic thermal cycler such as GeneAmp 2400 PCR system, Perkin-Elmer / Cetus.
  • the cycle is repeated 25 to 60 times under general PCR cycle conditions, but the number of cycles for amplification can be set to an appropriate number according to the purpose.
  • the conditions for the denaturation include, for example, denaturation 90 to 95 ° C 5 to 100 seconds, annealing 40 to 60 ° C 5 to 150 seconds, extension 65 to 75 ° C 30 to 300 seconds, preferably denaturation 94 ° C 15 seconds. , Annealing at 58 ° C for 15 seconds, extension 72. C A 45 second cycle can be mentioned, but the reaction key and time for annealing can be selected to appropriate values by experiments, and the denaturation reaction and extension reaction time also depend on the expected PCR product chain length. An appropriate value can be selected accordingly.
  • the annealing reaction is usually strongly preferable to be changed according to the Tm value of the hybrid between the primer and the type I DNA.
  • the extension time is usually about 1 minute per 100 bp of chain length, but it is possible to select a shorter time.
  • the obtained PCR product is usually subjected to 1-2 agarose gel electrophoresis, cut out from the gel as a specific band, and the DNA is extracted using a commercially available extraction kit such as gene clean kit (Bio 101).
  • the extracted DNA is digested with an appropriate restriction enzyme, purified if necessary, and further, if necessary, phosphorylated with 5 'T using T4 polynucleotide kinase or the like, and then pUC vector such as pUC18. Ligation to an appropriate Brass Vector, and transform appropriate Combinant cells.
  • the cloned PCR product has its:! ⁇ S sequence clarified.
  • the 5'-end of the cDNA of the gene is used.
  • the cDNA is obtained, and a primer designed based on the base sequence of the exon site at the 3 'end of the exon site of the exon site of the gene is used, and the 3' end of the cDNA of the gene is used.
  • primers designed using these primers and the nucleotide sequence of the 5'-end cDNA and the 3'-end cDNA of the cDNA of the gene obtained.
  • the first strand cDNA prepared by reverse transcriptase from mRNA isolated from human thread, especially human brain cDNA can also be obtained.
  • the primer at the 5 'end is selected so that it contains at least the initiation codon, or can be amplified so as to include the initiation codon. Is selected to be a force containing at least a stop codon, or to be able to amplify including the stop codon.
  • PCR can be performed as described above to obtain the full-length cDNA of the target 5 ?.
  • Preferred PCR cycle conditions include, for example, denaturation at 92 to 95 ° C for 10 to 20 seconds, annealing 55 A cycle of 60 ° C. for 10-30 seconds, extension 65-75 ° C. 150-300 seconds, more preferably denaturation 94 ° C. 15 seconds, annealing 58 ° C. 15 seconds, extension 68. C 4 minute cycle.
  • the obtained PCR product is cloned in the same manner as described above, and its nucleotide sequence is analyzed.
  • primers are designed based on the determined: ⁇ S sequence of DNA, and using these primers and cDNA libraries derived from various animal cells (for example, cDNA libraries derived from various human cells), By conducting screening, you can also obtain the target clone.
  • PCR amplification can be performed using these primers to obtain the target gene, novel fragments, fragments thereof, and the like. In this way, it is possible to search for PCR products that have homology to N-Tes, but are sequence-specific.
  • "oligonucleotide” is relatively short, single-stranded or :!
  • a polynucleotide of a chain preferably a polydeoxynucleotide, as described in Angew. Chem. Int. Ed. Engl., Vol. 28, p. 716-734 (1989). It can be chemically synthesized by any known method, for example, triester method, phosphite method, phosphoramidite method, phosphonate method and the like. It is generally well known that synthesis can be conveniently performed on a modified solid book, conveniently, for example, using an automated synthesizer; ⁇ it is commercially available. I have.
  • the oligonucleotide may contain one or more modified bases, for example, a naturally-occurring base such as inosine or a tritylated base. ,
  • the obtained PCR product is cloned, the nucleotide sequence of the obtained PCR product is determined, and a DNA fragment having a novel N-Tes and a gene sequence related thereto can also be obtained.
  • various cDNA libraries can be prepared using this DNA fragment as a probe. Screening can also be used to isolate the desired DNA.
  • Cloning of PCR products includes, for example, commercially available plasmids such as p-Direct (Clontech), pCR-Script TM SK (+) (Stratagene), GBM-T (Promega), pAmp TM (Gibco-BRL). Can be used.
  • cDNA can be obtained, for example, as follows.
  • Various human! ⁇
  • the isolation of mRNA can be performed by methods known in the art or methods substantially similar or modified thereto. See J. Sambrook et al., "Molecular Cioning", 2nd ed., Chapter. 7, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1989); D.. Glover et al. Ed., "DNA Cloning", 2nd ed., Vol. 1, (The Practical Approach Series), IRL Press, Oxford University Press (1995); L. Grossman et al. Ed., “Methods in Enzymology", Vol.
  • guanidine can be performed by methods such as the cesium chloride method, the guanidine thiocyanate method, and the phenol method.
  • Kits used for mRNA isolation include, for example, those commercially available from Pharmacia, Stratagene, Gibco-BRL, and the like; ⁇ .
  • RNA obtained can be purified using oligo (dT) -cellulose columns, spin columns, oligo (dT) -bound magnetic beads, etc. to obtain poly (A) + mRNA .
  • CDNA is produced using this mRNA and reverse transcriptase (RNA-dependent DNA polymerase).
  • an oligo (dT) primer can be used.
  • Oligo (dT) primers are preferably those having 12 to 18 T residues.
  • a synthetic oligonucleotide primer having a restriction enzyme site linked to the 5 'side of 12 to 18 T residues.
  • a primer is an Xba I oligo (dT) primer adapter.
  • this random hexamer primer can be formed in a worm or mixed with an oligo (dT) primer.
  • an RNase inhibitor such as RNasin (Boehringer Mannheim)
  • RNasin Boehringer Mannheim
  • cMA synthesis using mRNA and reverse transcriptase can be performed by a method known in the art or a method substantially similar to or a modification thereof. H. Land et al., Nucleic Acids Res., 9 : 2251, 1981; U. Gubler et al., Gene, 25: 263-269, 1983; SL Berger et al.
  • a cDNA library can be constructed by using a phage vector or a plasmid vector.
  • transformation of host cells such as fungi can be performed by, for example, the calcium method, the rubidium calcium method, the calcium / manganese method, the high-efficiency TFB method, and the FSB freezing cell method.
  • the method can be performed by a method known in the art or a method substantially similar thereto, such as a rapid colony method, an electorifice-portion (D. Hanah an, J. Mol. Biol., 166: 557, 1983, etc.) ).
  • RT-PCR reverse transcription PCR
  • RACE reverse transcript ion
  • a method that can isolate and purify mRNA from 1® cells or thread for example, using a commercially available kit such as REX kit, United States Biochemical; Glass MAX TM RNA spin cartridge system, Gibco-BRL, etc.
  • the obtained mRNA is reverse-transcribed using Oligo (dT) primer to synthesize 1st strand DNA, and then a homopolymer tail (for example, G residue) is added to the 3 ′ end of 1st strand DNA, or After attaching the adapter to the oligo (dT) primer and the oligo (dC) primer or adapter
  • the cDNA can also be amplified by PCR using a primer.
  • kits suitable for this include Superscript TM pre-amplification system (Gibco-BRL); cDNA Cycle TM kit (Invitrogen) and the like.
  • the plaque (or the nucleic acid itself) formed by the aife is transferred to a membrane such as a nylon filter, etc., by holding the inserted DNA, etc. After modification, immobilization, washing, etc., the DNA transferred to the membrane is reacted with the denatured labeled probe DNA fragment, if necessary, in a hybridization buffer.
  • the hybridization process is usually performed at about 35 ° C. to about 80 ° C., more preferably about 50 ° C.
  • the hybridization process is performed at about 55 ° C for about 18 hours.
  • the buffer for hybridization it is possible to use a buffer selected from those commonly used in the corresponding If.
  • a rapid hybridizatio buffer (Amersham) or the like.
  • An example of the denaturation treatment of the transferred film is a method of using an alkali denaturing solution. After the treatment, a treatment with a neutralizing solution or a buffer solution is preferable.
  • the immobilization treatment of the membrane is usually carried out at about 40 ° C.
  • the transferred membrane may be washed with a washing solution commonly used in this field, for example, 50 mM Tris-HC1 buffer containing 1 M NaCl, lm BDTA and 0.1% sodium dodecyl sulfate (SDS), and H8.0. It can be done by washing.
  • a washing solution commonly used in this field, for example, 50 mM Tris-HC1 buffer containing 1 M NaCl, lm BDTA and 0.1% sodium dodecyl sulfate (SDS), and H8.0. It can be done by washing.
  • a washing solution commonly used in this field, for example, 50 mM Tris-HC1 buffer containing 1 M NaCl, lm BDTA and 0.1% sodium dodecyl sulfate (SDS), and H8.0. It can be done by washing.
  • SDS sodium dodecyl sulfate
  • the membrane of the nylon filter may be used. it can.
  • the above-mentioned alkali denaturing solution, neutralizing solution, and buffer solution can be selected from those commonly used in the art, and the alkali denaturing solution may be, for example, 0.5M.
  • a solution containing NaOH and 1.5 M NaCl can be mentioned, and a neutralizing solution can be, for example, 0.5 M Tris-HCl buffer containing 1.5 M NaCl, ⁇ 8.0, and the like.
  • 2 XSSPE (0. 36 ⁇ NaCl 20 consideration NaH 2 P0 4 and 2m M EDTA) that force the like can ".
  • the transferred membrane is subjected to a pre-hybridization treatment, if necessary.
  • This pre-hybridization treatment may be performed, for example, using a pre-hybridization solution (50% formami de 5 X Denhardts overnight (0.2% serum albumin, 0.2% polyvinyl pyrrolidone), 5 XSSPE, 0. 1% SDS, 100 ⁇ g / ml heat-denatured salmon sperm DNA] etc.
  • the labeled probe DNA fragment used for hybridization may be, for example, heated at about 70 ° C. to about 100 ° C., preferably about 100 ° C. for about 1 minute to about 60 minutes, preferably for about 5 minutes. You can be powerful.
  • the hybridization can be carried out by a method known per se or a method analogous thereto.In the present specification, the stringent conditions refer to, for example, about 15 to about 50 mM, preferably, about sodium concentration.
  • the hybridized black (nucleic acid) is typically detected by photoradiography, and is used to detect plaque (nucleic acid) from methods used in the art. You can also.
  • the plaque (nucleic acid) corresponding to the detected signal is transferred to an appropriate buffer, for example, SM Intense Night (100 mM NaCl and 10 mM GS0 4 containing 50 taking into Tris-HCl buffer, H7. 5) was suspended in like, then the phage (may include nucleic acid itself) suspension was appropriately diluted and were infected with E. coli, the resultant E. coli To obtain a desired recombinant phage (nucleic acid) from the cultured E. coli.
  • SM Intense Night 100 mM NaCl and 10 mM GS0 4 containing 50 taking into Tris-HCl buffer, H7. 5
  • the above-mentioned probe DNA is applied, and the process of screening the target recombinant phage (nucleic acid) from the gene library or the cDNA library by the hybridization process is repeatedly performed. it can.
  • the target recombinant phage (nucleic acid) can be obtained by performing extraction treatment, centrifugation treatment, etc. from ⁇ cultivated E. coli.
  • the resulting phage particles can be purified and separated by methods commonly used in the art, for example, glycerol gradient fiber separation (Molecular clonin g, a laboratory manual, ed. T. Maniati s , Cold Spring Harbor Laboratory, 2nd ed. 78, 1989).
  • the DNA in a way that is commonly used in this the art can be purified and separated, for example, the resulting et phage TM solution (10 mM MgSO 4 containing 50 mM Tris-HCl buffer, H7. 8 ), And treated with DNase I and RNase A, and then add a mixture of 20 mM BDTA, 50 xg / ml Proteinase K and 0.5% SDS, etc., and incubate at about 65 ° (for about 1 hour, This was extracted with phenol and extracted with getyl ether, and the DNA was precipitated by ethanol precipitation.Then, the obtained DNA was washed with 70% ethanol and dried. H8.0) can be obtained, for example ..
  • DNA can also be obtained by sub-mouthing, for example, by using E. coli as a host. Using a brass vector, etc., can be used to perform the work.
  • the DNA obtained by the subcloning can be purified and separated in the same manner as described above by centrifugation, phenol extraction, ethanol precipitation, etc.
  • a clone containing the target DNA for example, a recombinant phage
  • the full length of the nucleotide sequence of the DNA insert isolated from the cloned recombinant phage Is 1510 bp, and its sequence is shown as SEQ ID NO: 1 in the Sequence Listing.
  • Testican-3 and HPTLG lacks the TY domain and CWCV domain present in Testican-3 and HPTLG, and lacks the Glycosaminoglycan attachment site present in Testican-3 and HPTLG. It was well accepted.
  • This putative protein is one of a new class of human Testicans and was named ⁇ -Tes. It is clear that N-Tes remains encodes a polypeptide belonging to a novel test family, and all recombinant plasmids produced using N-Tes And a transformant or transfectant obtained by transformation or transfection with the plasmid.
  • the nucleic acid having ⁇ or a part of the base sequence represented by SEQ ID NO: 1 in the sequence listing can also be obtained by DNA synthesis.
  • it is also possible to obtain a target rooster by using the synthetic fragments as described above as primers or probes.
  • the primer used in the PCR method is not particularly limited as long as it can amplify a DNA fragment containing the above site.
  • the primer is (a) an oligonucleotide having a nucleotide sequence corresponding to an arbitrary region of the nucleotide sequence shown in SEQ ID NO: 1 in the Sequence Listing and) SEQ ID NO: 1 in the Sequence Listing.
  • the oligonucleotide having a complementary nucleotide sequence to an arbitrary region of the nucleotide sequence shown in (1) can be identified, and more preferably (1) the nucleotide sequence shown in SEQ ID NO: 1 in the sequence listing.
  • the PCR conditions are not particularly limited, and may be well-known conditions that are usually performed.
  • PCR In PCR, one cycle consisting of thermal denaturation of DNA strands, annealing of the Braymer, and synthesis of complementary strands by polymerase is performed, for example, 10 to 50 times, preferably 20 to 35 times, and more preferably 25 to 30 times. It is repeated.
  • the DNA fragment obtained by the present invention is inserted into a suitable vector such as a plasmid pEX, pMAMneo, pKG5, etc., as described below. It can be expressed in host cells, for example, E. coli, yeast, CH0 cells, COS cells, and the like.
  • the DNA fragment may be expressed as it is or as a DNA fragment to which an appropriate control sequence is added, or may be inserted into an appropriate vector and introduced into an animal to express an N-Tes gene, for example, N-Tes.
  • a transgenic animal can be prepared by introducing the DM fragment into a fertilized egg of an animal such as a mouse.
  • Confirmation of the N-Tes gene product can be performed using animal cells suitable for the transfection of the N-Tes gene, such as 293T cells and COS 1 cells.
  • animal cells suitable for the transfection of the N-Tes gene such as 293T cells and COS 1 cells.
  • a method for introducing this gene into animal cells such as mammals a method known in the art or a method substantially similar thereto can be used. al., Virology, 52: 456, 1973, etc.), DEAE-dextran method (eg, D. Warden et al., J. Gen. Virol., 3: 371, 1968, etc.), electro-volatilization method (eg, E. Neumann et al., ⁇ J, 1: 841, 1982), microinjection method, ribosome method, virus infection method, phage particle method and the like.
  • the gene product produced by animal cells transfected with the N-Tes gene in this way can be Cut c
  • the N-Tes gene or the like may be used as a plasmid for ffiA.
  • Host cells commonly used in genetic engineering eg, prokaryotic hosts such as E. coli, Bacillus subtilis, yeast, CH0 cells
  • any eukaryotic cell host such as COS cell or insect cell host such as Sf21.
  • Such sequences may contain, for example, codons suitably modified for expression in the selected host cell, may have a restriction enzyme site, and may have Control sequences for promoting expression, promoter sequences, etc., which are useful for binding target genes, such as linkers and adapters, as well as controlling antibiotic resistance, metabolism, etc.
  • sequences useful for selection and the like including those coding for hybrid proteins and fusion proteins).
  • an appropriate promoter for example, a plasmid using Escherichia coli as a host, includes tryptophan promoter (trp), lactose promoter (lac), tryptophan 'lactose promoter (tac), and lipoprotein promoter (tac). lpp), sphage P L promoter, etc., and plasmids using animal cells as the host, such as SV40 rate promoter, MMTV LTR promoter, RSV LTR promoter, CMV promoter, SR promoter, etc., and yeast as the host. In this case, GAL1 and GAL10 promoters may be used.
  • Examples of plasmids using Escherichia coli as a host include, for example, pBR322, pUC18, pUC19, pUC118, pUC119, pSP64, pSP65, pTZ-18R / -18U, pTZ-19R / -19U, pGEM-3, pGEM-4, pGBM-3Z , PGEM-4Z, pGEM-5Zf (-), pBluescript KS TM, (Stratagene) and the like. Plasmid vectors suitable for expression in E.
  • Plasmids using animal cells as a host include SV40 vector, poliovirus vector, vaccinia virus vector, retrovirus vector, etc., for example, pcD, pcD-SR, CDM8, pCEV4, pME18S , PBCl 2BI, pSG5 (Stratagene) and the like.
  • Examples of plasmids using yeast as a host include Yip-type vector, YEp-type vector, Yp-type vector, and YCp-type vector, such as pGPD-2.
  • Host cells include the host cells ⁇ spores of Escherichia coli: lf ⁇ , for example, those derived from E. coli strain K12, such as 533, XL1-Blue-C600, DH1, DH5, DH11S, DH12S, DH5 o; DH10B, HB101, MC1061, JM109, STBL2, and B834 strains include BL21 (DE3) pLysS and the like.
  • the host cell is an animal cell: ⁇ , for example, COS-7 cells, COS-1 cells, CV-1 cells from African green monkey fibroblasts, COP cells from mouse fibroblasts, MOP cells, W0P cells, Chinese 'Examples include hamster cell-derived CH0 cells, CHO DHFR-cells, human He La cells, mouse cell-derived C127 cells, and mouse cell line IH 3T3 cells.
  • insect cells Bombyx mori nuclear polyhedrosis virus or a vector derived therefrom is used as a vector, and silkworm larvae or silkworm cultured cells, such as BM-N cells, can be used. It is also possible to use plant cells as host cells, which are widely known in the art, together with suitable vectors.
  • the genetic engineering method of the present invention includes modifying or converting a restriction enzyme, reverse transcriptase, or DNA fragment known or widely used in the art into a structure suitable for cloning.
  • DNA modifying and degrading enzymes DNA polymerase, terminal nucleotidyl transferase, DNA ligase, and the like can be used.
  • Restriction enzymes include, for example, RJ Roberts, Nucleic Acids Res., 13: rl65, 1985; S. Linn et al. Ed. Nucleases, p. 109, Cold Spring Harbor La b., Cold Spring Harbor, New York, 1982.
  • Reverse transcriptases include, for example, reverse transcriptase derived from mouse Moloney leukemia virus (MMLV) ⁇ reverse transcriptase derived from avian myeloblastosis virus (AMV). Is received.
  • MMLV mouse Moloney leukemia virus
  • AMV avian myeloblastosis virus
  • Suitable reverse transcriptases include MMLV RT (Gibco-BRL), Superscript RT plus (Life Technologies) and the like.
  • Examples of the DNA polymerase include Escherichia coli DNA polymerase, Klenow 'fragment which is a trigger thereof, Escherichia coli phage T4 DNA polymerase, Escherichia coli phage T7 DNA polymerase, and heat-resistant bacterium DNA polymerase.
  • Examples of the terminal nucleotidyl transferase include those described in R. Wu et al. Ed., "Methods in Enzymology", Vol. 100, p. 96, Academic Press, New York (1983). TdTase that adds a deoxynucleotide (d ⁇ P) to the OH terminus is exemplified.
  • DNA-modifying / degrading enzymes include exonuclease, endonuclease, and the like. Ze VI I, exonuclease, DNase I, nuclease Sl, Micrococcus nuclease and the like.
  • DNA ligases include, for example: DNA bacteria ligase, T4 DNA ligase and the like.
  • Suitable vectors for cloning a DNA gene to construct a DNA library include plasmid, sphage, cosmid, P1 phage, factor F, YAC, etc., and preferably phage-derived
  • Transformants transformed with an expression vector containing a nucleic acid encoding the protein of the present invention can stably maintain high expression ability by performing cloning repeatedly using an appropriate selection technique as necessary. To obtain a cell line.
  • the dhfr gene was used as a selective agent: ⁇ , by gradually increasing the MTX concentration and culturing to select a resistant strain, Amplifying the DNA encoding the protein of the present invention, it is possible to obtain a cell line that can obtain higher expression.
  • the transformant of the present invention is cultured under conditions in which a nucleic acid encoding the protein of the present invention can be expressed to produce and accumulate the desired product. You can power.
  • the transformant can be cultured in a medium used in the art.
  • a transformant using a prokaryotic host such as Escherichia coli or Bacillus subtilis, or a yeast as a host can suitably use a liquid medium.
  • the medium contains a carbon source, a nitrogen source, and others necessary for the growth of the transformant.
  • Carbon sources include, for example, gnorecose, dextrin, soluble starch, sucrose, etc.
  • Nitrogen sources include, for example, ammonium, nitric acid
  • the inorganic or inorganic substances such as the potato extract solution include canoledium chloride, sodium dihydrogen phosphate, magnesium chloride, calcium carbonate, and the like.
  • yeast, vitamins, casamino acids, growth promoting factors and the like may be added.
  • a drug such as 3-indolylacrylinoleic acid can be added in order to make the promoter work efficiently.
  • About 5 to 8 ⁇ ⁇ ⁇ of the medium is desirable.
  • cultivation of Escherichia coli is usually performed at about 15 to about 45 ° C for about 3 to about 75 hours, and if necessary, aeration and stirring may be applied.
  • a MEM medium containing about 5 to about 20% fetal bovine serum, a PRMI1640 medium, a DMEM medium, or the like is used as a medium.
  • the pH is about 6 to about 8.
  • Culture is usually performed at about 30 ° C to about 40 ° C for about 15 to about 72 hours. Aeration and stirring are added as necessary.
  • cells or cells are collected by a known method, and the cells are collected, suspended in an appropriate buffer, and then sonicated, lysozyme and / or freeze-thaw, etc.
  • itM can be used to obtain a crude extract by centrifugation or filtration after disrupting the cells.
  • Some buffer solutions include protein denaturants such as urea and guanidine hydrochloride, Triton X-100 (trade name), and Wien-80.
  • a surfactant such as (trade name) may be added.
  • 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 obtained in this manner can be purified by appropriately combining known separation and purification methods, for example, by the ammonium sulfate precipitation method. Salting out, cefade Gel filtration method using, for example, ion exchange chromatography using a carrier having a getylaminoethyl group or a carboxylmethyl group.
  • Hydrophobic chromatographic method dye gel chromatography, electrophoresis, dialysis, ultrafiltration, affinity chromatography, high-performance liquid chromatography, etc. It can be obtained by purification. Preferably, it can be purified and separated by a treatment such as polyacrylamide gel electrophoresis or affinity 'chromatography in which a ligand or the like is immobilized. For example, gelatin agarose 'affinity one' chromatography, heha. Lingarose 'Chromatography etc.' Furthermore, by using a method commonly used in genetic engineering based on the gene base sequence of N-Tes according to the present invention, one or more amino acids can be appropriately substituted in the amino acid sequence of N-Tes.
  • the obtained protein of the present invention can modify the amino acid residue contained therein by a dangling technique, and can also be used to prepare peptidases such as pepsin, chymotrypsin, and no. No ,.
  • the protein of the present invention has a C-terminus which is usually a carboxyl group (-C00H) or a phenol (-C00-), and the C-terminus is an amide (-CO ⁇ 2 ) or an ester (-COOR).
  • R in the ester e.g., methyl, E chill, n - propyl, isopropyl or n- butyl etc. - 6 alkyl group, For example, C 3 cyclopentyl, cyclohexylene, etc.
  • cyclohexyl - 8 cycloalkyl group for example, , phenyl, alpha - C G one 1 2 7 Li Ichiru group ⁇ such naphthyl example, benzyl, Fuweniru C such Fuwenechi le!
  • the amino group of the N-terminal methionine residue is protected with a protecting group (for example, d- 6 acyl such as C, 15 alkyl monocarbonyl group such as forminole group and acetyl). Group, etc.), N-terminal Glutamyl group generated by cleavage in the body, pyroglutamylated, Substituent on the il ⁇ side of the amino acid in the molecule (eg, -OH, -C00H , Amino group, imidazo Group, indole group, etc.
  • a protecting group for example, d- 6 acyl such as C, 15 alkyl monocarbonyl group such as forminole group and acetyl. Group, etc.
  • N-terminal Glutamyl group generated by cleavage in the body, pyroglutamylated
  • Substituent on the il ⁇ side of the amino acid in the molecule eg, -OH, -C
  • Guanijino group Chikaraku suitable protecting group (e.g., formyl group, those protected by such as a single 6 Ashiru group such Asechinore group), or a so-called sugar Tanno click proteins which sugar chains are bound Compound tano, such as.
  • suitable protecting group e.g., formyl group, those protected by such as a single 6 Ashiru group such Asechinore group
  • sugar Tanno click proteins which sugar chains are bound Compound tano, such as.
  • the quality is also included.
  • it is expressed as a fusion protein at the time of production by a genetic and recombinant method, and converted and processed into a substance having a biological activity substantially equivalent to that of natural N-Tes in vivo or in vitro. Is also good.
  • Such a fusion protein which can be used by a fusion production method commonly used in genetic engineering can be purified by affinity chromatography or the like utilizing the fusion protein.
  • Such fusion proteins include those fused to a histidine tag or ⁇ -galactosidase (-gal), maltose-binding protein (MBP), daltathione-S-transferase (GST), thioredoxin (TRX) or And those fused to the amino acid sequence of Cre Recombinase.
  • the polypeptide can be tagged with a heterogeneous epitope, so that purification by immunoaffinity chromatography using an antibody that specifically binds to the epitope can be accomplished.
  • the epitope tag includes, for example, AU5, c-Myc, CruzTag 09, CruzTag 22, CruzTag 41, Glu-Glu, HA, Ha.11, KT3, FLAG (registered trademark, SIMA -Aldrich), Omni-probe, S-probe, T7, Lex A, V5, VP16, GAL4, VSV-G. (Field et al., Molecular and Cellular Biology, 8: pp. 2159-2165 (1988); Evan et al., Molecular and Cellular Biology, 5: pp. 3610-3616 (1985); Paborsky et al. , Protein Engineering, 3 (6): pp.
  • the fusion protein may be a protein having a marker so as to be a detectable protein.
  • the detectable marker may be a biotin, a streptavidin-based Biot in Avi Tag, a fluorescent substance, or the like.
  • the fluorescent substance As the fluorescent substance,
  • GFP Green fluorescent protein
  • GFP variants such as EGFP (Enhanced-humanized GFP), rsGFP (red-shif t GFP), yellow fluorescent protein (YFP) , green fluorescent protein (green fluorescent protein: GFP), indigo fluorescent Tanno ⁇ 0 click proteins (cyan fluorescent protein: CFP), #fe fluorescent protein (blue fluorescent protein: BFP), derived Umishiitake (Reni lla reniformis) (Atsufumi Takawaki, Ed., Experimental Medicine Separate Volume, Experimental Lectures in the Post-Genome Era 3—GFP and Bio Imaging, Yodosha (2000)).
  • Detection can also be performed using an antibody that specifically recognizes the fusion tag (including a monoclonal antibody and its fragment).
  • a sequence of several markers for example, a fusion of a hexar histidine peptide can be obtained in order to preferably carry out purification.
  • Expression and purification of such fusion proteins can be conveniently performed using commercially available kits, or can be performed according to protocols specified by the kit manufacturer or kit distributor.
  • Modification and alteration of protein structure can be performed, for example, by referring to “The New Chemistry Laboratory Course 1, Protein VII, Protein Engineering” edited by The Biochemical Society of Japan, Tokyo Kagaku Dojin (1993), and the methods described there or It can be performed in the manner described in the literature used, or in a manner substantially similar thereto.
  • the biological activity may include having an immunological activity, for example, having antigenicity.
  • the modifications include deamination, hydroxylation, phosphorylation, methylation, acetylation, ring opening, ring closure, changing the number of sugar chains contained, and increasing or decreasing the number of sugar chains contained. Or substitution with a D-form amino acid residue.
  • the human-derived protein of the present invention is different from the natural protein in that at least one amino acid residue is different from the natural protein in terms of identity, and the position of one or more amino acid residues is different from the natural protein. It may be different.
  • the human-derived protein of the present invention is 1 or more amino acid residues (for example, 1 to 80, preferably 1 to 60, more preferably 1 to 40, more preferably 1 to 20, especially 1 to 10) Deficient ⁇ !
  • one or more of the amino acid residues in ⁇ force ⁇ placed page margin substituted with another residue, 1 or more (for example, 1-80, preferably 1-60, more preferably 1-40, More preferably, 1 to 20, especially 1 to 10 amino acid residues are added.
  • ⁇ for example, 1 to 80, preferably 1 to 60, more preferably 1 to 40, more preferably 1 to 20, especially Is 1-10, etc.
  • another residue for example, 1-80, preferably 1-60, more preferably 1-40, More preferably, 1 to 20, especially 1 to 10 amino acid residues are added.
  • the N-Tes of the present invention is also considered to include those having a primary structure conformation substantially equivalent to natural N-Tes or a part thereof.
  • the human-derived protein of the present invention is, for example, a protein having an amino acid sequence at positions 22 to 122 in the amino acid sequence represented by SEQ ID NO: 2 in the sequence listing; 60% or more than 70% homology to amino acid sequences selected from the group consisting of those having the amino acid sequence at position 1 and those having the amino acid sequence at positions 1 to 313 And more preferably those having a homologous amino acid sequence of 80% or 90% or more, particularly those lacking the Glycosaminoglycan attachment site.
  • a part of the human-derived protein of the present invention is a partial peptide of the human-derived protein (that is, a partial peptide of the protein), which is substantially equivalent to the N-Tes of the present invention. Any substance may be used as long as it has activity.
  • 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, more preferably 70 or more of the constituent amino acid sequences of the present invention ⁇ N-Tes.
  • Peptides having an amino acid sequence of preferably 100 or more, and at an age of 200 or more, are preferred, and they are preferably those corresponding to contiguous amino acid residues, or, for example, a sequence listing.
  • substantially equivalent means that the protein activities, eg, inhibitory activity, physiological activity, and biological activity, are substantially the same.
  • the meaning of the term includes the age having substantially the same activity.
  • the substantially same activity includes, for example, the inhibition of any one of bandits PS.
  • the activity and activity of inhibiting or inhibiting the ability to separate a synthetic substrate for any one of the activity and the solid PS can be mentioned.
  • the term “substantially the same activity” means that those activities are qualitatively the same, for example, physiologically, pharmacologically or biologically the same.
  • the activity such as the inhibitory activity against any one of the MMPs is equivalent (for example, about 0.001 to about 1000 times, preferably about 0.01 to about 100 times, more preferably about 0.1 to about 20 times). (More preferably, about 0.5 to about 2 times). Strong force These quantitative factors such as the degree of activity and the amount of protein may be different.
  • amino acid substitutions, deletions, or insertions often do not cause or significantly alter the physiological or biochemical properties of the polypeptide: ⁇ , its substitution ⁇ deletion, or The humanized polypeptide will be substantially identical to the one without such a deletion or insertion.
  • Substantially identical substitutions of amino acids in the amino acid sequence can be selected from other amino acids of the class to which the amino acid belongs.
  • non-polar (hydrophobic) amino acids include alanine, phenylalanine, leucine, isoleucine, valine, proline, tryptophan, methionine, and the like.
  • Polar (neutral) amino acids include glycine, cerith threonine, cysteine, and tyrosine.
  • Amino acids (basic amino acids) with a positive charge include arginine, lysine and histidine; and amino acids with a negative charge (acidic amino acids) include asparaginic acid and glutamic acid. No.
  • a method known in the field of peptide synthesis for example, a chemical synthesis method such as a liquid phase synthesis method or a solid phase synthesis method.
  • these methods include, for example, protein or peptide
  • an appropriately protected amino acid is sequentially bound to the desired amino acid sequence on the resin by various condensation methods known per se.
  • the condensation reaction preferably employs various activation methods known per se, and examples of the condensation reaction include, preferably, jj-carboximides such as hexylcarboximide.
  • the product having a power-protecting group can be obtained by removing the free protecting group to obtain the desired product.
  • the protein of the present invention and some of its peptides were obtained as free forms: ⁇ can be converted into a salt by a method known per se or a method analogous thereto, and At the age obtained as a salt, the salt can be converted into a sightseeing one 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 physiologically acceptable or pharmaceutically acceptable, but are not limited thereto.
  • salts examples include salts with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like, for example, nitric acid, gi, maleic acid, fumaric acid, succinic acid, citric acid, tartaric acid, and lincoic acid. And salts with organic acids such as benzoic acid, methanesulfonic acid, P-toluenesulfonic acid and benzenesulfonic acid. Examples of the salts further include ammonium salts, for example, salts with organic bases such as ethylamine, dimethylamine, trimethylamine, and hydroxethylamine.
  • fragments of the present invention and its mutants, modifications, and primers can be subjected to the separation / purification treatment described above.
  • fragments refer to the polypeptide of SEQ ID NO: 2, transcribed from the sequence of SEQ ID NO: 1 and are unspliced or specifically spliced.
  • fragment refers to the polypeptide encoded by the hnRNA or mRNA, or the polypeptide encoded by the digenomic DNA.
  • analogs include activatable proproteins and the like, where the proprotein portion is cleaved to produce an active polypeptide.
  • the polypeptide of the present invention is a recombinant polypeptide, It may be a natural or synthetic polypeptide. In certain preferred embodiments, this is a recombinant polypeptide.
  • the present invention thus provides a DNA sequence encoding the above-mentioned polypeptide, a N-Tes polypeptide having ⁇ or a part of a natural property, and a DNA encoding an analog or a fragment thereof. Also encompasses sequences.
  • the polynucleotide of the present invention may be a mature protein having an additional amino acid at the amino terminal or an additional amino acid at the carboxyl terminal, or a polypeptide endogenous to the mature protein (for example, having one or more polypeptide chains in a mature form.
  • a sequence may play a role in the processing of the precursor into the mature form of the protein, for example, tanna, It may be one that can facilitate the transport and transport of proteins, extend or shorten the length of a protein, or manipulate a protein to facilitate its detection or production.
  • the amino acids are processed by the fine ⁇ -enzyme and removed from the mature protein.
  • a precursor protein having a mature polypeptide fused to more than one prosequence can be an inactive 'f ⁇ f form polypeptide.
  • the precursor is usually activated, some or all of the prosequences can be removed prior to activation
  • Such precursors are commonly referred to as proproteins.
  • Polypeptides are mature proteins, Mature protein (the ability to be called a preprotein), a precursor of a mature protein having one or more prosequences that are not the leader sequence of the preprotein, or the leader sequence and
  • Prev mouth proteins which are precursors of proproteins having one or more prosequences. It may be quality. Also, the prosequence can be removed at a stage of processing that usually yields the active polypeptide and the mature polypeptide. Since the DNA sequence of the present invention has a '11 # related to the amino acid sequence of a mammalian protein which has not been known until now, the use of such ⁇ tfg is also encompassed by the present invention. Such uses include, for example, Tes and related proteins. Design of a probe for unitary and / or detection of genomic DNA and cDNA of a coding mammal, particularly preferably a human.
  • the DNA sequences of the present invention are useful as probes for the detection of genomic DNA and cDNA, for example, in mammals, particularly preferably mice and humans, encoding -Tes and related proteins. Probes can be labeled as necessary with reference to antibodies, if desired. In isolating the gene, a PCR method and a PCR method using reverse transcriptase (RT) (RT-PCR) can be used.
  • RT reverse transcriptase
  • the N-Tes cDNA and its related DNA are cloned and selected for specific sequence regions based on the amino acid sequence deduced from the sequenced N-TescDNA sequence, and a DNA primer is designed and chemically synthesized.
  • the obtained DNA primers can be used for the isolation and detection of N-Tes 3 gene using PCR, RT-PCR and other methods.
  • the expression of -Tes mRNA in human thread 1 ⁇ can be examined by Northern blot analysis on poly (A) + RNA from various paper tissues.
  • expression of N-Tes mRNA or N-Tes gene itself in human tissues by Northern blotting, Southern blotting, in situ hybridization, etc. can be used.
  • Detectable and involved in many normal cellular processes including intracellular protein metabolism, activation of hormone precursors, and bone modification in human fibres, the role of P-inhibition, Alzheimer's disease, lung It can contribute to the development of research on many diseases such as severe, rheumatoid arthritis, muscular dystrophy, osteoporosis, neurodegenerative diseases and cancer invasion and metastasis. It can also be used for genetic diagnosis of diseases related to N-Tes. Such diagnosis can be used to diagnose abnormalities in nucleic acids encoding the N-Tes and related proteins, such as damage, mutation, decreased expression, overexpression, and the like.
  • N-Tes and its related proteins, fragments thereof, and nucleic acids (including mRNAs and oligonucleotides), including DNA, disclosed in the present specification may be used in the form of a networm or organically. It can be applied to genomics and proteomics techniques in combination with the techniques described below (antisense method, antibodies including monoclonal antibodies, transgenic animals, etc.). For example, the -Tes variant can be used for functional analysis using dominant negative effects. Ma There is also an application to RNAi (RNA interference) technology using fffl of strand RNA (dsRNA).
  • dsRNA RNA interference
  • gene polymorphisms based on one i ⁇ S polymorphism SNP; single nucleotide polymorphisms
  • nucleic acid arrays and protein arrays were analyzed using fffl analysis, gene function analysis, protein-protein interaction analysis, It will be possible to analyze related diseases, and to treat diseases.
  • SNP single nucleotide polymorphism
  • a cDNA library is used, or the DNA obtained by the PCR technology is arranged at a high density using a spotting device near the base, and the sample is analyzed using hybridization.
  • the arraying is performed by using a needle or a pin, or by using an ink-jet printing technique, etc., to attach DNA to a unique position on a substrate such as a slide glass, a silicon plate, a plastic plate, etc. The ability to do so.
  • Observing a signal obtained as a result of hybridization on the nucleic acid array obtains data.
  • the signal may be a signal obtained from a label such as a fluorescent dye (eg, Cy3, Cy5, BODIPY, FITC, Alexa Fluor dyes (trade name), Texas red (trade name), etc.).
  • a laser-scanner or the like may be used for the tent, and the obtained data may be processed by a computer system equipped with a program according to an appropriate algorithm.
  • protein array technology may utilize tagged recombinantly expressed protein products, including two-dimensional electrophoresis (2-DE), mass spectrometry (MS) including enzymatic digestion fragments (including MALDI-T0F analysis, including techniques such as electrospray ionization (ESI) and matrix-assisted laser desorption / ionization (LDI) , A BSI-3 series Shito analyzer, an ES ion trap analyzer, etc.), dyeing technology, isotopic ⁇ * (awareness and angled corner, image processing technology, etc. can be used) Therefore, the present invention may also include ⁇ -Tes-related software, databases, etc.
  • 2-DE two-dimensional electrophoresis
  • MS mass spectrometry
  • enzymatic digestion fragments including MALDI-T0F analysis, including techniques such as electrospray ionization (ESI) and matrix-assisted laser desorption / ionization (LDI) , A BSI-3 series Shi
  • the DNA obtained by the present invention may be used I let the animal move the car Therefore, it is advantageous to use the DNA as a DNA fragment or by binding the DM downstream of a promoter capable of being expressed in animal cells, eg, introducing N-Tes DNA into mice: i ⁇ ⁇ N-Tes DNA from animals with high homology to this
  • a gene construct that is linked to the downstream of various promoters capable of expressing E. coli in animal cells is microinjected into fertilized eggs of humans, such as mouse fertilized eggs, to introduce genes that produce high levels of N-Tes (transgenetics).
  • a mouse can be created. The mouse is not particularly limited to a pure mouse.
  • a virus-derived promoter, a ubiquitous expression promoter such as metamouth thionein, etc. can be preferably used, etc.
  • the N-Tes DNA can also be introduced, or the recombinant N-Tes DNA can be recombined with a recombinant retrovirus and used.
  • the mouse fertilized egg into which the image DNA has been introduced is capable of growing a foster mother mouse such as ICR.
  • DNA obtained in the present invention eg, DNA encoding N-Tes
  • DNA encoding N-Tes DNA encoding N-Tes
  • the presence of the DNA encoding N-Tes in the germinal cells of the animal after DNA transfer means that the offspring of the animal produce the N-Tes-encoding DNA in all of its germinal and somatic cells. Means to have.
  • the offspring of this type of animal that has inherited the heritage have the potential to express the N-Tes in all of its germinal and somatic cells.
  • the animal After confirming that the N-Tes DNA-introduced animal stably retains the gene by breeding, the animal can be reared and subcultured in the usual breeding environment as the DNA-bearing animal. Furthermore, by crossing male and female animals having the DNA of interest, homozygous animals having the transgene in both homologous chromosomes are obtained, and all the progeny are obtained by crossing the 13 ⁇ 4Purn animals. Can be propagated to carry the DNA. Since the N-Tes protein is highly expressed in the animal into which the N-Tes DNA has been introduced, the animal is useful as an animal for screening for a blocking ij (inhibitor) against the N-Tes protein. It is also useful as an antisense oligonucleotide capable of inhibiting the expression of N-Tes residues, for example, as a screening animal for antisense DNA.
  • This transgenic animal can also be used as a cell source for ligament culture. .
  • analyze proteins related to MT1-MMP or MT3-MMP activity inhibition by directly analyzing DNA or RNA in the tissues of transgenic mice or by analyzing protein tissues expressed by genes. I can do it.
  • the N-Te s-containing paper-woven cells are cultured by standard paper-woven culture techniques, and the cells are cultured for, for example, brain, thymus, testis, brain, intestine, kidney, and other cells derived from paper. Can study the function. In addition, the use of these cells can contribute to the development of 13 ⁇ 4, for example, to enhance the functions of various types of paper. If high-expressing cell strains are available, N-Tes can be isolated and purified therefrom.
  • transgenic mice and the like include, for example, Brinster, RL, et al., Proc. Natl. Acad. Sci. USA, 82: 4438, 1985; Costantini, F. & Jaenisch, R. (eds): The method can be carried out by a method described in a literature such as Genetic manipulation of the early mammalian embryo, Cold Spring Harbor Laboratory, 1985 or a method described in a literature cited therein, or a modification method thereof. The ability to create a mutant mouse (knockout mouse) that has a mutation in the gene obtained in the present invention (eg, DNA encoding mouse N-Tes corresponding to N-Tes) and does not express mouse N-Tes at all.
  • a mutant mouse knockout mouse
  • I can do it.
  • a targeting vector having a gene can be constructed.
  • the gene cassette to be inserted includes a DT-A cassette, a tk cassette, a lacZ cassette and the like in addition to the neo resistance gene cassette.
  • the targeting vector is opened linearly, and the established mouse embryonic stem cells (embryonic stem cells: ES cells) are guided by electrophoresis and further cultured to select ES cells that have acquired neo resistance. I do.
  • Cells can be selected and prepared from mouse strains such as 129, C57BL / 6, F1 C57BL / 6 XCBA) mice.
  • ES cells that have acquired neo resistance are mouse N-Tes genetic ⁇ ! It is assumed that homologous recombination has occurred with the targeting vector containing the gene cassette in the region.k At least one of the mouse N-Tes gene alleles is destroyed and the mouse N-Tes cannot be expressed normally. . For sorting, it depends on the inserted cassette In addition, the introduction of the mutation can be confirmed by using a method such as PCR, Southern hybridization or Northern hybridization.
  • Mutant-introduced ES cells are injected into 8-cell stage embryos taken from C57BL / 6, BALB / c, ICR mice, etc., cultured for one day, and the cells that develop in the cells are transplanted to a foster parent such as ICR. Can grow up to.
  • Born offspring mice are chimeric mice derived from ES cells with mutations and normal inn ⁇ ! Pi. The degree to which cells derived from ES cells are included is determined by the coat color of the individual. Therefore, it is desirable that ES cells and Shuku-Pi are capable of combining strains with different hair colors. Mutations in the resulting chimeric mice are heterozygous, and homozygous mutant mice can be obtained by crossing them appropriately.
  • the homozygous mutant mouse obtained in this way is disrupted only in the mouse N-Tes gene in all of the 3 ⁇ 4 cells and somatic cells, does not express mouse N-Tes at all, and is subcultured. Descendants also have a similar expression system. ⁇
  • This knockout mouse clarifies the role of N-Tes in the life cycle of an individual, such as development, growth, reproduction, aging, and death, and the function of ⁇ -Tes in various organs and threads, compared to normal mice. Useful. It can also be applied to the development of pharmaceuticals related to MMP P. Knock-out mice can be used not only as model animals but also as a cell source for paper culture, and can be used to provide N-Tes functional keratosus at the cell level. Techniques related to knockout mice and the like are described, for example, in Mansour, S.
  • Oligonucleotides can be designed and synthesized based on the cloned or determined nucleotide sequence of the DNA encoding N-Tes.
  • Such oligonucus Reotide is capable of hybridizing with the mRNA of the N-Tes gene, and has the ability to inhibit the function of the mRNA, or the interaction with N-Tes-related iimRNA. It can regulate and control the expression of N-Tes gene.
  • Oligonucleotides complementary to the selected sequence of the N-Tes-related gene, and oligonucleotides that can specifically hybridize to the N-Tes-related gene are available in vivo and in vitro.
  • corresponding refers to a nucleotide, nucleotide sequence or a specific sequence of nucleic acid, including the residue Has homology to or is complementary to
  • correspondence between a nucleotide, a base sequence or a nucleic acid and a peptide (protein) usually refers to the amino acid of a peptide (protein) in the ⁇ derived from the nucleotide (nucleic acid) sequence or its complementarity. ing.
  • the terminal untranslated region, the terminal palindrome region, and the terminal hairpin loop can be selected as a preferred sentence size region. Any region within the region can be selected as a target.
  • the relationship between the target nucleic acid and an oligonucleotide complementary to at least a part of the image region means the relationship between the oligonucleotide and the oligonucleotide capable of hybridizing with the target, which is defined as "antisense". It can be powerful.
  • Antisense oligonucleotides include 2-deoxyxyl-D-ribose, polydoxynucleotides, and D-ribose, polydeoxynucleotides, purines or pyrimidine bases.
  • Other types of polynucleotides that are glycosides, or other polymers with non-nucleotide backbones eg, commercially available protein nucleic acids and synthetic sequence-specific nucleic acid polymers
  • other polymers containing key bonds Lima provided that the polymer contains nucleotides having a configuration that allows base pairing and base attachment as found in DNA and RNA).
  • RNA hybrids can be double-stranded DNA, single-stranded DNA, double-stranded RNA, single-stranded RNA, and even DNA: RNA hybrids, and can be unmodified polynucleotides or unmodified oligonucleotides. Leotide, or any other known modification, such as a labeled, capped, methylated, or one or more natural nucleotides known in the art.
  • Substituted, modified with an intramolecular nucleotide for example, having an electrical bond (eg, methylphosphonate, phosphotriester, phosphoramidate, olebamate, etc.), a charged bond or a sulfur-containing bond (eg, phosphorothioate
  • an electrical bond eg, methylphosphonate, phosphotriester, phosphoramidate, olebamate, etc.
  • a charged bond or a sulfur-containing bond eg, phosphorothioate
  • a sulfur-containing bond eg, phosphorothioate
  • proteins nucleases, nucleases' inhibitors, toxins, antibodies, signal peptides, poly-L-lysine, etc.
  • sugars eg, monosaccharides
  • an intergallant compound for example, Lysine, psoralen, etc.
  • chelating compounds eg, metals, metals with high dimensional activity, boron,
  • nucleoside include not only those containing known purine and pyrimidine bases, but also those containing other decorated 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 be modified at the sugar moiety, e.g., where one or more hydroxyl groups have been replaced with a halogen and a force, a (fatty) group, or an ether, amine, etc. It may have been converted to a functional group.
  • the antisense nucleic acid of the present invention is RNA, DNA, or a modified nucleic acid.
  • nucleic acids that have been subjected to ⁇ are those that are resistant to sulfur and nucleic acid derivatives of nucleic acids, and to the angle of separation of polynucleoside and polynucleonucleosides. It is not limited to that.
  • the antisense nucleic acid of the present invention can be preferably designed according to the following policy. That is, the antisense nucleic acid in the cell is made more stable, the cell permeability of the antisense nucleic acid is increased, the affinity for the target sense strand is increased, and if the toxic nucleic acid is Makes toxicity less.
  • the antisense nucleic acids of the present invention may have altered or modified sugars, bases, or linkages, may be in special forms such as ribosomes, microspheres, or may be applied by gene therapy. Or can be given in an added form.
  • additional forms include polycations, such as polylysine, that act to neutralize the charge on the phosphate backbone, enhance interactions with cell membranes, and increase nucleic acid uptake.
  • Hydrophobic substances such as lipids (eg, phospholipids, cholesterol, etc.) can be mentioned.
  • Preferred lipids to be added include cholesterol and its derivatives (eg, cholesteryl oral form, cholic acid, etc.).
  • Such a substance can be attached to the 3 'end or 5' end of a nucleic acid, and can be attached via a base, a sugar, or a nucleoside bond in a nucleic acid.
  • Other groups are cap groups specifically arranged at the 3 'end or 5' end of nucleic acids, and are strongly used for degradation by nucleases such as exonuclease and RNase! Can be Such capping groups include, but are not limited to, hydroxyl-protecting groups known in the art, such as glycols such as polyethylene glycol and tetraethylene 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 genetic expression system of the present invention, or the N-Tes in vivo or in vitro translation system. .
  • the nucleic acid can be applied to cells by various methods known per se. As described above, a method of transferring the N-Tes gene and the recombinant DNA into a host, expressing N-Tes, and obtaining a desired N-Tes is based on the research results of the present inventors. Thus, according to the present invention, recombinants or transfectants that substantially express the N-Tes gene, methods for producing the same, and uses thereof are also used.
  • the present invention has a maraudal P inhibitory activity belonging to the Testican family and lacks G1 ycosaminoglycan attachment site and / or lacks TY domain or CTCV d a protein or a salt thereof, which is a kind of polypeptide lacking omain and having substantially the same activity as natural human N-Tes, more preferably N-Tes or a salt thereof; A polypeptide having at least a portion of the protein having substantially the same activity, or at least a part of the protein having substantially the same primary structure conformation, or a polypeptide such as Escherichia coli or a mammalian cell. »Regarding nucleic acids such as DNA and RNA that can be expressed in organisms.
  • nucleic acids are (a) a sequence capable of coding for the amino acid sequence represented by SEQ ID NO: 2 in the sequence listing or a sequence complementary thereto, (b) the DNA sequence of (a). Or a sequence capable of hybridizing with the fragment thereof, and (c) a sequence having a degenerate code capable of hybridizing to the sequence of (a) or (b).
  • stringent conditions can be strongly applied as conditions for high presidency.
  • Prokaryotes such as Escherichia coli which can be transformed with such nucleic acids and can express the polypeptide of the present invention, as well as organisms such as mammalian cells, constitute '1' of the present invention.
  • the object of the present invention is to use the -Tes gene, a probe derived therefrom, or, if necessary, an inhibitor against N-Tes, to obtain N-Tes or N-Tes in the test sample.
  • An object of the present invention is to provide an excellent method for separating the gene and the production cell, and a kit therefor. It is understood that the present invention includes all aspects of such reagent kits capable of separating N-Tes or their preferences, and further producing cells, by Nenrophoresis.
  • an object of the present invention is to perform intracellular tannos by sorting N-Tes or its gene, and further producing cells, using the U-method.
  • bandits Ps in many normal cellular processes, such as clast metabolism, activation of hormone precursors, and bone modification, Alzheimer's disease, pulmonary wing weight, rheumatoid arthritis, muscular dystrophy, osteoporosis, It is an object of the present invention to provide a method for monitoring many diseases such as neurodegenerative diseases and cancer invasion and metastasis, and to provide a diagnostic agent.
  • I ⁇ in the medical-physiological field, research on cell and paper tissue of humans and other animals such as tumors and cancers, analysis, measurement, etc. It is understood to be included within that embodiment of the invention.
  • the N-Tes or a salt thereof of the present invention has a solid Ps inhibitory activity, and is an indispensable factor in, for example, protein metabolism, antigen presentation, bone modification, hormone precursor activity, etc. in vivo. Conceivable.
  • the protein is considered to be useful for the treatment of N-Tes-related dysfunction diseases, such as N-Tes dysfunction, N-Tes expression deficiency, and N-Tes gene deficiency.
  • N-Tes-related dysfunction diseases such as N-Tes dysfunction, N-Tes expression deficiency, and N-Tes gene deficiency.
  • the use of a drug containing N-Tes, mutants, modifications, and primers can bring a disease patient due to inadequate N-Tes activity to a healthy state. .
  • the polypeptide of the present invention is one of protease inhibitors, and has a reduced protease inhibitory activity of the protein; treatment and Z or prevention of various diseases caused by t ⁇ . It is useful as a drug such as an agent.
  • the polypeptide of the present invention has enhanced MMP ecchiamasia for tissues and proteins: it is useful as a medicament such as an agent for treating and / or preventing various diseases caused by ti ⁇ .
  • the biological activity of the cells is not sufficient due to lack or abnormalities: (B) administering the nucleic acid such as sub-A of the present invention to the patient to express the protein or the like of the present invention in a living body; (C) transplanting cells into which the nucleic acid such as the DNA of the present invention can be expressed so that the protein of the present invention can be replenished into the living body, etc. Or improve the symptoms.
  • the compound (agonist or promoter) or a salt thereof that promotes a function such as a biological activity of N-Tes (eg, protease inhibitory activity) of the present invention or a salt thereof is defective in N-Tes function.
  • a medicament useful as a therapeutic or Z or prophylactic agent for various diseases such as symptoms, lung weight, muscular dystrophy, osteoporosis, Alzheimer's disease, m. Disease, rheumatoid arthritis and cancer invasion / metastasis.
  • a compound (antagonist or inhibitor) or a salt thereof that inhibits a function such as a biological activity of N-Tes (eg, a protease inhibitory activity) of the present invention or a salt thereof may be used for trans-Tes dysfunction.
  • N-Tes is involved in the expression of the activity of banded Ps such as MT1-banded P and MT3-banded-P, and has an inhibitory activity against them. It is useful as a medicament for the treatment of diseases caused by excessive degradation by fractions Ps such as T3-MMP etc.
  • the polypeptides such as N-Tes of the present invention are useful as the N-Tes etc. of the present invention.
  • a polypeptide such as N-Tes of the present invention a protein such as N-Tes of the present invention using a partial peptide or a salt thereof, a partial peptide or a salt thereof, etc.
  • the substrate was inoculated to, for example, (i) the protein of the present invention, a part of the peptide, or a salt thereof (which may include a transformant expressing the protein, the same applies hereinafter), etc .: ff ⁇ and (ii) a substrate and a test for the protein of the present invention, some of its peptides or their salts, and the like! ⁇
  • Contacted material Compare with 13 ⁇ 4. Specifically, in the above-mentioned screening, the biological activity (for example, protease activity) is measured and compared.
  • the substrate may be any substrate as long as it can be a substrate such as knitted Ps.
  • it can be used by selecting from protein substances such as casein, collagen, and synthetic oligopeptides which are used for the purpose of determining the protein activity.
  • Substrates can be used.
  • the substrate can be used as it is, and is preferably labeled with a fluorescent, enzymatic or enzymatic substance such as fluorescein. Try! ⁇ : Examples of materials include proteins, peptides, non-peptide compounds, and synthesis Examples include fermentation products, plant extracts, extracts of animals and the like, cell extracts, and the like. Examples of test compounds converted into test samples are preferably PP and closed (J The compound may include a compound having inhibitory activity against P.
  • a synthetic compound which may be a novel compound or a known compound. It 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). In addition, it is possible to use various labels, buffer systems, etc., etc., or to perform the operations according to the operations described there. Treatment with an activating agent The precursor or the latent form can be pre-converted to the active form. The measurement is usually performed using a buffer such as Tris-HCl buffer or phosphate buffer that does not adversely affect the reaction. In a liquid or the like, for example, pH of about 4 to about 10 (preferably, about pH 6 to about 8) can be applied.
  • a buffer such as Tris-HCl buffer or phosphate buffer
  • the ⁇ -Tes of the present invention or a polybenth having substantially the same activity as the ⁇ -Tes of the present invention is added to the ordinary conditions and procedures of each method, taking into account ordinary technical considerations of those skilled in the art. It is only necessary to construct a measurement system related to peptides or peptides. For details of these general technical means, it is possible to refer to reviews, written books, etc. [For example, Methods in Enzymology, Vol. 1, 2, 5 & 6 (Preparation and Assay of Enzymes); J, Vol. 3 (Preparation and Assay of Substrates); Id., Vol. (Special Techniques for the Bnzymologist); Id., Vol.
  • Test compounds such as peptides, proteins, non-peptides, non-peptide compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts, etc. Promotes the function of the protein etc. of the present invention, Is a compound that inhibits, for example, a pharmaceutically acceptable salt And the like.
  • salts with inorganic bases examples thereof include salts with organic donkeys, salts with inorganic acids, salts with organic acids, and salts with donkey or acidic amino acids.
  • the salt with an inorganic assalt include an alkali metal salt such as a sodium salt and a potassium salt, an alkaline earth metal salt such as a canoresium salt and a magnesium salt, and an aluminum salt and an ammonium salt.
  • Preferred examples of the salt with an organic base include, for example, trimethylamine, triethylamine, pyridine, picoline, 2,6-lutidine, ethanolamine, diethanolamine, triethanolamine, cyclohexinoleamine, dicyclohexane.
  • Salts with xylamine, ⁇ , ⁇ '-dibenzylethylenediamine and the like Suitable examples of the salt with an inorganic acid include, for example, salts with hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, and the like.
  • Preferred examples of the salt with an organic acid include, for example, H-acetic acid, acetic acid, propionic acid, fumaric acid, oxalic acid, tartaric acid, maleic acid, cunic acid, succinic acid, apple apple, methanesulfonic acid, benzenesulfonic acid, Salts with benzoic acid and the like can be mentioned.
  • the salt with a basic amino acid include, for example, salts with arginine, lysine, orditin and the like.
  • Preferred examples of the salt with a basic amino acid include, for example, aspartic acid, glutamic acid, etc. And salts thereof.
  • the term “antibody” may be used in a broad sense, and may be a single monoclonal antibody or various epitopes against a desired N-Tes polypeptide and a related peptide fragment. And monovalent or multivalent antibodies, polyclonal antibodies and monoclonal antibodies, and further include native (intact) molecules and their fragments and antibodies.
  • the term "antibody” is to be construed in the same manner for the ⁇ antibody as for the antibody to the desired N-Tes polypeptide and related peptide fragments.
  • Monoclonal antibodies raised against the antigenic substance can be produced using any method that allows for the production of antibody molecules to be produced by a series of cell lines during culture.
  • Each monoclonal antibody contains a population of antibodies that are identical, except that only a small number of naturally occurring variants may be present.
  • Monoclonal antibodies have high specificity and are directed against sites with a single antigenicity. When compared to a conventional (polyclonal) antibody preparation, which typically contains a variety of antibodies directed against different antigenic determinants (epitopes), each monoclonal antibody will Are directed against a single antigen determination S. In addition to its specificity, monoclonal antibodies are synthesized by hybridoma culture and are excellent in that they have no or little other immunoglobulins. Monoclonal antibodies include, but are not limited to, hybrid antibodies and recombinant antibodies.
  • variable domain domains with constant region domains (eg, humanized antibodies) or replace light chains with heavy chains, regardless of their origin or immunoglobulin class I subclass, as long as they exhibit the desired biological activity.
  • constant region domains eg, humanized antibodies
  • immunoglobulin class I subclass e.g. humanized antibodies
  • the ability to replace, replace one chain with another, or fuse it with a heterogeneous protein eg, US Pat. No. 4,816,567; Monoclonal Antibody Production Techniques and Appl. i cat ions, pp. 79-97, Marcel Dekker, Inc., New York, 1987).
  • Examples of suitable methods for producing monoclonal antibodies include the hybridoma method (G. Kohler and C. Milstein, Nature, 256, pp. 495-497 (1975)); human B cell hives. Lidoma method (Kozbor et al., Immunology Today, 4, pp. 72-79 (1983); Kozbor, J. Immunol., 133, pp. 3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63, Marcel Dekker, Inc., New York (1987); Trioma method; EBV-hybrid method (Cole et al., Monoclona 1 Antibodies and Cancer Therapy, Alan R. Liss , Inc., pp.
  • the monoclonal antibodies according to the present invention may be heavy chains and / or light chains, as long as they exhibit the desired biological activity, of antibodies derived from a particular species or belonging to a particular antibody class or subclass.
  • a ⁇ chimera '' which is identical or homologous to the corresponding sequence, while the remainder of the chain is identical or homologous to the corresponding sequence of an antibody derived from another species or belonging to another antibody class or subclass.
  • antibodies immunoglobulins
  • the monoclonal antibody of the present invention was obtained using a cell fusion technique using myeloma cells (such as G. Kohler and C. Milstein, Nature, 256, pp. 495-497 (1975)). It may be a monoclonal antibody, which can be produced, for example, by the following steps.
  • N-Tes polypeptide or a fragment thereof derived from the N-Tes polypeptide may be used as the antigen.
  • the determined amino acid of N-Tes may be used.
  • an appropriate oligopeptide can be chemically synthesized and used as an antigen.
  • the antigen may be mixed with an appropriate adjuvant as it is to form a immunogenic conjugate which can be used for immunizing animals.
  • a peptide used as a license is a fragment of N-Tes or a synthetic polypeptide fragment obtained by selecting a specific sequence region based on the amino acid sequence, designing the polypeptide and chemically synthesizing it. It may be.
  • the fragment is bound to various carriers such as hapten-tanno via a suitable condensing agent to form a hapten-tanno. This can be used to design a monoclonal antibody capable of reacting only with a specific sequence (or recognizing only a specific sequence).
  • a cysteine residue or the like is added to the designed polypeptide in advance, so that the preparation of the 3 ⁇ -soluble conjugate can be performed in a forehead.
  • the carrier proteins In binding to carrier proteins, the carrier proteins can be activated first. For such activation, the ability to guide an activating binding group can be cited.
  • Examples of the activated linking group include: (1) an activated ester or an activated carboxyl group, for example, a nitrophenyl ester group, a pentafluorophenyl ester group, a tribenzotriazole ester group, an N-succinimide ester group, etc. (2) An activated dithio group, for example, a 2-pyridyldithio group.
  • Examples of carrier proteins include polypeptides such as keyhorn oleoresin 'limpet' hemocyanin (KLH), bovine serum albumin (BSA), ovalbumin, glopurine and polylysine, and bacterial cell components such as BCG.
  • Immunization can be carried out by methods known to those skilled in the art. For example, 'TT Kosei, other editions, real, animal ⁇ ! Ed., Seikagaku Experimental Lecture 5, licensing research method, Tokyo Kagaku Dojin, 1986, The Japanese Biochemical Society, Ed., Shinsei Chemistry Experimental Lecture 12, Molecular Immunology, Antigen, Antibody, Complement, Tokyo Kagaku Dojin , 1992 etc. can be carried out according to the method described.
  • the immunizing agent is immunized by injecting the immunizing agent into a mammal or the like one or more times (with adipants as needed).
  • the immunizing agent and / or adjuvant is measured by subcutaneously measuring or intrasternally measuring a mammal a plurality of times.
  • the immunizing agent include those containing the above-mentioned antigen peptide or its related peptide fragment.
  • the immunizing agent is a protein that is known to be immunogenic in the mammal to be immunized.
  • the conjugate may be formed with (for example, the above-mentioned carrier proteins) and subjected to ⁇ ffl.
  • adjuvants include mouth adjuvant, Ribi adjuvant, pertussis vaccine, BCG, Lipid KA, ribosome, hydroxylamine, silica, and the like. Immunization is performed on mice, hams such as BALB / c, etc. This is done using stars and other suitable animals.
  • the dose of the antigen is, for example, about 1 to 400 g / animal with respect to the mouse.
  • the booster is repeated about 2 to 10 times in the air, subcutaneously, intravenously, or intramuscularly.
  • mice in addition to BALB / c mice, F1 mice of BALB / c mice and other mice can be used. If necessary, an antibody titer can be prepared, and the antibody titer can be measured to confirm the degree of animal immunity.
  • the antibody of the present invention may be obtained from the thus obtained and immunized animal, and includes, for example, polyclonal anti-antibodies and the like.
  • a cell line that does not produce immunoglobulin can be selected.
  • ⁇ 3-NS-Ag4-1 NS-1, Bur. J. Immunol , 6: 511-519, 1976
  • SP-2 / 0-Agl4 SP-2, Nature, 276: 269 -270, 1978
  • P3-X63-Ag8_Ul derived from mouse myeloma MOPC-21 cell line ( P3U1, C urr. Topics Mi crobiol. Immunol., 81: 1-7, 1978)
  • P3-X63-Ag8 X63, Nature, 256: 495-497, 1975
  • P3-X63-Ag8-653 653, J.
  • the 8-azaguanine-resistant mouse myeloma cell line is prepared by adding a substance such as penicillin or amikacin, fetal calf serum (FCS), etc. to a cell medium such as Dulbecco's MEM medium (DMEM medium) or RPMI-1640 medium.
  • DMEM medium Dulbecco's MEM medium
  • FCS fetal calf serum
  • RPMI-1640 medium Dulbecco's MEM medium
  • the ability to be passaged in a medium supplemented with azaguanine eg, 5-45 zg / ml
  • the ability to prepare the required number of cell lines by passage in normal medium 2-5 days prior to cell fusion.
  • the cell strain used should be thawed completely at 37 ° C, rinsed at least 3 times in a normal medium, such as RPM 1640, and then cultured in a normal medium to obtain the required number of cells.
  • the stock may be prepared. .
  • Cell fusion between antibody-producing cells and myeloma cells should be thawed completely at 37 ° C, rinsed at least 3 times in a normal medium, such as RPM 1640, and then cultured in a normal medium to obtain the required number of cells.
  • the stock may be prepared. .
  • An animal for example, a mouse immunized according to the above step 2 is excised 2 to 5 days after the final immunization, and a spleen cell suspension is obtained.
  • lymph node cells from various parts of the body can be obtained and used for cell fusion.
  • the spleen cell suspension obtained and the myeloma cell line obtained according to step 3 above are placed in a cell culture medium such as a minimum essential medium (MEM medium), DMEM medium, RPMI-1640 ⁇ ground, and cell fusion is performed.
  • a cell culture medium such as a minimum essential medium (MEM medium), DMEM medium, RPMI-1640 ⁇ ground
  • An agent such as polyethylene glycol is added.
  • 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 the amount can be further increased to 1,000.
  • -4,000 polyethylene glycol can be more preferably used.
  • the concentration of polyethylene glycol in the fusion medium can be, for example, 30-60%. If necessary, for example, dimethylsulfoxide can be reduced to promote fusion.
  • the ratio of the spleen cells (lymphocytes) used for the fusion: myeloma cell line is, for example, 1: 1 to 20: 1, but is more preferably 4: 1 to 7: 1. can do.
  • the fusion reaction is performed for 1-10 minutes, and then a cell culture medium, such as RPM 1640 medium is added.
  • the fusion reaction can be performed multiple times. After the fusion reaction, the cells are separated by centrifugation and transferred to a selection medium.
  • MEM medium for example, MEM medium containing FCS containing hypoxanthine (H), aminopterin (A) and thymidine (T), i-field such as RPMI-1640 ⁇ field, so-called HAT ⁇ field.
  • the method of replacing the selective medium is generally the same as adding the same volume as the volume dispensed to the culture plate the next day, and then replacing the HAT medium by half every 1 to 3 days. Ffi can be done by adding ⁇ H to this.
  • aminopterin is removed, and the medium can be exchanged every 1 to 4 days in a so-called HT ⁇ ground.
  • a feeder for example, mouse thymocytes can be used, which is preferable.
  • the ability of the hybridoma to grow and the culture supernatant of the culture medium to be analyzed using a measurement system such as radioimmunoassay (RIA), enzyme immunoassay (ELISA), or fluorescence immunoassay (FIA), or fluorescence induction In cell sorting (FACS), etc., use a specific fragment peptide
  • a measurement system such as radioimmunoassay (RIA), enzyme immunoassay (ELISA), or fluorescence immunoassay (FIA), or fluorescence induction In cell sorting (FACS), etc.
  • RIA radioimmunoassay
  • ELISA enzyme immunoassay
  • FACS fluorescence immunoassay
  • screening is performed by measuring the target antibody using a labeled anti-mouse antibody.
  • Cloning hybridomas producing the desired antibody can be performed by the ability to pick up colonies in an agar medium, or by the limiting dilution method. It can be more preferably performed by the limiting dilution method. Cloning should be performed multiple times.
  • the obtained hybridoma strain is cultured in an appropriate medium such as MEM ⁇ field or RPMI-1640 ⁇ field containing FCS, and it is possible to obtain the desired monoclonal antibody from Lb culture. . : In order to obtain the antibody of the above, ascites of the hybridoma is strongly mentioned. This: transplants each hybridoma and hybridoma into the peritoneal cavity of a thread-compatible animal that is syngeneic to an animal derived from an i3 ⁇ 4myeloma cell, the ability to proliferate, or transplants each hybridoma into, for example, a nude mouse. Then, the antibody can be obtained by collecting the monoclonal antibody produced in the ascites of the fiber.
  • an appropriate medium such as MEM ⁇ field or RPMI-1640 ⁇ field containing FCS
  • the animals can transfer mineral oils such as pristane (2,6,10,14-tetramethylpentadecane) into the air after transplantation of the hybridomas.
  • the hybridomas can grow and ascites Can also 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, etc., 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 is 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 an antigen or antigen fragment (for example, a synthetic peptide, a recombinant antigen protein or peptide, a site specifically recognized by an antibody, etc.) is immobilized, and affinity in which protein A is immobilized is preferred.
  • affinity chromatography in which an antigen or antigen fragment (for example, a synthetic peptide, a recombinant antigen protein or peptide, a site specifically recognized by an antibody, etc.) is immobilized, and affinity in which protein A is immobilized is preferred.
  • Niti Chromatography I Hydroxia. Tight
  • nucleic acid encoding the monoclonal antibody can be isolated by a conventional method, for example, by using an oligonucleotide probe capable of specifically binding to the residue encoding the heavy or light chain of the mouse antibody.
  • the DNA can be placed in a hotel such as a COS.
  • the DNA can be modified, for example, by replacing the sequence of a homogenous mouse with a sequence encoding the constant region domain of a human heavy chain or light chain (Morrison et al., Proc. . Natl.
  • Antibodies can also be modified by applying chemical protein syntheses, including the use of the following condensing agents, to prepare chimeric antibodies or hybrid "antibodies.
  • Humanized antibodies can be performed by techniques known in the art (eg, Jones et al., Nature, 321: pp. 522-525 (1986); Riechmann et al., Nature, 332: Verhoeyen et al., Science, 239: pp. 1534-1536 (1988))
  • Human monoclonal antibodies can also be prepared by techniques known in the art.
  • Human myeloma cells for producing ⁇ Human / mouse heteromyeloma cells are known in the art (Kozbor, J. Immunol.,
  • Antibodies can be converted to any known assay, such as competitive binding assays, direct and indirect sandwich assays, and immunoprecipitation: ⁇ (Zola, Monoclonal Antibodies: A Manual of Techniques, pp). 147-158 (CRC Press, Inc., 1987) Any method known in the art for conjugating antibodies to detectable groups can be used, for example, David et al. , Biochemistry, Vol. 13, pp. 1014-1021 (1974); Pain et al, J. Immunol. Meth., 40: pp. 219-231 (1981); and "Methods in Enzymology", Vol. 184, pp. 138.
  • the antibody to be labeled use is made of the IgG fraction, and the specific binding ⁇ 15Fab 'obtained by reducing after reducing pepsin. Examples of labeling of these are: Enzymes (peroxidase, alkaline phosphatase) Or 3-D-galactosidase), iridescent substances, fluorescent substances, radioactive alleles, etc.
  • the arrogant and SU determination in the present invention is based on Immo staining, for example, a thread, Staining, immunoassay, for example, competitive or noncompetitive immunoassay, can be performed using radioimmunoassay, ELISA, FIA, etc., B-F separation may be performed, Alternatively, it is possible to carry out the measurement without performing the method, preferably, immunoassay, enzyme immunoassay J, and fluorescence immunoassay, and furthermore, a sandwich type assay, for example, a sandwich type assay.
  • one is an antibody against the N-Tes of the present invention and its related peptide fragment
  • the other is an antibody against the C-terminal residue of N-Tes
  • one is detectably labeled. Immobilize on the solid phase other antibodies that can recognize the antibody.Incubation treatment is performed to react the sample with the labeled antibody and the immobilized antibody as necessary, and then the unbound antibody is separated and labeled. Measure the object. The amount of label measured is proportional to the amount of antigen, ie, the N-Tes polypeptide fragment KJ ⁇ .
  • insolubilized antibody or labeled antibody Depending on the river drunk, it is called a simultaneous sandwich-type assembly, a forward sandwich-type assembly, or a reverse sandwich-type assembly.
  • washing, freshness, shaking, filtration or pre-extraction of antigens, etc. are employed in these measurement processes under certain circumstances.
  • Other measurement conditions such as the concentration of a particular buffer, temperature, or incubation time, can be varied according to factors such as the concentration of the antigen in the sample, the nature of the sample, and the like.
  • a person skilled in the art can perform a measurement by selecting an optimum condition effective for each measurement while using a normal experimental method.
  • carrier various types of carriers that are used for antigen-antibody reactions and the like are known, and according to the present invention, it is needless to say that these known mediums can be selected.
  • glass for example, activated glass, porous glass, silica gel, silica-alumina, alumina, magnetized iron, magnetized alloys and other inorganic materials, polyethylene, polypropylene, polyvinyl chloride.
  • high-quality organic substances such as denatured cellulose, crosslinked dextran, nylon, etc., polyurethane, polyepoxy resin, etc., and those obtained by emulsion polymerization, cells, erythrocytes, etc., if necessary. And those having a functional group introduced by a silane coupling agent.
  • filter paper beads, inner walls of test containers, such as test tubes, titer plates, titer cells, glass cells, cells made of synthetic materials such as synthetic resin cells, glass rods, rods made of synthetic material, and thicker ends
  • a solid material such as a rod that is thinned or thinned, a rod that has a rounded or flat projection at the end, or a rod that has been shaped like an acknowledgment.
  • An antibody can be bound to these carriers, and preferably a monoclonal antibody that specifically reacts with the antigen obtained in the present invention can be bound.
  • the binding between the carrier and those involved in the antibody reaction can be performed by physical methods such as adsorption, chemical methods using a condensing agent, activated substances, etc.
  • the labeling can be performed by a method using a chemical binding reaction, such as an enzyme, an enzyme substrate, an enzyme inhibitor, a prosthesis, a capture enzyme, an enzyme precursor, an apoenzyme, a fluorescent substance, a dye substance, Examples include luminescent compounds, luminescent substances, coloring substances, magnetic substances, metal particles, radioactive substances such as gold colloid, and the like.
  • enzymes include SfeR enzyme, reductase, oxidase, and other oxidases, such as transferases that transfer amino, carboxyl, methyl, acyl, and phosphate groups.
  • Hydrolytic enzymes that hydrolyze ester bonds, glycosidic bonds, ether bonds, peptide bonds, etc., lyases, isomerases, ligases and the like can be mentioned.
  • the enzyme can be used arbitrarily by using a complex fiber enzyme. For example, simple cycling can be used.
  • Representative enzyme labels include peroxidase such as horseradish peroxidase, galactosidase such as E. coli 3-D-galactosidase, maleate dehydrogenase, glucose-6-phosphate dehydrogenase, glucose oxidase, and glucoamylase.
  • Alkaline phosphatase such as glycerol, acetylcholinesterase, catalase, yeast small intestine alkaline phosphatase, and Escherichia coli alkaline phosphatase.
  • alkaline phosphatase invitation of 4-methylphenylphenol phosphate, etc., and phosphorylation of phenol, such as ditrophenylphosphonate.
  • NADP-based enzymatic cycling system NADP-based enzymatic cycling system
  • luciferin induction It can be measured by the light, luminescence, etc. generated by using a substrate such as body or dioxetane.
  • Noreciferin and luciferase systems can also be used.
  • a force plate ⁇ , It reacts with hydrogen peroxide to generate oxygen, so that oxygen can be supplied to several electrodes.
  • the electrodes used are glass electrodes, ion electrodes using insoluble materials, and so on. 3 ⁇ 4 Electrodes, polymer membrane electrodes, etc. can also be used.
  • Enzyme labeling can be replaced with biotin-labeled enzyme and enzyme-labeled avidin (streptavidin).
  • the sign can be a number of different signs.
  • Such ⁇ can also allow multiple measurements to be taken continuously or discontinuously, and simultaneously or separately.
  • Labels and detectable signals can be obtained using commercially available kits that are suitable for them, and can be performed according to protocols specified by the kit manufacturer or kit distributor.
  • the formation of a signal includes 4-hydroxyphenylsulfuric acid, 1,2-phenylenediamine, tetramethylbenzidine and the like, as well as horseradish peroxidase, campbellifrinoregalactoside, and nitrofuran.
  • nilgalactoside and other enzyme reagents such as 3-D-galactosidase and glucose-6-phosphate 'dehydrogenase can also be used (for J, quinol compounds such as hydroquinone, hydroxybenzoquinone and hydroxyanthraquinone)
  • Thiol compounds such as lipoic acid and gnorethation, phenol-inducing compounds, and ferrocene-inducing compounds can be strongly used as they can be formed by the action of enzymes and the like.
  • fluorescein isothiocyanate such as rhodamine dithiothionate, tetramethyl rhodamine isothiocyanate, etc.
  • one rhodamine derivative dansyl chloride, dansyl fluoride Fluorescamine, phycopyriprotein, ataridinium salt, noremiferin, norreciferase, luminocorinase such as etaolin, imidazole, oxalate, dilute chelates, coumarin-inducing compounds and the like.
  • the reaction between a thiol group and a maleimide group, the reaction between a pyridyl disulfide group and a thiol group, and the reaction between an amino group and an aldehyde group can be carried out in parallel.
  • the method can be selected from known methods or methods that can be easily performed by those skilled in the art, and further, modified methods thereof and applied.
  • a condensing agent that can be used in the production of the above-described insoluble complex, a condensing agent that can be converted into a bond with a carrier, and the like can be used.
  • condensing agent examples include formaldehyde, glutaraldehyde, hexamethylene diisocyanate, hexamethylene diisothiocyanate, ⁇ , ⁇ '-poly (methylene bis-doacetamide), ⁇ , ⁇ '-ethylene bismaleimide, ethylene glycol 1-Rubis-succinimidyl succinate, bis-diazobenzidine, 1-ethyl-3- (3-dimethylaminopropinole) carbodiimide, succinimidyl 3- (2-pyridyl dithio) propionate (SPDP), N-succinimidyl 4- (N-maleidomethyl) cyclohexane-1_carboxylate (SMCC), N-sulfosuccinimidinole 4-(N-maleimidomethyl) cyclohexane-1-carboxylate, N-succinimidyl ( 4- (4-acetyl) amino benzoate, N-
  • a target antibody such as a monoclonal antibody in which a substance to be measured is labeled with an enzyme and the like and an antibody bound to a carrier can be sequentially reacted with each other, and can be simultaneously reacted. You can also. Lli depends on the type of carrier system chosen. If sensitized plastic or other beads are used, first place a labeled antibody, such as a monoclonal antibody labeled with an enzyme, together with the sample containing the substance to be measured in an appropriate test tube. Thereafter, the measurement can be performed by adding beads such as the sensitized plastic.
  • an immunoassay is used.
  • a solid support made of polystyrene or polycarbonate, which adsorbs proteins such as antibodies well, is used.
  • Various materials and forms such as kits, polypropylene or polyvinyl Beauneles, microplates, sticks, fine particles or test tubes can be arbitrarily selected and manipulated.
  • the measurement can be performed in an appropriate buffer system so as to maintain the optimum pH, for example, about pH 4 to about 9.
  • buffers include, for example, acetate buffers, citrate buffers, phosphate buffers, tris buffers, triethanolamine buffers, borate buffers, glycine buffers, carbonate buffers Agents, Tris-hydrochloride buffer ff ij, and the like.
  • the buffers can be mixed and used in any desired ratio.
  • the antigen-antibody reaction is preferably performed at a temperature between about 0 ° C and about 60 ° C.
  • Antibodies such as monoclonal antibodies labeled with enzymes, etc. and antibodies bound to the carrier, and the incubation of the substance to be measured, can be performed until the target is reached.
  • the solid phase and the liquid phase can be separated and the reaction can be stopped after a limited incubation treatment at a time earlier than the time when S3 ⁇ 4 ⁇ ⁇ tan ⁇ is achieved.
  • the measurement operation can be powerfully performed using an automated detector, and a luminescence detector, a photo detector, or the like is used to detect a display signal generated when the substrate is converted by the action of an enzyme. Can also be measured.
  • the antigen-antibody reaction it is possible to stabilize the label used for the wisteria, the substance to be measured, and the enzyme, and to take appropriate measures to stabilize the antigen-antibody reaction itself.
  • proteins, stabilizing agents, surfactants, chelating agents, etc. are used to remove abnormal reactions and reduce inhibitory effects, or to activate measurement reactions. It can also be added at midnight.
  • ethylenediamine tetrasalt (BDTA) is preferred.
  • Collagen, gelatin can be processed by These methods are not particularly limited and can be used as long as the purpose is to prevent nonspecific binding reactions.
  • Samples to be measured by the measurement method of the present invention include, but are not limited to, Nada in any form, a colloid reservoir, a body sample, and the like; preferably a biological sample, such as thymus, testicle, intestine, intestine, body, and brain.
  • a biological sample such as thymus, testicle, intestine, intestine, body, and brain.
  • Breast, Ovarian, Colorectal, Rectal, Blood, Serum, Plasma, Synovial Fluid, Cerebrospinal Fluid, Saliva, Amniotic Fluid, Urine, Other Body Fluids, Cell Culture, Cell Culture, «Homodunate, Biopsy Examples include a tissue and a cell.
  • the term "substance" of the present invention relates to a substance having substantially the same activity.
  • Epitope mapping can also be performed using the anti-N-Tes antibody of the present invention, in particular, a monoclonal antibody. It can be performed.
  • Antibodies against N-Tes and its related peptide fragments can be used to detect and measure phenomena such as inhibition of the activity of MT-band Ps by N-Tes, as well as to detect or measure various physiologically active substances caused by MMPs activity (ii). And / or useful for measurements.
  • m especially monoclonal antibodies, can be used to (i) detect disorders, abnormalities and Z or disease associated with the degradation of paper or protein by banding Ps, or (ii) detect cells or cells caused by paper or protein degradation by MMPs. Crushing, cell migration, invasion, play Z or metastasis or the possibility thereof, and / or (iii) SSK formation of cells, migration, invasion of cells such as cells, blood cells, etc.
  • Useful for detecting play Z or metastasis or its potential It can be used to determine the degree of cancer mobility, invasiveness, chemotaxis and / or metastasis.
  • the inhibition of the activity of ⁇ -diet by ⁇ -Tes is detected and / or measured. It can be used as a monitor for determining the effects of ij, elimination, and Z or immunosuppressants. Further, in the present invention, it is possible to detect and measure Z and Z or a method of measuring the angle of division of paper woven or protein by ⁇ - ⁇ and Z or MT3-band P, and the power for the method.
  • the antibodies (including monoclonal antibodies) used in the present invention include known antibodies, those obtained by applying various known methods for producing the antibodies, and the methods for preparing the antibodies described above. And those obtained according to the above.
  • the active ingredient of the present invention [for example, (a) the N-Tes polypeptide, a part of the peptide or a salt thereof, a peptide related thereto, or the like; (b) the N-Tes or N-Tes polypeptide (C) the antibody of the present invention, a fragment thereof (including a monoclonal antibody) or its trigger, and (d) inhibition of the activity of MT-MPs by N-Tes.
  • a phenomenon is a compound or a salt thereof that suppresses and inhibits or inhibits the biological activity of a paper weave or a protein, and (e) an antisense oligonucleotide to a nucleic acid such as the DNA of the present invention)
  • Use as a pharmaceutical 1)
  • "MP inhibitors or their salts, etc. are usually mixed with wimps or pharmacologically acceptable paint adjuvants and administered as pharmaceutical pirates or pharmaceutical preparations
  • oral It is administered in the form of a pharmaceutical preparation suitable for administration, topical administration, parenteral administration and the like, and may be in any administration form (including inhalation or rectal administration) depending on the purpose.
  • the activity of the present invention is determined by combining mmm (an anticancer agent), wisteria transfer inhibition u, angiogenesis inhibitor, Alzheimer's preparation ij, joint destruction prevention u, immunosuppressive and z, or an immunosuppressant. Can also be used.
  • Promising drugs pile cancer drugs
  • s-segment relocation inhibitor angiogenesis inhibitor
  • Alzheimer's return U joint destruction return U
  • demonstrable iJ and immunosuppressants anyone can use it without restriction, for example, one can choose from those known in the art.
  • Parenteral dosage forms may also include direct, topical, transdermal, intravenous, intramuscular, subcutaneous, intradermal or intradermal administration directly to the affected area, including: ⁇ Is also preferred.
  • mammals including humans (eg, intracellular, tissue, intravenous, intramuscular, subcutaneous, intradermal, M empty, pleural cavity)
  • Intrathecal intrathecal, infusion, ⁇ 1easy, trauma, eardrops, eye drops and nose, teeth, skin and mucous membranes).
  • Specific forms of pharmaceutical preparations include ⁇ preparations, dispersion preparations, semi-solid preparations, granular preparations, molded preparations, leaching preparations, and the like.
  • examples include tablets, coated tablets, sugar-coated preparations, and pills.
  • Soft Capsenole Microcapsules, Uniform powders, Powders, Granules, Fine granules ij, Dimensions, Solutions, Elixirs, Emulsions, Irrigation agents, Syrups, Solutions, Emulsions, Suspensions , Liniments, lotions, aerosols, sprays, inhalants, sprays, ointments, plasters, patches, pasta, cataplasms, creams, oils, suppositories (eg rectal suppositories), tinctures Powder, lyophilizer, gel preparation, etc. for skin preparations, skin solutions, eye drops, nasal drops, ear drops, liniments, infusions, and solutions.
  • Soft Capsenole Microcapsules, Uniform powders, Powders, Granules, Fine granules ij, Dimensions, Solutions, Elixirs, Emulsions, Irrigation agents, Syrups, Solutions, Emulsions, Suspensions
  • compositions may be formulated according to conventional methods. For example, as needed, physiologically acceptable carriers, pharmaceutically acceptable carriers, adjuvants, shellfish release agents, excipients, diluents, flavoring agents, flavors, sweeteners, vehicles, preservatives, if necessary , Stabilizers, binders, pH regulators, buffers, surfactants, bases, solvents, fillers, extenders, solubilizers, solubilizers, isotonic agents, emulsifiers, emulsifiers Agents, dispersants, thickeners, gelling agents, curing agents, absorbents, adhesives, elasticizers, plasticizers, disintegrants, propellants, preservatives, antioxidants, sunscreens, humectants, emollients,
  • an antistatic agent, a soothing agent, or the like in combination with an insect or in combination with the protein or the like of the present invention, a single state required for a generally accepted preparation can be obtained. Can be manufactured.
  • Formulations suitable for parenteral use include sterile solutions of the active ingredient with water or other pharmaceutically acceptable vehicles, or suspensions, such as agents.
  • water, Dextrose solution, solutions of other related sugars, glycols such as ethanol, propylene glycol, and polyethylene glycol are examples of preferred liquid carriers for liquor.
  • a carrier such as distilled water, Ringer's solution, or physiologic solution, a suitable dispersing agent or wetting agent and a suspending agent, and the like may be used, and the method may be performed in a manner known in the art. , Such as suspensions and emulsions.
  • aqueous liquid for measurement examples include menstrual fluid, isotonic solution containing glucose and other auxiliary agents (eg, D-sorbitol, D-mannitol, sodium chloride, etc.), and are pharmacologically acceptable.
  • auxiliary agents eg, D-sorbitol, D-mannitol, sodium chloride, etc.
  • solubilizers such as alcohols (eg, ethanol), polyalcohols (eg, propylene glycol, polyethylene glycol, etc.), nonionic surfactants (eg, polysorbate)
  • oily liquid examples include sesame oil and soybean oil, and may be used in combination with benzyl benzoate, benzyl alcohol, or the like as a dissolution aid.
  • buffers eg, phosphate buffer, sodium acetate buffer, etc.
  • soothing agents eg, benzalkonium chloride, proforce hydrochloride, etc.
  • stabilizers eg, , Human serum albumin, polyethylene glycol and the like
  • preservatives for example, benzyl alcohol and phenol
  • antioxidants such as ascorbic acid, absorption promoters and the like.
  • the prepared liquid is usually filled into an appropriate sample.
  • a sterile pharmaceutically acceptable liquid such as water, ethanol or oil
  • a sterile pharmaceutically acceptable liquid such as water, ethanol or oil
  • a sterile pharmaceutically acceptable liquid such as water, ethanol or oil
  • oily vehicle or solvent used in the preparation include natural or synthetic, semi-synthetic mono- or di- or triglycerides, and natural, semi-synthetic or synthetic oils and fats or fatty acids.
  • Vegetable oils such as oil, corn oil, soybean oil, and sesame oil.
  • this size preparation usually contains 0.1 to 10 S »% of the compound of the present invention! ⁇ Can be prepared to contain.
  • Formulations suitable for topical use include, for example, mouthwashes, dentifrices, oral sprays, inhalants, ointments, dental fillings, dental coatings, dentistry Pastes, suppositories and the like.
  • Mouthwashes and other dental agents are prepared by a conventional method using a pharmacologically acceptable carrier.
  • a pharmacologically acceptable carrier As an oral spray or an inhalant, the compound of the present invention itself or a pharmacologically acceptable inert carrier may be dissolved in a night air for an aerosol or nebulizer, or it may be used as a fine powder for inhalation. Can be administered.
  • 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 by a conventional method .
  • Chemicals for topical application to teeth and skin can be formulated into suitably sterilized water or non-aqueous release agents or suspensions.
  • Additives include buffers such as sodium bisulfite or sodium edetate; acetic acid or phenylino nitrate! ⁇ R
  • Preservatives including bactericidal and antifungal agents such as silver, benzalkonium chloride or black hexidine, and thickeners such as hypromelrose.
  • the suppository may be a carrier well known in the art, preferably a non-irritating suitable excipient, such as polyethylene glycols, lanolin, cocoa butter, fatty acid triglyceride, etc. Is prepared by a conventional method using a liquid that melts in the rectum and releases the drug, but is usually prepared to contain the present compound in the range of 0.1 to 95 fi *% . Depending on the agent and concentration, the drug can be suspended or dissolved in the agent. Auxiliary agents, such as local preservatives and buffers, can be dissolved in shellfish.
  • Formulations suitable for oral use include, for example, solid compositions such as tablets, pills, capsules, powders, granules and troches, and liquid compositions such as solutions, syrups and suspensions. Is mentioned.
  • a formulation auxiliary known in the art and the like are used. Locks and pills can also be manufactured with enteric coating.
  • the preparation is in the form of a capsule, the above-mentioned type of material may further contain a liquid carrier such as oil and fat.
  • the nucleic acid such as the DNA of the present invention is used as a therapeutic and Z 'or prophylactic agent as described above: ⁇ , the nucleic acid can be used in insects or as a genetic and recombinant technique as described above.
  • nucleic acid such as DM of the present invention can be administered by a commonly known method, and may be used as it is, or by using an appropriate auxiliary or It can be formulated and used together with a physiologically acceptable carrier and the like, and can be administered as a pharmaceutical composition or a pharmaceutical preparation as described above. Also, 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 and the number of administrations depend on the tt3 ⁇ 4lj, age, weight, general health, condition, diet, administration of the treated patient. It will depend on time, mode of administration, rate of drug administration, drug combination, and the severity of the condition being treated by the patient at that time, and will take into account these and other factors.
  • their additives and preparation methods are described, for example, in the Japanese Pharmacopoeia Editorial Manual Editing Committee, 14th edition IE IE Pharmacopoeia, June 27, 2001, published by Hirokawa Corporation. Bookstore; Takashi Ichigase et al.
  • the activity of the present invention is to inhibit the activity of MMPs, particularly Mil-solid P, MT3-fraction P or the activation of ⁇ Ps, and to suppress and Z or inhibit the biological activity.
  • MMPs particularly Mil-solid P, MT3-fraction P or the activation of ⁇ Ps
  • the active ingredient of the present invention includes, for example, (a) N-Tes, a mutant polypeptide thereof, a part of the polypeptide or a salt thereof, and (b) DNA encoding the N-Tes, N (C) the antibody of the present invention, a partial fragment thereof (including a monoclonal antibody) or a derivative thereof, and (d) MT_reaction by N-Tes.
  • an active 'f raw P ⁇ of P such, such compounds or salts thereof to inhibit and 7 or inhibit the ivy biological activity are encompassed; TL Ru.
  • the activity of the present invention also includes an antibody, for example, a monoclonal antibody.
  • the active ingredient of the present invention is expected to be useful for suppressing or inhibiting the processing of proteins or proteins by MMPs, for example, the processing of proteins by at least ⁇ -substituted P or MT3- ⁇ P.
  • the activity is ⁇
  • it is useful for suppressing the expression of P-9 and MMP-2 activities, and is ⁇ -MMP or MT3-MMP inherited? ⁇
  • It is useful for prevention or treatment of disorders, abnormalities and Z or diseases related to protein processing by Ps in the current cell. It is also expected to be useful for controlling, for example, suppressing the migration, invasion, migration, and / or metastasis of tumor cells and the like involving Ps.
  • N-Tes and its related peptides are useful for controlling and / or inhibiting the migration, invasion and / or metastasis of evil Sffi ulcers, i.e., cancer, angiogenesis inhibitors, SH inhibitors and / or cancer It can be expected as a transfer inhibitor. It is also useful for the prevention or treatment of disorders, abnormalities and / or diseases related to the processing of blood cells by solid Ps, and can be expected as anti-inflammatory agents and Z or immunosuppressants. Furthermore, it can be expected as Alzheimer's Ito, joint destruction; ⁇ IJ. Furthermore, in the present invention, (a) a sequence ranging from the 22nd amino acid residue to the 312th amino acid residue in the amino acid sequence of N-Tes:
  • the substance thus obtained is also within the scope of the concept of the present invention, and can be treated as the activity of the present invention.
  • (ii) at least one of the constituent amino acid residues is replaced with a D-form amino acid residue.
  • Unamplified cDNA Library (derived from human fetal kidney) from EdgBioSysterns was used.
  • Stratagen cDNA synthesis kit, expression plasmid it is also possible to use a combination senoré.
  • Escherichia coli of 30 clones from a human fetal kidney-derived cDNA expression library was used as a pool and cultured at 37 ° C for 16 hours at 37 ° C in TB ⁇ ⁇ . Purified.
  • Approximately 20,000 293 cells per well were plated in a 96-well microplate in DMEM containing 5% fetal bovine serum and cultured for 24 hours.
  • ⁇ -9, ⁇ -2, MT1-MMP full-length cDNA was inserted into the pSG5 (Stratagene) cloning site to create an expression vector, and 3 ng of MMP-9 expression plasmid per well, MP-2 22 ng of the expression plasmid and 33 ng of the T1-MMP expression plasmid were transfused together with 200 ng of the library DNA by the calcium phosphate method.
  • SDS-PAGE sample buffer (10 mM Tris-HC1 buffer, H6.8, 20% glycerol, 0.24 mg / ml BPB, 1% SDS). After heating at 37 ° C for 30 minutes, 5 microliters were subjected to gelatin zymography to detect latent type, intermediate, and activated MP-9 and MMP-2. By primary screening, a population of gene plasmids that suppressed the production of activated SMMP-2 was identified from the library.
  • a population of the bovine gene plasmid obtained by the primary screening was introduced into E. coli XL-Blue (Stratagene), and cultured on an LB plate containing ampicillin for 24 hours. Fifty colonies of Escherichia coli were separated one by one, cultured in a TB medium at 37 ° C for 16 hours, and a plasmid was purified from 1.5 ml of the cultured E. coli by the alkali-SDS method.
  • Figure 1 shows the data of the microplate 8 wells.
  • lane 4 the production of intermediates and activated ⁇ P-2 is suppressed.
  • the single plasmid added to the wells in which the production of activated MM P-2 was suppressed was analyzed using the LI-COR DNA Sequencer Model.
  • the base sequence of the inserted DNA was determined using 4200.
  • the nucleotide sequence of the determined DNA is described in Rooster column number: 1 in the Rooster column list.
  • a PCR primer with a complementary sequence corresponding to positions 451-473 of N-Tes cDNA and two bases for improving the recognition sequence of restriction enzyme BglII and BglI cleavage A PCR primer with a complementary sequence corresponding to positions 451-473 of N-Tes cDNA and two bases for improving the recognition sequence of restriction enzyme BglII and BglI cleavage
  • PCR amplification was performed using pEAK8 containing N-Tes cDNA as type III. As a result, a fragment of about 500 bases was obtained. This DNA fragment was quenched with EcoRI and Bglll, and ligated with pSG-FLAG vector digested with EcoRI and Bglll and DNA ligase.
  • pSG-FLAG is a pSG5 with a nucleotide sequence coding for a FLAG peptide (DYKDDDK) and a stop codon inserted at the 3 'end of the closing site. Expression tanno ,. FLAG peptide can be added to the N-terminal side of the protein.
  • pSG-N-Tes-de-FLAG was obtained by an alkali-SDS method and a CsClM core purification method.
  • pSG- N- Tes - del - proteins expressed in FLAG is a 12 Amino acid residues derived from Met 1 of N-Tes to Gly 1 2 2 from to 122 amino acid residues and Bgl ll recognition sequence and a FLAG sequence 134 amino Encodes a protein consisting of an acid residue (SEQ ID NO: 5 in the sequence listing).
  • N-Tes-del-FLAG 21 amino acid residues Ala 2 1 from Met 1 is the leader sequence, are cut and removed as they are secreted from the cells, the recombinant protein of 113 amino acid residues is produced.
  • the protein produced by this expression plasmid is referred to as N-Tes-del-FLAG.
  • N - TES-DEL-FLAG is, N - Tes at the C-terminus of the (101 amino acid residues, from Ala 2 2 Gly 1 2 2) having a FLAG peptide tag.
  • N-Tes-de FLAG suppresses P-2 activation via ⁇ -MMP and MT3-MP
  • Lane 1 Marauder P- 2: 25 ng; pSG5: 475 ng.
  • Lane 2 Customer P- 2: 25 ng; MT1-MMP: 50 ng; pSG5: 425 ng ⁇
  • Lane 3 Marauder P- 2: 25 ng; T1-M P: 50 ng; pSG-N-Tes-del-FLAG: 425 ng, lane 4: MP-2: 25 ng; pSG5: 475 ng ⁇ 5: MMP- 2: 25 ng; MT3-MMP: 50 ng; pSG5: 425 ng;
  • Lane 6 MMP- 2: 25 ng; MT3-MMP: 50 ng; pSG-N-Tes-del-FLAG; 425 ng.
  • CDNA was synthesized from total RNA extracted from the tumor site (lane T) and the site not containing ffi3 ⁇ 4 (lane N) of Gliomas patients using reverse transcriptase Superscript (GIBC0-BRL) and random primers (Takara Shuzo). This type is referred to as ⁇ type Mar
  • SEQ ID NO: 6 corresponds to positions 1008 to 1027 of SEQ ID NO: 1 in the Sequence Listing
  • SEQ ID NO: 7 corresponds to a sequence complementary to positions 1308 to 1289 of SEQ ID NO: 1 in the Sequence Listing Is what you do.
  • PCR reaction for example, using a GeneAmp 2400 PCR system (Perkin Elmer / Cetus), denaturation (94 ° C, 15 seconds), annealing (60. C, seconds) and extension (72. C, 15 seconds) This was performed in 40 cycles. PCR products were analyzed on a 2.5% aka "rose gel.
  • the K used for immunization it is possible to use a synthetic peptide designed based on the amino acid sequence of SEQ ID NO: 2 in the sequence listing, including the fusion recombinant N-Tes obtained in Difficult Example 2 and the like.
  • the antigen used for immunization is recombinant N-Tes obtained by linking the cDNA obtained in Example 1 to an animal cell expression vector and expressing it in CH0 cells, COS cells, etc. It is also possible.
  • These antigenic proteins can be purified by ion exchange, gel filtration or various other types of chromatography. Immunized with purified M antigen by a general method, antibody-producing cells are induced, and antibody-producing cells can be obtained as hybridomas by cell fusion.
  • the strain can be cloned and cloned as a monoclonal antibody-producing hybridoma based on its ability to purify the immunity.
  • a haptenylated synthetic peptide having an amino acid sequence specific to N-Tes can be used as an immunogen for obtaining an N-Tes-specific monoclonal antibody.
  • the C-terminus contains Gly-Lys-Arg [rooster j number: 10].
  • Cys-Phe-Gin-Arg-Gin-Gin-Gly-Lys-Arg S sequence number : 11] is a suitable source of immunity.
  • a characteristic sequence is selected from the amino acid sequence of human N-Tes described in SEQ ID NO: 2 and synthesized.
  • Peptides are synthesized by the Fmoc-bop method using a peptide synthesizer (Peptide Synthesizer-1 9600, MiniGen / Biosearch).
  • a cysteine is introduced at the N-terminus of the polypeptide.
  • the synthesized peptide is purified by high performance liquid chromatography using Bondasphere, C18 column (Waters).
  • the antigen was conjugated with cysteine albumin (BSA) via a cysteine residue.
  • BSA cysteine albumin
  • EMCS N- (£ -maleimidecaproyloxy) -succinimide
  • the mixture was allowed to react at 30 ° C for 30 minutes, and then the above mixture was purified with a 0.1 M phosphate buffer, H7.0 purified Sephadex G-25 (Pharmacia) gel column (diameter 13 mm). Gel filtration is performed on the thigh (length: 120 thighs).
  • the polypeptide synthesized in the above (b) is dissolved in 0.1 M phosphate buffer, H7.0, and approximately 50-fold mixed with maleimide-bound BSA. That is, the polypeptide is mixed with maleimido-bound BSA and incubated at 4 ° C for 20 hours to prepare a BSA-polypeptide complex.
  • the resulting BSA-polypeptide complex is diluted with 0.1 M phosphate buffer, pH 7.0, dispensed in 150 il portions, and stored frozen at -30 ° C.
  • the BSA-polypeptide complex prepared in the above (c) was intraperitoneally administered together with complete Freund's adjuvant to 6-week-old Balb / c female mice for the first immunization. Approximately on the 18th day, the BSA-polypeptide complex dissolved in 0.1 M phosphate buffer, pH 7.5 is injected into the mouse that had been initially immunized, and boosted. Furthermore, about 52 words, a BSA-polypeptide complex dissolved in 0.1M phosphate buffer and PH7.5 is intravenously administered for final immunization. Four days later, the spleen is removed and a spleen cell suspension is prepared.
  • RPMI-1640 medium RPMI-1640 (Flow Lab.) With sodium bicarbonate (21 ⁇ 2M), sodium pyruvate (lmM), potassium benicillin G (50 U / ml), and amikacin sulfate (100 zg / ml). Then, sterile filtration is performed with a 0.2 zm Toyo Membrane Filter.
  • NS-1 medium Add fetal serum (FCS, MA Bioproducts) filtered and sterilized to the RPM 1640 medium described above to a concentration of 15% (v / v).
  • PEG-4000 solution Prepare a serum-free medium by adding polyethylene glycol-4000 (PEG-4000, Merk & Co.) To RPMI-1640 ⁇ ground to 50% (w / w).
  • nucleated splenocytes (viable cell ratio 100%) and myeloma cells (viable cell ratio 100) prepared in the above (d) are fused at a ratio of approximately 5: 1 to 10: 1 by the following procedure. Wash the polypeptide-free cell suspension and myeloma cells with RPMI1640 medium. Next, the cells are suspended in the same medium and mixed with nucleated splenocytes and myeloma cells for fusion. That is, approximately 8.0 ⁇ 10 8 nucleated splenocytes are mixed with approximately 8.0 ⁇ 10 7 myeloma cells.
  • RPMI-1640 medium containing 50 PEG-4000 heated to 37 ° C determines the volume so that the number of myeoma cells is approximately 3 ⁇ 10 7 cells / mL
  • Resuspend and disperse cells add twice the volume of RPMI-1640i medium heated to 37 ° C twice the volume of the added RPMI-1640 medium containing 50% PEG-4000.
  • 7 times the volume of RPMI-1640 ⁇ ground containing 50% PEG-4000 containing PEG-4000 supplemented with calorie is dripped without vigorous stirring to disperse the cells.
  • HAT medium The hypoxanthine (100 M), aminopterin (0.4 M) and thymidine (16 M) are further added to the NS-1 medium described in the above (e).
  • HRP horseradish persian oxidase
  • Cappel horseradish persian oxidase
  • TMB 3,3,5,5'-tetramethylbenzidine
  • NS - li ⁇ prepared cloning medium approximately containing 10 7 mouse thymocytes as 1 ml per Fi one da one, 5 per Ueru the hybridoma in 96 well microwell, one, 0.5 I will be individual And add to 36, 36, and 24 wells, respectively.
  • days 5 and 12 add about 0.1 ml of NS-1 medium to all wells.
  • the ELISA described in (f) is performed on the group in which macroscopically sufficient growth of the hybridoma was observed, and the group in which the colony formation negative well was 50% or more. When all the tested wells are not cut off, select 4 to 6 wells with 1 colony in the antibody well and re-cloning. Finally, hybridomas producing monoclonal antibodies against each polypeptide are obtained.
  • the supernatant of the hybridoma obtained in the above section (g) is added to a polystyrene 96-well plate coated with each polypeptide according to the above-mentioned ELISA.
  • an isotype-specific mouse heron anti-mouse IgG antibody Zymed Lab.
  • the horseradish peroxidase-labeled goat anti-Peacock IgG H + L
  • the horseradish peroxidase-labeled goat anti-Peacock IgG H + L
  • mice with pristane before advance one week High Priestess dormer 10 7 obtained was administered thigh-vivo (Balb / c system, female, 6 weeks old) likewise administered in S Sosora, after 1-2 weeks
  • ascites can be obtained from ascites containing 4-7mg / ml monoclonal antibody.
  • a sandwich EIA system capable of specifically detecting and measuring human N-Tes by a combination of two suitable antibodies from the control-Tes antibody prepared in Example 5 can be constructed.
  • the EIA system can use either the one-step method or the two-step method, and the labeled antibodies are not limited to Fab and -HRP.
  • the purpose of the measurement is the reaction buffer and the reaction conditions. Can be adjusted to shorten or extend according to
  • human N-Tes which is a standard product, can be purified from a silkworm culture supernatant, a yeast culture supernatant, or a recombinant expressed by the method described in Example 2 or other methods. Purification is achieved by a combination of ion exchange, gel filtration, affinity chromatography using an anti-human N-Tes monoclonal antibody, or a variety of other affinity chromatography.
  • Anti-human N-Tes monoclonal antibody is digested for 24 hours at 37 ° C by adding 2% (W / W) of pepsin to 0.1 female acid buffer containing 0.1 M NaCl and H4.2. 3M Tris-HCl, pH 7.5 is added to the dish to stop the reaction.
  • Separate the F (ab ') 2 fraction by gel filtration using an Ultrogel AcA54 column that has been renewed with 0.1 M phosphate buffer and H7.0. To this F (ab ') 2 fraction was added cysteamine hydrochloride to a final concentration of 0.01M, reduced at 37 ° C for 1.5 hours, and 0.1M phosphate buffer containing 5m EDTA, H6.
  • Separate the Fab 'fraction by gel filtration using a wool-mouth gel AcA54 force ram that has been converted to a tan.
  • the Fab 'fraction and the maleimide-labeled HRP were mixed at equimolar proportions and reacted at 4 ° C for 20 hours.Then, unreacted with 10-fold mono-I * N-ethyl maleimide of Fab' Blocks thiol groups. This is subjected to gel filtration with a ureto-mouth gel AcA54 column prepared with 0.1 M phosphate buffer and H6.5 to collect Fab'-HRP-labeled antibodies. Add 0.1% BSA and 0.001% chlorhexidine to this and store at 4 ° C.
  • Antibody binding microplates 0. 05% Tween20, prepared, 0. 1M NaCl, 5 m CACH containing Tris-HC1 buffer, washed 3 times with P H8. 0, the standard antigen and the labeled antibody mixture by 100 zL / Ueru Added. After reacting at room temperature for 1 hour, wash three times with Tris-HC1 buffer containing 0.05% Tween20, 0.1 M NaCl, 5 mM CaC and H8.0. Next, 0.01% 3,3 ', 5,5'-tetramethylbenzidine dissolved in 0.1 M acetate buffer (PH5.5) containing 6% dimethylformamide and 0.005% hydrogen peroxide was added. Add 100 ⁇ L per well, and after reacting at room temperature for 20 minutes, add 100 L of 2N sulfuric acid to stop the reaction. Measure the 450 nm of this reaction mixture using a microplate reader to obtain a standard curve.
  • Samples to be measured include human serum, spinal fluid, plasma, synovial fluid, urine, saliva, and other human-derived components, extracts of various human fibres, and cell extracts of various cultured cells such as human and recombinant. It is prepared from the culture supernatant.
  • Each test sample is subjected to the above-mentioned one-step sandwich EIA instead of the standard human N-Tes, and the reaction proceeds simultaneously with the human N-Tes. Calculate the amount of human N-Tes contained in the measurement sample by fitting to the standard curve obtained from the measurement sample.
  • Example 7 Cloning of N-Tes
  • E.coli of 30 clones of a human fetal kidney-derived cDNA expression library constructed with PEKA8 was used as a pool, and a pool of E. coli I medium (1 M Tris-HCl, 1.25% tryptone, 2.5% yeast extract, 125 ni
  • the cells were cultured at 37 ° C for 16 hours in NaCl, 0.4% glycerol; pH 7.2), and the plasmid was purified by ⁇ cultivated Escherichia coli 2 m aliquot (an alkaline method using polyethylene glycol and a rapid method).
  • Approximately 50,000 293T cells per well in a 96-well microplate were seeded with a thigh containing 0.1% semen (0.1 mL / well) and cultured for 24 hours.
  • a full-length cDNA of MMP-9, solid P-2, and MT1-MMP was inserted into the pSG5 (Stratagene) cloning site to create an expression vector, and 3 ng of pro-solid P-9 expression plasmid was added per well.
  • 20 ng of the proband P-2 expression plasmid and 30 ng of the MT1-MMP expression plasmid were transfected together with 100 ng of the above library DNA using Trans IT LT1 into the fetus I ⁇ (Mirus).
  • E. coli XL-IBlue (Stratagene) and cultured for 24 hours on an LB plate containing ampicillin.o 50 E. coli colonies were separated one by one and incubated at 37 ° C in ⁇ I medium. After culturing for 16 hours, plasmid was purified from 2 ml of cultured E. coli.
  • pro-MMP-9, pro-P-2, and MT-P expression plasmids and purified plasmids were introduced into 293T cells, and the latent form, intermediate, and intermediate were analyzed by gelatin zymography. Activation P-9 and MMP-2 were detected.
  • a single plasmid that suppressed the production of activated ⁇ 3 ⁇ 4 ⁇ -2 was identified. Square beveling with gelatin zymography for any of the 12 samples showed that no 64 kDa active ftS intermediate ⁇ -2 was found in Samples 3, 5, and 6 at all.
  • the plasmid used in these lanes has a 1.4 kb insertion sequence, and the base sequence of DNA obtained using the LI-COR DNA Sequencer Model 4200L (S) -2 was (Acc ession No. AB056866) o In this sequence, the first 1038 residues on the 5th side are the same as Test ican 3 except that they lack the 311-319 force of Testican 33 ⁇ 4S sequence (Accession No. 016950). It was.
  • N-Tes was found on the chromosome 4 genome database (BAC clone RP11-44 0L30, Accession No. AC020599) because the 3 'portion of N-Tes and the cDNA i ⁇ S sequence of Testican 3 were strong. Testican 3
  • the 21 amino acid residues at the N-terminal end of N-Tes are hydrophobic signal peptide, and the 22-85 amino acid residues are unique to Testican 1, Testican 2 and Testican 3 It was a rooster.
  • the amino acid residues 86 to 191 were in the cysteine-rich follistatin-like (FS) domain, and the amino acid residues 87 to 311 were in the extracellular Ca 2 + Itoyoshigo (EC) domain.
  • Testican 3 has a 1; 1 ⁇ 1 "0 £ 101) 111 (TY) -like domain and two sugar chain modification sites on the same-terminal side.
  • Example 8 Recombinant Testican 1, Testican 2, Testican Cloning 3 and BM-40
  • Plasmids expressing Testican 1, Testican 2, Testican 3 and BM40 with FLAG epitope added to the C-terminal side were prepared as follows. A DNA fragment encoding the multicloning site and the FLAG epitope (Asp-Tyr-Lys-Asp-Asp-Asp-Lys [SEQ ID NO: 12]) was primed from the pFLAG-CTC plasmid (IBI FLAG Biosystems, NY). Prepared by PCR with N26 and C24 (IBI FLAG Biosystems, NY).
  • cDNA was synthesized from total RNA derived from human or mouse placenta using 8 mer random primers and ReverTra Ace reverse transcriptase (T0Y0B0, Japan).
  • Primer for Human Testican 1, Human Testican 2, Human Testican 3 and mouse BM-40 GeneBank TM accession numbers: CAA51999, CAA04774, 016950 and 009242.
  • Testi can 1-5 'GCAGATCTAGCTCGAGCAACTCGGACTAG [SEQ ID NO: 13] Testi can 1-3, GGAGATCTCCATATGTACCCGACCTCATC 3 ⁇ 4 Own column number 14] Testi can 2-5' CAGGTCGAAnCAGACCACGATGCG [SEQ ID NO: 15] Tes ti can 2-3 'GGAGATCTCGATCCGCCCC No.
  • Tes ti can 3-5 'AAAGCAGCGAGnGGCAGAG 3 ⁇ 4 Own column number 17) Tes ti can 3-3' CATGMTTCMTGTATACATCATGGTC 3 ⁇ 4 Own column number 18] BM-40-5 ': GAGGGATCCCAGCATCATGAGGGC [SEQ ID NO: 19] BM-40-3' : GCAGGATCCGTGAACmGATCAC [SEQ ID NO: 20]
  • Each cDNA fragment amplified by PCR was cut at the BamHI or BglII site in the primer and inserted into the Bgl11 site of pSG-FLAG.
  • the Testican3 cDNA was inserted into the SmaI and BglII sites of pSG-FLAG.
  • the base sequence of each cDNA fragment was confirmed by the LI-COR DM sequencer.
  • Each expression vector was called pSG-Testcan 1 -FLAG, pSG-Testican2-FLAG, pSG-Testican3-FLAG and pSG-BM-40-FLAG.
  • PSG-N-Tes-FLAG and pSG-Testican 3-FLAG were introduced into 293T cells, and the distribution of N-Tes and Testic an 3 was examined.
  • FLAG epitopes added to N-Tes and Testican 3 were found in the lysates and culture supernatants of the transfected cells. The molecular weights were 37 kDa and 60 kDa, respectively.
  • Testican 3-FLAG was determined to be a soluble protein because N-Tes-FLAG was not detected in ECM of transfected cells and N-Tes-FLAG was not detected. .
  • Example 9 Binding of N-Tes and ⁇ -MP by immunoprecipitation
  • 293T cells were seeded into a 60 mm diameter culture dish, 1 pSG-N-Tes-FLAG and pSG-SG-band P expression plasmid were introduced, and 48 hours later 100 zCi / ml Pro-mix L -( 35 S) Labeled with in vitro cell labeling mix (Amersham Pharmacia Biotech) for 4 hours.
  • Cells were prepared using RIPA buffer (150 mM NaCl, 5 mM EDTA, 1% Trito This was dissolved in n-X100, 10 mM Tris-HCl buffer containing 0.1% SDS, pH 7.4), and centrifuged at 15,000 rpm for 10 minutes to obtain a supernatant.
  • irCMTl-MMP antibody 114-1F2 (Daiichi Fine Chemicals) or anti-FLAG antibody M2 (Sima) was added to the supernatant, and the mixture was incubated at 4 ° C for 16 hours.
  • the antigen-antibody complex was recovered with 20 L of Protein-G Sepharose 4B (Amersh am Pharmacia Biotech) and subjected to 12% SDS-PAGE. The gel was dried and the radioactivity was detected with a Bio-image Analyzer BAS1000 (Fuji Film).
  • the ⁇ - ⁇ monoclonal antibody precipitated 50 kDa and 47 kDa C MTl- ⁇ from cells into which only the ⁇ -MMP gene had been introduced.
  • N-Tes-FLAG was not precipitated by this antibody from cells into which only the FLAG transfection was introduced.
  • the anti-FLAG antibody precipitated 35 kDa N-Tes-FLAG from the cells into which the N-Tes-FLAG gene was introduced.
  • the anti-FLAG antibody did not precipitate MT1-secreted cells from the cells carrying the MT1-MP gene.
  • MT1-1P was precipitated from cells co-transfected with N-Tes-FLAG and MT1-wake P gene. did.
  • MT3- ⁇ P also co-precipitated with N-Tes-FLAG.
  • N-Tes deficient! CDNA fragments of variants 85, 97 and 122 were amplified by PCR with the 5 'primer of ⁇ 8 and the primers described below.
  • the 122C / S mutant cDNA was prepared by replacing four cystine residues with serine residues by continuous PCR using the following primers.
  • the underline is the base into which the mutation was introduced.
  • C / Sl-2 AAGGATCCMGCnAAAGATGAAMGTAG [SEQ ID NO: 23]
  • C / S3 CGCCATAAAGTMGCAnGCTCAAG [SEQ ID NO: 24]
  • C / S4 CAGACTGCAGTCAGCAHAGTCACC [SEQ ID NO: 25]
  • Testican 2 chimeric cDNA fragment was prepared. Insert the C-terminal cDM fragment of Test i can 2 starting from No. 550 with Testican 2-3 'primer and
  • AAGGATCCCTGCCAGAAGATGAAGTGCAGC (amplification number: 26) was used for amplification.
  • the C-terminal side fragment of N-Tes cDNA (# 359 # -1046) was digested with BamHI and BglII, and the amplified fragment was inserted.
  • Testican2 / N-Tes chimeric cDNA fragment was prepared.
  • the cDNA fragment of the N-terminal side cDNA (No. 279-550) of Testican 2 was amplified using Testican 2-5 'primer and AGG COTGGTGGTATCCAGGGCTTCAT No. 27 starting from No. 550]. ⁇ Replace the widened fragment with the N-terminal side of N-Tes cDNA (No. 1-550) /
  • Testican 1 and Testican 3 strongly inhibited the activation of pro-reading II-2 by ⁇ - ⁇ and ⁇ ⁇ 3-MMP, whereas the expression of Testican 2 and BM-40 showed no inhibitory activity. Deletion dissociations were made to determine the N1-Tes MT1-banded P and ti3 ⁇ 4T3-MMMP P and toxic domains. The 122 amino acid residue ( ⁇ 122) on the N-terminal side of N-Tes had the same ability to inhibit the activation of pro-P-2 as wild-type N-Tes.
  • Deletion impairments consisting of the N-side 97 and 85 residues ( ⁇ 97, ⁇ 85) inhibited ffll--and MT3-MMP at lower levels than wild-type.
  • those lacking residues 33-84 of N-Tes lost P and no harmful activity.
  • N-Tes / Tes-2 consisting of the FS, EC, TY, and C-terminal domains of Testican 2 with the signal and unique domain of N-Tes showed sufficient inhibitory activity.
  • N-Tes (Tes-2 / N_Tes) replaced with the signal of an 2 and the unique domain lost the inhibitory activity.
  • Human glioma fiber was prepared from 10 patients together with a normal brain thread associated with removal of the brain.
  • the specimen was excised, rapidly frozen in liquid nitrogen, and kept at -80 ° C. In addition, they were fixed with 4% paraformaldehyde fixative for 18 to 24 hours (4 ° C) for yarn and weave inspection. Sections were stained with hematoxylin and eosin and examined microscopically.
  • Brain S Fuji's ⁇ is described in Kleihues, P., Burger, PC, Schei thauer, BW., "Histol ogical Typing of Tumours of the Central Nervous System", Berl in, Springer-Verlag, 1993, p. 11-20. The determination was performed according to the described criteria. No chemotherapy or radiation therapy was given to any tissue prior to resection.
  • RT-N-Tes-5 'GAGTGGTGCTACTGCTTCCA self sequence number 6] RT-N-Tes-3, GTGCHCnGGTGGCTCATA [SEQ ID NO: 7] RT-Tes3-5' TGCTAnGGACCAGTCAGAGCTC [SEQ ID NO: 28] RT-Tes3-3 'TCCAACACTGCCATGACACTGTG (Sequence (No. 29) (3) Result
  • N-Tes and Testican 3 mRNA levels in normal brain and glioma tissue were examined by semi-RT-PCR. In normal brain, either -Tes or Yestican 3 In addition, the force was detected.
  • N-Tes mRNA in the three strains T98 and U87 was significantly lower in U251 cells, which were comparable to normal brain.
  • no glioma cell lines expressing detectable levels of Test Can 3 were present.
  • the expression level of Testican3 and N-Tes of the strain, n 4) was determined by semi-real-time RT-PCR.) As bad tt ⁇ increased, the expression level of N-Tes decreased. Admitted.
  • Return fe 12 Inhibition of cancer invasion by N-Tes or Testican 3
  • pSG-Testican3-FLAG, pSG-N-Tes-FLAG and pSG-N-Tes-del-FLAG were each cut with Xbal, and the early SV40 promoter of pSG5 and the rabbit / 3-globin intron II
  • the fragment containing the T7 bacteriophage promoter, the respective cDNA and the polyadenylation signal was cut out and inserted into pCEP4 (Invitrogen) which had been cut with Xbal in advance.
  • the resulting expression vectors were designated as pCEP-Testican3-FLAG, pCEP-N-Tes-FLAG and pCEP-N-Tes-del-FLAG.
  • pCBP-Testican3-FLAG and pCEP-N-Tes-FLAG were introduced into MDCK cells or U251 glioma cells carcinomatized with erbB2 and selected under 400 ⁇ g / mL hygromycin B.
  • a type I collagen gel (Nitta Gelatin) was prepared.
  • a 6-L collagen gel containing 3,000 U251 cells was placed in a 35-diameter culture dish in which 1 mL of collagen was gelled, and sandwiched with 1 mL of collagen gel. The gel was added with 2 niL of medium and cultured for 5 days.
  • collagen containing approximately 3,000 cells was gelled in a culture dish with a diameter of 35, and 2 mL of medium was added to the culture dish, followed by i-culture for 7 days.
  • N-Tes-FLAG or Testican 3-FLAG expression plasmid was introduced into erbB2-carcinated MDCK cells (MDCK-erbB2) that express MT1-MP and infiltrate and grow in collagen gel, and compare the properties after introduction did. No morphological changes were induced by the N-Tes-FLAG or TesUcan 3-FLAG gene guide. Parental cell line or empty vector-derived AMDCK-erbB2 cell line showed invasive growth in collagen gel.Notably, introduction of N-Tes FLAG or Testcan 3-FLAG residue Later, the invasive viability decreased.
  • Example 13 Purification of recombinant Test i can 3, N-Tes and M-Tes-del
  • Recombinant Test i can 3, N-Tes and Tes-del expression system
  • pCEP-Testican 3-FLAG, pCEP N-Tes-FLAG and pCEP-N-Tes-del-FLAG were introduced into 293E BNA cells, and selected in the presence of 00 ag / mL of igromycin B, and Tes ti can Cell lines producing 3-FLAG, N-Tes FLAG and "N-Tes-de FLAG were obtained.
  • Each of the expression strains was inoculated into a bottle with a bottle, and cultured in DMEM containing 10% FCS until confluence.
  • the nutrient solution was replaced with FMEM-free DMEM, and after surrounding culture for 2 days, ⁇ ⁇ 3 ⁇ 4 ⁇ was recovered.
  • 0.5 mL of the culture supernatant was concentrated, and expression was confirmed by Western blotting using an anti-FLAG antibody.
  • N-Tes From the amino acid sequence of N-Tes, the following sequence was selected as a sequence specific to N-Tes.
  • CFQRQQGKR [SEQ ID NO: 11) This polypeptide was synthesized by the Fmoc-bop method. The synthesized peptide was purified to more than 80-90% purity by reverse phase HPLC.
  • Antigen conjugates were prepared by combining each polypeptide with cysteine serum albumin (BSA) via cysteine residues added to the peptide FN terminus.
  • BSA cysteine serum albumin
  • the BSA-polypeptide prepared in the previous section (2) was subcutaneously administered (0.15 mg / bird) to two Japanese female white rabbits together with complete Freund's adjuvant, and immunized for the first time. After 2 weeks, 4 weeks, 6 weeks, 8 weeks, and 10 weeks, these rabbits were boosted with 0.3 mg / bird BSA-polypeptide complex to the endothelium together with Freund's adjuvant, and whole blood was collected after 11 weeks. Was performed. The obtained blood was centrifuged to obtain “Egret!” & 1 containing an anti-BSA-polypeptide complex polyclonal antibody.
  • ELISA was performed on a 96-well plate on which the immunized BSA-polypeptide complex had been immobilized, and the titer of ML purified obtained from whole blood collection was measured to confirm the antibody.
  • An expression vector that expresses a fusion protein with a His tag added to the C-terminal side of N-Tes Inoculated with pCTCN-TesHis-introduced Escherichia coli JM109 into 4 mL of LB medium containing 50 g / mL Ampicillin, 37 ° C For 16 hours to prepare a primary culture. This was re-inoculated into 400 mL of LB medium containing 50 g / mL Ampicillin, and cultured at 37 ° C for about 3 hours until mid-log phase. IPTG (final concentration: 0.1 lmM) was added, and the culture was continued for another 4 hours to induce the recombinant fusion protein.
  • the insoluble ' ⁇ living inclusion bodies prepared from the above-mentioned thread-recombinant large-scale bacteria were treated with 50 mM phosphate buffer ( ⁇ 7.5) containing 8 M urea, 0.5 M NaCl, and 10 mM imidazole.
  • the resulting solution was subjected to a soluble gelling and subjected to a Ni ++ Chelating Sepharose gel column. After washing, eluted with 50 mM phosphate buffer (pH 7.5) containing 8 urea, 0.5 M NaCl, 0.5 imidazole, and 1% SDS, 0.53 ⁇ 4 2-mercaptoethanol was added to the purified fraction.
  • 50 mM phosphate buffer pH 7.5
  • SDS 0.53 ⁇ 4 2-mercaptoethanol
  • Booster boost 20 days later 41.1 g / 185 ⁇ 1 / mouse 0 air administration
  • ELISA Immobilized antigen coated with 100 ng / well of solid phase.
  • the cell extract fraction prepared from 293EBNA cells transfected with pCEP-Testican3-FLAG ⁇ pCEP-N-Tes-FLAG and pCEP-N-Tes-del-FLAG described in Example 13 was subjected to 12% SDS.
  • the culture was added as the primary antibody, and anti-mouse Ig (G + A + M) -HRP was used as the secondary antibody and stained. All clones had improved potency with recombinant Test Can 3 -FLAG and -Tes-FLAG. None of the three clones examined reacted with N-Tes-de FLAG.
  • N-Tes which has an inhibitory effect on Ps fractions such as MT1-MP and T3-banded P, is expected to be effective in suppressing cancer invasion / metastasis, angiogenesis, etc., and amyloid precursor degradation It is also expected to be effective in suppressing the progression of Alzheimer's disease by suppression.
  • Antibodies against N-Tes are also expected to be used as diagnostics.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Medicinal Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Animal Behavior & Ethology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Physical Education & Sports Medicine (AREA)
  • Rheumatology (AREA)
  • Biomedical Technology (AREA)
  • Orthopedic Medicine & Surgery (AREA)
  • Neurology (AREA)
  • Genetics & Genomics (AREA)
  • Neurosurgery (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Psychiatry (AREA)
  • Microbiology (AREA)
  • Biotechnology (AREA)
  • Molecular Biology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • Pain & Pain Management (AREA)
  • Hospice & Palliative Care (AREA)
  • Immunology (AREA)
  • Peptides Or Proteins (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

L'invention porte sur l'élucidation des gènes participant à la régulation des MMP, tels que les MT1-MMP et les MT3-MMP et sur la recherche et le développement de différentes activités biologiques et de phénomènes biologiques auxquels les MMP sont associés. En raison de sa séquence caractéristique (par exemple l'absence de site de fixation du glycosaminoglycan) et de sa capacité d'inhibition des activités des MT1-MMP et MT3-MMP, le N-Tes peut servir à la recherche et au développement de médicaments utiles.
PCT/JP2001/011529 2000-12-27 2001-12-27 Nouveaux peptides et leurs activites WO2002057448A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
JP2002558500A JPWO2002057448A1 (ja) 2000-12-27 2001-12-27 新規ペプチド及びその活性

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP2000-398817 2000-12-27
JP2000398817 2000-12-27

Publications (1)

Publication Number Publication Date
WO2002057448A1 true WO2002057448A1 (fr) 2002-07-25

Family

ID=18863704

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP2001/011529 WO2002057448A1 (fr) 2000-12-27 2001-12-27 Nouveaux peptides et leurs activites

Country Status (2)

Country Link
JP (1) JPWO2002057448A1 (fr)
WO (1) WO2002057448A1 (fr)

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998046757A2 (fr) * 1997-04-15 1998-10-22 Genetics Institute, Inc. Proteines secretees et polynucleotides codant ces proteines
WO1999023110A1 (fr) * 1997-10-31 1999-05-14 Eli Lilly And Company Gene hptlg issu de l'hypothalamus humain
WO1999046281A2 (fr) * 1998-03-10 1999-09-16 Genentech, Inc. Nouveaux polypeptides et acides nucleiques les codant
WO2000032221A2 (fr) * 1998-12-01 2000-06-08 Genentech, Inc. Promotion et inhibition de l'angiogenese et de la vascularisation cardiaque
WO2000053756A2 (fr) * 1999-03-08 2000-09-14 Genentech, Inc. Polypeptides secretes et transmembranaires et acides nucleiques codant ces polypeptides

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998046757A2 (fr) * 1997-04-15 1998-10-22 Genetics Institute, Inc. Proteines secretees et polynucleotides codant ces proteines
WO1999023110A1 (fr) * 1997-10-31 1999-05-14 Eli Lilly And Company Gene hptlg issu de l'hypothalamus humain
WO1999046281A2 (fr) * 1998-03-10 1999-09-16 Genentech, Inc. Nouveaux polypeptides et acides nucleiques les codant
WO2000032221A2 (fr) * 1998-12-01 2000-06-08 Genentech, Inc. Promotion et inhibition de l'angiogenese et de la vascularisation cardiaque
WO2000053756A2 (fr) * 1999-03-08 2000-09-14 Genentech, Inc. Polypeptides secretes et transmembranaires et acides nucleiques codant ces polypeptides

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
NAKADA M. ET AL.: "Suppression of membrane-type 1 matrix metalloproteinase (MMP)-mediated MMP-2 activation and tumor invasion by testican 3 and its splicing variant gene product, N-tes", CANCER RESEARCH, vol. 61, no. 24, 15 December 2001 (2001-12-15), pages 8896 - 8902, XP002909003 *
YAMANAKA H. ET AL.: "Expression and tissue localization of membrane-types 1,2 and 3 matrix metalloproteinases in rheumatoid synovium", LABORATORY INVESTIGATION, vol. 80, no. 5, May 2000 (2000-05-01), pages 677 - 687, XP002909002 *

Also Published As

Publication number Publication date
JPWO2002057448A1 (ja) 2004-05-20

Similar Documents

Publication Publication Date Title
JP3837494B2 (ja) 可溶型ｒａｇｅタンパク質
US20050042222A1 (en) Methods of diagnosing thyroid carcinoma
JP2008131944A (ja) 白血球免疫グロブリン様受容体（ｌｉｒ）と命名される免疫調節剤ファミリー
CN102712687A (zh) 作为补体激活的抑制剂的masp同种型
JP2002543787A (ja) 白血球免疫グロブリン様受容体（ｌｉｒ）と命名された免疫調節因子のファミリー
RU2153885C2 (ru) Способ осуществления селективного метионинового голодания клеток в организме млекопитающего, полинуклеотид, каталитически активный полипептид рекомбинантной метазы
JPH10210982A (ja) 新規なタンパク質
JP4295516B2 (ja) ガストロカインおよびインヒビターを含むその誘導ペプチド
JP3800175B2 (ja) 新規なアグリカナーゼ
JP4841054B2 (ja) 新規インスリン／ｉｇｆ／リラキシンファミリーポリペプチドおよびそのｄｎａ
JP2003189874A (ja) ガレクチン−９活性制御剤
JP2000050885A (ja) カテプシンに対する抗体およびそれらの利用
JPH05501951A (ja) 組換えヒト甲状腺ペルオキシダーゼ
JP4171228B2 (ja) 可溶型ｒａｇｅ測定法
JP2001231578A (ja) Ｉｌ−１ファミリーに属する蛋白質
WO2007037245A1 (fr) Polypeptide présentant une activité anti-angiogénique
WO2002057448A1 (fr) Nouveaux peptides et leurs activites
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
US6800473B1 (en) Human cathepsin L2 protein, gene encoding said protein and use thereof
WO2004048565A9 (fr) Proteine associee a l'apoptose et son utilisation
JP2003000249A (ja) クローディンによるＭＴ−ＭＭＰｓを介したｐｒｏＭＭＰ−２活性化
JP2006180813A (ja) アミノペプチダーゼｏおよびその利用
WO2004040302A1 (fr) Regulation des interactions entre rapl et rapi
JP2004329040A (ja) ポリセラーゼ−ｉ及びその利用

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BY BZ CA CH CN CO CR CU CZ DE DK DM DZ EC EE ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NO NZ OM PH PL PT RO RU SD SE SG SI SK SL TJ TM TN TR TT TZ UA UG US UZ VN YU ZA ZM ZW

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): GH GM KE LS MW MZ SD SL SZ TZ UG ZM ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE TR BF BJ CF CG CI CM GA GN GQ GW ML MR NE SN TD TG

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
WWE Wipo information: entry into national phase

Ref document number: 2002558500

Country of ref document: JP

REG Reference to national code

Ref country code: DE

Ref legal event code: 8642

122 Ep: pct application non-entry in european phase