WO2002057448A1 - Novel peptides and activities thereof - Google Patents

Novel peptides and activities thereof 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
French (fr)
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/en
Publication of WO2002057448A1 publication Critical patent/WO2002057448A1/en

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

Genes participating in the regulation of the activities of MMPs such as MT1-MMP and MT3-MMP are clarified and thus various biological activities and physiological phenomena in which MMPs participate are researched and developed. Because of having a characteristic sequence (for example, lacking the glycosaminoglycan attachment site) and being capable of inhibiting the activities of MT1-MMP, MT3-MMP, etc., N-Tes is useful in the research and development of useful drugs.

Description

明細書  Specification
新規べプチド及びその活'性 技術分野  New peptides and their activities
本発明は、 新規に同定されたポリヌクレオチド （あるいは核酸） 及びポリぺプ チド （あるいはタンパク質） （Xはその D あるいはその塩；該ポリヌクレオ チド （あるいは核酸） 及びポリペプチド （あるいはタンパク質） の変異体及び誘 m ；該ポリヌクレオチド （あるいは核酸） 及びポリぺプチド （あるいはタンパ ク質） 、 並びにそれらの変異体及び誘 の製造法；該ポリペプチド （あるいは タンパク質） （又はその一部） に対する抗体、 該抗体を用いた免疫学的測定法及 びそれに^する 該ポリペプチド （あるいはタンパク質） のァゴニスト及 びアンタゴニスト、 それらに関連したスクリ一ニンク"法及びスクリ一ニングキッ ト；並びに該ポリヌクレオチド （あるいは核酸） 、 ポリぺプチド （あるいはタン パク質） 、 変異体、 誘観本、 ァゴニスト及びアン夕ゴニストの用途に関するもの である。 背景技術  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.
マトリックスメタロプロテアーゼ類 (MPs) は、 細胞外マトリックスと基底膜 成分のさまざまな構成タンノ、°ク質を分解する Mlf依存性のェンドぺプチダーゼの ファミリーである。 これらの酵素群は、 正常な胚の発生、 骨の成長あるいは創傷 の治癒など、 結合纖の再構成に関連している（Woessner, J. F. , FASEB J. , 5， 2145-2154 (1991) ; Matrisian, L. . , BioEssays, 14, 455-463 (1992)； Birke ( - Hansen, H. , Moore W. G. I.， Bodden, M. K. , Windsor, L. J. , Birkedal-Hans en, B. , DeCarlo, A. , and Engler, J. A. , Cri t. Rev. Oral Biol. Med. , 4, 19 7-250 (1993)) 。 また、 ァテローム性動脈硬化症 (Henney, A. . , Wakeley, P. R. ， Davies, M. J. , Foster, K. , Hembry, R. , Murphy, G.， and Humphries, S. , P roc. Natl. Acad. Sci. USA, 88， 8154-8158 (1991))、 肺翅重 (Hautamaki, R. D. , Kobayashi, D. K. , Senior, R. M. , and Shapiro, S. D. , Science, 277, 2002-2 004 (1997)) 、 リウマチや生関節炎 (Murphy, G.， and Hembry, R. M. , J. Rheumato 1,， 19， 61-64 (1992)) および癌の浸潤 '転移 （Stetler- Stevenson, . G. , Azn avoorian, S.， and Liotta, L. A., Annu. Rev. Cell Biol., 9， 541-573 (1993) ) などさまざまな病的な過程における関与が知られるようになってきた。 今日までに、 アミノ酸レベルまで角？!斤された 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)； 璧 - 11 (stromelysin-3)； MMP - 12 (macrophage elastase)； 删 P- 13 (collagenase - 3) ； MP- 14 (MT1-MMP)； MMP- 15 (MT2-MMP)； MMP- 16 (MT3-MMP)； MMP- 17 (MT4-MMP) ； MMP- 18 (collagenase- 4); MMP-19; MMP-20 (enamelysin); MMP- 24 ( T5-M P)； MMP-25 (MT6-MMP) など） 力く知られている（Woessner, J. F., FASEB J.， 5， 2145 -2154 (1991); Matrisian, L. M.， BioEssays, 14, 455-463 (1992)； Birkedal- Hansen, H.， Moore W. G. I. , Bodden, . K. , Windsor, L. J. , Birkedal-Hansen, B.， DeCarlo, A., and Bngler, J. A., Crit. Rev. Oral Biol. Med., 4, 197 - 25 0 (1993)； Gururajan, R. , Grenet, J. , Lahti, J. M. , and Kidd, V. J. , Genomi cs， 52， 101-106 (1998)； Velasco, G.， Pendas, A.M., Fueyo, A., Knauper, V .， urphy, G. , and Lopez Otin， C. , J, Biol. Chem. , 274, 4570-4576 (1999) ； Pei D.， Cell Res., 9, 291-303 (1999)) 。 1次ネ ¾t、 基質特異性および細胞 分布により、 これらの匪 Psは少なくとも 4種のサブフアミリー： コラゲナ一ゼ、 ゼラチナーゼ、 スト口メライシンおよび膜 MMPs(MT-匪 Ps) に分類されている。 Matrix metalloproteases (MPs) 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. Med., 4, 197-250 (1993)), and atherosclerosis (Henney, A., Wakeley, PR, Davies, MJ, Foster, K. , Hembry, R., Murphy, G., and Humphries, S., Proc. Natl. Acad. Sci. USA, 88, 8154-8158 (1991)), Lung wing weight (Hautamaki, RD, Kobayashi, DK) , Senior, RM, and Shapiro, SD, Science, 277, 2002-2 004 (1997)), rheumatism and arthritis (Murphy, G., and Hembry, RM, J. Rheumato 1, 19, 61-64 (1992)) and cancer invasion 'metastasis (Stetler-Stevenson, G., Azn avoorian, S., and Liotta, LA, Annu. Rev. Cell Biol., 9, 541- 573 (1993)), and its involvement in various pathological processes has become known. By today, horns up to the amino acid level? ! 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); 删 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, H., Moore WGI, Bodden, .K., Windsor, LJ, Birkedal-Hansen, B., DeCarlo, A., and Bngler, JA, Crit. Rev. Oral Biol. Med. Gururajan, R., Grenet, J., Lahti, JM, and Kidd, VJ, Genomics, 52, 101-106 (1998); Velasco, G., Pendas, AM. , Fueyo, A., Knauper, V., urphy, G., and Lopez Otin, C., J, Biol. Chem., 274, 4570-4576 (1999); Pe. i D., Cell Res., 9, 291-303 (1999)). 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).
MT-MMPs サブファミリ一は、 最も新しく MMPsのサブクラスとして報告されたも ので、 讓 Psに保存された領域に対するディジヱネレ一トプライマーと RT- PCRによ つてこれまで 5種を超えるメンバ一 MMP， MT2-MMP, MT3- MP, T4-MMP, MT5 -画 Pなど） 力く単離同定されている（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. Biochem., 231, 602-608 (1995)； Takin o， T.， Sato, H. , Shinagawa, A.， and Seiki, M.， J. Biol. Chem. , 270, 2301 3-23020 (1995)； Puente X. S. , Pendas, A.M., Llano, E., Velasco G., and Lo pez-Otin, C.， Cancer Res, 56, 944-949 (1996)； 特開 2000- 270874号； Pei, D ·， J. Biol. Chem. , 274, 8925-8932, 1999; Kaj ita, M. et al. , FEBS Lett. , 457, 353-356, 1999) 。 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. Biochem., 231, 602-608 (1995); Takin o, T., Sato, H., Shinagawa, A., and Seiki, M., J. Biol. Chem., 270, 2301 3-23020 (1995); Puente XS, Pendas, AM, Llano, E. , Velasco G., and Lo pez-Otin, C., Cancer Res, 56, 944-949 (1996); JP-A-2000-270874; Pei, D., J. Biol. Chem., 274, 8925-8932, 1999; Kajita, M et al., FEBS Lett., 457, 353-356, 1999).
MT-MMPs は、 多くの匪 Psに特徴的なホモぺキシンドメインの後方に、 単一の膜 貫通領域と短い細胞内テールを持つ I型の膜タンパク質である。 さらに、 これら はプロべプチドと活性ドメインの間に^ S性ァミノ酸の挿入カ洪通して し、 フューリン (furin) あるいはフユ一リン様酵素による切断で、 これらの膜タンパ ク質の活性化がおきる（Pei， D,， and Weiss, S. J. , J. Biol. Chem. , 271, 913 5-9140 (1996)； Sato, H. , Kinoshi ta, T.， Takino, T. , Nakayama, K. , and Se iki, M.， FEBS Lett. , 393， 101-104 (1996)； Cao, J. , Rehemtulla, A:， Bahou ， W. , and Zucker, S.， J. Biol. Chem. , 271, 30174-30180 (1996))。  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. Chem., 271, 913 5-9140 (1996); Sato, H., Kinoshita, T., Takino, T., Nakayama, K., and Seiki, M., FEBS Lett., 393, 101-104 (1996); Cao, J., Rehemtulla, A :, Bahou, W., and Zucker, S., J. Biol. Chem., 271, 30174-30180 (1996)).
Testicanは、 精巣で最初に発見されたプロテオグリカンで、 ヒト精巣プラズマ よりコンドロイチン硫酸/へノ N0ラン硫酸プ口テオグリカンとして単離された （B i ochem. J. , 288， 565-569 (1992)) 。 そのアミノ酸酉 Jは、 ヒト精巣 cDNAより推 論され (Bur. J. Bi ochem, 214: 347-350 (1993))、 Β -40/secreted protein, acidic and rich in cysteine (SPARO/osteonectin protein fami ly (Bio chem.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)), Β-40 / secreted protein, acidic and rich in cysteine (SPARO / osteonectin protein family). (Bio chem.
J.， 288, 565-569 (1992), FEBS Lett. , 414, 557-561 (1997))と類似のドメイ ン構造を持っていること力く示されている。 Testicanは、 マルチドメイン構造を持 つている。 Testicanの局在は、 脳の限定された領域であり、 シナプス後部に存在 する。 このことは、 Testicanが、 レセプタ一活性、 神経調節、 シナプスの形成性 、 あるいは神糸圣伝達などに関与することが考えられる。 J., 288, 565-569 (1992), FEBS Lett., 414, 557-561 (1997)). 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及びその関連物質としては、 Testican- 1 (Bur. J. Bi ochem, 214： 34 7-350 (1993)) 、 Testican - 2 (Journal of Neurochemistry, Vol. 73: 12 - 20 (1 999)) 、 Testican - 3などの Testican類のほ力、、 ΒΜ-40/SPARC/osteonectin, QR1, SCl/hevin, tsc36/Fl ikなどが知られ、 それらのタンパク質のうちには、 特徵的 なドメイン構造、 ィ列えば extracellular Ca"" -binding domain (EC domain), f oll istatin-l ike domain (FS domain)の 力認められ、 あるものには、 thyrog lobul in-l ike domain (ΊΎ domain) や Glycosaminoglycan attachment si te 力く ¾している。特に、 Testican-3は、 36個のアミノ酸をコードしている遺伝子 に由来し、 マルチドメイン構造をもっている。 その特徵的なドメインとしては、 Kazal-type domain (アミノ酸酉己列の第 148 - 183位） で、 セリンプロテア一ゼイン ヒビターとして働く foll istatin様ドメイン、 そして TY domain (Thyroglobul in type I repeats, アミノ酸酉己列の第 317- 380位， 特には第 338- 380位） で、 シ スティンプロテア一ゼインヒビ夕一、 へパリンの結合パ一トナーとして働く ドメ インが挙げられる。 Testican - 3は、 Glycosaminoglycan attachment si te 持つ ていること力認められる。 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, ΒΜ-40 / SPARC / osteonectin, QR1, SCl / hevin, tsc36 / Flik, etc. are known, and among those proteins, there are special domain structures, In the case of extracellular Ca "" -binding domain (EC domain), foll istatin-like domain (FS domain), and some are thyrog lobul in-like domain (ΊΎ domain) and Glycosaminoglycan attachment si te ¾ In particular, 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). He ranks 317-380, especially 338-380), and has a domain that acts as a heparin-binding partner, including cysteine protein and zeinhibi. Testican-3 is recognized as having a Glycosaminoglycan attachment site.
これと類似したアミノ酸配列を持つものに、 HPTLG (W099/23110 A1)がある。 この HPTLG は、 ヒトネ!^下部からの HPTLG遺伝子に由来するもので、 細胞の粘着 、 移動、 増殖等に関与すると考えられている。 Testican様タンパク質であること から、 脳において成長因子のレセプターとして働くこと力推測される。 HPTLG は マルチドメィン構造をもっており、 その |½勺なドメィンとしては、 Kaza卜 type domain (アミノ酸酉己列の第 136 - 182位） で、 QR1、 SC1 や foll istatinのような タンパク分解阻害活性が知られ、 そして、 CWCV domain (アミノ酸酉己列の第 332- 38 1位） で、 これは、 例えば、 insul in-l ike growth factors を結合すること力知 られている。 HPTLG も Glycosaminoglycan attachment si te を持っていること力、 認、められる。 これまでの固 P類及び/又は膜型匪 P類と癌との関係については、 例えば、 プ ロゼラチナ一ぜ Aの活性化 (Okada ら， Bur. J. Biochem. , 194， pp721-730, 1990 ) 、 MT1-MMP による固 P - 2の活性化 （Satoら， Nature, 370, pp61-65, 1994) な どによる関係が示唆されて 、るが、具体的な関与にっレては未だ不明である。 こうした細胞外マトリックスや基劇誠分のさまざまな構成タンパク質に対し て活性を有する鮮 sは、 その活性の亢進などにより様々な病的な症状や発症に影 響を及ぼしているだけでなく、 癌などの転移'浸潤、 さらには癌組 辺での血 管新生などに関与していること力示唆されている。 従って、 MMPsの活性調節に関 与している遺伝子ゃタンパク質として如何なるもの;^存在し、 どのような機構で それらカ售 Psの活性の制御に関与しているなどを解明すること力く求められている 発明の開示 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. And, in the 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. Fresh s, which has activity against various extracellular matrices and various constituent proteins, is not only affecting various pathological symptoms and onset due to its increased activity, It has been suggested that it is involved in invasion of metastases such as metastasis, as well as angiogenesis in cancer. Therefore, there is a strong need to elucidate the genes involved in the regulation of the activity of MMPs and what proteins exist as ^ proteins, and what mechanisms are involved in the regulation of the activity of Ps. ing Disclosure of the invention
発明者等は、 匪 Psの活性調節に関与している遺伝子のスクリーニング系を構築 し、 ヒト由 伝子につき、 讓 Psの活性調節に関与している遺伝子の探索をおこ なった結果、 Testicanと呼ばれる一群の遺伝子群と極めて類似してはいるカ^ T 1-MMPや MT3-删 P に対して阻害活性を有し、 特徴ある構造を有する新規な遺 及びそれがコ一ドする新規なポリぺプチドを見出した。  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- 删 P, which are very similar to a group of genes called I found a peptide.
すなわち、 本発明は、  That is, the present invention
〔 1〕 （A) ヒト由来の N- Tes ポリぺプチド若しくは  [1] (A) Human-derived N-Tes polypeptide or
(B) (0 該 N- Tes のアミノ酸配列と少なくとも 60%の相同性を有し且つ (B) (0 having at least 60% homology with the amino acid sequence of the N-Tes;
(i i) (a; Glycosaminoglycan attachment
Figure imgf000006_0001
(ii) (a; Glycosaminoglycan attachment
Figure imgf000006_0001
(b) TY d隠 inや CWCV domainを欠いている、  (b) Lack of TY d hidden in or CWCV domain,
(c) C末端が特徴的な配列 Gly- Lys- Argを有している、 及び (c) the C-terminal has a characteristic sequence Gly-Lys-Arg, and
(d) N - Tesのアミノ酸配列のうちの少なくとも 5〜313個の遮铳したァ ミノ酸残基を有する (d) having at least 5-313 blocked amino acid residues in the amino acid sequence of N-Tes
カヽら成る群から選ばれた特徵を有しているもの Having features selected from the group consisting of
であることを樹敫とするポリぺプチド又はその塩； A polypeptide or a salt thereof, which is a tree;
〔 2〕 配列表の配列番号： 2で表されるァミノ酸配列のうち、  [2] Of the amino acid sequence represented by SEQ ID NO: 2 in the sequence listing,
(i) 少なくとも連镜した 5 〜70個のァミノ酸残基を有するもの、 (i) having at least 5 to 70 consecutive amino acid residues,
(i i) 少なくとも連镜した 71〜150個のァミノ酸残基を有するもの、 (ii) having at least 71 to 150 consecutive amino acid residues,
(i i i) 少なくとも連続した 151 〜313個のァミノ酸残基を有するもの、  (iii) having at least 151 to 313 consecutive amino acid residues,
(iv) 少なくとも第 22番目〜第 122番目のァミノ酸配列を有するもの、  (iv) having at least the 22nd to 122nd amino acid sequence,
(V) 第 22番目〜第 313番目のァミノ酸配列を有するもの、 (V) those having the 22nd to 313rd amino acid sequences,
(vi) 第 1番目〜第 313番目のァミノ酸配列を有するもの、 及び  (vi) those having the 1st to 313rd amino acid sequences, and
(vi i) それらのいずれか一つと実質的に同等のァミノ酸配列を有するもの からなる群から選ばれたポリぺプチドであることを特徴とする上記 〔 1〕 記載の ポリぺプチド又はその塩；  (vi i) The 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. ;
〔 3〕 上記 〔 1〕 または 〔 2〕 記載のポリぺプチドの部分べプチドまたは その塩； [3] Partial peptide or the polypeptide of the above [1] or [2] Its salt;
〔4〕 下記の性質：  [4] The following properties:
1) MT- MMP類の MMP類活性化能を抑制する機能を有し、 且つ  1) MT- MMPs have the function of suppressing the ability to activate MMPs, and
2) (i) 配列表の配列番号： 2で表されるアミノ酸配列において、 Ala2 2〜G1 y1 2 2のァミノ酸配列のうち、 少なくとも 5〜101個の連铳したァミノ酸残基を有 するもの、 及び 2) (i) sequence listing SEQ ID NO: in the amino acid sequence represented by, among Ala 2 2 ~G1 y 1 2 2 of Amino acid sequences, the Amino acid residues at least 5 to 101 amino communicating gun Have, and
(i i) N-Tesのアミノ酸配列のうちの少なくとも 5〜313個の連镜したアミ ノ酸残基を有するもの  (ii) N-Tes amino acid sequence having at least 5 to 313 consecutive amino acid residues
から成る群から選ばれたものである Selected from the group consisting of
ことを特徴とするポリぺプチド又はその塩。 Or a salt thereof.
〔5〕 (i) MT-■類が ΜΊΊ-置又は MT3-置である及び Z又は（i i) 匪 P類 カ镇 P- 2であることを 1敷とする上記 〔4〕 のポリペプチド又はその塩；  [5] The polypeptide or the polypeptide of [4] above, wherein (i) the MT-II is a ΜΊΊ-position or an MT3-position and Z or (ii) a marauding P-type is 镇 P-2. Its salt;
〔 6〕 上記 〔 1〕〜〔 5〕 の ゝずれか一記載のポリぺプチドをコ一ドする 塩基配列を有することを特徵とする核酸；  [6] a nucleic acid having a base sequence encoding the polypeptide according to any one of [1] to [5];
〔 7〕 配列表の配列番号： 1で表される塩基配列のうち、 オープンリーデ ィングフレーム部分またはそれと実質的に同等な ¾S配列を有することを特徴と する上記 〔6〕 記載の核酸；  [7] the nucleic acid of the above-mentioned [6], which has an open reading frame portion or a ΔS sequence substantially equivalent thereto in the nucleotide sequence represented by SEQ ID NO: 1 in the sequence listing;
〔8〕 上記 〔6〕 または 〔7〕 記載の核酸を含有することを樹敫とするベ クタ一；  [8] A vector which is a tree containing the nucleic acid according to the above [6] or [7];
〔9〕 上記 〔6〕 または 〔7〕 記載の核酸あるいは上記 〔8〕 記載のべク 夕一を含有することを特徵とする形質転換体；  [9] a transformant comprising the nucleic acid of [6] or [7] or the vector of [8];
〔10〕 宿主钿胞が大腸菌、 酵母、 293T細胞、 CH0細胞および COS細胞から なる群から週 れたものであることを とする上記 〔9〕 記載の形質転換体； [10] the transformant of the above-mentioned [9], wherein the host cell is selected from the group consisting of Escherichia coli, yeast, 293T cells, CH0 cells and COS cells;
〔11〕 上記 〔10〕 記載の形質転換体によって発現させて得たものであるこ とを 1敷とする上記 〔1〕〜〔5〕 のいずれか一記載のポリペプチド； [11] the polypeptide of any one of the above-mentioned [1] to [5], wherein the polypeptide is one obtained by being expressed by the transformant of the above-mentioned [10];
〔12〕 配列表の配列番号： 1で表される塩基配列のうち、 オープンリーデ ィングフレーム部分又はその一部あるいはそれと実質的に同等な塩基配列を PCR 増幅するに有効なプライマ一；  [12] a primer effective for PCR-amplifying an open reading frame part or a part thereof or a base sequence substantially equivalent thereto among the base sequence represented by SEQ ID NO: 1 in the sequence listing;
〔13〕 次のものに対する抗体： (i) 上記 〔 1〕 記載のポリぺプチドまたはその塩、 [13] Antibodies to: (i) the polypeptide according to the above (1) or a salt thereof,
(i i) 上記 〔2〕 記載のポリペプチドまたはその塩、  (i i) the polypeptide of the above (2) or a salt thereof,
(i i i)上記 〔3〕 記載の部分ペプチドまたはその塩、  (iii) the partial peptide or a salt thereof according to the above (3),
(iv) 上記 〔4〕 記載のポリペプチドまたはその塩、 あるいは  (iv) the polypeptide of the above (4) or a salt thereof, or
(V) 上記 〔 5〕 記載のポリぺプチドまたはその塩； (V) the polypeptide of the above-mentioned [5] or a salt thereof;
〔14〕 上記 〔1〕〜〔5〕 のいずれか一記載のポリペプチドまたはその塩 と特異的に免疫反応する抗体を測定 として用いることを特徴とする上記 〔 1 〕〜〔5〕 のいずれか一記載のポリペプチドまたはその塩の免疫学的測定方法； [14] Any of the above-mentioned [1] to [5], wherein an antibody that specifically immunoreacts with the polypeptide of any one of the above [1] to [5] or a salt thereof is used as a measurement. A method for immunologically measuring the polypeptide or a salt thereof according to one of the above;
〔15〕 上記 〔 1〕〜〔 5〕 の 、ずれか一記載のポリぺプチドまたはその塩 と特異的に免疫反応する抗体を含むことを特徴とする上記 〔1〕〜〔5〕 のいず れか一記載のポリぺプチドまたはその塩の免疫学的測定試薬； [15] Any of the above [1] to [5], which comprises an antibody specifically immunoreactive with the polypeptide or the salt thereof according to any one of the above [1] to [5]. The reagent for immunologically measuring the polypeptide or a salt thereof according to any one of the above items;
〔16〕 上記 〔1〕 〜〔5〕 のいずれか一記載のポリペプチド、 その一部の ペプチドまたはそれらの塩；あるいは上記 〔6〕 または 〔7〕 記載の核酸；ある いは上記 〔13〕 記載の抗体を含有していることを特徵とする医薬；  [16] the polypeptide of any one of the above [1] to [5], a partial peptide thereof or a salt thereof; or the nucleic acid of the above [6] or [7]; or the above [13] A medicament characterized in that it contains the described antibody;
〔17〕 (i) MT1-MMP又は MT3-壓 P活性阻害剤、  (17) (i) MT1-MMP or MT3-pressure P activity inhibitor,
(i i) 癌の浸潤、 転移抑制及び/又は阻害剤、  (ii) cancer invasion, metastasis inhibitor and / or inhibitor,
(i i i)血管新生阻害剤、  (i i i) angiogenesis inhibitor,
(iv) アルツハイマー治療剤、 及び  (iv) Alzheimer's therapeutic agent, and
(V) 関節破壊治翻 (V) Joint destruction
から成る群から遷ま'れたものであることを特徵とする上記 〔16〕 記載の医薬； 〔18〕 上記 〔1〕〜〔5〕 のいずれか一記載のポリペプチド、 その一部の ぺプチドまたはそれらの塩の生物学的活性を促進または阻害する化^!またはそ の塩を含有していることを樹敫とする医薬； 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;
〔19〕 上記 〔1〕〜〔5〕 のいずれか一記載のポリペプチド、 その一部の ぺプチドまたはそれらの塩の生物学的活性を促進または阻害する化合物のスクリ 一ニンク、'方法およびスクリーニングキット；  [19] A screening method for compounds that promote or inhibit the biological activity of the polypeptide according to any one of the above [1] to [5], a part of the peptide or a salt thereof, a method and screening. Kit;
〔20〕 國 Psから成る群から選ばれたものの発現プラスミ ドと検体遺伝子を 宿主細胞に導入し、 発現する画 P についての検出を指標とすることを顿敷とする 画 Psから成る群から選ばれたものの活性調節に関与する遺好のスクリ一ニング 方法およびスクリーニングキット； [20] 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;
〔21〕 画 P- 2、 MT1-MMP発現プラスミ ドと検体プラスミ ドを 293T細胞など の宿主細胞に導入し、 例えば潜在型、 中間体、 活性化議 P- 2 を検出にかけるこ とを樹敫とする上言己 〔20〕 記載のスクリーニング方法およびスクリーニングキッ ト ；及び  (21) Transformation of the expression plasmid P-2, MT1-MMP expression plasmid and the specimen plasmid into host cells such as 293T cells, and the detection of latent, intermediate, and activated P-2 The screening method and screening kit according to [20]; and
〔22〕 下記の指標：  [22] The following indicators:
(a) N-Tes の発現量が正常部位に比べ低いこと、  (a) the expression level of N-Tes is lower than that in a normal site,
(b) N-Tes ポリペプチド又はそれから誘導されたペプチド断片、  (b) an N-Tes polypeptide or a peptide fragment derived therefrom,
(c) N-Tes遺伝子又はそれから誘導された mRNAなどの核酸、 及び  (c) N-Tes gene or nucleic acid such as mRNA derived therefrom, and
(d) i CN-Tes抗体  (d) i CN-Tes antibody
カヽら成る群から選ばれたものを指標として翻することを樹敫とするグリォ一マ の検出方法を i¾共する。 別の態様では、 本発明は The method of detecting gloma is based on the fact that the index is selected from the group consisting of birds. In another aspect, the invention relates to
〔23〕 MT1-MMP及び Z又は MT3- MMP に対して阻害活性を有することを特徴 とする上記 〔 1〕 〜 〔 5〕 のいずれか一記載のポリぺプチド又はその塩；  [23] the 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;
〔24〕 MT1-MMP及び: /又は MT3- MMP に対して阻害活性を有するポリぺプチ ド又はタンパク質を産生する機能を有する上記 〔6〕 又は 〔7〕 記載の核酸； [24] the nucleic acid of the above-mentioned [6] or [7], which has a function of producing a polypeptide or a protein having an inhibitory activity on MT1-MMP and / or MT3-MMP;
〔25〕 MT1-MMP及び Z又は MT3- MMP に対して阻害活性を有するポリぺプチ ド又はタンパク質を産生する機能を有する上記 〔8〕 記載のベクタ一； [25] the vector of the above-mentioned [8], which has a function of producing a polypeptide or a protein having an inhibitory activity on MT1-MMP and Z or MT3-MMP;
〔26〕 MT1-MMP及びノ又は MT3-固 P に対して阻害活性を有するポリぺプチ ド又はタンパク質を産生する機能を有する上記 〔9〕 記載の形質転換体；  [26] the transformant of the above-mentioned [9], which has a function of producing a polypeptide or a protein having an inhibitory activity on MT1-MMP and MT3-solid P;
〔27〕 抗体が、 Testican-3 及び HPTLG と実質的に^反応はしないもの であることを とする上記 〔13〕 記載の抗体；  [27] the antibody of the above-mentioned [13], wherein the antibody does not substantially react with Testican-3 and HPTLG;
〔28〕 モノクローナル抗体であることを特徴とする上記 〔1¾ 記載の抗体  [28] the antibody of the above-mentioned [1], which is a monoclonal antibody;
〔29〕 少なくとも ^¾の¾¾ - Tes モノクローナル抗体を使用することを特 徵とする上記 〔14〕 記載の測定方法； [29] the method of the above-mentioned [14], wherein a monoclonal antibody of at least ^ ¾-¾¾-Tes is used;
〔30〕 少なくとも ¾の抗 N- Tes モノクロ一ナル抗体を含有することを特 徵とする上記 〔15〕 記載の測定 ；及び [30] Contains at least ¾ anti-N-Tes monoclonal antibody And the measurement according to the above [15]; and
〔31〕 本発明の活性成分を使用することを ：とするグリォーマの検出用 ¾¾¾びその測定方法を樹共する。 本発明のその他の目的、 樹敫、 優秀 tt¾びその有する観点は、 以下の記載より 当業者にとっては明白であろう。 しかしながら、 以下の記載及び具体的な実施例 等の記載を含めた本件明細書の記載は本発明の好ましい態様を示すものであり、 説明のためにのみ示されているものであることを理解されたい。本明細書に開示 した本発明の意図及び範囲内で、 種々の変化及び Z又は改変 （あるいは修飾） を なすことは、 以下の記驗び本明細書のその他の部分力、らの知識により、 当業者 には額に明らかであろう。本明細書で弓 I用されて 、る全ての特許文南扱び参考 文献は、 説明の目的で弓 1用されているもので、 それらは本明細書の "^としてそ の内容はここに含めて解釈されるべきものである。  [31] A method for detecting glioma using the active ingredient of the present invention and a method for measuring the same. Other objects, advantages, and aspects of the present invention will be apparent to those skilled in the art from the following description. However, it is understood that the description of the present specification, including the following description and the description of specific examples, etc., shows preferred embodiments of the present invention and is given only for explanation. I want to. Various changes and modifications or alterations (or modifications) within the spirit and scope of the present invention disclosed herein will be made by the following tests and other knowledge of the present specification. It will be obvious to those skilled in the art. All patent documents referred to as bow I in this specification are used for bow 1 for explanation purposes, and they are referred to as "^" in this specification. It should be interpreted including.
本明細書において、 用語 「及び/又は」 とは、 （1)併合的接続関係と （2)選択 的接続関係の両方が存在することを意味しており、 例えば「治療及び Z又は予防 」 の: t では （1)治療及び予防並びに （2)治療又は予防の両方を包含する意味で ィ¾¾されている。 その他においても用語「及び/又は」 は同様に （1)併合的接続 9係と （2)選択的接続関係の両方を包含する意味で使用されている。 図面の簡単な説明  As used herein, 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. BRIEF DESCRIPTION OF THE FIGURES
図 1は、 MMP-9、 MMP-2、 MT1-MMP発現プラスミ ドと試験検体プラスミドを 29 3T細胞に導入し、 ゼラチンザィモグラフィーにて潜在型、 中間体、 活性化 M P- 9、 MMP-2を検出にかけた結果を示す。  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.
図 2は、 Testican-1, Testican 2、 Testican - 3および HPTLGと、 N-Tes とのァ ミノ酸酉己列に関するホモロジ一を示す。  FIG. 2 shows the homology of Testican-1, Testican 2, Testican-3 and HPTLG, and N-Tes with respect to the amino acid rooster.
図 3は、 ゼラチンザィモグラフィ一分析した、 N- Tes- de卜 FLAGによる MT1-画 P 、 MT3- MPを介した匪 P- 2活'性化抑制の結果を示す。  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.
図 4は、 グリォーマにおける N-Tes発現検索の結果を示す。  FIG. 4 shows the results of an N-Tes expression search in glioma.
図 5は、 MT- MMPと N- Tesの結合性の調査結果を示す。 図 6は、 Testican 3と N - Tesの発現レベルを半 的 RT- PCRで角斜斤した結果を 示す。 NB: 正常脳繊； LGA:髓應蠊； M: 未分化星細胞腫； 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 : グリア芽細胞腫； Meta: 脳へ転移した ¾ϋ器癌及び GCL :グリオ一マ細胞株 発明を実施するための最良の形 II GB: Glioblastoma; Meta: Cancer of the brain metastasized to the brain and GCL: Glioma cell line Best mode for carrying out the invention II
本発明に従い、 Testicanフアミリーの一種で、 匪 P阻害活性を有するヒト由来 の N- Tes ポリぺプチド若しくは該 N - Tes ポリぺプチドのァミノ酸酉 と少なくと も 60%の相同性を有し且つ删 P阻害活性あるいは同等の抗原性を有するものであ るべプチドまたはその塩、 そのポリべプチドの特徵的な部分べプチドまたはその 塩、 それらをコードする遺伝子、 例えば DNA、 RNAなど、 その遺伝子を遺伝 且 換え技林 ίで ί喿作すること力可能なように含有しているべクターあるいはブラスミ ド、 こうしたベクターなどで形質転換された宿主細胞、 さらにはその遺伝子を発 現するトランスジエニックマウス等のトランスジエニック動物、 その遺伝子を特 異的に不活性化したノックアウトマウス等のノックアウト動物、 その形質転換細 胞を、 培養して該ポリペプチドまたはその塩を製造する方法、 こうして得られた 該ポリべプチドまたはその塩やそのポリべプチドの特徵的な部分べプチドまたは その塩を用いて得られた抗体、 特にはモノクローナル抗体、 その抗体を産生する ハイプリドーマ細胞、 該単離された遺伝子、 例えば DNA、 RNAなどをプローブと して用いたり、 あるいは該抗体を用いた測定 ·診断手段並びに カ^ ί共される ο さらには、 本明細書で開示され説明されている活す«分の利用を樹共し、 例え ば、 該活誠分を含有する医薬あるいは藤などが樹共され、 そうした活性成分 を用いた疾患、 疾病あるいは異常な状態の治療及び Ζ又は予!^法、 さらにはス クリ一ニング方法などが樹共される。 本明細書において 「N- Tes」 とは、 プロテオグリカンである Testican類に関連 したべプチドであって、 本発明で開示されている新規なぺプチドを指している。 該 N-Tes は、 313個のアミノ酸残基からなるペプチドであり、 その C末端側には 樹敫的な配列 Gly3 " -Lys3 1 2-Ar 3 1 3を有しており、 Testi can- 3や HPTLGに存在す る TY domainや CWCV domainを欠いたもので、 さらに Testi can- 3や HPTLGに存在 している Glycosaminoglycan attachment si teを欠いていることを としてい る。 該 N - Tes は、 MT-MMPs P且害活性を有し、 特に、 ΜΉ -匪 P， MT3-副 Pに対して強 い阻害活性を有し、 さらに MT3-匪 P に対してより強い阻害活性を有する。 この阻 害活性としては、 例えば、 MT-MMPsカ領 P- 2 を活性化することを阻害することが 挙げられる。 この MT-固 Ps に対する阻害活性は、 配列表の配列番号: 1の第 22番目 〜第 122番目のァミノ酸配列部分内にあると予測され、 これらアミノ酸配列また はそれと実質的に同等なアミノ酸配列を有するポリペプチドは、 MT-MMPs に対す る阻害活性を有することを意味する。 これは、 また、 例えば、 HPTLG は MT-匪 Ps に対する阻害活性を有することを意味する。 さらに、 testican- 3は、 これら酉己列 に 3アミノ酸残基の挿入がある力 1651； &11-3も同様に1^-圖?3 に対する阻害活 性を有するものと考えられる。 According to the present invention, 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べ 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. Vector or plasmid containing a gene so that it can be genetically and genetically engineered in a genetic engineering plant, a host cell transformed with such a vector, and a transgenic gene expressing the gene. 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. A method for producing a polypeptide or a salt thereof, an antibody obtained by using the polypeptide or a salt thereof or a special partial peptide of the polypeptide or a salt thereof, particularly a monoclonal antibody, 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. Utilization of active ingredients disclosed and described in the specification, for example, drugs or wisteria containing the active ingredients are used, and diseases, diseases or abnormalities using such active ingredients are used. The treatment of various conditions and the Ζ or 予 method, as well as the screening method, are also used. As used herein, “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敫的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 匪 -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. This also means that, for example, HPTLG has inhibitory activity against MT-banded Ps. Furthermore, 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.
本明細書で用いる用語 「ポリペプチド」 としては、 以下に記載するような如何 なるポリぺプチドを指すものであつてもよい。 ポリぺプチドの基本的な構造は周 知であり、 当該技術分野にお 、て非常に数多くの参考書及びその他の刊行物に記 載がある。 こうしたことに鑑み、 本明細書で用いる用語 「ポリペプチド」 は、 ぺ プチド結合又は修飾したべプチド結合により互 、に結合しているような 2個又は それ以上のァミノ酸を含む任意のぺプチド又は任意のタンパク質を意味する。 本 明細書で用いる用語 「ポリペプチド」 としては、 当該分野において通常、 例えば ぺプチド、 ォリゴぺプチドあるいはぺプチドォリゴマーとも称せられる短い鎖の もの、 及びタンパク質と一般的に言われ、 多くの形態のもの力 口られている長い 鎖のものの両方を意味してよい。 ポリペプチドは、 しばしば、 通常、 20種の天然 型アミノ酸 （天然に存在しているアミノ酸： あるいは遺伝子でコードされるアミ ノ酸） と称されるアミノ酸 （20個存在している） 以外のアミノ酸を含有していて もよい。 ポリぺプチドは、 また末端ァミノ酸残基を含めて、 その多くのアミノ酸 残基が翻訳された後にプロセッシング及びその他の改変 （あるいは修飾） される といつた天然の工程によるのみならず、 当業者に周知の化学的改変技術によつて も、 上記のポリペプチドはそれが改変 （修飾） できることは理解されよう。 該ポ リペプチドに加えられる改変 （修飾） については、 多くの形態のもの力く知られて おり、 それらは当該分野の基礎的な参考書及びさらに詳細な論文並びに多数の研 究文献にも詳しく記載されており、 これらは当業者に周知である。 幾つかのとり わけ常套的な改変，修飾としては、 例えばグリコシル化、 脂質結合、 硫酸化、 グ ルタミ,ン酸残基の 7一カルボキシル化、 水酸化及び ADP-リボシル化等が挙げられ 、 例えば Τ· B. Creighton, Proteins-Structure and Molecular Properties, Se cond Edi tion, W. H. Freeman and Company, New York, (1993)； B. C. Johnson (Ed. ), Posttranslational Covalent Modif ication of Proteins, Academic Pre ss， New York, (1983) (Wold, F.， "Posttranslational Protein Modif ications ： Perspective and Prospects", pp. 1-12)； Seif ter et al. , "Analysis for Pr otein Modif ications and nonprotein cofactors", Meth. Bnzymol. , 182: 626- 646 (1990) ; Rattan et al. , "Protein Synthesis: Posttranslational Modif ic ation and Aging", Ann. N. Y. Acad. Sci. , 663 : p. 48-62 (1992)等の記載を参 照できる。 本発明の 「ポリペプチド」 としては、 特には N- Tes及びその関連ポリペプチド を包含する。 該 N- Tes及びその関連ポリペプチドとしては、 ヒト由来のもの力く挙 げら 匪 Ps阻害活性、 例えば Ml - MMP， MT3 -匪 Pなどに対して阻害活性を有する もの力く挙げら† 代表的には Glycosaminoglycan attachment si te を欠いている 及び/又は TY domainや CWCV domainを欠いたもの力く挙げられ、 より具体的には 、 配列表の配列番号： 2で表されるアミノ酸配列のうち、 少なくとも第 311 位〜 第 313位のァミノ酸配列を有するもの、 第 22位〜第 122位のァミノ酸配列を有す るもの、 同第 22位〜第 313位のァミノ酸酉 B^ijを有するもの、 同第 1位〜第 313位 のアミノ酸配列を有するもの、 及びそれらのいずれか一つと少なくとも 60% より 高い相同性、 好ましくは 70%以上の相同性、 さらに好ましくは 80%以上の相同性 、 また好ましくは 85%以上の相同性、 もつと好ましくは 90%以上の相同性、 より 好ましくは 95%以上の相同性、 特に好ましくは 97%以上の相同性を有し且つ應 P P且害活性ある 、は同等の抗原性などといつた実質的に同等の生物学的活性を有す るアミノ酸配列を有するもの力くすべて挙げられる。 本発明のヒト N-Tes としては、 MT1-匪 P， MT3-匪 Pなどの阻害活性を有し、 Test ican- 3などの Testicanファミリ一のバリアントの一種であり、 Glycosaminoglyca n attachment si te を欠いたもので且つ新規なアミノ酸配列を有するものであれ ばよい。 より好ましくは、 本発明のぺプチドとしては、 Testicanファミリ一と少 なくとも 60% より高 、相同性を持つァミノ酸配列を有するもののうち、 glycosam inoglycan attachment si teを欠いたもの力挙げられ、 特には配列表の配列番号 ： 2で表されるアミノ酸配列のうち、 少なくとも第 311位〜第 313位のアミノ酸 配列部位、 第 22位〜第 122位のアミノ酸配列部位あるいはそれの主要部位 （例え ば、 該第 311位〜第 313位を含む連続したアミノ酸残基、 あるいは該第 22位〜第 122位のうちの違続したアミノ酸残基 5個以上、 好ましくは 10個以上、 また好ま しくは 20個以上、 さらに好ましくは 30個以上、 より好ましくは 40個以上、 また好 ましくは 50個以上、 さらに好ましくは 60個以上、 もっと好ましくは 70個以上、 ま た好ましくは 80個以上、 さらに好ましくは 90個以上、 もっとも好ましくは 100個 以上、 また好ましくは 110個以上） を有するもの力く挙げられる。 代表的には、 本 発明のペプチドは、 配列表の配列番号： 2で表されるアミノ酸配列のうち、 少な くとも第 22位〜第 313位のアミノ酸配列を有するもの、 及びそれらのいずれか一 つと実質的に同等のァミノ酸配列を有するものからなる群から選ばれたものであ る。 さらに本発明のペプチドとしては、 配列表の配列番号： 2で表されるァミノ 酸配列の一部または^ ¾を有していてもよい。 こうした配列を有するものはすべ て包含されてよい。 本明細書中、 「相同性」 とは、 ポリペプチド配列 （あるいはアミノ酸配列） 又 はポリヌクレオチド配列 （あるいは塩基配列） における 2本の鎖の間で該鎖を構 成している各ァミノ酸残基同志又は各塩基同志の互いの適合関係において同一で あると決定できるようなものの量 （数） を意味し、 二つのポリぺプチド配列又は 二つのポリヌクレオチド配列の間の配列相関性の を意味するものである。 相 同性は容易に算出できる。 二つのポリヌクレオチド配列又はポリぺプチド配列間 の相同性を測定する方法は数多く知られており、 「相同性」 （ 「同一性」 とも言 われる） なる用語は、 当業者には周知である (例えば、 Lesk, A. Μ· (Ed. ), Com putational Molecular Biology, Oxford University Press, New York, (1988)； Smi th, D. W. (Ed. ), Biocomputing: Informatics and Genome Projects, Acad emic Press, New York, (1993) ; Gr i f in, A. M. & Gri f in, H. G. (Ed. ), Compu ter Analysis of Sequence Data: Part I, Human Press, New Jersey, (1994)； von Heinje, G.， Sequence Analysis in Molecular Biology, Academic Press, New York, (1987)； Gribskov, M. & Devereux, J. (Ed. ), Sequence Analysis P rimer, M Stockton Press, New York, (1991) 等） 。 二つの酉己列の相同性を測定 するのに用いる一般的な方法には、 Martin, J. Bishop (Ed. ), Guide to Huge C omputers, Academic Press, San Diego, (1994)； Cari l lo, H. & Lipman, D.， S I層 J. Appl ied Math. , 48 : 1073 (1988) 等に開示されているもの力く挙げられる が、 これらに限定されるものではない。相同性を測定するための好ましい方法と しては、 試験する二つの配列間の最も大きな適合関係部分を得るように設計した もの力挙げられる。 このような方法は、 コンピュータ一プログラムとして組み立 てられているもの力《挙げられる。二つの配列間の相同性を測定するための好まし いコンピュータ一プログラム法としては、 GCGプログラムパッケージ （Devereux ， J. et al.， Nucleic Acids Research, 12(1) : 387 (1984))、 BLASTPヽ BLASTN 、 FASTA (Atschul, ' S. F. et al.， J. Molec. Biol. , 215 : 403 (1990)) 等が挙 げられる力、 これらに限定されるものでなく、 当該分野で公知の方法を使用する こと力くできる。 本発明の N- Tes をコ一ドする遺伝子は、 代表的には配列表の配列番号： 2で表 されるぺプチド及びその一部の連続したァミノ酸配列をコードする塩基配列を含 有するもの、 例えば、 配列表の配列番号： 1で表される塩基配列の少なくとも 10 38-1046位により構成される塩基配列を含有するもの、 配列表の配列番号： 1で 表される塩基配列の少なくとも m- 473位により構成される塩基配列を含有する もの、 及ぴ 列表の配列番号： 1で表される塩基配列の 108 - 110位の ATGから 10 47-1049位の TGA より構成される塩基配列を含有するもの （1047-1049 の終止コ ドン TGA は、 TAAまたは TAGであってもよい） であること力くできるし、 配列表の 配列番号： 1で表される 配列の 171位から 1049位の塩基配列を含有するもの 、 塩基配列に開始コドン、 例えば、 Met をコードするコドン （及び終止コドン） を ¾口したもの、 また、該塩基配列がコ一ドするタンパク質と少なくとも 80%の 相同性を有するァミノ酸酉 を持ち且つ MT1-删 P， MT3 - MMPなどの讓 Psの少なくと も一つに対し阻害活性を有するかあるいは同等の抗原性などのそれと実質的に同 等の生物学的活性を有するぺプチドをコ一ドするといつたそれと同効の 配列 を含有するものであれば如何なるものであってもよい。該 N-Tesをコードする遺 伝子は、 一本鎖 DNA、 二本鎖 DNA、 RNA、 DNA:RNAハイプリッド、 合成 DNAなど の核酸であり、 またヒトゲノム DNA、 ヒトジヱノミツク DNAライブラリー、 ヒト 紙織'細胞由来の cDNA、 合成 DNAのいずれであってもよい。該 N-Tesをコードす る遺 の塩基配列は、修飾（例えば、 ira、 除去、 置換など） されることもで き、 そうした修飾されたものも包含されてよい。 さらには、 以下説明するように 、 本発明の核酸は、 本発明のペプチドあるいはその一部をコードするものであつ てよく、好ましいものとしては DNAが挙げられる。 また上記 「同効の塩基配列」 とは、 例えばストリンジヱントな条件で配列表の配列番号： 1の塩基配列うちの 連徺した 5個以上の塩基配列、 好ましくは 10個以上の塩基配列、 より好ましくは 15個以上の塩基配列、 さらに好ましくは 20個以上の 配列とハイブリダイズし 、 N-Tes と実質的に同等のアミノ酸酉 をコードするものなどが挙げられる。 配列表の配列番号： 1で表される塩基配列またはそれと同効の塩基配列を含有 する本発明の DNAは、 例えば以下に示す方法によって取得できる。 As used herein, the term "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. B. Creighton, Proteins-Structure and Molecular Properties, Second Edition, WH Freeman and Company, New York, (1993); BC Johnson (Ed.), Posttranslational Covalent Modification of Proteins, Academic Press, New York , (1983) (Wold, F., "Posttranslational Protein Modifications: Perspective and Prospects", pp. 1-12); Seifter et al., "Analysis for Preterin Modifications and nonprotein cofactors", Meth. , 182: 626-646 (1990); Rattan et al., "Protein Synthesis: Posttranslational Modification and Aging", Ann. NY Acad. Sci., 663: p. 48-62 (1992). I can illuminate. The “polypeptide” of the present invention particularly includes N-Tes and related polypeptides. Examples of the N-Tes and its 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. More specifically, among the 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. Is the amino acid sequence represented by SEQ ID NO: 2 in the sequence listing, at least the amino acid sequence portion at positions 311 to 313, the amino acid sequence portion at positions 22 to 122, or a major portion thereof (for example, Consecutive amino acid residues including the positions 311 to 313, or 5 or more, preferably 10 or more, and preferably 20 amino acids that are intermittent among the 22nd to 122nd positions Or more, more preferably 30 or more, more preferably 40 or more, further preferably 50 or more, further preferably 60 or more, more preferably 70 or more, and still more preferably 80 or more. Ku is 90 or more, most preferably 100 or more, and preferably include rather force those having more than 110 pieces). Typically, 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. As used herein, 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). It means the amount (number) of bases or bases that can be determined to be the same in the mutual matching relationship, and the sequence correlation between two polypeptide sequences or two polynucleotide sequences. Is what you do. The homology can be easily calculated. Many methods for measuring the homology between two polynucleotide or polypeptide sequences are known, and the term "homology" (also referred to as "identity") is well known to those skilled in the art ( For example, Lesk, A. Μ · (Ed.), Computational Molecular Biology, Oxford University Press, New York, (1988); Smith, DW (Ed.), Biocomputing: Informatics and Genome Projects, Acad emic Press, New York, (1993); Gr if in, AM & Griffin, HG (Ed.), Computer Analysis of Sequence Data: Part I, Human Press, New Jersey, (1994); von Heinje, G., Sequence Analysis in Molecular Biology, Academic Press, New York, (1987); Gribskov, M. & Devereux, J. (Ed.), Sequence Analysis Primer, M Stockton Press, New York, (1991), etc.). A general method used to determine the homology of two roosters is Martin, J. Bishop (Ed.), Guide to Huge Computers, Academic Press, San Diego, (1994); , H. & Lipman, D., SI layer J. Applied Math., 48: 1073 (1988), and the like, but are not limited thereto. 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力 Forces such as BLASTN and FASTA (Atschul, 'SF et al., J. Molec. Biol., 215: 403 (1990)), and the like, but are not limited to these. Can be used powerfully. 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. For example, those containing a nucleotide sequence composed of at least positions 1038 to 1046 of the nucleotide sequence represented by SEQ ID NO: 1 in the sequence listing, and at least m -A nucleotide sequence comprising the nucleotide sequence consisting of position 473, and a nucleotide sequence consisting of ATG at position 108-110 to TGA at position 10 47-1049 in the nucleotide sequence represented by SEQ ID NO: 1 in the sequence listing (The stop codon TGA of 1047-1049 may be TAA or TAG.) It is possible to increase the number of 171 to 1049 of the sequence represented by SEQ ID NO: 1 in the sequence listing. Those containing the base sequence, 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- 删 P, MT3-MMP, etc. Any peptide containing a sequence having the same effect as that obtained by coding a peptide having an inhibitory activity against or having substantially the same biological activity as that of antigenicity It may be. 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. Further, as described below, 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.
なお、 遺伝 換え技術は、 例えば Sambrook, E. F. Fri tsch & T. Mania t is, "Molecular Cloning: A Laboratory Manual (2nd edi tion)", Cold Spring Genetic recombination techniques are described, for example, in Sambrook, E. F. Fritsch & T. Mania isis, "Molecular Cloning: A Laboratory Manual (2nd edition)", Cold Spring
Harbor Laboratory Press, Cold Spring Harbor, New York (1989)； D. M. Glo ver et al. ed.， "DNA Cloning", 2nd ed. , Vol. 1 to 4, (The Practical Appr oach Series), IRL Press, Oxford Universi ty Press (1995)；日本生化学会編、Harbor Laboratory Press, Cold Spring Harbor, New York (1989); DM Glo ver et al. Ed., "DNA Cloning", 2nd ed., Vol. 1 to 4, (The Practical Approach Series), IRL Press, Oxford University Press (1995); Biochemical Society of Japan,
「続生化学実験講座 1、遺伝子研究法 I I」 、 東京化学同人（1986) ;日本生化学会 編、 「新生化学実験講座 2、 核酸 I I I (組換え DNA技術） 」 、 東京化学同人（19 92) ; R. Wu ed. , "Methods in Bnzymology", Vol. 68 (Recombinant DNA), Acad emi c Press, New York (1980)； R. Wu et al. ed.， "Methods in Bnzymology", Vol. 100 (Recombinant DNA, Part B) k 101 (Recombinant DNA, Part C)， Acad emi c Press, New York (1983)； R. Wu et al. ed. , "Methods in Enzymology", Vol. 153 (Recombinant DNA, Part D), 154 (Recombinant DNA, Part E) k 155 (Recombinant DNA, Part F)， Academi c Press, New York (1987)； J. H, Mi l ler ed. , "Methods in Enzymology", Vol. 204, Academi c Press, New York (1991) ； R. Wu et al. ed. , "Methods in Enzymology", Vol. 218, Academic Press, N ew York (1993)などに記載の方法あるいはそこで引用された文献記載の方法ある いはそれらと実質的に同様な方法や改変法により行うこと力できる （それらの中 にある記載はそれを参照することにより本明細書の開示に含められる） 。 クローニングされて 、るヒト由来の cDNAラィブラリ一、例えば種々のヒト由来 の組織あるいは培養細胞 （特には、 ヒ卜の腎臓、 脳、 松果体、 下垂体後葉、 網膜 、 胸腺、 精巣、 卵巣、 子宮、 腸、 心臓、 肝臓、 塍臓、 小腸、 昜などの糸職.細 胞等） cDNAライブラリーから得られた cDNA挿入酉 を持つプラスミ ドを、 適当な 検知系によって、 MMPsのうちの少なくとも一つの活性を抑制していることを指標 にして選別を行う。 具体的には、 例えは IMP- 9， MP- 2及び Ml匪 P の全長の cDNA を発現するべクタ一と共に、 試験されるべき該 cDNAライブラリーから得られた cD NA挿入配列を持つプラスミ ドを、 適当な宿主細胞にトランスフヱクトし、 例えば 潜在型、 中間体、 活性化 MMMP- 9， MMP- 2を検出にかけ、 活性化型 MMP- 9 あるいは 活す生化型固 P- 2 の産生を抑制する遺伝子プラスミ ド集団を検索する。 "Seismic Chemistry Experiment Course 1, Gene Research Method II", Tokyo Chemistry Dojin (1986); edited by The Biochemical Society of Japan, "New Course Chemistry Experiment Course 2, Nucleic Acid III (Recombinant DNA Technology)", Tokyo Chemical Dojin (1992) R. Wu ed., "Methods in Bnzymology", Vol. 68 (Recombinant DNA), Acad mic Press, New York (1980); R. Wu et al. Ed., "Methods in Bnzymology", Vol. 100. (Recombinant DNA, Part B) k101 (Recombinant DNA, Part C), Acad em Press, New York (1983); R. Wu et al. ed., "Methods in Enzymology", Vol. 153 (Recombinant DNA, Part D), 154 (Recombinant DNA, Part E) k 155 (Recombinant DNA, Part F), Academic Press, New York (1987); J. H., Miller ed., "Methods in Enzymology", Vol. 204, Academic Press, New York (1991); R. Wu et al. ed., "Methods in Enzymology", Vol. 218, Academic Press, New York (1993), or the method described in the literature cited therein, or a method substantially similar thereto or a modified method. (The statements therein are included in the disclosure herein by reference thereto). Human cloned cDNA libraries, such as various human-derived tissues or cultured cells (particularly human kidney, brain, pineal gland, posterior pituitary gland, retina, thymus, testis, ovary, uterus , Gut, heart, liver, kidney, small intestine, and others. Cells, etc.) 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.
この検索手法は、 繰り返し行なうことができる。 こうして同定された遺伝子に つき、 その配列決定する。 こうして同定された配列に基づいて適切なプライマー を設計'合成し、 ^によっては、 同定された配列のオリジンである動物由来の cDNAライブラリ一を用いるなどし、 目的の酉己列をポリメラ一ゼ ·チヱイン · リア クシヨン（polymerase chain reaction: PCR)増幅する。 得られた DNA断片はそれ をプローブに種々のヒト糸職あるいは培養細胞等から構築されたヒトジヱノミツ ク DNAライブラリーあるいはヒト由来 cDNAラィブラリーをスクリーニングし、 プ ローブにノヽィブリダイズするクロ一ンを選択し、 該クローン中の DNAの揷人配列 の塩基配列を;^し、 新規な N- Tes及びそれに関連する遺ィ 5?配列を有する DNA 断片を決定 ·取得することもできる。 必要に応じて該クローン中の DNAの挿入配 列はサブクローニングすること力できる。 こうして解斤された新規な遺伝子を有 していると考えられる DNA配列を基に、 該 N- Tes及びそれに関連する遺伝子配列 をコードする遺伝子を取得することも可能である。 また、 角?！斤された該新規な N- Tes及びそれに関連する遺伝子配列 酉 」） を基にセンスブライマ一とアンチ センスプライマーをデザィンし、 合成することができる。 センスプライマ一は、 好ましくは角?！斤された該所望のぺプチドと推定される遺伝子の 5'端側のェクソン 部位から選んで合成すること力くでき、 7ンチセンスブラィマ一は、 好ましくは解 析された該所望のぺプチドと推定される遺伝子の 3'端側のェクソン部位から選ん で合成すること力くでき、 より子ましくは該センスブラィマ一合成に利用したェク ^？位以外から選ぶことか'できる。 遺伝子の cDNAは、 その全長を一度に入手することを目指してもよいが、 解析さ れたェクソン部位 のェクソン部位） を利用して、 のプライマーをデザ インして合成し、 ネ纖の PCRをデザインして行い （必要に応じて塩基配列決定さ れた DNA断片を角 I斤して、 当該遺伝子の cDNAの全塩基配列を決定し、 それに基づ いてクロ一ニングし） 、 得られた DNA断片から当該遺伝子の cDNAを得ること力で きる。 プライマ一は、 好ましくは 5個以上の塩基からなるオリゴヌクレオチド、 より好ましくは 18〜25個の塩基からなるォリゴヌクレオチドが挙げられる。 ブラ ィマーの作製は、 当該分野で知られた方法で行うこと力くでき、 代表的にはフォス フォジエステル法、 フォスフォ トリエステル法、 フォスフォアミダイ卜法などに より合成でき、 例えば自動 DNA合成装置、 例えば、 model 381A DNA synthesizerThis search technique can be repeated. 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. If necessary, insert the DNA into the clone. Rows can be subcloned. It is also possible to obtain a gene encoding the N-Tes and a gene sequence related thereto based on the DNA sequence considered to have a novel gene that has been unwound in this manner. Another corner! Based on the new N-Tes and its related gene 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. It is possible to select and synthesize from the exon site at the 3 'end of the putative gene, and more preferably to use it for the synthesis of the sense primer. You can choose from other than 'places'. 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. Typically, the primer can be synthesized by a phosphodiester method, a phosphotriester method, a phosphoramidite method, and the like. Equipment, for example, model 381A DNA synthesizer
(Appl ied Bi osys terns) などを用いて合成できる。 cDNAライブラリーと前記のセ ンスプライマ一及びアンチセンスプライマ一を用いて PCRを行い、 cDNAを増幅す ることもできる。 また、 当該遺伝子の取得には、 上記のようにして同定されたク 口一ンから特異的なハイブリダイゼ一ションプ口一ブを調製し、 ヒト由来 DNA ラ イブラリ一をスクリーニングし、 プローブにハイブリダイズするクロ一ンを選択 することにより行うことができる。 プローブなどを放射性同位体などによって標 識するには、 市販の標識キット、 例えばランダムプライムド DM ラベリングキッ ト (Boehdnger Mannheim)などを! して行うこと力できる。 例えば、 random- ρ rimingキット （Pharmacia LKB， Uppsala) などを使用して、 プローブ用 DNAを [ a-3 2P] dCTP (Amer sham)などで標識し、 放射活性を持つプロ一ブを得ること力くで きる。 さらに麵として用いる cDNAライブラリ一は、 巿販の種々の紙熾由来 cDNAライ ブラリーを直 することもでき、 例えば Stratagene, Invi trogen, Clontech などから市販された cDNAライブラリ一を用いること力《できる。典型的な例では、 該標識 DNA断片とヒト紙織 ·細胞から調製した遺伝子ライブラリ一、 例えばヒト PI artif icial chromosomeジヱノミックライブラリー (Human Genome Mapping Re source Center), ヒト脳 cDNAライブラリ一 （例えば、 Clontechなどから入手可能 ) を用い、 ノ、イブリダィゼーシヨンを行う。 ヒト脳 cDNAライブラリ一は、 例えば 、 i gU0などのファージ中に構築することができ、 それを大腸菌 C600hil 株など の宿主大腸菌に感染させ、 ブラ一クを形成させて得ることができる。 必要に応じ て該ク口一ン中の cDNAの挿入配列はサブク口一ニングすることができる。 決定さ れた塩基配列を基にして、 目的とする DNAを単離することもできる。 (Applied Biosys terns). PCR can be carried out using the cDNA library and the above-mentioned sense primer and antisense primer to amplify the cDNA. To obtain the gene, 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. In order to label a probe or the like with a radioisotope or the like, a commercially available labeling kit such as a random primed DM labeling kit (Boehdnger Mannheim) can be used. For example, random-ρ riming kit (Pharmacia LKB, Uppsala) using such a DNA probe labeled with a [a- 3 2 P] dCTP ( Amer sham), cut with rather force to obtain a professional part with radioactivity. Further, the cDNA library used as 麵 can be prepared from various commercially available cDNA libraries. For example, 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.
塩基配列の決定は、 ダイデォキシ法、 例えば M13ダイデォキシ法など、 Maxam- Gi lbert法などを用いて行うこと力くできる力く、 市販のシ一クェンシングキット、 例えば Taqダイプライマ一サイクルシークェンシングキット （Appl ied Biosyste ms) 、 Sequenase v 2. 0 ki t などを用いたり、 自動塩基酉 ij決 置、 例えば蛍 光 DNA シーケンサ一装置 （例えば、 odel 373A, Appl ied Biosys terns) などを用 レ、て行うこと力く出来る。 ダイデォキシ法に用いられるポリメラ一ゼとしては、 例 えば、 DNA ポリメラーゼ Iのクレノー ·フラグメント、 層逆転写酵素、 Taq DN A ポリメラ一ゼ、 T7 DNAポリメラ一ゼ、 修飾 T7 DNA ポリメラ一ゼなどが挙げら れる。 本明細書中、 「ポリメラ一ゼ 'チェイン' リアクション (Polymerase Chain Re action) 」 又は 「PCR」 とは、 一般的に、 米国特許第 4683195号明細書などに記 載されたような方法を指し、 例えば、 所望のヌクレオチド配列をインビトロで酵 素的に増幅するための方法を指している。 一般に、 PCR法は、 铸型核酸と優先的 にハイプリダイズすることのできる 2個のオリゴヌクレオチドプライマ一を^ ffl して、 プライマ一伸長合成を行うようなサイクルを繰り返し ί亍うことを含むもの である。 典型的には、 PCR法で用いられるプライマーは、 ^内部の増幅される べきヌクレオチド配列に対して相補的なブラィマ一を使用すること力くでき、 例え ば、 該増幅されるべきヌクレオチド配列とその両端において相補的である力、、 あ るいは該増幅されるべきヌクレオチド配列に,しているものを好ましく使用さ れ得る。 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. Examples of 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. In the present specification, "Polymerase Chain Reaction" or "PCR" generally refers to a method as described in U.S. Pat. For example, it refers to a method for enzymatically amplifying a desired nucleotide sequence in vitro. In general, 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. Typically, 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.
PGR反応は、 当該分野で公知の方法あるいはそれと実質的に同様な方法や改変 法により行うことができる力く、 例えば R. Saiki, et al. , Science, 230： 1350， 1985 ; R. Saiki, et al. , Science, 239 : 487, 1988 ； H. A. Erl i ch ed.， PCR Technology, Stockton Press, 1989 ； D. M. Glover et al. ed.， "DNA Cloning", 2nd ed. , Vol. 1, (The Practical Approach Series), IRL Press, Oxford Universi ty Press (1995) ； M. A. Innis et al. ed. , "PCR Protocols : a guide to methods and appl ications", Academic Press, New Yor k (1990)) ; . J. McPherson, P. Quirke and G. R. Taylor (Ed. ), PCR: a pra ctical approach, IRL Press, Oxford (1991) ; M. A. Frohman et al. , Proc. N atl. Acad. Sci. USA, 85, 8998-9002 (1988) などに記載された方法あるいはそ れを修飾したり、 改変した方法に従つて行うこと力くできる。 また、 PCR法は、 そ れに適した市販のキットを用いて行うことができ、 キット製造業者あるいはキッ ト販売業者により明らかにされているプロトコルに従って実施することもできる  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. Quirke and GR Taylor (Ed.), PCR: apractical approach, IRL Press, Oxford (1991); MA Frohman et al., Proc. Natl. Acad. Sci. USA, 85, 8998- 9002 (1988), etc. or by modifying or modifying the method. In addition, the 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.
代表的な i ^には、 例えば となる 1st strand DNAとプライマーとを、 10 X 反応緩衝液 (Taq DNA ポリメラ一ゼなど DNA ポリメラーゼに添付されている） 、 dNTPs ( デォキシヌクレオシド三リン酸 dATP, dGTP, dCTP, (ΠΤΡの混合物） 、 Ta q DNAポリメラ一ゼ及び脱イオン蒸留水と混合する。 混合物を、 例えば、 GeneAm p 2400 PCR system, Perkin-Elmer/Cetus などの自動サーマルサイクラ一を用い て一般的な PCRサイクノレ条件下にそのサイクルを 25〜60回繰り返すが、 増幅のた めのサイクル数は舰目的に応じて適当な回数とすること力《できる。 PCRサイク ル条件としては、 例えば、 変性 90〜95°C 5〜100秒、 アニーリング 40〜60°C 5〜 150秒、 伸長 65〜75°C 30〜300秒のサイクル、 好ましくは変性 94 °C 15秒、 ァニ一リング 58 °C 15秒、 伸長 72 。C 45秒のサイクルが挙げられるが、 ァニ —リングの反応鍵及び時間は¾»験によって適当な値を選択できるし、 変性 反応及び伸長反応の時間も、 予想される PCR産物の鎖長に応じて適当な値を選択 できる。 アニーリングの反応 は、 通常プライマーと铸型 DNA とのハイブリツ ドの Tm値に応じて変えること力く好ましい。 伸長反応の時間は、 通常 lOOObpの鎖長 当たり 1 分禾號がおおよその目安であるが、 より短い時間を選択することも により可能である。 得られた PCR産物は、 通常 1~2¾ ァガロースゲル電気泳動にかけて、 特異な バンドとしてゲルから切り出し、 例えば、 gene clean ki t (Bio 101)などの市販 の抽出キットを用いて DNAを抽出する。 抽出された DNA は適当な制限酵素で切断 し、 必要に応じ精製処理したり、 さらには必要に応じ 5' ¾を T4ポリヌクレオチ ドキナーゼなどによりリン酸ィヒした後、 pUC18 などの pUC系べクタ一といった適 当なブラスミ ドべクターにライゲーシヨンし、 適当なコンビ一タント細胞を形質 転換する。 クロ一ニングされた PCR産物はその:!^ S配列を角晰される。 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 ΠΤΡ), 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.
また、 当該遺 fe^のェクソン部位のうち角斜斤した 5'端側のェクソン部位の塩基 配列に基づ 、てデザィンされたブラィマ一を利用して、 当該遺伝子の cDNAの 5'端 側の cDNAを取得し、 一方当該遺伝子のェクソン部位のうち角晰した 3'端側のェク ソン部位の塩基配列に基づいてデザィンされたプライマーを利用して、 当該遺伝 子の cDNAの 3'端側の cDNAを取得し、 次に必要に応じてこれらプライマーや得られ た当該遺伝子の cDNAの 5'端側の cDNA並びに 3'端側の cDNAの塩基配列の'髒 gを利用 してデザインしたプライマ一を用い、 ヒトの糸職、 特には、 ヒト脳糸職などから 単離した mRNAから逆転写酵素により作製された 1st strand cDNAを铸型にして PC R により増幅して、 当該遺 fe?の cDNAを得ることもできる。  In addition, based on the base sequence of the exon site at the 5'-end of the exon site of the exon of the subject fe, using the designed primer, 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. And then, if necessary, 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.
5'端側のブライマ一としては、 少なくとも開始コドンを含有する力、、 あるいは 該開始コドンを含めて増幅できるように選択し、 また 3'端側のブライマ一として は、 少なくともストップコドンを含有する力、、 あるいは該ストップコドンを含め て増幅できるように選択すること力 子ましい。 当該遺ィ 5?の全長の cDNAを得るに あたり、 PCR は上記したようにして行なうこと力でき、 また好ましい PCRサイク ル条件としては、 例えば、 変性 92〜95°C 10〜20秒、 アニーリング 55〜60°C 10 〜30秒、 伸長 65〜75°C 150〜300秒のサイクル、 より好ましくは変性 94 °C 15 秒、 ァニーリング 58 °C 15秒、 伸長 68 。C 4分のサイクルが挙げられる。 得られた PCR産物は、 上記と同様にしてクローニングしてその塩基配列を解析 され される。 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.
また決定された DNAの:^ S配列を基にプライマ一をデザインし、 これらプライ マーと各種の動物細胞由来の cDNAライブラリー （例えば、 各種のヒ卜細胞由来の cDNAライブラリ一） を用いて、 スクリーニングを fiうことにより、 同様に目的と するクローンを得ること力できる。 またこれらプライマ一を用いて PCR増幅を行 い、 目的とする遺伝子や、 新規な遺^、 それらの断片などを得ること力できる 。 こうして N - Tes と相同性を有するが、 配列カ渐規な PCR産物を検索することも できる。 本明細書中、 「ォリゴヌクレオチド」 とは、 比較的短 、一本鎖又は:！^:鎖のポ リヌクレオチドで、 好ましくはポリデォキシヌクレオチドが挙げられ、 Angew. C hem. Int. Ed. Engl. , Vol. 28, p. 716-734 (1989) に記載されているような既知 の方法、 例えば、 トリエステル法、 ホスファイ ト法、 ホスホアミダイ ト法、 ホス ホネート法などの方法により化学合成されること力できる。 通常合成は、 修飾さ れた固体 本上で合成を便利に行うこと力くできること力く知られており、 例えば 、 自動化された合成装置を用いて行うこと;^でき、 該装置は市販されている。 該 ォリゴヌクレオチドは、 一つ又はそれ以上の修飾された塩基を含有していてよく 、 例えば、 イノシンなどの天然にお 、ては普通でない塩基あるいはトリチル化さ れた塩基などを含有してし、てよ 、。  In addition, 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. In addition, 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. As used herein, "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. ,
得られた PCR産物をクローニングし、 得られた PCR産物の塩基配列を決定し、 新規な N- Tes及びそれに関連した遺伝子配列を有する DNA断片を取得することも できる。 また、 この DNA断片をプローブに同様にして種々の cDNAライブラリ一を スクリーニングし、 目的とする DNAを単離することもできる。 PCR産物のクロ一 ニングには、 例えば、 p- Direct (Clontech), pCR- Script™ SK(+) (Stratagene ), GBM-T (Pr omega), pAmp™ (Gibco- BRL)などの市販のプラスミ ドベクタ一を 用いることが出来る。 cDNAライブラリ一を構築するためには、 cDNAを人手する必要があるが、 これは 、 例えば次のようにして得られる。種々のヒトの！ Ιϋあるいは培養細胞から mRNA を単離する。 mRNAの単離は、 当該分野で公知の方法あるいはそれと実質的に同様 な方法や改変法により行うこと力くできる力く、 J. Sambrook et al. , "Molecular CI oning", 2nd ed. , Chapter 7, Cold Spring Harbor Laboratory, Cold Spring H arbor, N. Y. (1989) ； D. . Glover et al. ed. , "DNA Cloning", 2nd ed. , V ol. 1， (The Practi cal Approach Series), IRL Press, Oxford Universi ty Pre ss (1995) ； L. Grossman et al. ed.， "Methods in Enzymology", Vol. 12, Pa rt A & B, Academic Press, New York (1968)； S. L. Berger et al. ed.， "Met hods in Enzymology", Vol. 152, p. 33 & p. 215, Academic Press, New York (1 987) ; Biochemistry, 18 : 5294-5299， 1979などに記載の方法、 ί歹 [1えばグァニジン —塩化セシウム法、 チォシアン酸グァニジン法、 フエノール法などの方法で行う こと力出来る。 mRNAの単離に用いられるキットとしては、 例えば、 Pharmacia, S tratagene, Gibco- BRLなどから市販されているもの；^挙げられる。 必要に応じ、 得られた全 RNA はオリゴ (dT)—セルロースカラム、 スピンカラム、 オリゴ (dT)結 合磁性ビーズなどを ί®1して精製してポリ （A)+ mRNAを得ること力出来る。 この mRNA及び逆転写酵素 (RNA依存性 DNA ポリメラーゼ） を用いて、 cDNAを作製 する。逆転写反応では、 オリゴ (dT)プライマ一を用いることができる。 オリゴ (d T)プライマ一は、 好適には 12〜18個の T残基を持つものカ^ fflできる。指向性ク ローニングを行う には、 12〜18個の T残基の 5'側に制限酵素部位を 結した 合成ォリゴヌクレ才チドプライマ一を用いることも好ましい。 こうしたプライマ 一の例としては、 Xba I オリゴ (dT)プライマーアダプターなどが挙げられる。 ま サマープライマーを用いると、 mRNAの 5'末端側が得られる可能性 力く増大し、 このランダムへキサマープライマーは期虫で、 あるいはオリゴ (dT)プ ライマ一と混合して できる。 逆転写 では、 必要に応じて RNase阻害剤、 例えば、 RNasin (Boehringer Mannheim)を加えること力くできる。 mRNA及び逆転写 酵素を用いての cMA合成は当該分野で公知の方法あるいはそれと実質的に同様な 方法や改変法により行うことができる力く、 H. Land et al. , Nucleic Acids Res. ， 9 : 2251, 1981 ; U. Gubler et al. , Gene, 25 : 263-269, 1983 ; S. L. Berge r et al. ed. , "Methods in Enzymology", Vol. 152, p. 307, Academic Press, New York (1987) などに記載の方法が挙げられる。 得られた cDNAはそれを基にして、 ファージベクタ一、 プラスミ ドベクターを使 用するなどして cDNAライブラリーを構築できる。 またファージベクタ一をィ¾¾す る以外で、 昜菌などの宿主細胞の形質転換をするには、 例えばカルシウム法、 ルビジウム カルシウム法、 カルシウム/マンガン法、 TFB高効 去、 FSB 凍結 コンビテント細胞法、 迅速コロ二一法、 エレクト口ポレーシヨンなど当該分野で 知られた方法あるいはそれと実質的に同様な方法で行うこと力できる （D. Hanah an, J. Mol. Biol. , 166 : 557， 1983 など） 。 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. In the same manner, 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 ™ SK (+) (Stratagene), GBM-T (Promega), pAmp ™ (Gibco-BRL). Can be used. In order to construct a cDNA library, it is necessary to manually prepare cDNA, which can be obtained, for example, as follows. Various human! Ιϋ Alternatively, isolate mRNA from cultured cells. 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. 12, Part A & B, Academic Press, New York (1968); SL Berger et al. ed., "Met hods in Enzymology", Vol. 152, p. 33 & p. 215, Academic Press, New York (1 987); Biochemistry, 18: 5294-5299, 1979. For example, 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; ^. If necessary, the total 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). In the reverse transcription reaction, an oligo (dT) primer can be used. Oligo (dT) primers are preferably those having 12 to 18 T residues. To perform directional cloning, it is also preferable to use a synthetic oligonucleotide primer having a restriction enzyme site linked to the 5 'side of 12 to 18 T residues. One example of such a primer is an Xba I oligo (dT) primer adapter. The possibility of obtaining the 5 'end of mRNA using summer primers Strongly amplified, this random hexamer primer can be formed in a worm or mixed with an oligo (dT) primer. For reverse transcription, an RNase inhibitor, such as RNasin (Boehringer Mannheim), can be added as needed. 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. Ed., "Methods in Enzymology", Vol. 152, p. 307, Academic Press, New York (1987). Based on the obtained cDNA, a cDNA library can be constructed by using a phage vector or a plasmid vector. In addition to using phage vectors, 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.) ).
目的とする DMを単離するためには、 逆転写 PCR (polymerase chain reaction coupled reverse transcript ion; RT-PCR) 、 RACE urapid ampl if icat ion of c DNA ends) を適用することが出来る。 RACEは、 例えば、 M. A. Innis et al. ed. ， "PCR Protocols" (M. A. Frohman, "a guide to methods and appl ications ') , pp. 28-38, Academic Press, New York (1990) などに記載された方法に従って 行うことができる。 RT- PCR産物はプラスミ ドベクタ一にクロ一ニングすることが でき、 それを高効率のコンビ一タント細胞に導入できる。  In order to isolate the target DM, reverse transcription PCR (polymerase chain reaction coupled reverse transcript ion; RT-PCR) and RACE urapid amplification of cDNA ends can be applied. RACE is described, for example, in MA Innis et al. Ed., "PCR Protocols" (MA Frohman, "a guide to methods and applications"), pp. 28-38, Academic Press, New York (1990). The RT-PCR product can be cloned into a plasmid vector, which can be introduced into highly efficient recombinant cells.
更に、 1®の細胞あるいは糸職から mRNAを単離精製できる方法、 例えば、 REX ki t, Uni ted States Biochemical； Glass MAX™ RNA spin cartridge system, Gibco-BRL などの市販のキットを利用し、 得られた mRNAをォリゴ (dT)プライマー を用いて逆転写して、 1st strand DNAを合成し、 ついで 1st strand DNAの 3'末端 にホモポリマーテール（例えば、 G残基） を付けた後、 あるいは該 DNAにァダプ ターを付けた後、 オリゴ (dT)プライマー-とオリゴ (dC)プライマ一あるいはァダプ タ一プライマ一を用いて cDNAを PCR増幅することもできる。 これに適した市販の キットとしては、 Superscript™ pre- ampl if ication system (Gibco-BRL) ; cDN A Cycle™ ki t (Invi trogen) などが挙げられる。 ノ、ィブリダイゼ一ションは、 所定の挿入 DNAを保持するなどしてし、る aife物に より形成されたプラーク （核酸自体でもよい） をナイロンフィルタ一などの膜に 車云写せしめ、 必要に応じ変)«理、 固定化処理、 洗浄処理などを施した後、 その 膜に転写せしめられたものを、 必要に応じ変成させた標識プローブ DNA断片と、 ハイプリダイゼ一ション用バッファ中で反応させて行われる。 ハイブリダイゼー シヨン処理は、 普通約 35°C〜約 80°C、 より好適には約 50°C〜約 65°Cで、 約 15分〜 約 36時間、 より好適には約 1 時間〜約 24時間行われる力 «：最適な条件を選択 して行うことができる。 例えば、 ハイブリダイゼ一ション処理は、 約 55°Cで約 18 時間行われる。 ハイブリダイゼ一シヨン用バッファとしては、 当該分 Ifで普通に ィ されるものの中から選んで用いることができ、 例えば、 Rapid hybridizatio n buffer (Amersham) などを用いること力《できる。 転写した膜の変成処理として は、 アルカリ変性液を する方法が挙げられ、 その処理後中和液や緩衝液で処 理するのカ 子ましい。 また膜の固定化処理としては、 普； S約 40°C〜約 100° よ り好適には約 70°C〜約 90°Cで、 '約 15分〜約 24時間、 より好適には約 1 時間〜約 4 時間べーキングすることにより行われるが、 子ましい条件を選択して行うこ と力くできる。 例えば、 フィルタ一を約 80°Cで約 2時間べ一キングすることにより 固定化が行われる。 転写した膜の洗浄処理としては、 当該分野で普通に使用され る洗浄液、 例えば 1M NaCl, lm BDTAおよび 0. 1% sodium dodecyl sulfate (S DS) 含有 50mM Tris - HC1緩衝液， H8. 0 などで洗うことにより行うこと力くできる 。 ナイロンフィルタ一などの膜としては、 当該分野で普通に使用されるものの中 から選んで用いること力くでき、 例えば、 ナイロンフィルタ一 [ハイボンド （Hybo nd) - N、 Amersham] などを挙げること力《できる。 上記アルカリ変性液、 中和液、 緩衝液としては、 当該分野で普通に使用される ものの中から選んで用いること力くでき、 アルカリ変性液としては、 例えば、 0. 5M NaOHおよび 1. 5M NaCl を含有する液などを挙げること力でき、 中和液としては 、 例えば、 1. 5M NaCl 含有 0. 5M Tris-HCl 緩衝液， ρΗ8. 0 などを挙げることが でき、 緩衝液としては、 例えば、 2 XSSPE (0. 36Μ NaCl 20慮 NaH2P04および 2m M EDTA) などを挙げること力《できる。 またハイブリダイゼ一ション処理に^:ち 寺異的なハイブリダイゼーション反応を防ぐために、 必要に応じて転写した 膜はプレハイブリダィゼーシヨン処理すること力く好ましい。 このプレハイブリダ ィゼ一ション処理は、 例えば、 プレハイブリダイゼーション溶液 [50% formami de 5 XDenhardt s溜夜 (0. 2 %ゥシ血清アルブミン、 0. 2 % polyvinyl pyrro l idone), 5 XSSPE, 0. 1 % SDS, 100 ^g/ml熱変性サケ精子 DNA]などに浸し、 約 35°C〜約 50°C、 好ましくは約 42°Cで、 約 4〜約 24時間、 好ましくは約 6〜約 8 時 間反応させることにより行うこと力できる力 こうした条件は当業者であれば適 宜実験を繰り返し、 より好ましい条件を決めること力くできる。 ハイブリダィゼ一 シヨンに用いる標識プローブ DNA断片の は、 例えば、 約 70°C〜約 100 °C、 好 ましくは約 100 °Cで、 約 1分間〜約 60分間、 好ましくは約 5分間加熱するなどし て行うこと力くできる。 なお、 ハイブリダイゼ一ションは、 それ自体公知の方法あ るいはそれに準じた方法で行うことができるが、 本明細書でストリンジヱン卜な 条件とは、 例えばナトリゥム濃度に関し、 約 15〜約 50mM、 好ましくは約 19〜約 40 mM、 より好ましくは約 19〜約 20mMで、 温度については約 35〜約 85°C、 好ましくは 約 50〜約 70°C、 より好ましくは約 60〜約 65°Cの条件を示す。 、ィブリダイゼーション完了後、 フィルタ一を十分に洗浄処理し、 特異的な ィブリダイゼーション反応をした標識プローブ DNA断片以外の標識プローブを取 り除く。 フィルターの洗浄処理は、 当該分野で普通に使用されるものの中から選 んで用いて行うこと力くでき、 例えば、 0. 1 % SDS含有 0. 5 XSSC ( 0. 15M NaCU 15mM クェン酸） 溜夜などで洗うことにより実施できる。 Furthermore, 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 ™ 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. Commercially available kits suitable for this include Superscript ™ pre-amplification system (Gibco-BRL); cDNA Cycle ™ kit (Invitrogen) and the like. In the hybridization, 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. Is The hybridization process is usually performed at about 35 ° C. to about 80 ° C., more preferably about 50 ° C. to about 65 ° C., for about 15 minutes to about 36 hours, more preferably about 1 hour to about 24-hour force «: It can be done by selecting the optimal conditions. For example, the hybridization process is performed at about 55 ° C for about 18 hours. As the buffer for hybridization, it is possible to use a buffer selected from those commonly used in the corresponding If. For example, it is possible to use 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. to about 100 ° C., more preferably about 70 ° C. to about 90 ° C., for about 15 minutes to about 24 hours, more preferably about It is performed by baking for 1 hour to about 4 hours, but it can be more powerful to select appropriate conditions. For example, immobilization is performed by baking the filter at about 80 ° C for about 2 hours. 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. As the membrane of the nylon filter or the like, it is possible to use a membrane selected from those commonly used in the field. For example, the membrane of the nylon filter [Hybond-N, Amersham] 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, ρΗ8.0, and the like. as the liquid, for example, 2 XSSPE (0. 36Μ NaCl 20 consideration NaH 2 P0 4 and 2m M EDTA) that force the like can ". In addition, in order to prevent an unusual hybridization reaction in the hybridization treatment, it is strongly preferable that 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. at about 35 ° C to about 50 ° C, preferably about 42 ° C, for about 4 to about 24 hours, preferably about 6 to about The force that can be achieved by reacting for 8 hours Under these conditions, those skilled in the art can determine the more preferable conditions by repeating experiments as appropriate. 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. About 19 to about 40 mM, more preferably about 19 to about 20 mM, and a temperature of about 35 to about 85 ° C, preferably about 50 to about 70 ° C, more preferably about 60 to about 65 ° C. Is shown. After the completion of the hybridization, the filter is thoroughly washed, and the labeled probe other than the labeled probe DNA fragment that has undergone the specific hybridization reaction is removed. Washing of the filter can be carried out using a selection from those commonly used in the field, for example, 0.5 XSSC (0.15M NaCU 15mM citrate) containing 0.1% SDS. It can be carried out by washing with etc.
ハイブリダイズしたブラ一ク （核酸） は、 代表的にはォ一トラジォグラフィ一 により検出することができる力、 当該分野で用いられる方法の中から «：選択し てプラーク （核酸） 検出に用いることもできる。 検出したシグナルに相当するプ ラ一ク （核酸） を、 適切な緩衝液、 例えば、 SM激夜 ( lOOmM NaCl および 10mM M gS04含有 50慮 Tris-HCl 緩衝液、 H7. 5 ) などに懸濁し、 ついでこのファージ （ 核酸自体を含めてよい） 懸濁液を適度に希釈して、 大腸菌に感染させ、 得られた 大腸菌を培養して、 その培養された大腸菌から目的組換え体ファージ （核酸） を 得る。 なお、 必要に応じて上記プローブ DNAを删して、 ハイブリダィゼ一ショ ン処理により遺伝子ラィブラリ一や cDNAラィブラリ一から目的組換え体ファ一ジ (核酸） をスクリーニングする処理は、 繰り返して行うこと力くできる。 また目的 組換え体ファージ （核酸） は、 ±咅養された大腸菌から抽出処理、 遠心分離処理な どを施して得ることができる。 得られたファージ粒子は、 当該分野で普通に棚される方法で精製分離するこ と力くでき、 例えば、 グリセロールグラジェント纖心分離法 (Molecular clonin g, a laboratory manual, ed. T. Maniati s, Cold Spring Harbor Laboratory, 2nd ed. 78, 1989) などにより精製すること力《できる。 ファージ粒子からは、 当 該分野で普通に使用される方法で DNAを精製分離することができ、 例えば、 得ら れたファージを TM溶液 (10mM MgS04含有 50mM Tris-HCl 緩衝液、 H7. 8 ) などに 懸濁し、 DNase I および RNase Aなどで処理後、 20mM BDTA、 50 xg/ml Protein ase K及び 0. 5 %SDS混合液などを加え、 約 65° (、 約 1 時間保温した後、 これを フエノール抽出ジェチルエーテル抽出後、 エタノ一ル沈殿により DNAを沈殿させ 、 次に得られた DNAを 70%エタノールで洗浄後乾燥し、 TE鎌 (lOmM EDTA含有 10mM Tri s-HCl 緩衝液、 H8. 0 ) に溶解するなどして得られる。 また、 目的とし てし、る DNA は、 サブク口一ニングなどにより に得ることも可能であり、 例え ばサブク口一ニングは、 宿主として大腸菌を用いブラスミ ドべクタ一などを用し、 て行うこと力くできる。 こうしたサブクロ一ニングにより得られた DNA も、 上記と 同様にして遠心分離、 フエノール抽出、 エタノール沈殿などの方法により精製分 離できる。 こうして本発明に従って、 目的とする DNAを含有するクローン （例えば、 組換 ぇ体ファ一ジなどとして) を得ることができる。 例えば、 このクローン化した組 換え体ファージより単離された DNAィンサ一卜の配列^された塩基配列の全長 は 1510bpであり、 その配列は配列表の配列番号： 1で示されたもの力く得られて 、 ること力認められる。 同定された DNA配列中には、 推定 313個のアミノ酸をコー ドするオープンリ一ディングフレームの存在が認められ、 その推定されるァミノ 酸配列は、 配列表の配列番号： 2で示されるようなものと認められる。 GenBank ™ /EMBL DNA Data Baseなどを使用したホモロジ一検索の結果、 Testican-1 (Ge nBank Accession No. AF231124)、 Testican-2 (GenBank Accession No. AJ0014 53)、 Testi can-3 (GenBank Accession No. MJ001454)および HPTLG (蘭/ 231 10) のアミノ酸配列と高い相同性が認められた力、 その C末端側には特徵的な配 列 Gly3 1 ' - Lys3 1 2-Arg3 1 3を有しており、 さらに Testi can- 3や HPTLG に存在する T Y domainや CWCV domainを欠いたものであって、 また Testican- 3や HPTLG に存在 している Glycosaminoglycan attachment si te を欠いているとの樹敫カく認められ た。 この推定されるタンパク質は、 新規なヒト Testican類の一つであり、 それを ΓΝ-Tes 」 と名付けた。 そして、 N-Tes遺^は、 新規な Testi canフアミリーに 属するポリペプチドをコードしていることは明白であり、 N- Tes遺 feiを用いて 作製した組換え体プラスミ ドは全て新規な組換え体であり、 そのプラスミ ドで形 質転換あるいはトランスフヱタトされ得られた形質転換体あるいはトランスフエ 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. It is to be noted that, if necessary, 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. In addition, 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). From phage particles, 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 .. For the purpose of the present invention, 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. Thus, according to the present invention, a clone containing the target DNA (for example, For example, a recombinant phage) can be obtained.For example, 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. In the identified DNA sequence, the presence of an open reading frame encoding an estimated 313 amino acids was confirmed. The deduced amino acid sequence was as shown in SEQ ID NO: 2 in the sequence listing. Is recognized. As a result of homology search using GenBank ™ / EMBL DNA Data Base, etc., Testican-1 (GenBank Accession No. AF231124), Testican-2 (GenBank Accession No. AJ0014 53), Testican-3 (GenBank Accession No. MJ001454) and HPTLG (Ran / 231 10) have high homology to their amino acid sequences, and have a special sequence Gly 3 1'-Lys 3 12- Arg 3 13 at the C-terminal side. It also 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 ΓΝ-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.
)新規なものである。 配列表の配列番号: 1で示される塩基配列の^^あるいは一部を有する核酸は、 ィ匕学合成によって得ることも可能である。 その: !f^断片を化学合成し、 それらを 酵素により結合することによつてもよい。 また、 ィ匕学合成断片を上記したように して、 プライマーあるいはプローブとして用いて目的とする酉己列を得ることも可 能である。 PCR法で用いるプライマ一としては、 上記の部位を含む DNA断片を増 幅できるものであれば、 特に限定されない。 代表的には、 プライマ一は （a)配列 表の配列番号: 1に示された塩基配列のうちの任意の領域に相当する塩基配列を有 するォリゴヌクレオチド及び )配列表の配列番号: 1に示された塩基配列のうち の任意の領域に対する相補塩基配列を有するォリゴヌクレオチドを ί¾¾すること 力《でき、 より好ましくは (1) 配列表の配列番号: 1に示された塩基配列のうちの 5' 端側の任意の領域に相当する塩基配列を有するォリゴヌクレオチド及び （2)配列 表の配列番号: 1に示された塩基配列のうちの 3'端側の任意の領域に対する相補塩 基配列を有するオリゴヌクレオチドを使用すること力でき、 例えば、 3〜100個 、 好ましくは 10〜50個、 さらに好ましくは 15〜35個のヌクレオチドを含有するも の力挙げられる。 また、 PCR条件も特に限定されず、 通常行われる公知の条件で よく、 例えば、 上記した文献の記載を参考に選択すること力くできる。 PCR におい ては、 DNA鎖の熱変性、 ブラィマーの了ニーリング及びポリメラーゼによる相補 鎖の合成からなる一つのサイクルが、 例えば、 10〜50回、 好ましくは 20〜35回、 より好ましくは 25〜30回繰り返して行われる。 本発明で得られた DNA断片を、 下記で詳しく説明するような適当なベクタ一、 例えば、 プラスミ ド pEX、 pMAMneo、 pKG5などのベクダ一に糸1¾み、 下記で詳し く説明するような適当な宿主細胞、 例えば、 大腸菌、 酵母、 CH0細胞、 COS細胞 などで発現させること力できる。 また、 該 DNA断片は、 そのままあるいは適当な 制御配列を付加した DNA断片として、 または適当なベクタ一に糸 み、 そして動 物に導入して、 N-Tes遺伝子、 例えば、 N-Tes を発現するトランスジヱニック動 物を作成すること；^できる。 動物としては、 哺乳動物力く挙げられ、 例えば、 マウ スヽ ラット、 ゥサギ、 モルモット、 ゥシなどが挙げられる。 好ましくは、 マウス などの動物の受精卵に該 DM断片を導入して、 トランスジヱニック動物を作成す ることができる。 ) It is new. 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. The: You can also chemically synthesize! F ^ fragments and link them with enzymes. In addition, 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. Typically, 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. An oligonucleotide having a nucleotide sequence corresponding to an arbitrary region at the 5 'end of the oligonucleotide and (2) the sequence It is possible to use an oligonucleotide having a complementary base sequence to an arbitrary region on the 3 ′ end side of the base sequence shown in SEQ ID NO: 1 in the table, for example, 3 to 100, preferably 10 to Those containing 50, more preferably 15 to 35 nucleotides. In addition, the PCR conditions are not particularly limited, and may be well-known conditions that are usually performed. 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. Create transgenic animals; Examples of animals include mammals such as mouse rats, egrets, guinea pigs, and sea lions. Preferably, a transgenic animal can be prepared by introducing the DM fragment into a fertilized egg of an animal such as a mouse.
N-Tes遺伝子産物の確認を、 N-Tes遺伝子をトランスフエクシヨンした、 293T 細胞、 COS 1 細胞などのそれに適した動物細胞などを用いて行うこと力できる。 この外«伝子を哺乳動物などの動物細胞に導入する方法としては当該分野で知 られた方法あるいはそれと実質的に同様な方法で行うこと力でき、 例えばリン酸 カノレシゥム法 （例えば、 F. L. Graham et al.， Virology, 52: 456, 1973など） 、 DEAE- デキストラン法 （例えば、 D. Warden et al.， J. Gen. Virol. , 3 : 371 , 1968など） 、 エレクトロボレ一シヨン法 （例えば、 E. Neumann et al. , ΒΜΒΟ J, 1 : 841, 1982 など） 、 マイクロインジヱクシヨン法、 リボソーム法、 ウィル ス感染法、 ファージ粒子法などが挙げられる。 こうして N- Tes遺伝子をトランス フエクションされた動物細胞の産生する遺伝子産物は、 それを角浙することもで きる c 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. As 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., ΒΜΒΟ 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
N-Tes遺伝子など （本発明で得られた DNAなど） を, ffiAむプラスミ ドとしては 遺伝子工学的に常用される宿主細胞 （例えば、 昜菌、 枯草菌等の原核細胞宿主 、 酵母、 CH0細胞、 COS細胞等の真核細胞宿主、 Sf21等の昆虫細胞宿主） 中で該 DNA力く発現できるプラスミ ドであればどのようなプラスミ ドでもよい。 こうした 配列内には、 例えば選択した宿主細胞で発現するのに好適に修飾されたコドンが 含まれていることができるし、 制限酵素部位が設けられていることもできるし、 目的とする遺伝子の発現を^ にするための制御配列、 促進配列など、 目的とす る遺伝子を結合するのに役立つリンカ一、 アダプタ一など、 さらには抗生物質耐 性などを制御したり、 代謝を制御したりし、 選別などに有用な配列 （ハイプリ ド タンパク質や融合タンパク質をコードするものも含む） 等を含んでいることがで きる。 The N-Tes gene or the like (such as the DNA obtained in the present invention) 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) And 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. And sequences useful for selection and the like (including those coding for hybrid proteins and fusion proteins).
好ましくは、 適当なプロモータ一、 例えば 昜菌を宿主とするプラスミ ドでは 、 トリプトファンプロモータ一（trp) 、 ラクトースプロモータ一（lac) 、 トリプ トフアン 'ラク ト一スプロモータ一（tac) 、 リポプロテインプロモーター（lpp) 、 スファージ PL プロモータ一等を、 動物細胞を宿主とするプラスミ ドでは、 SV 40レートプロモータ一、 MMTV LTRプロモーター、 RSV LTR プロモータ一、 CMV プ 口モータ一、 SR プロモーター等を、 酵母を宿主とするプラスミ ドでは、 GAL1、 GAL10 プロモータ一等を删し得る。 大腸菌を宿主とするプラスミ ドとしては、 例えば 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 ™、 (Stratagen e)などが挙げられる。 大腸菌での発現に適したプラスミ ドベクタ一としては、 pA S、 pKK223 (Pharmacia), pMC1403、 pMC931、 pKC30、 pRSBT-B (Invi trogen)な ども挙げられる。 動物細胞を宿主とするプラスミ ドとしては、 SV40ベクタ一、 ポ リオ一マ ·ウィルスベクタ一、 ワクシニア ·ウィルスベクター、 レトロウイルス ベクタ一などが挙げられ、 例えば pcD、 pcD - SR 、 CDM8、 pCEV4、 pME18S、 pBCl 2BI 、 pSG5 (Stratagene) などが挙げられる。 酵母を宿主とするプラスミ ドとし ては、 Yip型べクタ一、 YEp型べクタ一、 Y p型べクタ一、 YCp型べクタ一など 力く挙げられ、 例えば pGPD-2などが挙げられる。 宿主細胞としては、 宿 ±田胞が大 腸菌の: lf^、 例えば大腸菌 K12株に由来するもの力挙げられ、 例えは 533、 XL 1- Blueヽ C600、 DH1、 DH5、 DH11S、 DH12S、 DH5 o;、 DH10B、 HB101、 MC1061 、 JM109、 STBL2、 B834株由来としては、 BL21(DE3)pLysSなどが挙げられる。 宿 主細胞が動物細胞の:^、 例えばアフリカミ ドリザル線維芽細胞由来の COS- 7細 胞、 COS- 1 細胞、 CV- 1細胞、 マウス線維芽細胞由来の COP細胞、 MOP細胞、 W0P 細胞、 チャイニーズ'ハムスター細胞由来の CH0細胞、 CHO DHFR-細胞、 ヒト He La細胞、 マウス細胞由来 C127細胞、 マウス細胞由刺 IH 3T3細胞などが挙げられ る。 昆虫細胞としては、 カイコ核多角体病ウィルス （Bombyx mori nuclear poly hedrosis virus) あるいはそれに由来するものをベクターとし、 カイコ幼虫ある いはカイコ培養細胞、 例えば BM - N細胞などを用いること力挙げられる。植物細胞 を宿主細胞として使用することも可能であり、 それに適するベクタ一と共に、 そ れらは当該分野で広く知られている。 本発明の遺伝子工学的手法にぉ 、ては、 当該分野で知られたあるいは汎用され ている制限酵素、 逆転写酵素、 DNA断片をクローン化するのに適した構造に修飾 したりあるいは変換するための酵素である DNA修飾 ·分解酵素、 DNAポリメラ一 ゼ、 末端ヌクレオチジルトランスフェラーゼ、 DNA リガーゼなどを用いることが 出来る。 制限酵素としては、 例えば、 R. J. 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 ; R. J. Roberts, D. Mace l is, Nucle ic Acids Res. , 19 : Suppl. 2077, 1991などに記載のもの力く挙げられる。 逆転写 酵素としては、 例えばマウスモロネィ白血病ウィルス （mouse Moloney leukemia virus ; MMLV) 由来の逆転写酵素 (reverse transcriptase)^ ニヮトリ骨髄芽球 症ウィルス （avian myeloblastosis virus ; AMV)由来の逆転写酵素など力く挙げら れる。 逆転写酵素は、 RNase H欠損体などは好ましく用いること力でき、 特には RNase H活性を欠いた修飾 MMLV RT力く好ましく翻でき、 さらには熱安定性の高 いもの力 子ましい。 適した逆転写酵素としては、 MMLV RT (Gibco-BRL) 、 Supers cript RT plus (Li fe Technologies) などが挙げられる。 Preferably, 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 ™, (Stratagene) and the like. Plasmid vectors suitable for expression in E. coli include pAS, pKK223 (Pharmacia), pMC1403, pMC931, pKC30, pRSBT-B (Invitrogen), and the like. 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. As 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. RJ Roberts, D. Macelis, Nucleic Acids Res., 19: Suppl. 2077, 1991. Reverse transcriptases include, for example, reverse transcriptase derived from mouse Moloney leukemia virus (MMLV) ^ reverse transcriptase derived from avian myeloblastosis virus (AMV). Is received. As the reverse transcriptase, an RNase H-deficient mutant or the like can be preferably used. The power of things is strong. Suitable reverse transcriptases include MMLV RT (Gibco-BRL), Superscript RT plus (Life Technologies) and the like.
DNAポリメラーゼとしては、 例えば大腸菌 DNA ポリメラーゼ、 その誘 本であ るクレノウ 'フラグメント、 大腸菌ファ一ジ T4 DNAポリメラーゼ、 大腸菌ファ― ジ T7 DNAポリメラ一ゼ、 耐熱菌 DNA ポリメラ一ゼなどが挙げられる。 末端ヌクレ ォチジルトランスフェラ一ゼとしては、 例えば R. Wu et al. ed.， "Methods in Enzymology", Vol. 100, p. 96， Academic Press, New York (1983) に記載の 3， -OH末端にデォキシヌクレオチド (d丽 P)を付加する TdTaseなどが挙げられる。 DN A修飾 ·分解酵素としては、 ェキソヌクレアーゼ、 エンドヌクレア一ゼなどが挙 げられ、 例えばへビ毒ホスホジエステラーゼ、 脾臓ホスホジエステラーゼ、 大腸 菌 DNAェキソヌクレア一ゼ I、 大腸菌 DNAェキソヌクレア一ゼ I I I 、 大腸菌 DNA ェキソヌクレア一ゼ VI I、 スェキソヌクレア一ゼ、 DNase I 、 ヌクレア一ゼ Sl、 ミクロコッカス (Micrococcus) ヌクレアーゼなど力く挙げられる。 DNA リガーゼ としては、 例えば:^昜菌 DNA リガーゼ、 T4 DNAリガ一ゼなどが挙げられる。 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 丽 P) to the OH terminus is exemplified. Examples of 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.
DNA遺伝子をクローニングして DNA ライブラリ一を構築するのに適したべクタ —としては、 プラスミ ド、 スファ一ジ、 コスミ ド、 P1ファージ、 F因子、 YAC な どが挙げられ、 好ましくはスファ一ジ由来のべクタ一が挙げられ、 例えば Charon 4A、 Charon 21 Aヽ A tlO, A gtlU DASH I K A FIXI I , ス EMBL3、 λ ΖΑΡΙ Ι ™ (Stratagene) などが挙げられる。 本発明のタンパク質をコードする核酸を含有する発現ベクターで形質転換され た形質転換体は、 必要に応じて適当な選択マ一力一を用い、 繰り返しクローニン グを行うことにより、 高い発現能を安定して有する細胞株を得ること力くできる。 例えば、 宿主細胞として動物細胞を用いた形質転換体において、 dhfr遺伝子を選 択マ一力一として利用した:^、 MTX濃度を徐々に上げて培養し、 耐性株を選択 することにより、 本発明のタンパク質をコードする DNAを増幅させ、 より高い発 現を得られる細胞株を得ること力くできる。 本発明の形質転換体は、 本発明のタン パク質をコードする核酸が発現可能な条件下で培養し、 目的物を生成、 蓄積せし めること力できる。 該形質転換体は、 当該分野で沉用されている培地中で培養す ることができる。 例えば、 大腸菌、 枯草菌等の原核細胞宿主、 酵母などを宿主と している形質転換体は、 液体培地を好適に使用すること力できる。 培地中には、 該形質転換体の生育に必要な炭素源、 窒素源、 無 «その他が含有せしめられる 。 炭素源としては、 たとえばグノレコース、 デキストリン、 可溶性澱粉、 ショ糖な ど、 窒素源としては、 たとえばアンモニゥム 、 硝酸 ί¾|、 コーンスチープ' リカ一、 ペプトン、 カゼイン、 肉エキス、 麦芽エキス、 大豆粕、 ノ レイショ抽出 液などの無機または有»質、 無機物としては， 例えば、 塩化カノレシゥム、 リン 酸二水素ナトリウム、 塩化マグネシウム、 炭酸カルシウムなどが挙げられる。 ま た、 酵母、 ビタミン類、 カザミノ酸、 生長促進因子などを添加してもよい。 また 、 必要によりプロモーターを効率よく働力、せるために、 例えば、 3 一インドリ ル ァクリノレ酸のような薬剤を加えることができる。 培地の ρΗは約 5〜 8力望ま しい。 . 培養は、 例えば大腸菌では通常約 15〜約 45°Cで約 3〜約 75時間行い、 必要によ り、 通気や攪拌を加えることもできる。 宿主が動物細胞である形質転換体を培養 する際、 培地としては、 たとえば約 5〜約 20%の胎児牛血清を含む MEM ±咅地、 PR MI1640培地、 DMEM培地などが用いられる。 pHは約 6〜約 8であるの力 子ましい。 培養は通常約 30°C〜約 40°Cで約 15〜約 72時間行 必要に応じて通気や攪拌を加 える。 上記培養細胞から抽出するに際しては、 ±咅體、 公知の方法で菌体あるい は細胞を集め、 これを適当な緩衝液に懸濁し、 超音波、 リゾチームおよび/また は凍結融解などによって菌体あるいは細胞を破壊したのち、 遠心分離やろ過によ り粗抽出液を得る方法などを itMいることができる。 緩衝液の中には尿素や塩 酸グァニジンなどの蛋白変性剤や、 トリ トン X- 100 (商品名） 、 ッウイーン - 80Suitable 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 For example, Charon 4A, Charon 21A ヽ AtlO, AgtlU DASH IKA FIXI I, EMBL3, λΖΑΡΙΖΑΡΙ ™ (Stratagene) and the like. 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. For example, in a transformant using an animal cell as a host cell, 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. For example, 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 Examples of the inorganic or inorganic substances such as the potato extract solution include canoledium chloride, sodium dihydrogen phosphate, magnesium chloride, calcium carbonate, and the like. In addition, yeast, vitamins, casamino acids, growth promoting factors and the like may be added. If necessary, a drug such as 3-indolylacrylinoleic acid can be added in order to make the promoter work efficiently. About 5 to 8 ρ 培 地 of the medium is desirable. For example, 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. When culturing a transformant in which the host is an animal cell, for example, 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. When extracting from the above cultured cells, 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. Alternatively, 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.
(商品名） などの界面活性剤を加えてあってもよい。 培養液中に目的生成物力く分 泌される齢には、 培養終了後、 それ自体公知の方法で菌体あるいは細胞と上清 とを分離し、 上清を集める。 このようにして得られた培養上清、 あるいは抽出液 中に含まれる目的生成物は、 自体公知の分離 ·精製法を適切に組み合わせてその 精製を行なうこと力でき、 例えば硫酸ァンモニゥム沈殿法などの塩析、 セフアデ ックスなどによるゲルろ過法、 例えばジェチルァミノェチル基あるいはカルボキ シメチル基などを持つ担体などを用いたィォン交換ク口マトグラフィ一法、 例え ばブチル基、 ォクチル基、 フェニル基など疎水性基を持つ担体などを用 、た疎水 性クロマトダラフィ一法、 色素ゲルク口マトグラフィ一法、 電気泳動法、 透析、 限外ろ過法、 ァフィ二ティ 'クロマトグラフィ一法、 高速液体ク口マトグラフィ 一法などにより精製して得ることができる。 好ましくは、 ボリァクリルアミ ドゲ ル電気泳動、 リガンドなどを固定化したァフィニティー'クロマトグラフィ一な どで処理し精製分離処理できる。 例えば、 ゼラチンーァガロース 'ァフィ二ティ 一'クロマトグラフィー、 へハ。リンーァガロース 'クロマトグラフィ一などが '挙 げられる。 さらに、 本発明に係わる N- Tes の遺伝子塩基配列を基に遺伝子工学的に常用さ れる方法を用いることにより、 N- Tes のァミノ酸配列中に適宜、 1個ないし複数 個以上のアミノ酸の置 ^ 欠失、 挿入、 転移あるいは付加したごとき変異を導入 した相当するタンパク質を製造することができる。 こうした変異 ·変換 ·修飾法 としては、 日本生化学会編、 「続生化学実験講座 1、 遺伝子研究法 I I 」、 pl05A surfactant such as (trade name) may be added. At the age at which the target product is strongly secreted into the culture solution, after the culture is completed, 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. ^ Corresponding proteins into which mutations such as deletion, insertion, transposition or addition have been introduced can be produced. Such mutation, conversion, and modification methods are described in The Biochemical Society of Japan, “Seismological Chemistry Laboratory Course 1, Gene Research Method II”, pl05.
(広瀬進） 、 東京化学同人 (1986) ; 日本生化学会編、 「新生化学実験講座 2、 核 酸 Ι Π (組換え DNA技術） 」 、 p233 (広瀬進） 、 東京化学同人 (1992)； R. Wu, L . Grossman, ed.， "Methods in Enzymology", Vol. 154, p. 350 & p. 367， Aca demic Press, New York (1987)； R. Wu, L. Grossman, ed. , "Methods in Bnzym ology", Vol. 100， p. 457 & p. 468, Academic Press, New York (1983)； J. A . Wells et al. , Gene, 34: 315， 1985 ; T. Grundstroem et al. , Nuclei c Aci ds Res. , 13 : 3305, 1985 ; J. Taylor et al. , Nucleic Acids Res. , 13 : 8765,(Susumu Hirose), Tokyo Kagaku Doujin (1986) ; edited by The Biochemical Society of Japan, "New Lecture on Experimental Chemistry 2, Nucleic Acid II (Recombinant DNA Technology)", p233 (Susumu Hirose), Tokyo Kagaku Dojin (1992); R Wu, L. Grossman, ed., "Methods in Enzymology", Vol. 154, p. 350 & p. 367, Aca demic Press, New York (1987); R. Wu, L. Grossman, ed., " Methods in Bnzymology ", Vol. 100, p. 457 & p. 468, Academic Press, New York (1983); J. A. Wells et al., Gene, 34: 315, 1985; T. Grundstroem et al. , Nucleic Acids Res., 13: 3305, 1985; J. Taylor et al., Nucleic Acids Res., 13: 8765,
1985 ; R. Wu ed. , "Methods in Enzymology", Vol. 155， p. 568, Academic Pr ess, New York (1987)； A. R. Ol iphant et al. , Gene, 44: 177， 1986 などに 記載の方法が挙げられる。 例えば合成オリゴヌクレオチドなどを利用する位置指 定変 入法 （部位特異的変異導入法） (Zol ler et al. , Nucl. Acids Res. , 10 ： 6487, 1987 ; Carter et al. , Nucl. Acids Res. , 13 : 4331, 1986)， カセット 変 入法 (cassette mutagenesis : Wel ls et al. , Gene, 34: 315， 1985), 制 限咅位選択変 ¾¾Λ法 (restriction selection mutagenesis : Wells et al. , P hi los. Trans. R. Soc. London Ser A, 317 : 415， 1986)，ァラニン'スキャン二 ング法 (Cunningham k Wel ls, Science, 244: 1081-1085, 1989), PCR変異導人 法， PCR メガプライマ一法， Kunkel法， dNTP [ S]法 （Eckstein),亜硫酸ゃ亜硝 酸などを用し、る領繊旨定変異導人法等の方法が挙げられる。 さらに得られた本発明の夕ンパク質は、 ィ匕学的な手法でその含有されるァミノ 酸残基を修飾することもできるし、 ぺプチダ一ゼ、 例えばぺプシン、 キモトリプ シン、 ノ、。ノ、。イン、 ブロメライン、 ェンドぺプチダ一ゼ、 ェキソぺプチダーゼなど の酵素を用いて修飾したり、 部分分解したりしてその誘 本などにすることがで きる。 本発明のタンパク質は、 C末端が通常カルボキシル基 (- C00H) または力ノレ ボキシレート （- C00— ) である力く、 C末端がアミ ド (- CO丽 2)またはエステル (- CO OR) であってもよい。 ここでエステルにおける R としては、 例えば、 メチル、 ェ チル、 n -プロピル、 イソプロピルもしくは n-ブチルなどの -6アルキル基、 例え ば、 シクロペンチル、 シクロへキシルなどの C 3— 8 シクロアルキル基、 例えば、 フエニル、 α —ナフチルなどの CG一 1 2 7リ一ル基ヽ 例えば、 ベンジル、 フヱネチ ルなどのフヱニルー C！— 2アルキル基もしくは 一ナフチノレメチルなどの 一ナフ チル -C,— 2 アルキル基などの C7— 1 4 ァラルキル基のほ力、、 経口用エステルとして 汎用されるピノくロイルォキシメチル基などが用いられる。 本発明のタンノ、°ク質が C末端以外にカルボキシル基 （またはカルボキシレート） を有している#^r、 力 ルポキシル基がァミ ドィ匕またはエステル化されているものも本発明のタンパク質 に含まれる。 この のエステルとしては、 例えば上記した C末端のエステルな ど力用いられる。 さらに、 本発明のタンパク質には、 上記したタンパク質において、 N末端のメ チォニン残基のァミノ基が保護基 （例えば、 ホルミノレ基、 ァセチルなどの C ,一 5 アルキル一カルボニル基などの d— 6ァシル基など） で保護されているもの、 N端 側力性体内で切断され生成したグルタミル基がピログルタミル化したもの、 分子 内のァミノ酸の側 il±の置換基 （例えば、 -OH、 -C00H、 アミノ基、 イミダゾ一 ル基、 インドール基、 グァニジノ基など） 力く適当な保護基 （例えば、 ホルミル基 、 ァセチノレ基などの 一 6ァシル基など） で保護されているもの、 あるいは糖鎖が 結合したいわゆる糖タンノ ク質などの複合タンノ、。ク質なども含まれる。 また遺伝 且換え法で製造する時に融合タンパク質として発現させ、 生体内あるいは生体 外で天然の N - Tes と実質的に同等の生物学的活生を有して 、るものに変換 ·加工 してもよい。 遺伝子工学的に常用される融合産生法を用いること力できるカ^ こ うした融合タンパク質はその融^を利用してァフィ二ティクロマトグラフィー などで精製することも可能である。 こうした融合タンパク質としては、 ヒスチジ ンタグに融合せしめられたもの、 あるいは、 β - ガラクトシダ一ゼ （ - gal)、 マルトース結合タンパク （MBP), ダルタチオン- S-トランスフヱラ一ゼ（GST)、 チォレドキシン (TRX)又は Cre Recombinaseのアミノ酸配列に融合せしめられた ものなどが挙げられる。 同様に、 ポリペプチドは、 ヘテロジーニアスなェピトー プのタグを付加され、 該ェピトープに特異的に結合する抗体を用いてのィムノア フィニティ ·クロマトグラフィーによる精製をなし得るようにすることもできる1985; R. Wu ed., "Methods in Enzymology", Vol. 155, p. 568, Academic Press, New York (1987); AR Ol iphant et al., Gene, 44: 177, 1986. Method. For example, site-directed mutagenesis using a synthetic oligonucleotide (site-directed mutagenesis) (Zoller et al., Nucl. Acids Res., 10: 6487, 1987; Carter et al., Nucl. Acids Res. , 13: 4331, 1986), cassette mutagenesis (Wells et al., Gene, 34: 315, 1985). Restriction selection mutagenesis: Wells et al., Phi los. Trans. R. Soc. London Ser A, 317: 415, 1986), alanine's scanning method (Cunningham k Wel ls, Science, 244: 1081-1085, 1989), PCR mutation guide method, PCR megaprimer method, Kunkel method, dNTP [S] method (Eckstein), constant mutation using sulphite-nitrite, etc. There is a method such as a guide method. Furthermore, 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 ,. It can be modified with enzymes such as quinone, bromelain, endopeptidase, exopeptidase, etc., or partially degraded to produce its trigger. 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 丽2 ) or an ester (-COOR). You may. Here, as 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ヽsuch naphthyl example, benzyl, Fuweniru C such Fuwenechi le! - 2 alkyl group or single naphthyl -C such as single Nafuchinoremechiru, - 2 C 7 such as an alkyl group - such as 1 4 Pinoku Roy Ruo carboxymethyl groups commonly used as ho force ,, oral ester Ararukiru groups using Can be The tanno of the present invention, # ^ r having a carboxyl group (or carboxylate) other than the C-terminus other than the C-terminus, and those in which the carboxyl group is amide or esterified are also the proteins of the present invention. include. As this ester, for example, a force such as the above-mentioned C-terminal ester is used. Furthermore, in the protein of the present invention, in the above-mentioned protein, 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. 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. The quality is also included. In addition, 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 β-galactosidase (-gal), maltose-binding protein (MBP), daltathione-S-transferase (GST), thioredoxin (TRX) or And those fused to the amino acid sequence of Cre Recombinase. Similarly, 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.
。 より適した難態様においては、 該ェピト一プタグとしては、 例えば AU5， c - Myc, CruzTag 09, CruzTag 22, CruzTag 41, Glu-Glu, HA, Ha. 11， KT3, FLAG ( registered trademark, Si ma-Aldrich), Omni -probe, S - probe, T7， Lex A, V5 ， VP16, GAL4, VSV-Gなどが挙げられる。 (Field et al.， Molecular and Cel lu lar Biology, 8: pp. 2159-2165 (1988)； Evan et al. , Molecular and Cel lular Biology, 5 : pp. 3610-3616 (1985)； Paborsky et al. , Protein Engineering, 3(6) : pp. 547-553 (1990)； Hopp et al. , BioTechnology, 6 : pp. 1204-1210 (19 88) ; Martin et al. , Science, 255: pp. 192-194 (1992) ; Skinner et al. , J. Biol. Chem. , 266: pp. 15163-15166 (1991) ; Lutz-Freyermuth et al. , Proc. N atl. Acad. Sci. USA, 87: pp. 6393-6397 (1990)) など。酵母を利用した two- h ybrid法も利用できる。 さらに融合タンパク質としては、 検出可能なタンパク質 となるようなマーカ一を付されたものであることもできる。 より好適な実施態様 におし、ては、 該検出可能なマーカーは、 ビ才チン,ストレプトァビジン系の Biot in Avi Tag, 螢光を発する物質などであってよい。該螢光を発する物質としては . In a more suitable difficult mode, 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. 547-553 (1990); Hopp et al., BioTechnology, 6: pp. 1204-1210 (1988); Martin et al., Science, 255: pp. 192- 194 (1992); Skinner et al., J. Biol. Chem., 266: pp. 15163-15166 (1991); Lutz-Freyermuth et al., Proc. Natl. Acad. Sci. USA, 87: pp. 6393-6397 (1990)). A two-hybrid method using yeast can also be used. Further, the fusion protein may be a protein having a marker so as to be a detectable protein. In a more preferred embodiment, the detectable marker may be a biotin, a streptavidin-based Biot in Avi Tag, a fluorescent substance, or the like. As the fluorescent substance,
(Aequorea victorea)などの ¾ tクラゲ由来の Hfe螢光タンパク 質 (green fluorescent protein: GFP). それを改変した変異体 (GFPバリアント） 、 例えば、 EGFP (Enhanced- humanized GFP), rsGFP (red-shif t GFP), 黄色螢光 タンパク質 (yellow fluorescent protein: YFP), 緑色螢光タンパク質 (green fluorescent protein: GFP)，藍色螢光タンノヽ0ク質 (cyan fluorescent protein: CFP), #fe螢光タンパク質 (blue fluorescent protein: BFP), ゥミシィタケ ( Reni lla reniformis) 由来の GFPなどが挙げられる （宫脇敦史編、 実験医学別冊 ポストゲノム時代の実験講座 3— GFP とバイオイ一ジング、 羊土社 （2000年)）。 また、 上記融合タグを特異的に認識する抗体 （モノクローナル抗体及びそのフラグ メントを含む） を使用して検出を行うこともできる。 本発明の好ましい態様において、 精製を好適に実施するのに数つマーカ一配 列、 例えばへキサーヒスチジンぺプチドを融合したものなどが ^できる。 こう した融合タンパク質の発現及び精製は、 それに適した市販のキットを用いて行う こと力くでき、 キット製造業者あるいはキット販売業者により明らかにされている プロトコルに従って実施することもできる。 タンパク質の構造の修飾 ·改変など は、 例えば日本生化学会編、 「新生化学実験講座 1、 タンパク質 VI I、 タンパク 質工学」 、 東京化学同人 (1993)を参考にし、 そこに記載の方法あるいはそこで引 用された文献記載の方法、 さらにはそれらと実質的に同様な方法で行うこと力で きる。 また下記するようにその生物学的活性のうちには、 免疫的に活性、 例えば 抗原性を有するということも含まれてよい。 該修飾 '改変のうちには、 脱ァミノ 化、 ヒドロキシル化、 リン酸化、 メチル化、 ァセチル化、 開環、 閉環、 含有糖鎖 の を違うものに変えること、 含有糖鎖の数を増減すること、 D-体アミノ酸残 基への置換などであってもよい。 それらの方法は、 当該分野で知られている （例 えば、 T. E. Creighton, Proteins: Structure and Molecular Properties, pp. 79 - 86 W. H. Freeman & Co. , San Francisco, USA (1983)，等） 。 力、くして本発明のヒト由来のタンパク質は、 1個以上のァミノ酸残基が同一性 の点で天然のものと異なるもの、 1個以上のァミノ酸残基の位置が'天然のものと 異なるものであってもよい。 本発明のヒト由来のタンパク質は、 -Tes に な 了ミノ酸残基が 1個以上 （例えば、 1〜80個、 好ましくは 1〜60個、 さらに好ま しくは 1〜40個、 さらに好ましくは 1〜20個、 特には 1〜10個など） 欠けている 欠^!縁体、 贿のァミノ酸残基の 1個以上 （例えば、 1〜80個、 好ましくは 1 〜60個、 さらに好ましくは 1〜40個、 さらに好ましくは 1〜20個、 特には 1〜10 個など） 力《他の残基で置換されている置擬頁縁体、 1個以上 （例えば、 1〜80個 、 好ましくは 1〜60個、 さらに好ましくは 1〜40個、 さらに好ましくは 1〜20個 、 特には 1〜10個など） のアミノ酸残基が付加されている付加類縁体も包含する 。天然の N- Tes の特徵であるドメイン構造あるいは活性中心構造が膽されてい れば、 上記のごとき変異体は、 全て本発明に包含される。 また本発明の N-Tes は 天然の N- Tes と実質的に同等の一次構造コンフオメ一シヨンあるいはその一部を 有しているものも含まれてよいと考えられ、 さらに天然の N- Tes と実質的に同等 の生物学的活性を有しているものも含まれてよいと考えられる。 さらに天然に生 ずる変異体の一つであることもできる。本発明のヒト由来のタンパク質は、 例え ば、 配列表の配列番号： 2で表されるアミノ酸配列のうち、 第 22位〜第 122位の ァミノ酸配列を有するもの、 同第 22位〜第 313位のァミノ酸配列を有するもの、 及び同第 1位〜第 313位のァミノ酸配列を有するものからなる群から選ばれたァ ミノ酸配列に対し、 60%、 によっては 70% より高い相同性を有しているもの 力挙げられ、 より好ましくはそれに対し、 80¾あるいは 90%以上の相同アミノ酸 酉己列を有するもの力挙げられ、 特には Glycosaminoglycan attachment si te を欠 くもの力挙げられる。 本発明のヒト由来のタンパク質の一部のものとは、 該ヒト 由来のタンパク質の一部のペプチド （すなわち、 該タンパク質の部分ペプチド） であって、 本発明の N- Tes と実質的に同等な活性を有するものであればいずれの ものであってもよい。 例えば、 発明のタンパク質の部分ペプチドは、 本発明 ©N-Tes の構成ァミノ酸配列のうち少なくとも 5個以上、 好ましくは 20個以上、 さらに好ましくは 50個以上、 より好ましくは 70個以上、 もつと好ましくは 100個 以上、 ある齢には 200個以上のアミノ酸配列を有するペプチドが挙げられ、 好 ましくはそれらは連続したアミノ酸残基に対応するものである力、、 あるいは、 例 えば、 配列表の配列番号： 2で示されるァミノ酸配列のうち対応する領域に対す る相同性に関して、 上記と同様の相同性を有するもの力挙げられる。 本明細書において、 「実質的に同等」 とは蛋白質の活性、 例えば、 阻害活性、 生理的な活性、 生物学的な活性が実質的に同じであることを意味する。 さらにま た、 その用語の意味の中には、 実質的に同質の活性を有する齢を包含していて よ 該実質的に同質の活性としては、 例えば、 匪 PSのいずれか一つに対する阻 害活性、 固 PSいずれか一つ用の合成基質に対する分艇性を抑制あるいは阻害す る活性などを挙げること力くできる。 該実質的に同質の活性とは、 それらの活性が 性質的に同質であることを示し、 例えば、 生理的に、 薬理学的に、 あるいは生物 学的に同質であることを示す。 例えば、 MMPsのいずれか一つに対する阻害活性な どの活性が、 同等 （例えば、 約 0. 001〜約 1000倍、 好ましくは約 0. 01〜約 100倍 、 より好ましくは約 0. 1〜約 20倍、 さらに好ましくは約 0. 5〜約 2倍） であるこ と力 子ましい力 これらの活性の程度、 タンパク質の 量などの量的な要素は 異なっていてもよい。 次に、 アミノ酸の置換、 欠失、 あるいは挿入は、 しばしば ポリぺプチドの生理的な !生や化学的な特性に大きな変化を生ぜしめないし、 こ うした:^、 その置 ^ 欠失、 あるいは揷人を施されたポリペプチドは、 そうし た置 欠失、 あるいは挿入のされていないものと実質的に同一であるとされる であろう。 該アミノ酸配列中のアミノ酸の実質的に同一な置換体としては、 その アミノ酸が属するところのクラスのうちの他のアミノ酸類から選ぶことができう る。 例えば、 非極性 （疎水性） アミノ酸としては、 ァラニン、 フヱニルァラニン 、 ロイシン、 イソロイシン、 バリン、 プロリン、 トリプトファン、 メチォニンな どが挙げられ、 極性 (中性） アミノ酸としては、 グリシン、 セリス スレオニン 、 システィン、 チロシン、 ァスパラギ グルタミンなどが挙げられ、 陽電荷を もつアミノ酸 （塩基性アミノ酸） としては、 アルギニン、 リジン、 ヒスチジンな ど力挙げられ、 陰電荷をもつアミノ酸 （酸性アミノ酸） としては、 ァスパラギン 酸、 グルタミン酸などが挙げられる。 本発明のタンパク質及びその一部のぺプチドの合成には、 当該べプチド合成分 野で知られた方法、 例えば液相合成法、 固相合成法などの化学合成法を itfflする こと力くできる。 こうした方法では、 例えばタンパク質あるいはペプチド合 樹 脂を用い、 適当に保護したアミノ酸を、 それ自体公知の各種縮合方法により所望 のアミノ酸配列に順次該樹脂上で結合させていく。縮合反応には、 好ましくはそ れ自体公知の各種活性化 を用いる力 そうした としては、 例えばジシク 口へキシルカルポジイミ ドなどカルポジイミ ド類を好ましく^ j¾できる。 生成物 力保護基を有する: には、 遊保護基を除去することにより目的のものを得る こと力くできる。 (Aequorea victorea) and other Hfe fluorescent proteins derived from jellyfish Quality (green fluorescent protein: GFP). Mutants that have been modified (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ヽ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). In a preferred embodiment of the present invention, 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. As described below, 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. These methods are known in the art (eg, TE Creighton, Proteins: Structure and Molecular Properties, pp. 79-86 WH Freeman & Co., San Francisco, USA (1983), etc.). 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 ^! Analogue, one or more of the amino acid residues in 贿 (for example, 1 to 80, preferably 1 to 60, more preferably 1 to 40, more preferably 1 to 20, especially Is 1-10, etc.) 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. As long as the domain structure or active center structure, which is a characteristic of natural N-Tes, is breached, all of the above mutants are included in the present invention. 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. It is contemplated that those having substantially the same biological activity may be included. It can also be one of the naturally occurring variants. 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. For example, 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. With respect to the homology to the corresponding region in the amino acid sequence represented by SEQ ID NO: 2, those having the same homology as described above can be mentioned. As used herein, “substantially equivalent” means that the protein activities, eg, inhibitory activity, physiological activity, and biological activity, are substantially the same. In addition, 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. For example, 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. Second, 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. For example, 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. For synthesizing the protein of the present invention and some of its peptides, it is possible to use 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 Using a fat, 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.
本発明のタンパク質及びその一部のぺプチドは、 それが遊離型のものとして得 られた: ^には、 それ自体公知の方法あるいはそれに準じた方法で塩に変換する こと力でき、 またそれらは塩として得られた齢には、 それ自体公知の方法ある いはそれに準じた方法で遊离觀のものあるいは他の塩に変換すること力くできる。 本発明のタンパク質及びその一部のぺプチドの塩としては、 生理的に許容され るものあるいは医薬として許容されるもの力 子ましいが、 これらに限定されない 。 こうした塩としては、 例えば塩酸、 臭化水素酸、 硫酸、 硝酸、 リン酸などの無 機酸との塩、 例えは ¾酸、 ギ 、 マレイン酸、 フマール酸、 コハク酸、 クェン酸 、 酒石酸、 リンコ 、安息香酸、 メタンスルホン酸、 P-トルエンスルホン酸、 ベ ンゼンスルホン酸などの有機酸との塩などが挙げられる。 さらに該塩としては、 了ンモニゥム塩、 例えばェチルァミン、 ジメチルァミン、 卜リメチルァミン、 ヒ ドロキシェチルァミンなどの有機塩基との塩なども挙げられる。 こうした本発明の N_Tes及びその変異体、 修飾体、 誘 本などは、 上記で説明 したような分離 ·精製処理を施すことができる。 本発明では、 「断片」 、 「誘導 体」 及び 「類縁体」 なる用語は、 配列番号： 2のポリペプチド、 配列番号： 1の 配列から転写され且つスプライシングされていないか又は特異的にスプライシン グされた hnRNA又は mRNAによりコードされるポリぺプチド、 又はジヱノミック DN A によりコードされるボリペプチドに関連して、 その 「断片」 、 「誘 本」 又は 「類縁体」 と称した^、 このようなポリペプチドと本質的に同一の生物学的機 能又は活性を有しているポリペプチドを意味する。 従って、 類似体にはプロタン パク質部分が切断されて活'賊熟ポリべプチドを産生するような、 活性化できる プロ夕ンパク質等が包含される。 本発明のポリぺプチドは組換えポリぺプチド、 天然ポリぺプチド又は合成ポリぺプチドでよい。 特定の好ましい態様では、 これ は組換えポリぺプチドである。 一方では、 こうして本発明は上記したポリペプチドをコードする DNA配列、 そ して天然の特性の^^あるいは一部を有する N-Tes のポリペプチド、 さらにその 類縁体あるいは誘割本をコードする DNA配列も包含する。 本発明のポリヌクレオ チドは、 ァミノ末端に付加ァミノ酸又はカルボキシル末端に付加ァミノ酸を加え た成熟タンパク質、 又は成熟タンパク質に内在するポリペプチド （例えば、 成熟 形態で一つ以上のポリぺプチド鎖を有する のアミノ酸をコードして 、るも のであること力くできる。 このような配列は、 前駆体から成熟形態のタンパク質へ のプロセッシングにおいても何らかの働きをなすものであってよく、 例えば、 タ ンノ、°ク質の移動や輸送を促進したり、 タンパク質の を延長もしくは短縮し たり、 又はタンパク質を操作してその検出もしくは産生を容易にすることができ るものであってよい。 一般的には、 例えば、 イ taアミノ酸は、 細 β包酵素によりプ ロセッシングされ、 成熟タンパク質から取り除かれる。 1又はそれ以上のプロ配 列と融合した成熟形態ポリべプチドを有する前駆タンパク質ほ、 不活' f©f 態ポリ ペプチドであること力できる。 プロ配列が除去されると、 このような不活†生前,駆 体は、 通常活性化される。 プロ配列のいくつ力、又は全ては、 活性化の前に除去で きる。 通常、 このような前駆体はプ口タンパク質と称、される。 本発明のポリぺプ チドは、 成熟タンパク質、 リ
Figure imgf000041_0001
してある成熟タンパク質 （プレタ ンパク質と称すること力《できる） 、 プレタンパク質のリーダ一配列ではない 1又 はそれ以上のプロ配列を有する成熟タンパク質の前駆体、 又はリーダ一配列及び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. Examples of such salts 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. Such N_Tes of the present invention and its mutants, modifications, and primers can be subjected to the separation / purification treatment described above. In the present invention, the terms "fragment", "derivative" and "analog" refer to the polypeptide of SEQ ID NO: 2, transcribed from the sequence of SEQ ID NO: 1 and are unspliced or specifically spliced. In connection with the polypeptide encoded by the hnRNA or mRNA, or the polypeptide encoded by the digenomic DNA, the term "fragment", "attract" or "analog" is referred to as such, A polypeptide having essentially the same biological function or activity as the polypeptide is meant. Thus, 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. On the other hand, 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. Such 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. And the amino acids are processed by the fine β-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,
Figure imgf000041_0001
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
1又はそれ以上のプロ配列を有するプロタンパク質の前駆体であるプレブ口タン ノ、。ク質であってよい。 また、 該プロ配列は通常活 t娜態ポリペプチド及び成熟形 態ポリぺプチドを産み出すようなプロセッシングの段階で除去され得る。 本発明の DNA配列は、 これまで知られていなかった哺乳動物のタンパク質のァ ミノ酸配列に関する' 11#を樹共しているから、 こうした†tfgを利用することも本 発明に包含される。 こうした利用としては、 例えば Tes及び関連タンパク質を コ一ドする哺乳動物、 特に好ましくはヒトの、 ゲノム DNA及び cDNAの単离|¾び検 知のためのプローブの設計などが挙げられる。 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 Δtfg 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.
本発明の DNA配列は、 例えは - Tes及び関連タンパク質をコ一ドする哺乳動物 、 特に好ましくはマウスゃヒトの、 ゲノム DNA及び cDNAの単离|¾び検知のための プローブとして有用である。 プローブは、 必要に応じて、 抗体に関連して挙げら れている標識を付与しておくこと力くできる。 遺伝子の単離にあたっては、 PCR法 、 さらには逆転写酵素 （RT) を用いた PCR法 (RT-PCR) を禾拥することが出来る 。 N- Tes cDNA及びその関連 DNA は、 クローニングされ、 配列決定された N- Tes cD NA配列から推定されるアミノ酸配列に基づき特徵的な配列領域を選び、 DNAブラ イマ一をデザインして化学合成し、 得られた DNAプライマ一を用いて、 PCR法、 RT-PCR その他の方法を用いて N- Tes 関 3¾伝子の単離、 検出などに利用するこ とが出来る。 例えば、 -Tes mRNAのヒト糸 1ϋ中での発現を各種の紙織由来 poly ( A)+ RNA に対するノーザンブロット分析により検討すること力くできる。 本発明の cDNAをプローブとして用いれば、 例えばノ一ザン ·ブロティング、 サザン ·ブロ ティング、 in situハイブリダィゼ一シヨンなどによりヒト組織中での N- Tes mR NAの発現や N- Tes遺伝子自体などを検出'測定でき、 ヒト糸纖における細胞内タ ンパク質代謝、 ホルモン前駆体の活性化、 および骨の改変を含む、 多くの正常な 細胞のプロセスに関与する删 P阻害の役割、 アルツハイマー病、 肺 重、 リウマ チ性関節炎、 筋ジストロフィー、 骨粗鬆症、 神経変性疾患および癌の浸潤 '転移 の様な多くの疾患等の研究の発展に貢献できる。 N- Tes に関連した疾患の遺伝子 診断にも利用できる。 そうした診断は、 当該 N- Tes及び関連タンパク質をコード する核酸の異常、 例えば損傷、 突然変異、 発現低下、 発現過多などを診断するも のであること力くできる。 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. 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. For example, the expression of -Tes mRNA in human thread 1ϋ can be examined by Northern blot analysis on poly (A) + RNA from various paper tissues. When the cDNA of the present invention is used as a probe, 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.
本明細書中で開示した N- Tes及びそれに関連したタンパク質、 そのフラグメン ト、 さらには DNAを含めた核酸 (mRNAやオリゴヌクレオチドを含む） は、 それら を網虫あるいは有機的に使用し、 更には以下で説明する技術 （アンチセンス法、 モノクロ一ナル抗体を含めた抗体、 トランスジヱニク動物など） とも ite且合わ せて、 ゲノミックス及びプロテオミックス技術に応用できる。 例えば、 -Tes変 異体は、 ドミナントネガティブ効果を利用した機能解析にも利用可能である。 ま た、 鎖 RNA (dsRNA) をィ ffflしての RNAi (RNA interference) 技術への応用の 途もある。 かくして、一 i^S多型 (SNP; single nucleotide polymorphisms)を中 心とした遺伝子多型角斜斤、 核酸アレイ、 タンパク質アレイをィ ffflした遺ィ 解析、 遺伝子機能解析、 タンパク質間相互作用解析、 関連疾患解析、 疾患治療薬 角浙をすること力く可能となる。 例えば、 核酸アレイ技術では、 cDNAライブラリー を使用したり、 PCR技術で得た DNAを基ネ肚にスポッティング装置で高密度に配 置して、 ハイブリダイゼーションを利用して試料の解析が行われる。 該ァレイ化 は、 針あるいはピンを使用して、 あるいはインクジヱトプリンティング技術など でもって、 スライドガラス、 シリコン板、 プラスチックプレ一トなどの基板のそ れぞれ固有の位置に DNA力く付着せしめられることによりそれを実施すること力で きる。 該核酸ァレイ上でのハイブリダイゼーションの結果得られるシグナルを観 察してデータを取得する。 該シグナルは、 螢光色素などの標識 （例えば、 Cy3, C y5, BODIPY, FITC, Alexa Fluor dyes (商品名)， Texas red (商品名） など） よ り得られるものであつてよい。 嫩口にはレーザ一スキャナ一などを利用すること もでき、 得られたデータは適当なァルゴリズムに従つたプログラムを備えたコン ピューターシステムで処理されてよい。 また、 タンパク質アレイ技術では、 タグ を付された組換え発現タンパク質産物を利用してよく、 二次元電^動 (2 - DE)、 酵素消化フラグメントを含めての質量分析 （MS) (これにはエレクトロスプレーィ オン化法 (electrospray ionization: ESI), マトリックス支援レーザ一脱離ィォ ンィ匕法 natrix- assisted laser desorption/ionization: 退 LDI)などの技 ί'下了力く含 ま MALDI-T0F分析計、 BSI-3連四藤分析計、 ESトイオントラップ分析計な どを してよい） 、 染色技術、 同位 ί*(Τ識及び角斜斤、 画像処理技術などが利用 されることができる。 したがって、 本発明には上記で得られるあるいは利用でき る Ν - Tes に関連したソフトウェア、 データベースなども含まれてよい。 本発明で得られた DNA (例えば、 -Tes をコードする DNA ) を女像動物に車云移 させるにあたっては、 それを DNA断片としてあるいは該 DMを動物細胞で発現さ せうるプロモーターの下流に結合して用いるの力H に有利である。 たとえば、 マウスに N- Tes DNAを導入する: i§^、 これと相同性が高い動物由来の N- Tes DNA を動物細胞で発現させうる各種プロモーターの下流に結合した遺伝子コンストラ クトを、 文像動物の受精卵、 たとえばマウス受精卵へマイクロインジェクション することによって N- Tes を高産生する遺伝子導入 （トランスジヱニック） マウス を作出できる。 マウスとしては、 特に純系のマウスに限定されないが、 例えば、 C57BL/6, Balb/C, C3H， O^BL/e xDBA/^F, (BDF など力く挙げられる。 このプロ モーターとしては、 例えばウィルス由来プロモーター、 メタ口チォネイン等のュ ビキタスな発現プロモータ一などが好ましく使用しうる。 また該 N- Tes DNAを導 入する 、 組換えレトロウイルスに組み換えて、 それを用いて行うこともでき る。 好適には文像 DNAを導入されたマウス受精卵は、 例えば、 ICRのような仮親 のマウスをィ¾¾して生育せしめること力できる。 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). Thus, 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. For example, in the nucleic acid array technology, 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. Alternatively, 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 ί * (awareness and angled corner, image processing technology, etc. can be used) Therefore, the present invention may also include 、 -Tes-related software, databases, etc. obtained or usable above The DNA obtained by the present invention (eg, DNA encoding -Tes) 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. For example, C57BL / 6, Balb / C, C3H, O ^ BL / exDBA / ^ F, (BDF, etc. 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. Preferably, the mouse fertilized egg into which the image DNA has been introduced is capable of growing a foster mother mouse such as ICR.
受精卵細胞段階における本発明で得られた DNA (例えば、 N-Tes をコードする DNA ) の転移は、 対象動物の胚芽細胞および体細胞の全てに するように確保 される。 DNA転移後の作出動物の胚芽細胞において N-Tes をコードする DNA力く存 在することは、 作出動物の子孫が全てその胚芽細胞および体細胞の全てに該 N- Te s をコードする DNAを有することを意味する。遺 を受け継いだこの種の動物 の子孫はその胚芽細胞および体細胞の全てにおいて、 該 N- Tes を発現できる可能 性を有している。 該 N- Tes DNA導入動物は、 交配により遺伝子を安定に保持することを確認して 、 該 DNA保有動物として通常の飼龍境で飼育継代を行うこと力《できる。 さらに 、 目的 DNAを保有する雌雄の動物を交配することにより、 導入遺伝子を相同染色 体の両方に持つホモザィゴ一ト動物を取得し、 この 1¾隹の動物を交配することに よりすベての子孫が該 DNAを有するように繁殖継代すること力くできる。該 N- Tes DNA力導入された動物は、 該 N- Tes タンパク質が高発現させられているので、 該 N-Tes タンパク質に対する阻割 ij (インヒビター） のスクリーニング用の動物な どとして有用である。 また N- Tes遺 の発現を阻害することのできるアンチセ ンス ォリゴヌクレオチド、 例えば、 了ンチセンス DNA などのスクリ一ニング用 の動物などとして有用である。  Transfer of the DNA obtained in the present invention (eg, DNA encoding N-Tes) at the fertilized egg cell stage is ensured so as to be performed in all germ cells and somatic cells of the target animal. 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. 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 1¾Purn 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.
この遺伝子導入動物を、 糸且織培養のための細胞源としてィ¾¾することもできる 。 例えば、 遺^導入マウスの組織中の DNA もしくは RNAを直接分析するかある いは遺伝子により発現されたタンパク質組織を分析することにより、 MT1-MMPや MT3-MMP活性阻害に関連したタンパク質について分析すること力くできる。 該 N - Te s を有する紙織の細胞を標準紙織培養技術により培養し、 これらを麵して、 た とえば脳、 胸腺、 精巣、 脳、 腸、 腎臓やその他の紙織由来の細胞についてその機 能を研究すること力できる。 また、 その細胞を用いることにより、 たとえば各種 紙熾の機能を高めるような 1¾開発に資することも可能である。 また、 高発現細 胞株があれば、 そこから、 N- Tes を単離精製することも可能である。 トランスジ エニック マウスなどに関連した技術は、 例えば、 Brinster, R. L.， et al. ,； Proc. Natl. Acad. Sci. USA, 82: 4438, 1985 ; Costantini, F. & Jaenisch, R . (eds)： Genet i c manipulation of the early mammal ian embryo, Cold Spring Harbor Laboratory, 1985などの文献に記載の方法あるいはそこに引用された文 献に記載の方法、 さらにはそれらの改変法により行うことができる。 本発明で得られた遺伝子 （例えば、 N-Tes に相当するマウス N- Tes をコードす る DNA)に変異をもち、 マウス N- Tes を全く発現しない変異マウス （ノックアウト マウス） を作出すること力くできる。 たとえば、 該遺伝子の翻訳開始コドンの前後 4kb を含むおよそ 8kbのゲノム DNAの中央近傍に位置し翻訳開始コドンに近いェ クソンに neo耐性遺伝子- po 1 yA付加シグナルからなる遺伝子力セットを挿入した 変異遺伝子を持つタ一ゲティングベクターを構築することができる。 挿入する遺 伝子カセットは neo耐性遺伝子カセット以外に DT- Aカセット、 tkカセット、 lacZ カセットなどが挙げられる。 タ一ゲティングベクタ一を直鎖状に開き、 樹立した マウス胚性幹細胞 （embryoni c stem cel ls : ES細胞） にエレク トロボレ一シヨン で導人、 さらに培養して neo耐性を獲得した ES細胞を選別する。 細胞は 129、 C57BL/6、 F1 C57BL/6 XCBA)マウスなどのマウス系統から選択して調製すること 力くできる。 neo耐性を獲得した ES細胞は、 マウス N- Tes遺伝^！域において遺伝 子カセットを揷入したターゲティングベクタ一と相同組換えを起こしていると想 定さ k 少なくともマウス N- Tes遺伝子ァレルのうち一つは破壊され、 マゥス N - Tes を正常に発現できなくなる。 選別には挿入した遺^カセットによりそれぞ れ適当な方法が選択さ; T また、 変異の導入は PCR、 サザンハイブリダィゼ一シ ヨンあるいはノーザンハイブリダイゼーションなどの方法を用レ、て確認すること 力《でさる。 変異を導入した ES細胞は、 C57BL/6、 BALB/c、 ICR マウスなどから取り出した 8細胞期胚に注入、 1日培養し 胞に発生したものを ICRのような仮親に移植 することで個体まで生育させることができる。 生まれる子マウスは変異をもつ ES 細胞と正常な宿^!丕に由来するキメラマウスで、 ES細胞に由来する細胞がどの程 度含まれる力、は個体の毛色で判断する。 従って、 ES細胞と宿 ϋ丕は毛色の異なつ た系統の組合わせ力く望ましい。 得られたキメラマウスの変異はへテロであり、 こ れらを適宜交配することでホモ変異マウスを得ることができる。 このようにして 得られたホモ変異マウスは^ ¾細胞および体細胞の全てにおし、て、 マゥス N- Tes 遺伝子のみカ 壊され、 マウス N- Tes を全く発現せず、 繁殖継代される子孫もま た同様の表現系をもつ。 · This transgenic animal can also be used as a cell source for ligament culture. . For example, 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 1¾, 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. Techniques related to 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. I can do it. For example, a mutation in which a genetic force set comprising a neo resistance gene-po1yA addition signal is inserted into an exon located near the center of approximately 8 kb genomic DNA including 4 kb before and after the translation initiation codon of the gene and near the translation initiation codon 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 ¾ cells and somatic cells, does not express mouse N-Tes at all, and is subcultured. Descendants also have a similar expression system. ·
このノックアウトマウスは正常マウスとの比較において、 発生、 成長、 生殖、 老化および死など個体のライフサイクルにおける N-Tes の役割や各臓器、 糸 Ιϋに おける Ν- Tes の機能を角晰するのに有用である。 また、 MMP P且害に関連した医薬 品開発にも応用できる。 ノックァゥトマウスはこれらモデル動物としてだけでは なく、 紙織培養のための細胞源として使用することもでき、 細胞レベルでの N - Te s の機能角科斤などに供すること力くできる。 ノックアウトマウス等に関連した技術 は、 例えば、 Mansour, S. L響， et al. ,； Nature, 336: 348-352, 1988; Joyner, A. L.， ed.； Gene targeting, IRL Press, 1993; 相沢慎一， ジーンタ一ゲティ ング ES細胞を用し こ変異マゥスの作成， 羊土社， 1995などの文献に記載の方法あ るいはそこに引用された文献に記載の方法、 さらにはそれらの改変法により行う こと力できる。 本発明に従えば、 -Tes遺伝子の発現を阻害することのできる  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 Ν-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. L Hibiki, et al., Nature, 336: 348-352, 1988; Joyner, AL, ed .; Gene targeting, IRL Press, 1993; Shinichi Aizawa Gene-targeting ES cells are used to generate mutant mice, and the method described in the literature such as Yodosha, 1995, or the method described in the literature cited therein, or a modification thereof is used. I can do it. According to the present invention,-expression of the Tes gene can be inhibited
ォリゴヌクレオチド （核酸） を、 クローンィヒしたあるいは決定された N - Tes をコ —ドする DNAの塩基配列'辭に基づき設計し、 合成しうる。 そうしたオリゴヌク レオチド （核酸） は、 N- Tes遺伝子の mRNAとハイブリダィズすること力くでき、 該 mRNAの機能を阻害すること力《できる力、、 あるいは N - Tes 関: iimRNAとの相互作用な どを介して N- Tes遺伝子の発現を調節'制御すること力《できる。 N - Tes関連遺伝 子の選択された配列に相補的なオリゴヌクレオチド、 及び" N- Tes 関連遺伝子と特 異的にハイブリダイズすることができるオリゴヌクレオチドは、 生体内及び生体 外で N- Tes遺伝子の発現を調節 '制御するのに有用であり、 またそれに関連した 病気などの治療又は診断に有用である。 用語 「対応する」 とは、 遺 を含めた ヌクレオチド、 塩基配列又は核酸の特定の配列に相同性を有するあるいは相補的 であることを意味する。 ヌクレオチド、 塩基配列又は核酸とペプチド （タンパク 質） との間で 「対応する」 とは、 ヌクレオチド （核酸） の配列又はその相補体力、 ら誘導される ί にあるペプチド （タンパク質） のアミノ酸を通常指している。 当該遺伝子の 5，端ヘアピンループ、 5，端 6-ベ一スペア ' リピ一ト、 5，端非翻訳領 域、 ポリべプチド翻訳開始コドン、 タンパク質コ一ド領域、 0RF翻訳開始コドン 、 3，端非翻訳領域、 3，端パリンドローム領域、 及び 3，端ヘアピンループは、 好ま しい文寸象領域として選択しうる力 当該遺 内の如何なる領域も対象として選 択しうる。 目的核酸と、 文像領域の少なくとも一部に相補的なォリゴヌクレオチドとの関 係は、 対'象物とハイブリダイズすることができるォリゴヌクレオチドとの関係を 意味し、 それは、 「アンチセンス」 であるということ力くできる。 アンチセンス · ォリゴヌクレオチドは、 2 -デ才キシ- D- リボースを含有してレ、るポリデォキシヌ クレオチド、 D-リボースを含有してレ、るポリデォキシヌクレオチド、 プリン又は ピリミジン塩基の Ν-グリコシドであるその他のタイプのポリヌクレオチド、 ある いは非ヌクレオチド骨格を有するその他のポリマ一 （例えば、 市販のタンパク質 核酸及び合成配列特異的な核酸ポリマ一） 又はキ魏な結合を含有するその他のポ リマ一 （但し、 該ポリマーは DNAや RNA中に見出されるような塩基のペアリング や塩基の付着を許容する配置をもつヌクレオチドを含有する） なと'が挙げられる 。 それらは、 2本鎖 DNA， 1本鎖 DNA， 2本鎖 RNA， 1本鎖 RNA，さらに DNA: RNAハイブ リッドであること力くでき、 さらに非修飾ポリヌクレオチド又は非修飾ォリゴヌク レオチド、 さらには公知の修飾の 口されたもの、 例えば当該分野で知られた標' 識のあるもの、 キャップの付いたもの、 メチル化されたもの、 1個以上の天然の ヌクレオチドを類縁物で置換したもの、 分子内ヌクレオチド修飾のされたもの、 例えば 電結合 （例えば、 メチルホスホネート、 ホスホトリエステル、 ホスホ ルアミデート、 力ルバメートなど） を持つもの、 電荷を有する結合又は硫黄含有 結合 （例えば、 ホスホロチォェ一ト、 ホスホロジチォエートなど） を持つもの、 例えばタンパク質 （ヌクレア一ゼ、 ヌクレアーゼ 'インヒビター、 トキシン、 抗 体、 シグナルペプチド、 ポリ- L- リジンなど） や糖 （例えば、 モノサッカライド など） などの側鎖基を有しているもの、 インタ一ガレント化合物 （例えば、 ァク リジン、 プソラレンなど） を持つもの、 キレート化合物 （例えば、 金属、 方 id寸活 性をもつ金属、 ホウ素、 酸化性の金属など） を含有するもの、 アルキル化剤を含 有するもの、 修飾された結合を持つもの （例えば、 ァノマ一型の核酸など） で あってもよい。 ここで 「ヌクレオシド」 、 「ヌクレオチド」 及び 「核酸」 とは、 公知のプリン及びピリミジン塩基を含有するのみでなく、 ィ 飾されたその他の複 素環型塩基をもつようなものを含んで 、て良 、。 こうした修飾物は、 メチル化さ れたプリン及びピリミジン、 ァシル化されたプリン及びピリミジン、 あるいはそ の他の複素環を含むものであってよい。 修飾されたヌクレオシド及び修飾された ヌクレオチドはまた糖部分が修飾されていてよく、 例えば 1個以上の水酸基がハ ロゲンと力、、 脂) ¾基などで置換されていたり、 あるいはエーテル、 ァミンなど の官能基に変換されていてよい。 本発明のアンチセンス核酸は、 RNA、 DNA、 あるいは修飾された核酸である。 ィ誠 Φされた核酸の具体例としては核酸の硫黄誘 ί本やチォホスフヱ一ト誘 本、 そしてポリヌクレオシドアミ ドゃォリゴヌクレオシドアミ ドの分角军に抵抗 '性のも の力く挙げられる力 それに限定されるものではない。 本発明のアンチセンス核酸 は次のような方針で好ましく設計されうる。 すなわち、 細胞内でのァンチセンス 核酸をより安定なものにする、 アンチセンス核酸の細胞透過性をより高める、 目 標とするセンス鎖に対する親和性をより大きなものにする、 そしてもし毒性があ 核酸の毒性をより小さなものにする。 こうした修飾は当該分野で数多く知られており、 例えば J. Kawakami et al. , Pharm Tech Japan, 8 : 247, 1992; 8: 395, 1992; S. T. Crooke et al. ed. , An ti sense Research and Appl i cations, CRC Press, 1993などに開示がある。本発 明のアンチセンス核酸は、 変化せしめられたり、 修飾された糖、 塩基、 結合を含 有していて良く、 リボゾーム、 ミクロスフエアのような特殊な形態で ^された り、 遺伝子治療により適用されたり、 付加された形態で与えられることができう る。 こうした付加形態で用いられるものとしては、 リン酸基骨格の電荷を中和す るように働くポリリジンのようなポリカチオン体、 細胞膜との相互作用を高めた り、 核酸の取込みを増大せしめるような脂質 （例えば、 ホスホリピッド、 コレス テロールなど） といった疎水性のもの力く挙げられる。 付加するに好ましい脂質と しては、 コレステロールやその誘導体 (例えば、 コレステリルク口口ホルメ一ト 、 コール酸など） 力挙げられる。 こうしたものは、 核酸の 3 ' 端あるいは 5 ' 端 に付着させること力でき、 塩基、 糖、 ノ^内ヌクレオシド結合を介して付着させ ることができうる。 その他の基としては、 核酸の 3 ' 端あるいは 5 ' 端に特異的 に配置されたキヤップ用の基で、 ェキソヌクレアーゼ、 RNaseなどのヌクレア一 ゼによる分解を! Sitするためのもの力く挙げられる。 こうしたキヤップ用の基とし ては、 ポリェチレングリコ一ノレ、 テトラェチレングリコールなどのグリコールを はじめとした当該分野で知られた水酸基の保護基が挙げられる力^ それに限定さ れるものではない。 Oligonucleotides (nucleic acids) can be designed and synthesized based on the cloned or determined nucleotide sequence of the DNA encoding N-Tes. Such oligonucus Reotide (nucleic acid) 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. It is useful for controlling and controlling the expression of the gene, and is also useful for treating or diagnosing diseases associated with it.The term "corresponding" refers to a nucleotide, nucleotide sequence or a specific sequence of nucleic acid, including the residue Has homology to or is complementary to The “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 ί derived from the nucleotide (nucleic acid) sequence or its complementarity. ing. 5, end hairpin loop, 5, end 6-basespare 'repeat, 5, end untranslated region, polypeptide translation start codon, protein code region, 0RF translation start codon, 3, 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) or 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). They 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 Such as proteins (nucleases, nucleases' inhibitors, toxins, antibodies, signal peptides, poly-L-lysine, etc.) and sugars (eg, monosaccharides) Having a chain group, an intergallant compound (for example, Lysine, psoralen, etc.), containing chelating compounds (eg, metals, metals with high dimensional activity, boron, oxidizing metals, etc.), those containing alkylating agents, modified bonds (For example, a nucleic acid of an anoma type 1). Here, "nucleoside", "nucleotide" and "nucleic acid" 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. Specific examples of nucleic acids that have been subjected to Φ 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. Many such modifications are known in the art, for example, J. Kawakami et al., Pharm Tech Japan, 8: 247, 1992; 8: 395, 1992; ST Crooke et al. Ed., Antisense Research and Appl. It is disclosed in ications, CRC Press, 1993 and the like. 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. Such 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.
アンチセンス核酸の阻害活性は、 本発明の形質転換体、 本発明の生体内や生体 外の遺伝^現系、 あるいは N- Tes の生体内や生体外の翻訳系を用いて調べるこ とができる。 該核酸はそれ自体公知の各種の方法で細胞に適用できる。 以上述べた、 本発明者らの研究成果により N-Tes の遺伝子及び組換え DNA を宿主に移入し、 N-Tes を発現させ、 目的とする N- Tes を得る方法が樹共される 。 こうして本発明によれば、 N-Tes の遺伝子を実質的に発現する組換え体あるい はトランスフエクタント及びその製造法、 さらにはその用途も ί 共される。 別の面では、 本発明は Testi canフアミリーに属する匪 P阻害活性を有し且つ G1 ycosaminoglycan attachment si te を欠いている及び/又は TY domainや CTCV d omainを欠いているポリペプチドの一種であり且つ 天然のヒト N- Tes と実質的 に同等な活性を有することを 1敷とするタンパク質またはその塩、 より好ましく は N- Tes またはその塩と、 実質的に同等な活性を有する力、、 あるいは実質的に同 等の一次構造コンフオメ一ションを持つ該タンパク質の少なくとも一部あるいは ^を有するポリべプチドを、 大腸菌などの原核生物あるいは哺乳動物細胞など の »生物で発現させることを可能にする DNAや RNAなどの核酸に関するとする こと力くできる。 またこうした核酸、 特には DNA は、 （a)配列表の配列番号： 2で 表されるァミノ酸配列をコ一ドできる配列あるいはそれと相補的な配列、 (b)，該 (a) の DNA配列またはその断片とハイブリダィズすることのできる配列、 及び (c ) 該 (a) 又は (b) の配列にハイブリダィズすることのできる縮重コードを持った 配列であることができる。 ここでハイプリダイズの条件としては、 ストリンジヱ ントな条件であること力くできる。 こうした核酸で形質転換され、 本発明の該ポリ ぺプチドを発現できる大腸菌などの原核生物あるし、は哺乳動物細胞などの 生 物も本発明の' 1：をなす。 こうして典型的には本発明の目的は、 -Tes遺伝子、 それから誘導されたプロ —ブを用い、 あるいはさらに必要に応じ、 N-Tes に対する阻働質を用い、 被検 試料中の N- Tes あるいはその遺伝子、 さらには産生钿胞を嫩口 ·分別 する優 れた方法及びその為の キットを ί¾共することにある。本発明はこうした N- Te s あるいはその遺好、 さらには産生細胞を嫩ロ ·分別 することのできる試 薬キットのうちの各¾¾をすベてその 態様のうちに含むと理解される。 さら に本発明の目的は、 U己方法を用いて N- Tes あるいはその遺伝子、 さらには産生 細胞を嫩ロ ·分別 することにより、 細胞内タンノ、。ク質代謝、 ホルモン前駆体 の活性化、 および骨の改変など、 多くの正常な細胞のプロセスに関与する匪 Psの 役割、 アルツハイマー病、肺翅重、 リウマチ性関節炎、筋ジストロフィー、 骨粗 鬆症、 神経変性疾患および癌の浸潤 ·転移の様な多くの疾患などをモニタ一し得 る方法並びに あるいは診断剤を樹共することにある。 した力つて、 医学的- 生理学的分野における上記 I ^の各種利用、 腫瘍'癌といったヒトなどの動物の 細胞 ·紙織の研究 .解析 .測定などの目的で上記 を使用することはすべて本 発明のその実施態様のうちに含まれると理解される。 本発明の N- Tes あるいはその塩は、 固 Ps阻害活性を有し、 例えば生体内でタン パク質代謝、 抗原提示、 骨の改変、 ホルモン前駆体の活性ィ匕などにおいて不可欠 な因子であると考えられる。 そして、 該タンパク質は N- Tes «成症、 N-Tes発 現不全症、 N-Tes遺伝子欠損症など病状を呈する N- Tes 関連機能不全疾患の治療' に有用であると考えられる。 すなわち、 N- Tes、 変異体、 修飾体、 誘 本を含有 する医薬を用いれば、 N-Tes による活性が不充分であることに起因する疾患患者 を健常な状態にすること力河能である。本発明のポリペプチドは、 プロテア一ゼ 阻害剤の一つであり、 該タンパク質の発糧ゃプロテア一ゼ阻害活性が減少して し、る; t ^に生ずる種々の疾患の治療及び Z又は予防剤などの医薬として有用であ る。 また本発明のポリペプチドは、 組織やタンパク質に対する MMP分角鞭性が亢 進している: ti^に生ずる種々の疾患の治療及び/又は予防剤などの医薬として有 用である。 例えば、 生体内において N- Tes カ咸少あるいは欠損しているために、 細胞における当該生物学的活性が十分に得られて 、ないか、 あるいは正常でない 症状の患者の: には、 (A) 本発明のタンパク質等を該患者に投与することによ る力、、 (B) 本発明の副 Aなどの核酸を該患者に投与して、 生体内で本発明のタン パク質等を発現させることによるか、 (C) 本発明の DNAなどの核酸を発現可能に 導入した細胞を該患者に移植することによって、 生体内に本発明の夕ンパク質等 を補充する等して、 該患者における当該症状を改善したりする。 本発明の N- Tes の生物学的活性などの機能 （例えば、 プロテア一ゼ阻害活性な ど） を促進する化合物 （ァゴ二スト、 あるいは促進剤） 又はその塩は、 N-Tes機 能不全症状、 肺 重、 筋ジストロフィー、 骨粗鬆症、 アルツハイマー病、 m . 患、 リゥマチ性関節炎および癌の浸潤 ·転移などの各種の疾病の治療及び Z 又は予防剤として有用な医薬として ί®¾できる。一方、 本発明の N- Tesの生物学 的活性などの機能 （例えば、 プロテア一ゼ阻害活性など） を阻害する化合物 （ァ ンタゴ二スト、 あるいは阻害剤） 又はその塩は、 遷- Tes機能症、 肺翅重、 筋ジ ストロフィー、 骨粗鬆症、 アルツハイマー病、 神経変嫉患、 リウマチ性関節炎 および癌の浸潤 .転移などの各種の疾病の治療及び/又は予防剤などの 1S¾とし て使用できる。 例えば" N- Tes は、 MT1-匪 Pや MT3-匪 P などの匪 Psの活性発現に関 与し、 それらに対して阻害活性を有する。 したがって、 該 N - Tes は、 MT1 -画 P や T3-MMP などの画 Psによる過剰分解に起因する疾患の治療用医薬として有用であ る。 力、くして、 本発明の N - Tes などのポリペプチド等は、 本発明の N - Tes などのポ リペプチド等の、 生物学的活性などの機能 （例えば、 プロテア一ゼ阻害活性など ) を促進する化合物 （ァゴ二スト） や阻害する化合物 （アンタゴニスト） 又はそ れらの塩をスクリーニングするための として有用である。 かくして、 本発明 の N- Tes などのポリペプチド、 その一部のペプチド又はそれらの塩を用いた、 本 発明の N- Tes といったタンパク質、 その一部のペプチド又はそれらの塩などの生 物学的活性などの機能 （例えば、 プロテア—ゼ阻害活性など） を促進する化合物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. In another aspect, 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. Such nucleic acids, especially DNA, 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). Here, 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. Thus, typically, 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. Furthermore, 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. The role of 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. The various uses of 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. In other words, 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. In addition, 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 ^. For example, in patients with inadequate or aberrant symptoms of N-Tes in vivo, 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. It can be used as 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. On the other hand, 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. , Weight of pulmonary wings, muscular dystrophy, osteoporosis, Alzheimer's disease, neurodegenerative disease, rheumatoid arthritis It can be used as 1S¾ for therapeutic and / or prophylactic agents for various diseases such as cancer invasion and metastasis. For example, “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. For screening compounds (agonists), compounds (antagonists), or salts thereof that promote functions such as biological activity (eg, protease inhibitory activity) such as polypeptides. Thus, 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. Functions such as biological activity of For example, protease - like peptidase inhibitory activity) compounds which promote
(ァゴ二スト） や阻害する化合物 (アンタゴニスト) 又はそれらの塩のスクリー ニング方法も提供される。 Also provided are methods for screening (agonist), inhibitory compounds (antagonists), or salts thereof.
該スクリーニングでは、 例えば (i) 本発明のタンパク質、 その一部のペプチド 又はそれらの塩 （該タンパク質を発現する形質転換体を含んでいもよい、 以下同 様） などに基質を接虫させた: ff^と、 （i i)本発明のタンパク質、 その一部のぺプ チド又はそれらの塩などに基質及び試！^料を接触させた： 1¾との比較を行う。 具体的には、 上記スクリーニングでは、 当該生物学的活性 （例えば、 プロテア一 ゼ活性など） を測定して、 比較する。  In the screening, 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 1¾. Specifically, in the above-mentioned screening, the biological activity (for example, protease activity) is measured and compared.
基質としては、 編 Ps等の基質となることのできるものであれば何れのものであ つてよい。 例えば、 カゼイン、 コラーゲン、 合成オリゴペプチド類などのプロテ ァ一ゼ活性を l定する目的で ί®1されるもの中から選んで用いること力できるが 、 好ましくは置 s用の蛍 識合成べプチド基質などを删できる。基質は、 そ のまま使用できるカ^ 好ましくはフルォレツセインなどの堂光、 酵素や方谢性物 質で標識したものを できる。 試!^:料としては、 例えばタンパク質、 ペプチド、 非ペプチド性化合物、 合成 ィ匕"^、 発酵生産物、 植物抽出物、 動物などの應抽出物、 細胞抽出物などが挙 げられる。 試験試料に翻される試験化合物の例には、 好ましくは羅 P P且豁 (J、 國 P フアミリ一に対するインヒビタ一活性を有する化合物、 特には合成化合物を 含んでいてよい。 これら化合物は、 新規な化合物であってもよいし、 公知の化合 物であってもよい。 該スクリーニングは、 通常のプロテア一ゼ活性の測定法に準 じて実施することができ、 例えば Biochemi stry, 32, pp. 4330-4337 (1993) に示 されている方法などを参考にして行うこと力できる。 また、 各種標識、 緩衝液系 その«当な!^等を使用したり、 そこで説明した操作等に準じて行うこと力くで きる。 スクリ一二ングにあたっては、 應 Ps等をァミノフェニル酢酸水銀などの活 性化剤で処理したり、 その前駆体ある 、は潜在型のものを活性型のものに予め変 換しておくこともできる。 測定は通常トリス塩酸緩衝液、 リン酸塩緩衝液などの 反応に悪影響を与えないような緩衝液等の中で、 例えば、 pH約 4〜約 10 (好まし くは、 pH約 6〜約 8 ) にお 、て亍ぅこと力できる。 The substrate may be any substrate as long as it can be a substrate such as knitted Ps. For example, 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. famili, especially 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.
これら個々のスクリ一ニングにあたっては、 それぞれの方法における通常の条件 、 操作法に当業者の通常の技術的配慮を加えて、 本発明の Ν- Tes あるいはそれと 実質的に同等な活性を有するポリべプチドあるいはべプチドに関連した測定系を 構築すればよい。 これらの一般的な技術手段の詳細については、 総説、 成書など を参照すること力くできる 〔例えば、 Methods in Enzymology, Vol. 1， 2, 5 & 6 (Preparation and Assay of Enzymes)； |口 J書， Vol. 3 (Preparat ion and Assay of Substrates)； 同書， Vol. (Special Techniques for the Bnzymologist)； 同書， Vol. 19 (Proteolytic Enzymes) ;同書， Vol. 45 (Proteolytic Enzymes, Part B) ；同書， Vol. 80 (Proteolyt ic Enzymes, Part C) (以上、 Academic Pre ss社 （USA)発行） など参照〕 o 本発明のスクリーニング方法又はスクリーニングキットを用いて得られる化合 物又はその塩は、 上記した試験化合物、 例えば、 ペプチド、 タンパク質、 非ぺプ チト" '性化合物、 合成化合物、 発酵生産物、 細胞抽出液、 植物抽出液、 動物組織抽 出液など力、ら選ばれた化合物であり、 本発明のタンパク質等の機能を促進あるし、 は阻害する化合物である。該化合物の塩としては、 例えば、 薬学的に許容される 塩などが挙げられる。 例えば、 無機塩基との塩、 有機驢との塩、 無機酸との塩 、 有機酸との塩、 驢性または酸性アミノ酸との塩などが挙げられる。無機驢 との塩の好適な例としては、 例えば、 ナトリウム塩、 カリウム塩などのアルカリ 金属塩、 カノレシゥム塩、 マグネシウム塩などのアルカリ土類金属塩、 並びにアル ミニゥム塩、 アンモニゥム塩などが挙げられる。 有機塩基との塩の好適な例とし ては、 例えば、 トリメチルァミン、 トリェチルァミン、 ピリジン、 ピコリン、 2， 6-ルチジン、 エタノールァミン、 ジエタノールァミン、 トリエタノールァミン、 シクロへキシノレァミン、 ジシクロへキシルァミン、 Ν, Ν' - ジベンジルエチレンジ ァミンなどとの塩が挙げられる。 無機酸との塩の好適な例としては、 例えば、 塩 酸、 臭化水素酸、 硫酸、 リン酸などとの塩が挙げられる。 有機酸との塩の好適な 例としては、 例えば、 ギ H、 酢酸、 プロピオン酸、 フマル酸、 シユウ酸、 酒石酸 、 マレイン酸、 クェン酸、 コハク酸、 リンゴ遞、 メタンスルホン酸、 ベンゼンス ルホン酸、 安息香酸などとの塩が挙げられる。 塩基性アミノ酸との塩の好適な例 としては、 例えば、 アルギニン、 リジン、 オル二チンなどとの塩が挙げられ、 酸 性アミノ酸との塩の好適な例としては、 例えば、 ァスパラギン酸、 グルタミン酸 などとの塩が挙げられる。 本明細書中、 「抗体」 との用語は、 広義の意味で使用されるものであってよく 、 所望の N-Tes ポリぺプチド及び関連べプチド断片に対するモノクローナル抗体 の単一のものや各種ェピトープに対する特異性を持つ抗体組成物であつてよく、 また 1価抗体または多価抗体並びにポリクローナル抗体及びモノク口一ナル抗体 を含むものであり、 さらに天然型（intact)分子並びにそれらのフラグメント及び 誘 本も表すものであり、 F(ab' ) 2, Fab' 及び Fab といったフラグメントを包含 し、 さらに少なくとも二つの抗原又はェピト一プ （epi tope)結合部位を有するキ メラ抗体若しくは雑種抗体、 又は、 例えば、 クヮドローム (qimdrome), トリオ一 ム（triome)などの二重特異性!且換え抗体、 種間雑種抗体、 抗イディォタイプ抗体 、 さらには化学的に修飾あるいは加工などされてこれらの誘 本と考えられるも の、 公知の細胞融合又はハイプリ ドーマ技術や抗体工学を適用したり、 合成ある 、は半合 術を使用して得られた抗体、 抗体生成の観点から公知である¾*技 術を適用したり、 DNA組換え技術を用いて調製される抗体、 本明細書で記載し且 つ定義する標的抗原物質あるし、は標的ェピト一プに関して中和特性を有したりす る抗体又は結合特性を有する抗体を包含していてよい。 本明細書中 「抗体」 との 用語は、 所望の N- Tes ポリペプチド及び関連ペプチド断片に対する抗体と同様、 ίτΐΜΜΡ抗体についても同様な意味で解釈される。 抗原物質に対して作製されるモノクローナル抗体は、 ±咅養中の一連のセルライ ンにより抗体分子の産生を樹共することのできる任意の方法を用いて産生される 。 修觸吾 「モノクローナル」 とは、 実質上均質な抗体の集団から得られていると 、うその抗体の性格を示すものであつて、 何ら力、の特定の方法によりその抗体が 産生される必要があるとみなしてはならない。 個々のモノクローナル抗体は、 自 然に生ずるかもしれない変異体が僅かな量だけ ίしているかもしれないという 以外は、 同一であるような抗体の集団を含んでいるものである。 モノクローナノレ 抗体は、 高い特異性を持ち、 それは単一の抗原性をもつサイ卜に対して向けられ ているものである。 異なった抗原決定基 （ェピト一プ） に対して向けられた種々 の抗体を典型的には含んでいる通常の （ポリクロ一ナル） 抗体調製物と対比する と、 それぞれのモノクローナル抗体は当該抗原上の単一の抗原決 ¾Sに対して向 けられているものである。 その特異性に加えて、 モノクローナル抗体は、 ハイブ リ ドーマ培養により合成され、 他のィムノグロブリン類の «がないあるいは少 な 、点でも優れて 、る。 モノクロ一ナル抗体は、 、イブリッド抗体及びリコンビ ナント抗体を含むものである。 それらは、 所望の生物活性を示す限り、 その由来 やィムノグロブリンクラスゃサブクラスの に関わりなく、 可変領域ドメィン を定常領域ドメインで置き換えたり （例えば、 ヒト化抗体） 、 あるいは軽鎖を重 鎖で置き換えたり、 ある種の鎖を別の種の鎖でもって置き換えたり、 あるいはへ テロジーニアスなタンパク質と融合せしめたりして得ること力《できる （例えば、 米国特許第 4816567号； Monoclonal Antibody Production Techniques and Appl i cat ions, pp. 79-97, Marcel Dekker, Inc. , New York, 1987など） 。 In these individual screenings, the Ν-Tes of the present invention or a polybenth having substantially the same activity as the て -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. 19 (Proteolytic Enzymes); Id., Vol. 45 (Proteolytic Enzymes, Part B); See Ibid., Vol. 80 (Proteolytic Enzymes, Part C) (above, published by Academic Press (USA)), etc. o Compounds or salts thereof obtained by using the screening method or screening kit of the present invention are as described above. 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. Examples thereof include salts with inorganic bases, salts with organic donkeys, salts with inorganic acids, salts with organic acids, and salts with donkey or acidic amino acids. Preferable examples of 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, Ν, Ν'-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. Preferable examples of 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. As used herein, 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. Which includes fragments such as F (ab ') 2 , Fab' and Fab, and further comprises a chimeric or hybrid antibody having at least two antigens or epitope binding sites, or Bispecificity such as qimdrome, triome, etc.! Recombinant antibodies, interspecific hybrid antibodies, anti-idiotypic antibodies, and even chemically Antibodies obtained by applying known cell fusion or hybridoma technology or antibody engineering, or by using semi-synthesis or antibody production技 * techniques that are known from the perspective of Antibodies prepared by the application of recombinant DNA technology or recombinant DNA technology, antibodies that have neutralizing properties with respect to the target antigen as defined and defined herein, or Antibodies with binding properties may be included. As used herein, the term "antibody" is to be construed in the same manner for the {τ} 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. Shutaigo "Monoclonal", when obtained from a substantially homogeneous population of antibodies, indicates the nature of the lie antibody and requires the production of that antibody by any particular method. Do not assume that there is. 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. They replace 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. 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).
モノクローナル抗体を製造する好適な方法の例には、 ハイプリ ドーマ法 (G. K ohler and C. Mi lstein, Nature, 256, pp. 495-497 (1975))； ヒト B細胞ハイブ リ ドーマ法 (Kozbor et al.， Immunology Today, 4, pp.72-79 (1983) ; Kozbor, J. Immunol. , 133， pp. 3001 (1984)； Brodeur et al. , Monoclonal Antibody P roduction Techniques and Applications, pp. 51-63, Marcel Dekker, Inc. , Ne w York (1987)；トリオ一マ法； EBV-ハイブリ ド一マ法 （Cole et al. , Monoclona 1 Antibodies and Cancer Therapy, Alan R. Liss, Inc. , pp. 77-96 (1985)) (ヒ トモノクローナル抗体を産生するための方法) ;米国特許第 4946778号（単鎖抗体 の産生のための技術） 力く挙げられる他、 抗体に関して以下の文献が挙げられる： S. Biocca et al. , BMBO J, 9, pp. 101-108 (1990)； R. E. Bird et al , Scienc e， 242, pp.423-426 (1988)； M. A. Boss et al. , Nucl. Acids Res. , 12, pp. 37 91-3806 (1984)； J. Bukovsky et al. , Hybrid隠, 6， pp. 219-228 (1987)； M. DA励 et al. , Anal. Biochem. , 166, pp. 223-229 (1987)； J. S. Huston et al. ， Proc. Natl. Acad. Sci. USA, 85， pp. 5879-5883 (1988)； P. T. Jones et al. ， Nature, 321， pp. 522-525 (1986)； J. J. Langone et al. (ed. )， "Methods in Bnzymology", Vol. 121 (Immunochemical Techniques, Part I : Hybridoma Tec hnology and Monoclonal Antibodies), Academic Press, New York (1986)； S. Morrison et al. , Proc. Natl. Acad. Sci. USA, 81, p. 6851-6855 (1984)； V. T. Oi et al. , BioTechniques, 4, pp. 214-221 (1986) ; L. Riechmann et al. , Nature, 332, pp. 323-327 (1988)； A. Tramontano et al. , Proc. Natl. Acad. Sci. USA, 83, pp. 6736-6740 (1986)； C. Wood et al. , Nature, 314, pp. 446-4 49 (1985)； Nature, 314, pp. 452-454 (1985) あるいはそこで引用された文献 ( それらの中にある記載はそれを参照することにより本明細書の開示に含められる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. 77-96 (1985)) (Method for Producing Human Monoclonal Antibodies); US Pat. No. 4,946,778 (Technique for Production of Single Chain Antibodies) The following documents are mentioned: S. Biocca et al., BMBO J, 9, pp. 101-108 (1990); RE Bird et al, Science, 242, pp. 423-426 (1988); MA Boss et. Al., Nucl. Acids Res., 12, pp. 3791-3806 (1984); J. Bukovsky et al., Hybrid Hidden, 6, pp. 219-228 (1987); M. DA En et al., Anal. Biochem., 166, pp. 223-229 (1987); JS Huston et al., Proc. Natl. Acad. Sci. USA, 85, pp. 5879-5883 (1988); PT Jones et al., Nature, 321, pp. 522-525 (1986); JJ Langone et al. (Ed.), "Methods in Bnzymology", Vol. 121 (Immunochemical Techniques, Part I: Hybridoma Tec hnology and Monoclonal Antibodies Academic Press, New York (1986); S. Morrison et al., Proc. Natl. Acad. Sci. USA, 81, p. 6851-6855 (1984); VT Oi et al., BioTechniques, 4, pp. 214-221 (1986); L. Riechmann et al., Nature, 332, pp. 323-327 (1988); A. Tramontano et al., Proc. Natl. Acad. Sci. USA, 83, pp. 6736. -6740 (1986); C. Wood et al., Nature, 314, pp. 446-449 (1985); Nature, 314, pp. 452-454 (1985) or references cited therein (in which Certain descriptions are included in the disclosure of the present specification by reference thereto
) o 本発明に係るモノクローナル抗体は、 それら力所望の生物活性を示す限り、 重 鎖及び: 又は軽鎖の ϋ ^特定の種から誘導される又は特定の抗体クラス若しく はサブクラスに属する抗体の対応配列と同一又はホモローガスであるが、 一方、 鎖の残部は、 別の種から誘導される又は別の抗体クラス若しくはサブクラスに属 する抗体の対応配列と同一又はホモ口一ガスである、 「キメラ」 抗体 （免疫グロ ブリン） を特に包含する （米国特許第 4816567号明細書； orrison et al. , Pro c. Natl. Acad. Sci. USA, 81, p. 6851-6855 (1984)) 。 o 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. Specifically encompasses antibodies (immunoglobulins) (US Pat. No. 4,816,567; orrison et al., Pro. c. Natl. Acad. Sci. USA, 81, p. 6851-6855 (1984)).
以下、 モノクローナル抗体を例に挙げて、 抗体の作製につき詳しく説明する。 本発明のモノク口一ナル抗体は、 ミエローマ細胞を用いての細胞融合技術 (G. Kohler and C. Mi lstein, Nature, 256, pp. 495-497 (1975)など） を利用して得 られたモノクローナル抗体であってよく、 例えば次のような工程で作製できる。 Hereinafter, the production of the antibody will be described in detail using a monoclonal antibody as an example. 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.
1 . 免麵生碰の調製 1. Preparation of license
2 . 免;^性抗原による動物の免疫  2. Immunization of animal with ^^ antigen
3 . ミエ口一マ細胞 （骨髄騸田胞） の調製  3. Preparation of myeoma cells (bone marrow cells)
4 . 抗体産 細胞とミエロ一マ細胞との細胞融合  4. Cell fusion between antibody-producing cells and myeloma cells
5 . ハイブリ ドーマ （融合細胞） の選 びモノクロ一ンィ匕  5. Selection of hybrid doma (fused cells)
6 . モノクローナル抗体の製造  6. Production of monoclonal antibodies
1 . 免疫原性麵の調製 1. Preparation of immunogenicity
抗原としては、 上記で記載してあるように、 N- Tes ポリぺプチド又はそれ力、ら 誘導された断片を単離したものを用いることもできるカ^ 決定された N- Tes のァ ミノ酸配列'髒 Rを基に、 適当なォリゴぺプチドを化学合成しそれを抗原として利 用すること力できる。 代表的には配列表の配列番号: 2の  As described above, an 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. Based on the sequence 'R, an appropriate oligopeptide can be chemically synthesized and used as an antigen. Typically, SEQ ID NO: 2 in the sequence listing
(1) 少なくとも第 311位〜第 313位のアミノ酸配列；  (1) at least the amino acid sequence at positions 311 to 313;
(2) 第 22位〜第 122位のァミノ酸配列；  (2) an amino acid sequence at positions 22 to 122;
(3) 第 22位〜第 313位のアミノ酸配列； 及び  (3) the amino acid sequence of positions 22 to 313;
(4) 第 1位〜第 313位のァミノ酸配列  (4) Amino acid sequence at positions 1 to 313
から成る群から選ばれた領域に するァミノ酸残基のうちの連続した少なくと も 5個のァミノ酸を有するぺプチドが挙げられる。 And a peptide having at least five consecutive amino acids among the amino acid residues in the region selected from the group consisting of:
抗原は、 そのまま適当なアジュバントと混合して動物を免疫するのに使用でき るカ^ 免疫原性コンジュゲートなどにしてもよい。 例えば、 免麵として用いる ¾¾¾は、 N-Tes を断片化したもの、 あるいはそのアミノ酸配列に基づき 的な 配列領域を選び、 ボリぺプチドをデザィンして化学合成して得られた合成ポリぺ プチド断片であってもよい。 また、 その断片を適当な縮合剤を介して種々の担体 タンノ、"ク質類と結合させてハプテンータンノ、。ク質の如き免麵性コンジュゲ一ト とし、 これを用レ、て特定の配列のみと反応できる （あるいは特定の配列のみを認 識できる） モノクローナル抗体をデザインするのに用いることもできる。 デザィ ンされるポリぺプチドには予めシスティン残基などを付加し、 免 3 ^性コンジュ ゲー卜の調製を額にできるようにしておくこと力できる。 担体タンパク質類と 結合させるにあたっては、 担体タンパク質類はまず活性化されること力《できる。 こうした活性化にあたり活性化結合基を導人すること力挙げられる。 The antigen may be mixed with an appropriate adjuvant as it is to form a immunogenic conjugate which can be used for immunizing animals. For example, 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. In addition, 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. 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.
活性化結合基としては、 （1) 活性化エステルあるいは活性化カルボキシル基、 例えばニトロフヱニルエステル基、 ペンタフルオロフヱニルエステノレ基、 卜ベン ゾトリアゾ一ルエステル基、 N-スクシンイミ ドエステル基など、 (2) 活性化ジチ ォ基、 例えば 2-ピリジルジチォ基などが挙げられる。 担体タンパク質類としては 、 キーホーノレ' リンペッ ト 'へモシァニン （KLH)、 牛血清アルブミン （BSA)、 卵 白アルブミン、 グロプリン、 ポリリジンなどのポリぺプタイド、 細菌菌体成分、 例えば BCGなどが挙げられる。  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.
2 · .免疫原性抗原による動物の免疫 2.Immunization of animals with immunogenic antigens
免疫は、 当業者に知られた方法により行うこと力くでき、 例えば ' tt公繁、 他編、 実,胜物^!冓座 14、 免胜物 丸善株式会社、 昭和 60年、 日本生化学会編、 続生化学実験講座 5、 免胜化 开究法、 東京化学同人、 1986年、 日本生化学会 編、 新生化学実験講座 12、 分子免疫学 Ι Π、 抗原.抗体 .補体、 東京化学同人 、 1992年などに記載の方法に準じて行うこと力くできる。 免疫化剤を （必要に応じ アジパ'ントと共に） 一回又はそれ以上の回数哺乳動物などに ί¾寸することにより 免疫化される。代表的には、 該免疫化剤及び/又はアジバントを哺乳動物に複数 回皮下測あるいは翻空内 ¾寸することによりなされる。 免疫化剤は、上記抗原 ぺプチドぁる 、はその関連べプチド断片を含むものが挙げられる。 免疫化剤は、 免疫処理される哺乳動物にお 、て免疫原性であることの知られてレ、るタンパク質 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). Typically, the immunizing agent and / or adjuvant is measured by subcutaneously measuring or intrasternally measuring a mammal a plurality of times. Examples of 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.
(例えば上記担体タンパク質類など） とコンジユゲートを形成せしめて^ fflして もよい。 アジュバントとしては、 例えばフ口イン卜完全アジュバント、 リビ (Rib i)アジュバント、 百日咳ワクチン、 BCG、 リピッ KA、 リボソーム、 水酸ィ匕アル ミニゥム、 シリカなどが挙げられる。 免疫は、 例えば BALB/cなどのマウス、 ハム スター、 その他の適当な動物を使用して行われる。抗原の投与量は、 例えばマウ スに対して約 1〜400 g/動物で、 一般には宿主動物の腿空内や皮下に舗し 、 以後 1〜 4週間おきに、 好ましくは 1〜2週間ごとに翻空内、 皮下、 静脈内あ るいは筋肉内に追加免疫を 2〜10回程度反復して行う。 免疫用のマウスとしては BALB/c系マウスの他、 BALB/c系マウスと他系マウスとの F1マウスなどを用いるこ ともできる。 必要に応じ、 抗体価測定系を調製し、 抗体価を測定して動物免疫の 程度を確認できる。 本発明の抗体は、 こうして得られ免疫された動物から得られ たものであってよく、 例えば、 , ポリクローナル抗ィ本等を包含する。 The conjugate may be formed with (for example, the above-mentioned carrier proteins) and subjected to ^ ffl. Examples of 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. As a mouse for immunization, 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.
3 . ミエローマ細胞 （骨髄腸田胞） の調製 3. Preparation of myeloma cells
細胞融合に麵される無限増歹 i i能株 纏細胞株） としては免疫グロブリン を産生しない細胞株から選ぶこと力でき、 例えば Ρ3 - NS-卜 Ag4 - 1 (NS-1, Bur. J. Immunol. , 6 : 511 - 519， 1976)、 SP-2/0-Agl4 (SP-2, Nature, 276: 269 -27 0, 1978 ). マウスミエローマ MOPC- 21セルライン由来の P3- X63- Ag8_Ul (P3U1, C urr. topi cs Mi crobiol. Immunol. , 81 : 1-7, 1978 )、 P3-X63-Ag8 (X63, Natur e, 256 : 495-497, 1975 ) 、 P3-X63-Ag8-653 (653, J. Immunol. , 123: 1548-15 50, 1979) などを用いることができる。 8-ァザグァニン耐性のマウスミエローマ 細胞株はダルベッコ MEM培地（DMEM培地） 、 RPMI-1640培地などの細胞培地に、 例えばペニシリン、 アミカシンなどの ½物質、 牛胎児血清 (FCS) などを加え、 さらに 8—ァザグァニン （例えば 5〜45 zg/ml) を加えた培地で継代される力 細胞融合の 2〜 5日前に正常培地で継代して所要数の細胞株を用意すること力《で きる。 また使用細胞株は、 凍結保存株を約 37°Cで完全に解凍したのち RPMト 1640 i咅地などの正常 ±咅地で 3回以上洗净後、 正常培地で培養して所要数の細胞株を用 意したものであってもよい。 . 抗体産生細胞とミエロ一マ細胞との細胞融合  As an infinitely expanding cell line for cell fusion, a cell line that does not produce immunoglobulin can be selected. For example, Ρ3-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. Immunol., 123: 1548-1550, 1979). 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. 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
上記 2 . の工程に従い免疫された動物、 例えばマウスは最終免疫後、 2〜5日 後にその脾臓が摘出され、 それから脾細胞懸濁液を得る。脾細胞の他、 生体各所 のリンパ節細胞を得て、 それを細胞融合に ί¾¾することもできる。 こうして得ら れた脾細胞懸濁液と上記 3 . の工程に従い得られたミエローマ細胞株を、 例えば 最小必須培地 (MEM培地） 、 DMEM培地、 RPMI-1640 ±咅地などの細胞培地中に置き、 細胞融合剤、 例えばポリエチレングリコールを添加する。細胞融合剤としては、 この他各種当該分野で知られたものを用いることカでき、 この様なものとしては 不活性化したセンダイウィルス（HVJ : Hemagglutinat ing Virus of Japan)なども 挙げられる。 好ましくは、 例えば 30〜60%のポリエチレングリコールを 0. 5〜2 ml加えること力くでき、 分子量が 1, 000〜8，000のポリエチレングリコールを用い ること力くでき、 さらに 量が 1, 000-4, 000 のポリエチレングリコールがより 好ましく使用できる。 融合培地中でのポリエチレングリコールの濃度は、 例えば 30〜60%となるようにすること力 子ましい。 必要に応じ、 例えばジメチルスルホ キシドなどを少 fttaえ、 融合を促進することもできる。 融合に使用する脾細胞 ( リンパ球） ： ミエ口一マ細胞株の割合は、 例えば 1 : 1〜20 : 1とすること力く挙げら れるが、 より好ましくは 4: 1〜7 : 1 とすることができる。 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. In addition to spleen cells, lymph node cells from various parts of the body can be obtained and used for cell fusion. Thus obtained 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. An agent such as polyethylene glycol is added. As 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). Preferably, for example, 0.5 to 2 ml of 30 to 60% of polyethylene glycol can be added, 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.
融合反応を 1〜10分間行い、 次に RPMト 1640培地などの細胞培地を加える。 融 合反応処理は複数回行うこともできる。 融合反応処理後、 遠心などにより細胞を 分離した後選択用培地に移す。  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.
5 . ハイプリ ドーマ （S合細胞） の選嫩びモノクローンィ匕 5. Selection of Hypri Doma (S-cell)
選択用培地としては、 例えばヒポキサンチン (H) 、 アミノプテリン (A) 及びチ ミジン (T) を含む、 FCS含有 MEM培地、 RPMI-1640 ±咅地などの i咅地、 所謂 HAT±咅 地が挙げられる。 選択培地交換の方法は、 一般的には培養プレートに分注した容 量と等容量を翌日加え、 その後 1〜 3日ごとに HAT培地で半量ずつ交換するとい うように処理することができる力く、 ffiこれに ¾Hを加えて行うこともできる。 また融合後 8〜16日目には、 アミノプテリンを除レ、ナこヽ 所謂 HT±咅地で 1〜 4日ご とに培地交換をすること力くできる。 フィーダ一として、 例えばマウス胸腺細胞を 使用することもでき、 それが好ましい がある。  As the selection 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. No. 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 を H to this. On the 8th to 16th day after the fusion, aminopterin is removed, and the medium can be exchanged every 1 to 4 days in a so-called HT ± ground. As a feeder, for example, mouse thymocytes can be used, which is preferable.
ハイプリ ドーマの増殖のさ力、んな培養ゥ工ルの培養上清を、 例えば放射免疫分 析 (RIA) 、 酵素免疫分析 (ELISA) 、 蛍光免疫分析 (FIA) などの測定系、 あるいは 蛍光惹起細胞分离1¾置 (FACS)などで、 戸；?定の断片べプチドを として用いたり 、 あるいは標識抗マウス抗体を用いて目的抗体を測定するなどして、 スクリ一二 ングしたりする。 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 Alternatively, screening is performed by measuring the target antibody using a labeled anti-mouse antibody.
目的抗体を産生しているハイプリ ドーマをクロ一ニングする。 クローニングは 、 寒天培地中でコロニーをピック ·アップする力、、 あるいは限界希釈法によりな されうる。 限界希釈法でより好ましく行うこと力くできる。 クロ一ニングは複数回 行うこと力 子ましい。  Cloning hybridomas producing the desired antibody. Cloning 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.
6 . モノクローナル抗体の製造 6. Production of monoclonal antibodies
得られたハイプリ ドーマ株は、 FCS含有 MEM ±咅地、 RPMI-1640 ±咅地などの適当 な増翻培地中で培養し、 その培: ¾Lb清から所望のモノクローナル抗体を得るこ と力く出来る。 ： の抗体を得るためには、 ハイプリドーマを腹水化すること力く挙 げられる。 この: i¾ミエローマ細胞由来の動物と同系の糸 1織適合性動物の腹腔内 に各ノ、ィブリ ドーマを移植し、 増殖させる力、、 あるいは例えばヌ一ド 'マウスな どに各ハイプリ ドーマを移植し、 増殖させ、 言纖物の腹水中に産生されたモノク 口一ナル抗体を回収して得ることが出来る。 動物はハイプリ ドーマの移植に ち、 プリスタン （2， 6， 10, 14-テトラメチルペンタデカン） などの鉱物油を翻空内 投与しておくこと力でき、 その処理後、 ハイプリ ドーマを増殖させ、 腹水を採取 することもできる。 腹水液はそのまま、 あるいは従来公知の方法、 例えば硫酸ァ ンモニゥム沈殿法などの塩析、 セフアデックスなどによるゲルろ過法、 イオン交 換クロマトグラフィ一法、 電気泳動法、 透析、 限外ろ過法、 ァフィ二ティ 'クロ マトグラフィ一法、 高速液体クロマトグラフィ一法などにより精製してモノクロ 一ナル抗体として用いること力できる。 好ましくは、 モノクロ一ナル抗体を含有 する腹水は、 硫安分画した後、 DEAE—セファロ一スの如き、 陰イオン交換ゲル及 びプロテイン Aカラムの如きァフィ二ティ ·カラムなどで処理し精製分離処理で きる。 特に好ましくは抗原又は抗原断片 （例えば合成ペプチド、 組換え抗原タン パク質あるいはペプチド、 抗体が特異的に認識する部位など） を固定化したァフ ィニティ ·クロマトグラフィー、 プロティン Aを固定ィ匕したァフィ二ティ ·クロ マトグラフィ一、 ヒドロキシァハ。タイト ·クロマトグラフィーなど力く挙げられる また、 トランスジヱニックマウス又はその他の生物、 例えば、 その他の哺乳動 物は、 本発明の免疫原ポリペプチド産物に対するヒト化抗体等の抗体を発現する のに用いること力できる。 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 i¾myeloma 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. The animals can transfer mineral oils such as pristane (2,6,10,14-tetramethylpentadecane) into the air after transplantation of the hybridomas. After the treatment, 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. Preferably, 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. it can. Particularly preferably, 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 · Powerful such as chromatography Also, transgenic mice or other organisms, such as other mammals, can be used to express antibodies, such as humanized antibodies, to the immunogenic polypeptide products of the invention.
またこうして: に得られた抗体の配列を決定したり、 ハイプリ ドーマ株から 得られた抗体をコードする核酸配列を禾 (用して、 遺伝 且換え技術により抗体を 作製することも可能である。 当該モノク口一ナル抗体をコ一ドする核酸は、 例え ばマゥス抗体の重鎖や軽鎖をコードしている遺 に特異的に結合できるオリゴ ヌクレオチドプローブをィ魏するなどの慣用の手法で単離し配列 することが できる。一旦単離された DM は、 上記したようにして発現べクターに入れ、 CH0, It is also possible to determine the sequence of the antibody obtained in this way, or to produce the antibody by genetic and recombination techniques using the nucleic acid sequence encoding the antibody obtained from the hybridoma strain. The 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. Once isolated, DM is placed in an expression vector as described above, and CH0,
COSなどの宿 ±画包に入れることができる。 該 DNAは、 例えばホモジーニアスな マウスの配列に代えて、 ヒ卜の重鎮や軽鎖の定常領域ドメインをコードする配列 に置換するなどして修飾することが可能である （Morrison et al.， Proc. Natl.It 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.
Acad. Sci. USA, 81 : 6581， 1984)。 力、くして所望の結合特異性を有するキメラ 抗体ゃハイブリツド抗体も調製すること力く可能である。 また、 抗体は、 下記する ような縮合剤を用いることを含めた化学的なタンパク合 術を適用して、 キメ ラ抗体やハイプリッ 卜"抗体を調製するなどの修飾をすることも可能である。 ヒト化抗体は、 当該分野で知られた技術により行うこと力河能である （例えば 、 Jones et al. , Nature, 321 : pp. 522-525 (1986)； Riechmann et al. , Nature ， 332: pp. 323-327 (1988)； Verhoeyen et al. , Science, 239 : pp. 1534-1536 ( 1988))。 ヒトモノクローナル抗体も、 当該分野で知られた技術により行うことが 可能で、 ヒトモノクローナル抗体を生産するためのヒトミエロ一マ細胞ゃヒト · マウスへテロミエ口一マ細胞は当該分野で知られている （Kozbor, J. Immunol. ,Acad. Sci. USA, 81: 6581, 1984). It is also possible to prepare chimeric antibodies / hybrid antibodies having the desired binding specificity. 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 ゃ Human / mouse heteromyeloma cells are known in the art (Kozbor, J. Immunol.,
133， pp. 3001 (1984)； Brodeur et al. , Monoclonal Antibody Production Tec hniques and Appl ications, pp. 51-63, Marcel Dekker, Inc. , New York (1987) ) 。 ノくイスぺシフィックな抗体を製造する方法も当該分野で知られている （Mi l l stein et al. , Nature, 305: pp. 537-539 (1983)； WQ93/08829 ; Traunecker et al. , EMB0 J. , 10 : pp. 3655-3659 (1991) ; Suresh et al. , "Methods in Enzymo logy", Vol. 121, pp. 210 (1986)) 。 さらにこれら抗体をトリプシン、 ィン、 ぺプシンなどの酵素により処理し て、 ： ^により還元して得られる Fab Fab' F(ab' ) 2 といった抗体フラグメン トにして删してもよい。 133, pp. 3001 (1984); Brodeur et al., Monoclonal Antibody Production Technologies and Applications, pp. 51-63, Marcel Dekker, Inc., New York (1987)). Methods for producing non-specific antibodies are also known in the art (Millstein et al., Nature, 305: pp. 537-539 (1983); WQ93 / 08829; Traunecker et al., EMB0). J., 10: pp. 3655-3659 (1991); Suresh et al., "Methods in Enzymology", Vol. 121, pp. 210 (1986)). Further, these antibodies may be treated with an enzyme such as trypsin, ind or pepsin to obtain antibody fragments such as Fab Fab'F (ab ') 2 obtained by reducing with ^^.
抗体は、 既知の任意の検定法、 例えば競合的結合検定、 直接及び間接サンドィ ツチ検定、 及び免疫沈 P射：^に翻すること力くできる （Zola, Monoclonal Antib odies : A Manual of Techniques, pp. 147-158 (CRC Press, Inc. , 1987) 抗体を検出可能な原子団にそれぞれコンジユゲー卜するには、 当分野で知られ る任意の方法を使用することができ、 例えば、 David et al. , Biochemistry, 13 卷, 1014-1021頁 (1974)； Pain et al, J. Immunol. Meth. , 40： pp. 219-231 ( 1981) ;及び "Methods in Enzymology", Vol. 184, pp. 138-163 (1990) により記 載の方法が挙げられる。 標識物を付与する抗体としては、 IgG画分、 更にはぺプ シン消ィ匕後還元して得られる特異的結 ^¾15Fab'を用いること力できる。 これらの ： の標識物の例としては、 下記するように酵素 （ペルォキシダ一ゼ、 アルカリ ホスファタ一ゼあるいは /3-D- ガラクトシダーゼなど） 、 ィ匕学物質、 蛍光物質あ る 、は放射性同 素などがある。 本発明での傲口 · SU定は、 ィムノ染色、 例えば糸職ある 、は細胞染色、 ィムノ アツセィ、 例えば競合型ィムノアツセィまたは非競合型ィムノアツセィで亍うこ と力くでき、 ラジオィムノアツセィ、 ELISA， FIAなどを用いること力でき、 B- F分 離を行ってもよいし、 あるいは行わないでその測定を行うこと力できる。 好まし くは方 f免疫測定法、 酵素免疫沏 J定法、 螢光免疫測定法であり、 さらにサンドィ ッチ型ァッセイカく挙げられる。 例えばサンドィッチ型ァッセィでは、 一方を本発 明の N- Tes及びその関連べプチド断片に対する抗体とし、 他方を N- Tesの C末端 側残基に対する抗体とし、 そして一方を検出可能に標識化する。 同じ を認識 できる他の抗体を固相に固定化する。 検体と標識化抗体及び固相化抗体を必要に 応じ順次反応させるためィンキュベーシヨン処理し、 ここで非結合抗体を分離後 、 標識物を測定する。 測定された標識の量は抗原、 すなわち N- Tes ポリペプチド 断片 KJ^の量と比例する。 このアツセィでは、 不溶化抗体や、 標識化抗体の添加 の川醉に応じて同時サンドィッチ型ァッセィ、 フォワード （forward)サンドィッ チ型アツセィあるいは逆サンドィツチ型アツセィなどと呼ばれる。 例えば洗浄、 ί鮮、 震盪、 ろ過あるいは抗原の予備抽出等は、 特定の状況のもとでそれら測定 工程の中で 採用される。 特定の 、 緩衝液等の濃度、 温度あるいはインキ ュベーシヨン処理時間などのその他の測定条件は、 検体中の抗原の濃度、 検体試 料の性質等の要素に従い変えること；^できる。 当業者は通常の実験法を用いなが ら各測定に対して有効な最適の条件を ϋ¾選定して測定を行うこと力出来る。 抗原あるいは抗体を固相化できる多くの担体が知られており、 本発明ではそれ らから: ϋ¾選んで用いること力くできる。担体としては、 抗原抗体反応などに使用 されるもの力く種々知られており、 本発明にぉ 、ても勿論これらの公知のものの中 力、ら選んでィ できる。 特に好適に使用されるものとしては、 例えばガラス、 例 えば活性化ガラス、 多孔質ガラス、 シリ力ゲル、 シリカ一アルミナ、 アルミナ、 磁化鉄、 磁化合金などの無機材料、 ポリエチレン、 ポリプロピレン、 ポリ塩化ビ ニル、 ポリフッ化ビ二リデ ポリ酢酸ビニル、 ポリメタクリレート、 ポリスチ レン、 スチレンーブタジェン共重合体、 ポリアクリルァミ ド、 架橋ポリアクリル 了ミ ド、 スチレン一メタクリレート共重合体、 ポリグリシジルメタクリレート、 ァク口レイン一エチレンダリコールジメタクリレート共重合体など、 架橋化ァノレ ブミン、 コラーゲン、 ゼラチン、 デキストラン、 ァガロース、 架橋ァガロース、 セルロース、 微;結晶セルロース、 カルボキシメチルセルロース、 セルロースァセ テートなどの天然または変成セルロース、 架橋デキストラン、 ナイロンなどのポ リアミ ド、 ポリウレタン、 ポリエポキシ樹脂などの有機高^? %質、 さらにそれ らを乳化重合して得られたもの、 細胞、 赤血球などで、 必要に応じ、 シランカツ プリング剤などで官能性基を導入してあるもの力く挙げられる。 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. -163 (1990) As 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. Then, 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, and 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 ^. In this atsey, the addition of 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. For example, 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. Many carriers on which an antigen or an antibody can be immobilized are known, and in the present invention, they can be selected from: As the 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. Particularly preferably used are, for example, glass, for example, activated glass, porous glass, silica gel, silica-alumina, alumina, magnetized iron, magnetized alloys and other inorganic materials, polyethylene, polypropylene, polyvinyl chloride. Nyl, polyvinylidene polyvinyl acetate, polymethacrylate, polystyrene, styrene-butadiene copolymer, polyacrylamide, cross-linked polyacrylamide, styrene-methacrylate copolymer, polyglycidyl methacrylate, lactone -Ethylene dalicol dimethacrylate copolymer, etc., cross-linked anolevumin, collagen, gelatin, dextran, agarose, cross-linked agarose, cellulose, fine; crystalline cellulose, carboxymethyl cellulose, cellulose acetate, etc. Or 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.
さらに、 ろ紙、 ビーズ、 試験容器の内壁、 例えば試験管、 タイタープレート、 タイタ一ゥヱル、 ガラスセル、 合成樹脂製セルなどの合成材料からなるセル、 ガ ラス棒、 合成材料からなる棒、 末端を太くしたりあるいは細くしたりした棒、 末 端に丸し突起をつけたりあるいは偏平な突起をつけた棒、 '謝反状にした棒などの 固体物質 （物体） の表面などが挙げられる。 これら担体へは、 抗体を結合させること力でき、 好ましくは本発明で得られる 抗原に対し特異的に反応するモノクローナル抗体を結合させること力できる。担 体とこれら 抗体反応に関与するものとの結合は、 吸着などの物理的な手法、 あるいは縮合剤などを用いたり、 活性化されたものなどを用いたりする化学的な 方法、 さらには相互の化学的な結合反応を禾拥した手法などにより行うことが出 標識としては、 酵素、 酵素基質、 酵素インヒビター、 補欠分 ΐ¾、 捕酵素、 酵 素前駆体、 アポ酵素、 蛍光物質、 色素物質、 ィ ルミネッセンス化合物、 発光物 質、 発色物質、 磁気物質、 金属粒子、 例えば金コロイドなど、 放射性物質などを 挙げることができる。酵素としては、 SfeR素酵素、 還元酵素、 酸化酵素などの酸 ィ匕還元酵素、 例えばアミノ基、 カルボキシル基、 メチル基、 ァシル基、 リン酸基 などを転移するのを角蝶する転移酵素、 例えばエステル結合、 グリコシド結合、 エーテル結合、 ペプチド結合などを加水分解する加水分解酵素、 リア一ゼ、 イソ メラーゼ、 リガ一ゼなどを挙げること力くできる。酵素はネ纖の酵素を複合的に用 、て傲口に利用することもできる。 例えば键素的サイクリングを利用することも できる。 代表的な放射性物質の標 liffi同位体元素としては、 [3 2P]， [1 25 ι]， [1 3 1 ι]，In addition, 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 The surface of a solid material (object), 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. Examples of 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. The standard liffi isotope typical radioactive material, [3 2 P], [ 1 25 ι], [1 3 1 ι],
[3H], [1 4 C], [3 5S] などが挙げられる。 [ 3 H], [ 14 C], [ 35 S] and the like.
代表的な酵素標識としては、 西洋ヮサビペルォキシダーゼなどのペルォキシダ ーゼ、 大腸菌 3- D- ガラクトシダーゼなどのガラクトシダーゼ、 マレエート ·デ ヒドロゲナーゼ、 グルコース- 6- フォスフヱート ·デヒドロゲナーゼ、 グルコ一 ス才キシダーゼ、 グルコアミラ一ゼ、 ァセチルコリンエステラーゼ、 カタラーゼ 、 ゥシ小腸アル力リホスファターゼ、 大腸菌アル力リホスファターゼなどのアル カリフォスファターゼなどが挙げられる。  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.
アル力リホスファタ一ゼを用いた: 4-メチルゥンベリフヱリルフォスフエ ―トなどのゥンべリフエ口ン誘 本、 二トロフヱニルホスフヱ一トなどのリン酸 化フェノ一ル誘 本、 NADPを利用した酵素的サイクリング系、 ルシフエリン誘導 体、 ジォキセタン誘 本などの基質を使用したりして、 生ずる 光、 発光などに より測定できる。 ノレシフェリン、 ルシフヱラ一ゼ系を利用したりすることもでき る。 力タラ一ゼを用いた:^、 過酸化水素と反応して酸素を生成するので、 その 酸素を電極などで ί数口することもできる。 電極としてはガラス電極、 難溶性 莫 を用いるイオン電極、 翻！ ¾電極、 高分子膜電極などであることもできる。 酵素標識は、 ビォチン標識体と酵素標識ァビジン （ストレブトァビジン） に置 き換えることも可能である。標識は、 複数の異なった観の標識を使用すること もできる。 こうした^、 複数の測定を連続的に、 あるいは非連続的に、 そして 同時にあるいは別々に行うことを可能にすることもできる。 標識及び検知可能な 信号を得るには、 それに適した市販のキットを用いて行うこと力くでき、 キット製 造業者あるいはキット販売業者により明らかにされているプロトコルに従って実 施することもできる。 本発明にお 、ては、 信号の形成に 4 ヒドロキシフエニル酔酸、 1， 2-フヱニレン ジァミン、 テトラメチルベンジジンなどと西洋ヮサビ ·ペルォキシダ一ゼ、 ゥン ベリフヱリノレガラクトシド、 ニトロフヱニルガラクトシドなどと 3 - D-ガラクト シダ一ゼ、 グルコース- 6- リン酸'デヒドロゲナ一ゼなどの酵素試薬の組合わせ も禾 (J用でき、 ヒドロキノン、 ヒドロキシベンゾキノン、 ヒドロキシアントラキノ ンなどのキノール化合物、 リポ酸、 グノレタチオンなどのチオール化合物、 フエノ —ル誘 本、 フエロセン誘 本などを酵素などの働きで形成しうるもの力く使用で きる。 Using alkaline phosphatase: Invitation of 4-methylphenylphenol phosphate, etc., and phosphorylation of phenol, such as ditrophenylphosphonate. , 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. With the use of 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. ¾ 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. In the present invention, the formation of a signal includes 4-hydroxyphenylsulfuric acid, 1,2-phenylenediamine, tetramethylbenzidine and the like, as well as horseradish peroxidase, campbellifrinoregalactoside, and nitrofuran. Combination of 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.
蛍光物質あるし、は化学ルミネッセンス化合物としては、 フルォレセインイソチ オシァネート、 例えばローダミン Βイソチオシァネート、 テトラメチルローダミ ンイソチオシァネートなどのローダミン誘 #1本、 ダンシルクロリ ド、 ダンシルフ ルオリ ド、 フルォレスカミン、 フィコピリプロテイン、 アタリジニゥム塩、 ノレミ フェリン、 ノレシフェラーゼ、 エタオリンなどのルミノ一ノレ、 ィミダゾール、 シュ ゥ酸エステル、 希 ±ϋキレート化合物、 クマリン誘 本などが挙げられる。 標識するには、 チオール基とマレイミ ド基の反応、 ピリジルジスルフィ ド基と チオール基の反応、 了ミノ基とアルデヒド基の反応などを禾翻して行うこと力くで き、 公知の方法あるいは当該分野の当業者が容易になしうる方法、 さらにはそれ らを修飾した方法の中から its選択して適用できる。 また上記免 性複合体作 製に使用されることのできる縮合剤、 担体との結合に翻されることのできる縮 合剤などを用いることができる。 縮合剤としては、 例えばホルムアルデヒド、 グルタルアルデヒド、 へキサメチ レンジイソシァネート、 へキサメチレンジイソチオシァネート、 Ν， Ν' - ポリメチ レンビスョ一ドアセトアミ ド、 Ν, Ν' - エチレンビスマレイミ ド、 エチレングリコ 一ルビススクシニミジルスクシネート、 ビスジァゾベンジジン、 1-ェチル - 3-(3 - ジメチルァミノプロピノレ） カルボジィミ ド、 スクシンィミジル 3- (2- ピリジル ジチォ） プロピオネート（SPDP)、 N-スクシンィミジル 4 - (N- マレイミ ドメチル ) シクロへキサン- 1_ カルボキシレ一ト（SMCC)、 N-スルホスクシンイミジノレ 4 - (N- マレイミ ドメチル) シクロへキサン- 1- カルボキシレート、 N-スクシンイミ ジル （4- ョ一ドアセチル） ァミノベンゾエート、 N-スクシンィミジル 4 -（卜 マレイミ ドフヱニル） ブチレ一ト、 N- ( £—マレイミ ドカプロィルォキシ） コハ ク酸イミ ド (EMCS)， イミノチオラン、 S -ァセチルメル力プトコハク酸無水物、 メ チル- 3- (4'― ジチォピリジノレ） プロピオンイミデート、 メチル -4- メルカプトブ チリルイミデー卜、 メチル -3- メルカプトプロピオンイミデート、 N -スクシンィ ミジル- S- ァセチルメルカプトァセテ一トなどが挙げられる。 本発明の測定法によれば、 測定すべき物質を酵素などで標識したモノクローナ ル抗体などの標 ϋ抗体 と、 担体に結合された抗体とを順次反応させることが できるし、 同時に S¾させることもできる。 を加える) lli は選ばれた担体系 の型により異なる。 感作されたプラスチックなどのビーズを用いた場合には、 酵 素などで標識したモノクロ一ナル抗体などの標識抗体 を測定すべき物質を含 む検体試料と共に最初適当な試験管中に一緒に入れ、 その後該感作されたプラス チックなどのビーズを加えることにより測定を行うこと力できる。 As a fluorescent substance or a chemiluminescent compound, 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. For labeling, 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. In addition, 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. In addition, 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. Examples of the condensing agent include formaldehyde, glutaraldehyde, hexamethylene diisocyanate, hexamethylene diisothiocyanate, Ν, Ν'-poly (methylene bis-doacetamide), Ν, Ν'-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-succinimidyl 4-(-maleimide Phenyl) butyrate, N- (£ —maleimi docaproyloxy) succinic imid (EMCS), iminothiolane, S-acetylmercaptosuccinic anhydride, methyl-3- (4'-dithiopyridinole) propionimi Dating, methyl-4-mercaptobutyrimidate, methyl-3-mercaptopropionimidate, N-succinimidyl-S-acetylmercaptoacetate, and the like. According to the measurement method of the present invention, 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.
本発明の 法においては、 免疫学的測定法が用いられるが、 その際の固相担 体としては、 抗体などタンパク質を良く吸着するポリスチレン製、 ポリカーボネ イト製、 ポリプロピレン製あるいはポリビニル製のボーノレ、 マイクロプレート、 スティック、 微粒子あるいは試験管などの種々の材料および形態を任意に選択し 、 側すること力くできる。 In the method of the present invention, an immunoassay is used. In this case, 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.
測定にあたつては至適 pH、 例えば pH約 4〜約 9に保つように適当な緩衝液系中 で行うことができる。 特に適切な緩衝剤としては、 例えばアセテート緩衝剤、 ク ェン酸塩緩衝剤、 フォスフェート緩衝剤、 トリス緩衝剤、 トリエタノールァミン 緩衝剤、 ボレ一ト緩衝剤、 グリシン緩衝剤、 炭酸塩緩衝剤、 トリスー塩酸緩ff斉 ij などが挙げられる。 緩衝剤は互いに任意の割合で混合して用いること力くできる。 抗原抗体反応は約 0 °C〜約 60°Cの間の温度で行うこと力 子ましい。  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. Particularly suitable 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.
酵素などで標識されたモノク口一ナル抗体などの抗体 ¾¾¾び担体に結合せし められた抗体襲、 さらには測定すべき物質のインキュベーション処理は、 に達するまで行うこと力《できるカ、 抗原抗体 S¾の ¥ί奐 ϊが達成されるよりもずつ と早い時点で固相と液相とを分離して限定されたィンキュベ一シヨン処理の後に 反応を止めること力でき、 液相又は固相のレ、ずれかにおける酵素などの標識の存 在の驢を測ること力くできる。 測定操作は、 自動化された測識置を用いて行う こと力く可能であり、 ルミネセンス ·ディテクター、 ホト ·ディテクターなどを使 用して基質が酵素の作用で変換されて生ずる表示シグナルを検知して測定するこ ともできる。 抗原抗体反応においては、 それぞれ用いられる藤、 測定すべき物質、 さらに は酵素などの標識を安定化したり、 抗原抗体反応自体を安定化するように適切な 手段を講ずること力できる。 さらに、 異的な反応を除去し、 阻害的に働く影 響を減らしたり、 あるいは測定反応を活性化したりするため、 タンパク質、 安定 ィ匕剤、 界面活性化剤、 キレ一ト化剤などをィンキュベ一シヨン溜夜中に加えるこ ともできる。 キレート化剤としては、 エチレンジァミン四職塩 (BDTA) 力 り 好ましい。  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 S¾ ¥ ίtanί is achieved. Can be used to measure the presence of markers, such as enzymes, in some areas. 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. In 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. In addition, 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. As a chelating agent, ethylenediamine tetrasalt (BDTA) is preferred.
当該分野で普通に採用されて こりあるいは当業者に知られた 異的結合反 応を防ぐためのブロッキング処理を施してもよく、 例えば、 哺乳動物などの正常 血清タンパク質、 アルブミン、 スキムミルク、 乳発酵物質、 コラーゲン、 ゼラチ ンなどで処理すること力できる。非特異的結合反応を防ぐ目的である限り、 それ らの方法は特に限定されず用いること力出来る。 It may be subjected to blocking treatment commonly used in the art or known to those skilled in the art to prevent an aberrant binding reaction. , 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.
本発明の測定方法で測定される試料としては、 あらゆる形態の灘ゃコロイド 溜夜、 ¾体試料などがィ趟しうる力^好ましくは生物由来の試料、例えば胸腺 、 睾丸、 腸、 體、 脳、 乳癌、 卵巣癌、 結腸 ·直腸癌、血液、 血清、血漿、 関節 液、 脳脊髄液、 唾液、 羊水、 尿、 その他の体液、 細胞培養液、 ， 織培養液、 « ホモジュネート、生検試料、 組織、細胞などが挙げられる。  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. , 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.
これら個々の免疫学的測定法を含めた各種の分析 . 法を本発明の測定方法 に適用するにあたっては、特別の条件、操作等の設定は必要とされない。 それぞ れの方法における通常の条件、 操作法に当業者の通常の技術的配慮を加えて、 本 発明の当 !¾¾ "象物質ある 、はそれと実質的に同等な活性を有する物質に関連した 測定系を構築すればよい。 これらの一般的な技術手段の詳細については、 総説、 成書などを参照すること 力くできる 〔例えば、 入江 寛編， 「ラジオィムノアツセィ」 ， 講談社， 昭和 49年 発行；入江 寛編， 「続ラジオィムノアッセィ」 ， 講談社， 昭和 54年発行；石川 栄治ら編， 「酵素免疫測定法」 ， 医学書院， 昭和 53年発行；石川栄治ら編， 「酵 素免疫測定法」 （第 2版）， 医学書院， 昭和 57年発行；石川栄治ら編， 「酵素免 疫測定法」 （第 3版）， 医学書院， 昭和 62年発行； H. V. Vunakis et al. (ed. ) ， "Methods in Enzymology", Vol. 70 (Immunochemical Techniques, Part A), Academic Press, New York (1980)； J. J. Langone et al. (ed. )， "Methods in When applying various analytical methods including these individual immunological assay methods to the assay method of the present invention, no special conditions, operations, and the like need to be set. With the usual conditions and procedures in each method and the ordinary technical considerations of a person skilled in the art, the term "substance" of the present invention relates to a substance having substantially the same activity. For the details of these general technical means, it is possible to refer to reviews, written reports, etc. [For example, Hiroshi Irie, “Radio Imnoatsy”, Kodansha, Showa Published in 1949; edited by Hiroshi Irie, "Continuing Radio Imnoassy", Kodansha, published in 1979; published by Eiji Ishikawa et al., "Enzyme Immunoassay", Medical School, published in 1978; published by Eiji Ishikawa, et al., " Enzyme Immunoassay, 2nd Edition, Medical Shoin, published in 1982; Eiji Ishikawa et al., Ed., Enzyme Immunoassay Assay, 3rd Edition, Medical Shoin, 1987, HV Vunakis et al (ed.), "Methods in Enzymology", Vol. 70 (Immunochemical Techniques, Part A), Academic Press, New York (1980); J. J. Langone et al. (Ed.), "Methods in
Enzymology", Vol. 73 (Immunochemical Techniques, Part B), Academic Pres s, New York (1981)； J. J. Langone et al. (ed. ), "Methods in Enzymology",Enzymology ", Vol. 73 (Immunochemical Techniques, Part B), Academic Press, New York (1981); J. J. Langone et al. (Ed.)," Methods in Enzymology ",
Vol. 74 (Immunochemical Techniques, Part C)， Academic Press, New York ( 1981)； J. J. Langone et al. (ed. ), "Methods in Enzymology", Vol. 84 (I腿 画 chemical Techniques, Part D: Selected Immunoassays), Academic Press, New York (1982)； J. J. Langone et al. (ed. ), "Methods in Enzymology", Vo 1. 92 (Immunochemical Techniques, Part E: Monoclonal Antibodies and Gene ral Immunoassay Methods), Academic Press, New York (1983)； J. J. Langone et al. (ed. )， "Methods in Enzymology", Vol. 121 (Immunochemical Techniq ues, Part I： Hybridoma Technology and Monoclonal Antibodies), Academic P ress, New York (1986)； J. J. Langone et al. (ed. ), "Methods in Enzymolog y", Vol. 178 (Antibodies, Antigens, and Molecular Mimicry), Academic Pre ss, New York (1989)； M. Wi lchek et al. (ed. )， "Methods in Enzymology", V ol. 184 (Avidin-Biotin Technology), Academic Press, New York (1990)； J. J. Langone et al. (ed. ), "Methods in Enzymology", Vol. 203 (Molecular De sign and Model ing: Concepts and Appl ications, Part B: Anibodies and Anti gens, Nucleic Acids, Polysaccharides, and Drugs), Academic Press, New Yo rk (1991) などあるいはそこで引用された文献（それらの中にある記載はそれを 参照することにより本明細書の開示に含められる）〕 。 本発明の抗 N - Tes抗体、 特にモノクローナル抗体を用いて、 ェピトープマツピ ングを行うこともでき、 各ェピト一プを認識する抗体を用いれは "N- Tes及びその 関連べプチド断片などの検知 ·測定を行うことができる。 Vol. 74 (Immunochemical Techniques, Part C), Academic Press, New York (1981); JJ Langone et al. (Ed.), "Methods in Enzymology", Vol. 84 (I technique chemical Techniques, Part D: Selected). Immunoassays), Academic Press, New York (1982); JJ Langone et al. (Ed.), "Methods in Enzymology", Vo 1.92 (Immunochemical Techniques, Part E: Monoclonal Antibodies and General Immunoassay Methods), Academic Press. , New York (1983); JJ Langone et al. (ed.), "Methods in Enzymology", Vol. 121 (Immunochemical Technologies, Part I: Hybridoma Technology and Monoclonal Antibodies), Academic Press, New York (1986); JJ Langone et al. (ed. ), "Methods in Enzymolog y", Vol. 178 (Antibodies, Antigens, and Molecular Mimicry), Academic Press, New York (1989); M. Wilchek et al. (Ed.), "Methods in Enzymology", Vol. 184 (Avidin-Biotin Technology), Academic Press, New York (1990); JJ Langone et al. (Ed.), "Methods in Enzymology", Vol. 203 (Molecular Design and Modeling: Concepts and Appl. ications, Part B: Anibodies and Antigens, Nucleic Acids, Polysaccharides, and Drugs), Academic Press, New York (1991), and the literature cited therein. Included in the disclosure of the specification)]. 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.
N - Tes及びその関連ペプチド断片に対する抗体は、 N-Tes による MT-匪 P類の活 性阻害などの現象の検出及び Z又は測定、 さらには MMPs活性の ii¾により生ずる 各種の生理活性物質の検出及び/又は測定に有用である。 m , 特にモノクロ ーナル抗体は、 （i)匪 Psによる紙織あるいはタンパク質の分解が関連する障害、 異常及び Z又は疾患を検出したり、 （i i) MMPs による紙織あるいはタンパク質の 分解が引き起こす細胞の鶴化、細胞の移動、浸潤、遊 び Z又は転移あるい はその可能性を検出したり、 及び'/又は（i i i)細胞の SSK化、 細胞、 血液系 細胞などの細胞の移動、 浸潤、 遊 び Z又は転移あるいはその可能性を検出す るのに有用である。癌の移動性、 浸潤性、 走化 f级び /又は転移性の程度を知る のに挺用できると期寺される。  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.
本発明にしたがえば、 Ν- Tesによる ΜΤ-議 Ρ類の活性阻害を検出及び/又は測 定し、 抗癌剤、 癌転移阻害剤、 血管新生阻割 U、 アルツハイマー治歸 ij、 関節破 壊治歸 ij、消^^及び Z又は免疫抑制剤の効果判定モニタ―として使用すること 力可食 となる。 また、 本発明では、 ΜΉ-ΜΜΡ及び Z又は MT3-匪 Pによる紙織あるいはタンパク 質の分角親象の検出及び Z又は測定方法やそのための,力 ¾¾共できる。 According to the present invention, the inhibition of the activity of 議 -diet by Ν-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 ΜΉ-ΜΜΡ and Z or MT3-band P, and the power for the method.
本発明でィ する抗体 （モノクローナル抗体を含む） としては、 公知のものあ るいは公知の各種の該抗体の製造法を適用して得られるものを含む他、上記で説 明した抗体の調製法に準じて得られるものも包含される。 本発明の活性成分 〔例えば、 （a) N-Tes ポリぺプチド、 その一部のぺプチドま たはそれらの塩、 それに関連するペプチド等、 (b)該 N- Tes あるいは N- Tes ポリ ペプチドをコードする DNAなどの核酸等、 （c)本発明の抗体、 その 片 （モ ノクロ一ナノレ抗体を包含する） またはその誘 本、 (d) N-Tes による MT- MP類の 活性阻害などの現象ある 、は紙織あるいはタンパク質の分角親象といつた生物学 的活性を抑制及び Z又は阻害する化合物またはその塩、 (e)本発明の DNAなどの 核酸に対するアンチセンス 'オリゴヌクレオチドなど〕 を医薬として用いる： 1¾ 、 例えば " MP阻害剤またはそれらの塩等は、 通常弱虫或いは薬理的に許容される 各画剤補助剤と混合して、 医薬糸賊物又は医薬調製物などとして投与すること ができる。 好ましくは、 経口投与、 局所投与、 または非経口投与等の使用に適し た製剤調製物の形態で投与され、 目的に応じていずれの投与形態 （吸入法、 ある いは直腸投与も包含される） によってもよい。  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) For example, "MP inhibitors or their salts, etc. are usually mixed with wimps or pharmacologically acceptable paint adjuvants and administered as pharmaceutical pirates or pharmaceutical preparations Preferably, 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.
また、 本発明の活' 分は、 mmm (抗癌剤） 、 月藤移転阻割 u、血管新生 阻害剤、 アルツハイマー治獻 ij、 関節破壊治藤 u、 消 ϋ及び z又は免疫抑制剤 と配合して使用することもできる。抛鎮剤 （杭癌剤） 、 s重瘍移転阻害剤、 血管 新生阻害剤、 アルツハイマー治歸 U、 関節破壊治歸 U、 消類 iJや免疫抑制剤とし ては、 有利な働きを持つものであれば制限なく使用でき、 例えば当該分野で知ら れたものの中から選択すること力できる。 そして、 非経口的な投与形態としては、 局所、 経皮、静脈内、 筋肉内、 皮下、 皮内もしくは l空内投与を包含し得るカ^ 患部への直接投与も可能であり、 また ある:^には好適でもある。好ましくはヒトを含む哺乳動物に経口的に、 あるい は非経口的 （例、 細胞内、組織内、静脈内、筋肉内、 皮下、 皮内、 M空内、 胸腔 内、 脊髄腔内、 点滴法、 ¾1昜、 経戲昜、 点耳、 点眼や点鼻、 歯、 皮膚や粘膜への 塗布など） に投与すること力できる。具体的な製剤調製物の形態としては、 碰 製剤、 分散製剤、 半固形製剤、 粉粒体製剤、 成型製剤、 浸出製剤などが挙げられ 、 例えば、 錠剤、 被覆錠剤、 糖衣を施した剤、 丸剤、 トローチ剤、 硬カプセル剤In addition, 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. Preferably, it is orally or parenterally to 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 碰 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. Agent, troche, hard capsule
、 軟カプセノレ剤、 マイクロカプセル剤、 ¾Λ斉 粉末剤、 散剤、顆粒剤、 細粒斉 ij 、 ¾寸剤、 液剤、 エリキシル剤、 ェマルジヨン剤、 灌注剤、 シロップ剤、 水剤、 乳剤、 懸濁剤、 リニメント剤、 ローション剤、 エアゾール剤、 スプレー剤、 吸入 剤、 噴霧剤、 軟膏製剤、 硬膏製剤、 貼付剤、 パスタ剤、 パップ剤、 クリーム剤、 油剤、坐剤 （例えば、 直腸坐剤）、 チンキ剤、 皮膚用水剤、 点眼剤、 点鼻剤、 点 耳剤、 塗布剤、 輸液剤、 拥液剤などのための粉末剤、 凍結乾 剤、 ゲル調 製品等が挙げられる。 , 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.
医薬用の組成物は通常の方法に従って製剤化すること力くできる。例えば、 遊 必要に応じて、生理学的に認められる担体、 医薬として許容される担体、 アジュ バント剤、 貝離剤、補形剤、希釈剤、 香味剤、 香料、 甘味剤、 べヒクル、 防腐剤 、 安定化剤、 結合剤、 p H調節剤、 緩衝剤、 界面活性剤、 基剤、 溶剤、 充塡剤、 増量剤、 溶解補助剤、 可溶化剤、 等張化剤、 乳化剤、 懸濁化剤、 分散剤、 増粘剤 、 ゲル化剤、 硬化剤、 吸収剤、 粘着剤、 弾性剤、 可塑剤、 崩壊剤、 噴射剤、 保存 剤、 抗酸化剤、 遮光剤、 保湿剤、 緩和剤、 帯電防止剤、 無痛化剤などを 虫もし くは組合わせて用い、 それとともに本発明のタンパク質等を混和することによつ て、 一般に認められた製剤 に要求される単删«¾態にして製造することが できる。  Pharmaceutical 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, By using 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.
非経口的使用に適した製剤としては、 活性成分と、 水もしくはそれ以外の薬学 的に許容し得る媒体との無菌性碰、 または懸濁液剤など、 例えば測剤等が举 げられる。一般的には、 水、
Figure imgf000072_0001
デキストロ一ス水碰、 その他関連した糖 の溶液、 ェタノ一ノレ、 プロピレングリコール、 ポリエチレングリコールなどのグ リコール類が好ましい酒寸剤用液体担体として挙げられる。測剤を調製する際 は、 蒸留水、 リンゲル液、 生理餘液のような担体、 適当な分散化剤または湿化 剤及び懸濁化剤などを して当該分野で知られた方法で、 激夜、 懸濁液、 エマ ルジョンのごとき ¾寸しうる形に調製する。 測用の水性液としては、 例えば生理雄液、 ブドウ糖やその他の補助薬 （例 えば、 D-ソルビトール、 D-マンニトール、 塩化ナトリウムなど） を含む等張液な どが挙げられ、 薬理的に許容される適当な溶解補助剤、 たとえばアルコール （た とえばエタノールなど） 、 ボリアルコール （たとえばプロピレングリコーノレ、 ポ リエチレングリコールなど） 、 非イオン性界面活性剤 （たとえばポリソルベートFormulations suitable for parenteral use include sterile solutions of the active ingredient with water or other pharmaceutically acceptable vehicles, or suspensions, such as agents. Generally, water,
Figure imgf000072_0001
Dextrose solution, solutions of other related sugars, glycols such as ethanol, propylene glycol, and polyethylene glycol are examples of preferred liquid carriers for liquor. When preparing a measuring agent, 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. Examples of the aqueous liquid for measurement include menstrual fluid, isotonic solution containing glucose and other auxiliary agents (eg, D-sorbitol, D-mannitol, sodium chloride, etc.), and are pharmacologically acceptable. Suitable solubilizers, such as alcohols (eg, ethanol), polyalcohols (eg, propylene glycol, polyethylene glycol, etc.), nonionic surfactants (eg, polysorbate)
80™, HC0 - 50など） などと併用してもよい。 油性液としてはゴマ油、 大豆油な どが挙げられ、 溶解捕助剤として安息香酸ベンジル、 ベンジルアルコールなどと 併用してもよい。 また、 緩衝剤 （例えば、 リン酸塩緩衝液、 酢酸ナトリウム緩衝 液など） 又は浸透圧調節のための難、 無痛化剤 （例えば、 塩化べンザルコニゥ ム、 塩酸プロ力インなど） 、 安定剤 （例えば、 ヒト血清アルブミン、 ポリエチレ ングリコールなど) 、 保存剤 (例えば、 ベンジルアルコ一ノレ、 フエノーノレなど) 、 ァスコルビン酸などの酸化防止剤、 吸収促進剤などと配合してもよい。 調製さ れた ¾寸液は通常、 適当なァンプルに充塡される。 非経口投与には、 界面活性剤及びその他の薬学的に許容される助剤を加えるか 、 あるいは加えずに、 水、 エタノール又は油のような無菌の薬学的に許容される 液体中の溜夜あるいは懸濁液の形態に製剤化される。 製剤にィ¾¾される油性べヒ クルあるいは溶剤としては、 天然あるいは合成ある 、は半合成のモノあるいはジ あるいはトリグリセリ ド類、 天然、 半合成あるいは合成の油脂類あるいは脂肪酸 類が挙げられ、 例えばピーナッツ油、 トウモロコシ油、 大豆油、 ゴマ油などの植 物油が挙げられる。 例えば、 この细寸剤は、 通常本発明化合物を 0. 1 〜10S»% 禾！ ^含有するように調製されることができる。 80 ™, HC0-50 etc.). Examples of the oily liquid include sesame oil and soybean oil, and may be used in combination with benzyl benzoate, benzyl alcohol, or the like as a dissolution aid. In addition, buffers (eg, phosphate buffer, sodium acetate buffer, etc.) or difficulties for adjusting osmotic pressure, 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. For parenteral administration, with or without surfactants and other pharmaceutically acceptable auxiliaries, in a sterile pharmaceutically acceptable liquid such as water, ethanol or oil Alternatively, it is formulated in the form of a suspension. Examples of the 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. For example, this size preparation usually contains 0.1 to 10 S »% of the compound of the present invention! ^ Can be prepared to contain.
局所的、 例えば口腔、 又は剷昜的ィ顿に適した製剤としては、 例えば洗口剤、 歯磨き剤、 口腔噴霧剤、 吸入剤、 軟膏剤、 歯科充載 ϋ、 歯科コ一ティング剤、 歯 科ペースト剤、 坐剤等が挙げられる。 洗口剤、 その他歯科用剤としては、 薬理的 に許容される担体を用いて慣用の方法により調製される。 口腔噴霧剤、 吸入剤と しては、 本発明化合物自体又は薬理的に許容される不活性担体とともにエアゾー ル又はネブライザ一用の溜夜に溶解させるかあるいは、 吸入用微粉末として歯な どへ投与できる。 軟膏剤は、 通常删される基剤、 例えば、 軟膏基剤 （白色ヮセ リン、 パラフィン、 オリ一ブ油、 マクロゴール 400、 マクロゴール軟膏など） 等 を添加し、 慣用の方法により調製される。 歯、 皮膚への局所塗布用の薬品は、 適切に殺菌した水または非水貝離剤の碰 または懸濁液に調剤すること力できる。 添加剤としては、 例えば亜硫酸水素ナト リゥムまたはェデト酸ニナトリゥムのような緩衝剤；酢酸または硝酸フヱニノ！^ R 銀、 塩化ベンザルコニゥムまたはクロ口へキシジンのような殺菌および抗真菌剤 を含む防腐剤およびヒプロメルローズのような濃厚剤力く挙げられる。 Formulations suitable for topical use, for example, in the oral cavity or for oral 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. 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.
坐剤は、 当該分野において周知の担体、 好ましくは非刺激性の適当な補形剤、 例えばポリエチレングリコール類、 ラノリン、 カカオ脂、 脂肪酸トリグリセライ ド等の、 好ましくは常温では固体である力 管の温度では液体で直腸内で融解し 薬物を放出するものなどを使用して、 慣用の方法により調製されるが、 通常本発 明化合物を 0. 1 〜95fi*%禾號含有するように調製される。 删する W ^剤およ び濃度によって薬品は、 剤に懸濁させるかまたは溶解させること力できる。 局部麻猶 防腐剤および緩衝剤のような補助薬は、 貝離剤に溶解可能である。 経口的使用に適した製剤としては、 例えば錠剤、 丸剤、 カプセル剤、 粉末剤、 顆粒剤、 トローチのような固形組成物や、 液剤、 シロップ剤、 懸濁剤のような液 状組成物等が挙げられる。 製剤調製する際は、 当該分野で知られた製剤補助剤な どを用いる。 鍵剤及び丸剤はさらにェンテリックコ一ティングされて製造される こともできる。 調剤単 ί 態がカプセルである齢には、 前記タイプの材料にさ らに油脂のような液状担体を含有することができる。 さらに、 本発明の DNAなどの核酸を上記したような治療及び Z'又は予防剤とし て用いる:^、 該核酸はそれを與虫で用いることもできるし、 あるいは上記した ような遺伝 且換え技術で使用される適当なベクター、 例えばレトロウイルス由 来ベクターなどウィルス由来のベクタ一などに糸吉合させるなどして用いることが できる。 本発明の DMなどの核酸は通常の知られた方法で投与でき、 そのままで 、 あるいは、 例えば細胞内への摂取が促進されるように、 適当な補助剤あるいは 生理的に許容される担体などと共に、 製剤化されて用いること力でき、上記した ような、 医薬組成物又は医薬調製物などとして投与すること力《できる。 また遺伝 子治療として知られた方法を適用することもできる。 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. In preparing a formulation, a formulation auxiliary known in the art and the like are used. Locks and pills can also be manufactured with enteric coating. When 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. Further, 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. It can be used by combining it with a suitable vector used in the above, for example, a vector derived from a virus such as a retrovirus-derived vector. The 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.
本発明の活' W:分は、 その投与量を広範囲にわたつて選択して投与できる力 その投与 び投与回数などは、 処置患者の tt¾lj、年齢、 体重、 一般的健康、状態 、 食事、投与時間、 投与方法、 陋速度、 薬物の組み合わせ、 患者のその時に治 療を行なつて ヽる病状の程度に応じ、 それらあるいはその他の要因を考慮して決 められる。 医薬品製造にあたっては、 その添加剤等や調製法などは、 例えば日本薬局方解 説書編集委員会編、 第十四 ¾IE 日本薬局方解説書、 平成 13年 6月 27日発行、 株 式会社廣川書店；一番ケ瀬 尚 他編 医薬品の開発 12巻（製剤素剤 〔 I〕 ） 、 平成 2年 10月 15曰発行、 株式会ネ適川書店；同、 医薬品の開発 12巻 （製剤素材 〔 I I〕 ）平成 2年 10月 28日発行、 株式会ネ: h^J I嚐店などの記載を にしてそれら のうちから必要に応じて ifiS選択して適用すること力できる。  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 tt¾lj, 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. In the manufacture of pharmaceuticals, 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. Development of Pharmaceuticals, Volume 12, Pharmaceutical Ingredients [I], published October 15, 1990, Nei-Nyokawa Shoten Co., Ltd .; Development of Pharmaceuticals, Volume 12, Pharmaceutical Materials [II] Published on October 28, 1990, Stock Association: You can select and apply ifiS from among those listed below as necessary.
本発明の活 分は、 MMPs、 特には Mil-固 P， MT3-画 Pの活性阻害あるいは删 Ps の活性化阻害とレ、つた生物学的活性を抑制 び Z又は阻害する作用をもつもので あれば特に限定されないが、好ましくは有利な作用を持つもの力挙げられる。本 発明の活性成分は、 例えば、 （a) N-Tes、 その変異体ポリぺプチド、 その一部の ぺプチドまたはそれらの塩等、 (b)該 N- Tesをコ一ドする DNA、 N-Tes変異体ポ リペプチドをコードする DNAなどの核酸等、 （c)本発明の抗体、 その一部断片 （ モノクローナル抗体を包含する） またはその誘 ¾j本、 (d) N-Tes による MT_應 P類 の活' f生 P且害と 、つた生物学的活性を抑制及び 7又は阻害する化合物またはその塩 などが包含さ; TLる。本発明の活' ¾ ^分には、 ί¾ΜΡ抗体、例えばモノクローナル ί¾ΜΜΡ抗体も包含される。 本発明の活性成分は、 MMPsによる各 ®ft熾あるいはタンパク質のプロセッ グ、 例えば少なくとも ΜΤΊ-讓 Pあるいは MT3-應 Pによるタンパク質のプロセッシ ングを抑制あるいは阻害するのに有用と期待される。 また、 該活' ί«分は、 匪 Ps 、 例えば P- 9， MMP- 2活性発現の抑制に有用であり、 ΜΉ- MMPあるいは MT3- MMP 遺伝？ ¾現細胞における議 Psによるタンパク質のプロセッシングが関連する障害 、 異常及び Z又は疾患の予防あるいは治療に有用である。 また、 羅 Psが関与する 腫瘍細胞などの移動、 浸潤、 遊赴び/又は転移の制御、 例えば抑制に有用であ と期待される。 The activity of the present invention is to inhibit the activity of MMPs, particularly Mil-solid P, MT3-fraction P or the activation of 删 Ps, and to suppress and Z or inhibit the biological activity. There is no particular limitation as long as it is present, but a force having an advantageous effect is preferably mentioned. 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且害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 讓 -substituted P or MT3- 應 P. In addition, the activity is 分 For example, it is useful for suppressing the expression of P-9 and MMP-2 activities, and is ΜΉ-MMP or MT3-MMP inherited?議 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及びその関連べプチドは、 悪 'Sffi瘍、 すなわち癌の移動、 浸潤及び/又 は転移の ΡΜι¾び/又は抑制するのに有用で、 血管新生阻害剤、 抛 SH剤及び/ 又は癌転移抑制剤として期待できる。 また、 血液系細胞の、 固 Psによるプロセッ シングに関連する障害、 異常及び/又は疾患の予防あるいは治療にも有用で、 消 炎剤及び Z又は免疫抑制剤としても期待できる。 さらに、 アルツハイマー治藤 IJ 、 関節破壊治；^ IJなどとしても期待できる。 さらに、 .本発明では、 （a) N-Tesのアミノ酸配列中、 第 22番目のアミノ酸残基 〜第 312番目のァミノ酸残基の範囲の配列：  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:
(b) N-Tesのァミノ酸配列中、 第 1番目のァミノ酸残基〜第 312番目のァミノ酸 残基の範囲の配列： 及び  (b) a sequence ranging from the 1st amino acid residue to the 312th amino acid residue in the amino acid sequence of N-Tes: and
(c) N-Tes のアミノ酸配列中、 第 22番目のアミノ酸残基〜第 122番目のアミノ酸 残基の範囲の配列  (c) Sequence of the amino acid sequence of N-Tes ranging from the 22nd amino acid residue to the 122nd amino acid residue
から成る群から選ばれたものに基づいて肝設計を施して、 MMPsによる各種タン パク質のプロセッシングを抑制あるいは阻害する活性を有する物質を得るのに使 用できる。 こうして得られる物質も本発明の思想の範囲内のものであるし、 本発 明の活 ft ^分として扱うこと力くできる。該配列から特定の'機部分を選択し、 (i ) そのうちの薬理作用団をイソスターで置き換えることによりなされる力、、 (i i) 構成ァミノ酸残基の少なくとも 1個を D体のァミノ酸残基に置き換える力、、 (i i i) アミノ酸残基の側鎖を修飾する力、、 (iv) 該配列に存在するアミノ酸残基と は異なるアミノ酸残基を配置して連結するカヽ、 (v)立ィ: を角浙して mimic体 をデザィンすることなど、 当該分野で採用される技術を駆使して行うことができ る （例えば、 首藤 紘ー 編 医薬品の開発 7巻 ^設計） 、平成 2年 δ月 25 曰発行、株式会擴川書店及びそこで引用している文献や論文など） 。 そうした 技術のー咅 ^は、 上記で説明したものを含んでいる。 Can be used to obtain a substance having an activity of suppressing or inhibiting the processing of various proteins by MMPs by performing liver design based on a substance selected from the group consisting of: 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. (I) the ability to replace a pharmacological agent with an isostere, (ii) at least one of the constituent amino acid residues is replaced with a D-form amino acid residue. (Iii) the ability to modify the side chains of amino acid residues; (iv) the ability to arrange and link amino acid residues different from the amino acid residues present in the sequence; :: Designing a mimic body by using a technique such as 角 な ど 行 う 例 え ば 首 編 例 え ば 例 え ば 例 え ば 例 え ばδMarch 25, issued by the Japan Society of Enlargement and the literature and papers cited therein. Such Technology 咅 ^ includes those described above.
明細書及び図面において、 用語は、 IUPAC- IUB Commission on Biochemical No menclatureによる力、、 あるいは当該分野にお 、て慣用的に使用される用語の意味 に基づくものである。 実施例 。  In the specification and drawings, terms are based on the power of the IUPAC- IUB Commission on Biochemical No menclature or on the meaning of terms used conventionally in the art. Example .
以下に実施例を掲げ、 本発明を具体的に説明するが、 本発明はこれら実施例に 限定されず、 本明細書の思想に基づく様々な実 ¾研態が可能であることは理解さ れるべきである。  EXAMPLES Hereinafter, the present invention will be described in detail with reference to examples, but it is understood that the present invention is not limited to these examples, and that various types of research based on the idea of the present specification are possible. Should.
全ての截 fe例は、 他に詳細に記載するもの以外は、 標準的な技術を用いて実施 したもの、 又は実施することのできるものであり、 これは当業者にとり周知で慣 用的なものである。  All examples are, or will be, implemented using standard techniques, unless otherwise specified in detail, which are well known and conventional to those skilled in the art. It is.
なお、 以下の実施例において、 特に指摘が無い齢には、 具体的な操作並びに 処 件などは、 DNA クロ一ニングでは J. Sambrook, E. F. Fri tsch & T. Maniatis, "Molecular Cloning", 2nd ed. , Cold Spring Harbor Laboratory, Cold Spring Harbor, N. Y. (1989)及び D. M. Glover et al. ed. , "DNA Cloning", 2nd ed. , Vol. 1 to 4, (The Practical Approach Series), IRL Press, Oxford Universi ty Press (1995) ； 特に PCR法では、 H. A. Erl ich ed.， PCR Technology, Stockton Press, 1989 ； D. M. Glover et al. ed. , "DNA Cloning", 2nd ed.， Vol. 1， (The Practical Approach Series), IRL Press, Oxford Universi ty Press (1995) 及び M. A. Innis et al. ed.， "PCR Protocols", Academi c Press, New York (1990)に記載の方法に準じて行つ ているし、 また市販の TOあるいはキットを用いている:^はそれらに添付の指 示書 (protocols) や添付の薬品等をィ¾¾している。 W 1 ： N-Tes のクローニング  In addition, in the following examples, specific operations and treatments were not performed by DNA cloning according to J. Sambrook, EF Fritzsch & T. Maniatis, "Molecular Cloning", 2nd ed. , Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1989) and DM Glover et al. Ed., "DNA Cloning", 2nd ed., Vol. 1 to 4, (The Practical Approach Series), IRL Press, Oxford. University Press (1995); In particular, in the PCR method, HA Erlich ed., PCR Technology, Stockton Press, 1989; DM Glover et al. Ed., "DNA Cloning", 2nd ed., Vol. 1, (The Practical Approach Series), IRL Press, Oxford University Press (1995) and MA Innis et al. Ed., "PCR Protocols", Academic Press, New York (1990). In addition, commercially available TOs or kits are used: ^ indicates the attached instructions (protocols) or attached chemicals. W1: N-Tes cloning
1) 一次スクリーニング 1) Primary screening
cDNA発現ライブラリ一は、 EdgBioSys terns社の Unampl if ied cDNA Library (ヒ ト胎児腎臓由来） を使用した。 Stratagen社の cDNA合成キット、 発現プラスミ ド 、 コンビ一タントセノレを しても可能である。 As the cDNA expression library, Unamplified cDNA Library (derived from human fetal kidney) from EdgBioSysterns was used. Stratagen cDNA synthesis kit, expression plasmid However, it is also possible to use a combination senoré.
ヒト胎児腎臓由来 cDNA発現ライブラリ一の 30クローンの大腸菌を 1プールとし て TB±§地にて 37°Cで 16時間培養し、 培養大腸菌 1. 5 πύよりアルカリ- SDS法にてプ ラスミ ドを精製した。  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.
96穴マイクロプレートに 1穴あたりおよそ 20， 000個の 293Τ細胞を 5 %牛胎児血 清を含む DMEMで播き、 24時間培養した。 ΜΜΡ - 9、 删 Ρ- 2、 MT1-MMPの全長 cDNAを pSG5(Stratagene)のクローニングサイトに揷入した発現べクタ一を作成し、 1穴 あたり MMP- 9発現プラスミ ド 3 ng、 MP-2発現プラスミ ド 22 ng、 T1-MMP発 現プラスミ ド 33 ngを上記ライブラリー DNA 200 ngとともにリン酸カルシウム法 にてトランスフヱクシヨンした。 37°Cで 48時間培衝麦、 ±咅地を除き、 細胞を 1穴 あたり 50マイクロリットルの SDS- PAGEサンプルバッファー （10 mM Tris- HC1緩衝 液， H6. 8, 20% glycerol, 0. 24 mg/ml BPB, 1% SDS)で可溶ィ匕した。 37°Cで 30分 加温後、 5マイクロリットルをゼラチンザィモグラフィ一に供し、 潜在型、 中間 体、 活性化 ¾MP- 9、 MMP-2を検出にかけた。 一次スクリーニングにより、 ライ ブラリーより活性化 SMMP- 2の産生を抑制する遺伝子プラスミ ドの集団を特定し た。  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.全長 -9, 删 -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. Exclude the wheat and culture medium at 37 ° C for 48 hours, and remove the cells. Dissolve the cells in 50 μl of 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.
2) 二次スクリーニング 2) Secondary screening
一次スクリーニングで得られた礙甫遺伝子プラスミ ドの集団を大腸菌 XL- lBlue (Stratagene)に導入し、 アンピシリン含有 LBプレート上で 24時間培養した。 大腸 菌コロニ一 50個を 1個づっ分離し、 TB培地にて 37°Cで 16時間培養し、 培養大腸菌 1. 5 mlよりアルカリ - SDS法にてプラスミ ドを精製した。 前項記載の一次スクリ一 ニングと同様に應 P- 9， 匪 P- 2， MT1-MMP発現プラスミ ドと精製プラスミ ドを 293T 細胞に導入し、 ゼラチンザィモグラフィ一にて潜在型、 中間体、 活性化 MMP - 9 、 MMP-2 を検出にかけた。 活性化 MMP- 2の産生を抑制した単一プラスミ ドを同 疋した。  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. In the same manner as in the primary screening described in the previous section, P-9, maraudal P-2, MT1-MMP expression plasmid and purified plasmid were introduced into 293T cells, and the latent form, intermediate, Activated MMP-9 and MMP-2 were subjected to detection. A single plasmid that suppressed the production of activated MMP-2 was used.
図 1にマイクロプレート 8穴のデータ一を示す。 レーン 4では中間体及び活性 化 麵 P- 2 の産生が抑制されている。 この活性化 MM P- 2 の産生が抑制されたゥ エルに添加された単一プラスミ ドについて、 LI-COR DNA シークェンサ一 Model 4200を使用して挿入された DNAの塩基配列を決定した。 決定した DNAの塩基配列 を酉己列表の酉己列番号： 1 に記載した。 Figure 1 shows the data of the microplate 8 wells. In lane 4, the production of intermediates and activated 麵 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.
この遺 fe?は、 1510ベースペアの長さを有し、 313 アミノ酸残基からなるォ一 プンリ一ディングフレームをコ一ド (0RF) していた。 該 0RF にコ一ドされるァミ ノ酸配列を配列表の配列番号： に記載した。 ホモロジ一検索の結果、 Testican -1 (GenBank Accession No. AF231124), Testican-2 (GenBank Accession No. A ■1453), Testican-3 (GenBank Accession No. MJ001454)および HPTLG (W099 /23110) のアミノ酸配列の N末端側およそ 310残基と相同性が認められた （図 2 ) 。 この遺伝子を N- Tes と命名した。 m¾ 2 ： N-Tes の N末端 122 アミノ酸残基からなる組換えタンパク質発現ブラ スミ ドの構築  This elephant had a length of 1510 base pairs and encoded (0RF) an open reading frame of 313 amino acid residues. The amino acid sequence encoded by the 0RF is described in SEQ ID NO: in the Sequence Listing. As a result of the homology search, the amino acid sequences of Testican-1 (GenBank Accession No.AF231124), Testican-2 (GenBank Accession No.A ■ 1453), Testican-3 (GenBank Accession No.MJ001454) and HPTLG (W099 / 23110) Approximately 310 residues were homologous to the N-terminal side (Fig. 2). This gene was named N-Tes. m¾2: Construction of a recombinant protein expression plasmid consisting of the N-terminal 122 amino acid residues of N-Tes
1) N-Tes cDNAフラグメント (1-473) の PCR増幅 1) PCR amplification of N-Tes cDNA fragment (1-473)
N-Tes cDNAの 451- 473位に対応する相補鎖に制限酵素 Bgl I I の認識配列および Bgl l切断効率向上のため 2塩基をイ^)口した PCR プライマ一  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)
GGAGATCTTCCTGCnCTTTCATCCTGTGTG 〔配列番号： 3 ] GGAGATCTTCCTGCnCTTTCATCCTGTGTG [SEQ ID NO: 3]
を設計した。 cDNA発現ライブラリ一 pEAK8 プラスミ ド (EdgBioSystems) 内の T7プ 口モータ一に対応する T7プライマー Was designed. cDNA expression library-T7 primers corresponding to T7 promoters in pEAK8 plasmid (EdgBioSystems)
TAATACGACTCACTATAGG 〔配列番号： 4〕  TAATACGACTCACTATAGG [SEQ ID NO: 4]
と本プライマ一を用い、 N- Tes cDNA含む pEAK8 を铸型として PCR増幅を行った。 その結果、 約 500塩基の断片を得た。 この DNA断片を EcoRIと Bgl l lで消ィヒ後、 EcoRI と Bgl l l で消化した pSG-FLAGベクターと DNAリガ一ゼを用いて 結した。 pSG - FLAGは、 pSG5のクロ一ニングサイトの 3'末端側に FLAGペプチド (DYKDDDK) を コ一ドする塩基配列と終止コドンを揷入したもので、 リ一ディングフレームを合 わせることにより、 発現タンノ、。ク質の N末端側に FLAGぺプチドを付加することが できる。 これを大腸菌 XL- lBlueに導人、 増幅した後アルカリ- SDS法、 CsClM¾心 精製法により、 発現べクタ一 pSG- N - Tes- de卜 FLAGを得た。 pSG- N- Tes - del - FLAGで 発現するタンパク質は、 N-Tes の Met 1から Gly 1 2 2に由来する 122 アミノ酸残基と Bgl l l 認識配列および FLAG配列に由来する 12ァミノ酸残基をあわせた 134 ァミノ 酸残基からなるタンパク質 （配列表の配列番号： 5 ) をコードする。 うち、 Met 1 から Ala2 1 の 21アミノ酸残基は、 リーダー配列であり、 細胞より分泌される際に 切断除去され、 113 アミノ酸残基の組換えタンパク質が産生される。 この発現プ ラスミ ドにより産生されるタンパク質を N- Tes-del- FLAGとする。 N - Tes- del- FLAG は、 N - Tes (101アミノ酸残基、 Ala2 2 から Gly1 2 2) の C末端に FLAGペプチドタグ を有する。 Using this and this primer, 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. This was introduced into Escherichia coli XL-Blue, amplified, and then an expression vector 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). Among them, 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.
2) N- Tes- de卜 FLAGによる ΜΤΊ - MMP、 MT3- MPを介した議 P- 2活性化抑制 2) N-Tes-de FLAG suppresses P-2 activation via ΜΤΊ-MMP and MT3-MP
24穴プラスチックプレート （Nunc) に 50， 000個の 293T細胞を 5 %牛胎児血清加 DMEM 0. 5 ml で播き、 24時間後に匿 - 2 (25 ng)、 MT1- MP(50 ng)あるいは MT3- MMP (50 ng) 発現べクタ一と前項記載の 250 ngの pSG- N- Tes- de卜 FLAGを導入した o 36時間後に牛胎 JS^l清を含まない DMEM 100マイクロリットルと交換し、 12時間 後に 10マイクロリツトルを採取し、 5マイクロリットルの SDS-PAGEサンプルノくッ ファーを添カロ、 37°Cで 30分加温後、 ゼラチンザィモグラフィーゲルで分析した （ 図 3 ) 。 図 3中、 レーン 1：匪 P- 2: 25 ng; pSG5 : 475 ng. レーン 2：顧 P- 2: 2 5 ng; MT1-MMP: 50 ng; pSG5: 425 ngヽ レーン 3：匪 P- 2: 25 ng; T1-M P: 50 ng; pSG-N- Tes- del- FLAG: 425 ng、 レーン 4 : MP-2: 25 ng; pSG5 : 475 ngヽ レ —ン 5 : MMP- 2: 25 ng; MT3-MMP: 50 ng; pSG5: 425 ng, レーン 6 : MMP- 2: 25 ng; MT3-MMP: 50 ng; pSG-N- Tes- del- FLAG; 425 ngをそれぞれ示している。  Seed 50,000 293T cells on a 24-well plastic plate (Nunc) with 0.5 ml of DMEM supplemented with 5% fetal calf serum, and concealed 24 hours later-2 (25 ng), MT1-MP (50 ng) or MT3 -After introducing the MMP (50 ng) expression vector and 250 ng of pSG-N-Tes-deto FLAG described in the previous section, o After 36 hours, replace with 100 microliters of DMEM without bovine JS ^ l clarified, Twelve hours later, 10 microliters were collected, 5 microliters of an SDS-PAGE sample buffer was added, the mixture was heated at 37 ° C for 30 minutes, and analyzed by gelatin zymography gel (Fig. 3). In Fig. 3, Lane 1: Marauder P- 2: 25 ng; pSG5: 475 ng. Lane 2: Customer P- 2: 25 ng; MT1-MMP: 50 ng; pSG5: 425 ng ヽ 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 ヽ 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.
図 3中、 レーン 3および 6ではそれぞれ MT1-廳 Pおよび ¾1T3-讓 P を介した ΜΡ - 2の活性化が抑制された。 このことは、 Ν - Tes- de卜 FLAGか ¥Γ -匪 Ps の活性を阻害 することを示している。 さらに、 N- Tes-del- FLAGと同じ塩基配列を含む N- Tes、 Testican- 3および PTLG も MT - MPs の活性を阻害し、 MMP-2 の活性化を抑制する こと力く容易に类貢推できる。  In FIG. 3, in lanes 3 and 6, the activation of ΜΡ-2 via MT1-caffeine P and ¾1T3-case P, respectively, was suppressed. This indicates that it inhibits the activity of Ν-Tes-de-FLAG or ¥ Γ-banded Ps. In addition, N-Tes, Testican-3 and PTLG, which contain the same nucleotide sequence as N-Tes-del-FLAG, also inhibit MT-MPs activity and can easily and easily contribute to suppressing MMP-2 activation. I can guess.
3 ：グリオ一マにおける N- Tes発現 3: N-Tes expression in glioma
グリオ一マ患者の腫瘍部位 （レーン T)と ffi¾を含まない部位 （レーン N)から抽 出した全 RNAから逆転写酵素 Superscript (GIBC0-BRL) とランダムプライマー （ 宝酒造） を用いて cDNAを合成し、 このものを鐯型として N- Tes に特異的なプライ マー CDNA was synthesized from total RNA extracted from the tumor site (lane T) and the site not containing ffi¾ (lane N) of Gliomas patients using reverse transcriptase Superscript (GIBC0-BRL) and random primers (Takara Shuzo). This type is referred to as 鐯 type Mar
Forward Primer :  Forward Primer:
GAGTGGTGCTACTGCTTCCA 〔配列番号： 6 ]  GAGTGGTGCTACTGCTTCCA [SEQ ID NO: 6]
Reverse Primer :  Reverse Primer:
GTGCnCnGGTGGCTCATA 〔配列番号： 7〕  GTGCnCnGGTGGCTCATA [SEQ ID NO: 7]
を用いて PCRを行った。配列番号： 6は、 配列表の配列番号： 1の 1008- 1027位 に対応するもので、配列番号： 7は、配列表の配列番号： 1の 1308- 1289位に相 補的な配列に対応するものである。 PCR反応は、 例えば GeneAmp 2400 PCR シス テム (Perkin Elmer/Cetus)などを使用し、 変性 (94 °C， 15秒） 、 アニーリング (6 0 。C， 秒） および伸長 (72 。C, 15秒） の 40サイクルで実施した。 PCR産物は、 2. 5% ァカ"ロースゲルで分析した。 Was used to perform PCR. SEQ ID NO: 6 corresponds to positions 1008 to 1027 of SEQ ID NO: 1 in the Sequence Listing, and 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. For the 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.
結果を、 図 4に示す。 6名のグリオ一マ患者すべてで、 N-Tes発現は、 J®¾を 含まない部位（レーン N)に比べ腫瘍部位（レーン T)で有意に低かった （図 4 ) 。 このことより、 グリオ一マ悪性度と N-Tes発現低下が関連するものと考えられる  The results are shown in FIG. In all six glioma patients, N-Tes expression was significantly lower at the tumor site (lane T) than at the site without J®¾ (lane N) (Figure 4). This suggests that glioma grade is associated with decreased N-Tes expression.
H¾例 4 ： MT-顯 Pと N - Tes の結合 H¾Example 4: Combination of MT-P and N-Tes
直径 60 ramの培養プレートに 293T細胞をおよそ 50万個をまき、 24時間後に pSG5 、 MTl-MMPを発現する pSGMH- MMPおよび" N- Tes- del- FLAGを発現する pSG- N-Tes - de 卜 FLAGのそれぞれを、 (1) 4 s PSG5 (lane 1), (2) 2 zg pSGMTl-M P + 2 /g pSG5 (lane 2), (3) 2 g pSG- N- Tes- deト FLAG + 2ug pSG5 (lane 3), (4) 2u g pSG-N-Tes-del-FLAG + 2 zg pSGMTl-MMP plasmid (lane 4)， (5) 4 g pSG5 ( lane 5)， (6) 2 g pSG - N- Tes- del- FLAG + 2/ g pSG5 (lane 6)， (7) 2 / g pSGM Tl-MMP + 2 zg PSG5 (lane 7)及び (8) 2 iig pSG-N- Tes- del- FLAG + 2 //g pSGM Tl-MMP plasmid (lane 8) となるようにトランスフヱクシヨンし、 24時間後に 10 0マイクロキューリ一/ ml の3-35ラべルの11½1 101 11 + cysteineを添加し 4時 間培養した。細胞は、 1 mlの細胞溶解バッファ一(150 mM NaCK 1 ¾ Tri ton X-1 00 (ν/ν)、 1 ¾ sodium deoxycholate、 0· 1 % SDSおよび protease inhibi tor c ocktai l (Sigma) を含有した 50 mM Tris-HCl (pH 7. 5))で可溶化し、 15, OOOrpm で 10分間遠心して上清を回収した。 この上清を ant i MT1- MMP抗体 (113- 5B7 (第一 ファインケミカル） 、 レーン 1- 4)および ant i- FLAG抗体 (M2 (Stratagene)ヽ レ一 ン 5- 8)のそれぞれ 1マイクログラムと共に 12時間ィンキュベートした後、 Protei n A-Sepharose ゲル (20 マイクロリットル） と 1時間混合した。 ゲルを細胞溶解 バッファ一で 3回洗った後、 SDS-PAGE sample bufferを 10マイクロリツトル加え て、 100 °Cで 2分煮沸した後、 上清を 12% SDS-PAGE ゲルで展開し、 S- 35のシグ ナルを Fuj i BAS 1000で検出した （図 5 ) 。 Approximately 500,000 293T cells are seeded on a culture plate having a diameter of 60 ram, and after 24 hours, pSG5, pSGMH-MMP expressing MTl-MMP and pSG-N-Tes-de expressing "N-Tes-del-FLAG" (1) 4 s PSG5 (lane 1), (2) 2 zg pSGMTl-M P + 2 / g pSG5 (lane 2), (3) 2 g pSG-N- Tes- de FLAG + 2ug pSG5 (lane 3), (4) 2ug pSG-N-Tes-del-FLAG + 2 zg pSGMTl-MMP plasmid (lane 4), (5) 4 g pSG5 (lane 5), (6) 2 g pSG-N- Tes- del- FLAG + 2 / g pSG5 (lane 6), (7) 2 / g pSGM Tl-MMP + 2 zg P SG5 (lane 7) and (8) 2 iig pSG-N- Tes- del-FLAG + 2 // g pSGM Transform to become Tl-MMP plasmid (lane 8), 24 hours later 100 microcurie / ml 3-35 label 11-1 101 11 + cysteine The cells were cultured for 4 hours with 1 ml of cell lysis buffer (150 mM NaCK 1 ¾ Triton X-100 (ν / ν), 1 ¾ sodium deoxycholate, 0.1% SDS and protease inhibitor. 50 mM T containing tor cocktai l (Sigma) ris-HCl (pH 7.5)), 15, OOOrpm And centrifuged for 10 minutes to collect the supernatant. The supernatant was combined with 1 microgram each of the anti i MT1-MMP antibody (113-5B7 (Daiichi Fine Chemical), lanes 1-4) and anti-FLAG antibody (M2 (Stratagene) ヽ lanes 5-8). After incubating for 12 hours, the mixture was mixed with Protein A-Sepharose gel (20 microliter) for 1 hour. After washing the gel three times with the cell lysis buffer, add 10 microliters of SDS-PAGE sample buffer, boil at 100 ° C for 2 minutes, and develop the supernatant on a 12% SDS-PAGE gel. 35 signals were detected with the Fuji BAS1000 (Figure 5).
レーン 4では、 免疫沈降した MT1-固 P と結合した N- Tes 力共沈すること、 レー ン 8では、 免疫沈降した N- Tes と結合した ΜΉ- MMP力く共沈すること力観察された (図 5 ) 。 このことより、 N- Tes は、 ΜΉ- MMP と結合することにより、 ΜΉ-匪 P の MMP-2活性化能を抑制していると思われる。 実施例 5 ：モノクローナル抗体の作成  In lane 4, N-Tes co-precipitated with immunoprecipitated MT1-solid P was observed, and in lane 8, ΜΉ-MMP co-precipitated with immunoprecipitated N-Tes. (Figure 5). This suggests that N-Tes suppresses the ability of 匪 -band P to activate MMP-2 by binding to ΜΉ-MMP. Example 5: Preparation of monoclonal antibody
(a) 免疫源 (a) Immunogen
免疫に用いる K は、 難例 2などで得られる融合組換え N-Tes をはじめ、 配 列表の配列番号: 2のァミノ酸配列を基にデザィンされた合成べプチドを使用する こと力くできる。 さらに、 免疫に用いる抗原は、 実施例 1で得られた cDNAを動物細 胞発現べクタ一に連結し、 これを CH0細胞、 COS細胞などで発現させて得られる 組換え N- Tes を使用することも可能である。 これらの抗原タンパク質は、 イオン 交換、 ゲル濾過またはそれ以外の各種クロマトグラフィーによって精製できる。 精製した免; M抗原を一般的な方法で免疫し、 抗体産生細胞を誘導、 細胞融合 によりハイブリ ドーマとして抗体産生細胞を得ることができる。 さらに精製した 免翻麵に対する 性に基づいてクロ一ニング、 モノクローナル抗体産生ハ イブリ ドーマとして株化できる。  As 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. Furthermore, 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. Furthermore, the strain can be cloned and cloned as a monoclonal antibody-producing hybridoma based on its ability to purify the immunity.
また、 N - Tes特異的モノクロ一ナノレ抗体を得るための免疫源としては、 N-Tes に特徵的なァミノ酸配列を持つハプテン化合成べプチドが できる。  In addition, as an immunogen for obtaining an N-Tes-specific monoclonal antibody, a haptenylated synthetic peptide having an amino acid sequence specific to N-Tes can be used.
例えば、 C末端には Gly- Lys- Arg 〔酉 j番号： 10〕 を含むこと力特に好ましく 、 具体的には Cys- Phe- Gin- Arg- Gin- Gin- Gly- Lys-Arg S己列番号： 11〕 などが免 疫源として好適である。 (b) 抗原ポリペプチドの調製 For example, it is particularly preferable that the C-terminus contains Gly-Lys-Arg [rooster j number: 10]. Specifically, Cys-Phe-Gin-Arg-Gin-Gin-Gly-Lys-Arg S sequence number : 11] is a suitable source of immunity. (b) Preparation of antigen polypeptide
配列番号: 2に記載したヒト N- Tes のアミノ酸配列中より特徴的な配列を選択し 、 合成する。 ペプチドはペプチド合成機 （ペプチドシンセサイザ一 9600、 Mi l l iG en/Biosearch) を使用して、 Fmoc- bop法で合成する。 ポリペプチドの N末端には システィンを導入する。 合成したペプチドは Bondasphere, C18カラム （Waters ) を用いた高速液体ク口マトグラフィ一などにより精製する。  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).
(c) ポリペプチドと BSAの複合体の調製  (c) Preparation of complex of polypeptide and BSA
システィン残基を介してゥシ血清アルブミン （BSA)と結合させ、 抗原コンジュ ゲ一トとした。 10. lmgの BSAを lmLの 0. 1Mリン酸緩衝液、 H7. 0 に溶解したもの と 1. 14mgの EMCS(N- ( £ -maleimidecaproyloxy) - succinimide)を 24. 9 /L のジメチ ルホルムアミ ドに溶解したものと混合し、 30°C、 30分間反応させ、 ついで、 上記 の混合液を 0. 1Mリン酸緩衝液、 H7. 0で ¾Ιϊ化した Sephadex G - 25 (Pharmacia) ゲルカラム（直径 13腿、 長さ 120腿)でゲルろ過する。 前記 （b)で合成したポリべ プチドを 0. 1Mリン酸緩衝液、 H7. 0 に溶解し、 マレイミ ド結合 BSA に対しおおよ そ 50倍モノ Λを混合する。 すなわち、 ポリペプチドに対しマレイミ ド結合 BSAを 混合し、 4 °C、 20時間インキュベートし、 BSA -ポリペプチド複合体を調製する。 得られる BSA -ポリべプチド複合体を 0. 1Mリン酸緩衝液、 pH7. 0で希釈した後、 15 0 il ずつに分注して- 30 °Cで凍結保存する。  The antigen was conjugated with cysteine albumin (BSA) via a cysteine residue. 10.1 mg of BSA dissolved in 1 mL of 0.1 M phosphate buffer, H7.0 and 1.14 mg of EMCS (N- (£ -maleimidecaproyloxy) -succinimide) in 24.9 / L dimethylformamide 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.
(め 抗体産生細胞の調製 (1) Preparation of antibody-producing cells
前記 (c) で調製した BSA -ポリべプチド複合体を完全フロインドアジュバントと 共に 6週令 Balb/c雌マウスに 空内投与し、 初回免疫した。 おおよそ 18日目に 0. 1Mリン酸緩衝液、 pH7. 5 に溶解した BSA-ポリペプチド複合体を初回免疫したマウ スに 空内投与し、 追加免疫する。 さらにおおよそ 52曰目に 0. 1Mリン酸锾衝液、 PH7. 5 に溶解した BSA-ポリペプチド複合体を静脈内投与し、 最終免疫とする。 そ の 4 日後に脾臓を摘出し、 脾細胞懸濁液を調製する。  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.
(e) 細胞融合 (e) Cell fusion
細胞融合には、 以下の材料および方法を用いる。 RPMI-1640培地： RPMI- 1640 (Flow Lab. )に重炭酸ナトリウム （2½M) 、 ピルビン酸ナトリウム （lmM)、 ベニ シリン G カリウム （50U/ml) 、 硫酸アミカシン （100 zg/ml) をカロえ、 ドライア イスで pHを 7. 2 にし、 0. 2 zm東洋メンブレンフィルタ一で除菌ろ過する。 NS- 1 培地：上記 RPMト 1640培地に除菌ろ過したゥシ胎児血清 （FCS， M. A. Bioproduct s)を 15% (v/v)の濃度になるように加える。 PEG- 4000溶液： RPMI- 1640 ±咅地にポ リエチレングリコール- 4000 (PEG- 4000， Merk&Co. )を 50% (w/w)になるように 加えた無血清培地を調製する。 The following materials and methods are used for cell fusion. RPMI-1640 medium: RPMI-1640 (Flow Lab.) With sodium bicarbonate (2½M), 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).
8 -ァザグァニン耐性ミエローマ細胞 SP2 (SP2/0-Agl4) との融合は、 Selected Fusion with 8-azazanin-resistant myeloma cell SP2 (SP2 / 0-Agl4)
Method in cul ture immunology p351~372 ed. B. B. Mi shel l and S. N. Shi igi), W. H. Freeman and Company (1980)に記載の Oiらの方法を若干改変して行Method in culture immunology p351-372 ed.B.B.Mishell and S.N.Shiigi), W.H.Freeman and Company (1980)
Ό ο 前記 (d) で調製した有核脾細胞 （生細胞率 100 %) とミエローマ細胞 （生細胞 率 100 ) とをおおよそ 5 : 1〜10 : 1の比率で以下の手順で融合する。 ポリべプチ ド免 卑細胞懸濁液とミエローマ細胞をそれぞれ RPMI1640培地で洗浄する。 次に 同じ培地に懸濁し、 融合させるために有核脾細胞とミエローマ細胞を混合する。 すなわち、 おおよそ 4. 0 X 108 個の有核脾細胞に対しおおよそ 8. 0 X 107 個のミ エローマ細胞を混合する。 Ό ο The 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.
次に、 それぞれの細胞混合液を遠心分離により細胞を沈殿させ、 上清を完全に 吸引除去する。 沈殿した細胞に 37°Cに加温した 50 PEG- 4000含有 RPMI- 1640培地 (ミエ口一マ細胞がおおよそ 3 X 107 個/ mL となるよう体積を決定する） を滴下 し、 撹拌し、 細胞を再懸濁、 分散させる。 次に添加した 50%PEG- 4000含有 RPMI- 1 640培地の 2倍体積の 37°Cに加温した RPMI- 1640 i咅地を滴下する。 さらに添カロし た 50%PEG- 4000含有 RPMI- 1640 ±咅地の 7倍体積の RPMI- 1640 ±咅地を常に撹拌しな 力くら滴下し、 細胞を分散させる。 これを遠心分離し、 上清を完全に吸引除去する 。 次に、 ミエローマ細胞がおおよそ 3 X 106 個/ mL となるように 37°Cに加温した NS - 1培地を沈殿した細胞に速やかに加え、 大きい細胞塊を注意深くピベッティン グで分散する。 さらに同培地を加えて希釈し、 ポリスチレン製 96穴マイクロウェ ルにゥエル当りミエ口一マ細胞がおおよそ 6. 0 X 105 個となるように接種する。 それぞれの細胞を加えた上記マイクロウェルを 7 %炭酸ガス /93 %空気中で 37°C、 湿度 100 %で培養する。 Next, cells are precipitated by centrifugation of each cell mixture, and the supernatant is completely removed by suction. To the precipitated cells, add RPMI-1640 medium containing 50 PEG-4000 heated to 37 ° C (determine the volume so that the number of myeoma cells is approximately 3 × 10 7 cells / mL), and stir. Resuspend and disperse cells. Next, 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. In addition, 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. This is centrifuged, and the supernatant is completely removed by suction. Next, immediately add NS-1 medium heated to 37 ° C to the precipitated cells so that the number of myeloma cells is approximately 3 × 10 6 cells / mL, and carefully disperse the large cell mass by carefully pipetting. Further, the same medium is added and diluted, and a polystyrene 96-well microwell is inoculated so that the number of cells per mouse per well becomes approximately 6.0 × 10 5 . Incubate the microwells containing the cells in 7% carbon dioxide / 93% air at 37 ° C and 100% humidity.
(0 選択培地によるハイプリ ドーマの選択的増殖 (0 Selective growth of hybridoma in selective medium
(1)使用した培地は以下の通りである。  (1) The media used were as follows.
HAT培地：前記 (e) 項で述べた NS - 1培地に更にヒポキサンチン （100 〃M)、 アミ ノプテリン (0. 4 M)およびチミジン (16〃M)を加える。  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).
HTi咅地：アミノブテリンを除去した以外は上記 HAT i咅地と同一糸! ^のものである  HTi ground: Same thread as HAT i ground except that aminobuterin was removed! ^
(2)前記 (e) 項の培養開始後翌日 （1 日目） 、 細胞にパスツールピぺッ卜で HAT 培地 2滴 （約 0. 1 ml) を加える。 2、 3、 5、 8 日目に培地の半分 （約 0. 1 ml) を新し 、HAT培地で置き換え、 10曰目に培地の半分を新しレヽ HTi咅地で置き換える 。 ハイプリ ドーマの生育が肉眼にて認められた全ゥエルについて固相-抗体結合 テスト法 （ELISA)により劚生ゥエルを調べる。 まず、 20 炭酸緩衝液 (pH9. 6) で 希釈した抗原ポリぺプチドでポリスチレン製 96穴プレ一トをコート（lOOng/ ゥェ ル） し、 次に 0. 05% Tween20含有 PBS を用いて洗浄して未吸着のペプチドを除く 。 各ゥエルにハイプリ ドーマの生育が確認されたゥエルの培 ¾±清 0. 1 mlを添加 し、 室温で約 1 時間静置する。 洗浄後、 2次抗体として西洋わさびペル才キシダ —ゼ （HRP)標識ャギ抗マウス免疫グロブリン （Cappel) を加え、 さらに室温で約 1 時間静置する。 洗浄後、 基質である過酸化水素と 3， 3，，5， 5' -テトラメチルベン ジジン (TMB) を加え発色させる。 各ゥエルに 2N硫酸を加え発色反応を停止し、 発 色の禾 1^をマイクロプレート用吸光度測定機 (MRP-A4. 東ソ一） を用いて 492nm の吸 ¾gで測定する。 (2) On the next day (day 1) after the start of the culture described in the above (e), add 2 drops (about 0.1 ml) of HAT medium to the cells with Pasteur pipette. On days 2, 3, 5, and 8, half of the medium (about 0.1 ml) is replaced with HAT medium, and on the tenth, half of the medium is replaced with fresh HTi medium. For all cells in which the growth of the hybridoma was visually observed, examine the cells by solid phase-antibody binding test (ELISA). First, coat a 96-well plate made of polystyrene with antigen polypeptide diluted with 20 carbonate buffer (pH 9.6) (100ng / well), and then wash with PBS containing 0.05% Tween20. To remove unadsorbed peptides. To each well, add 0.1 ml of culture medium from which the growth of the hybridoma has been confirmed, and incubate at room temperature for about 1 hour. After washing, add horseradish persian oxidase (HRP) -labeled goat anti-mouse immunoglobulin (Cappel) as a secondary antibody, and allow to stand at room temperature for about 1 hour. After washing, hydrogen peroxide as a substrate and 3,3,5,5'-tetramethylbenzidine (TMB) are added to develop color. 2N Sulfuric acid is added to each well to stop the color reaction, and the color development is measured using a microplate absorbance meter (MRP-A4. Tosoichi) at an absorbance of 492 nm.
(g) ハイブリ ドーマのクロ一ニンク" (g) Hybrid Doma's Cloth "
上記 ω 項で得られた抗原ペプチドに対する陽性ゥエル中のハイプリ ドーマを The hybridoma in the positive well for the antigenic peptide obtained in the above
、 限界希釈法を用いてモノクロ一ン化する。 すなわち、 NS - li咅地 1 ml当りフィ一 ダ一としておおよそ 107 個のマウス胸腺細胞を含むクローニング培地を調製し、 96穴マイクロウェルにハイブリ ドーマをゥエル当り 5個、 1個、 0. 5個になるよ うに希釈し、 それぞれ 36穴、 36穴、 24穴に加える。 5 日目、 12日目に全ゥエルに 約 0. 1 mlの NS- 1培地を追加する。 クローニング開始後肉眼的に十分なハイプリ ド 一マの生育を認めたもの、 コロニー形成陰性ゥエルが 50%以上である群について (f)項に記載した ELISAを行う。 調べた全ゥエルが斷生でない齢、 抗体圇生ゥ エル中のコロニー数が 1 個のゥエルを 4〜6個選択し、 再クロ一ニングを行う。 最終的にそれぞれのポリぺプチドに対するモノクロ一ナル抗体を産生するハイブ リ ドーマを得る。 Monochrome using the limiting dilution method. That, NS - li咅地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. On days 5 and 12, add about 0.1 ml of NS-1 medium to all wells. After the start of cloning, 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.
(h) モノクローナル抗体のクラス、 サブクラスの決定  (h) Determination of monoclonal antibody class and subclass
前述した EL I SA に従 それぞれのポリぺプチドをコートしたポリスチレン製 96穴プレートに、 前記 (g) 項で得られたハイプリ ドーマの上清を加える。 次に PB S で洗浄後、 アイソタイプ特異的ゥサギ抗マウス IgG抗体 (Zymed Lab. ) を加え る。 PBS により洗浄後、 西洋わさびペルォキシダ一ゼ標識ャギ抗ゥサギ IgG (H +L)をカロえ、 基質として過酸化水素および 2, 2， - アジノ- ジ （3-ェチルベンゾチ 了ゾリン酸） を用いてクラス、 サブクラスを決定する。  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. Next, after washing with PBS, an isotype-specific mouse heron anti-mouse IgG antibody (Zymed Lab.) Is added. After washing with PBS, the horseradish peroxidase-labeled goat anti-Peacock IgG (H + L) is caloried, using hydrogen peroxide and 2,2, -azino-di (3-ethylbenzothiozolinic acid) as substrates. Determine the class and subclass.
(i) ハイプリ ドーマの i咅養とモノクローナノレ抗体の精製 (i) Hypuri doma i-culture and purification of monoclonal antibody
得られたハイプリ ドーマ細胞を NS - 1±咅地で培養し、 その上清からモノクロ一ナ ノレ抗体を得ること力できる。 また、 得られたハイプリ ドーマ 107 個を予め 1週間 前にプリスタンを腿空内投与したマウス （Balb/c系、 雌、 6週齡） に同じく S創空 内投与し、 1〜2週間後、 腹水中からも 4〜7mg/mlのモノクローナル抗体を含む 腹水を得ること力くできる。 得られた腹水を 40%飽和硫酸ァンモニゥムで塩析後、 IgG クラスの抗体をプロテイン A ァフィゲル （Bio- Rad)に吸着させ、 0. 1Mクェン 酸緩衝液、 PH5. 0で溶出することにより精製する。 実施例 6 ：サンドィツチ EIA The obtained hybridoma cells are cultured in NS-1 ± ground, and a monoclonal antibody can be obtained from the supernatant. Moreover, 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 However, ascites can be obtained from ascites containing 4-7mg / ml monoclonal antibody. The resulting ascites is salted out with 40% saturated ammonium sulfate, and IgG-class antibodies are adsorbed to protein A-affigel (Bio-Rad) and purified by elution with 0.1 M citrate buffer, PH 5.0. . Example 6: Sandwich EIA
下記の方法に従えば、 歸 fe例 5で調製した御 - Tes抗体から適当な 2種の抗体 の組み合わせによってヒト N- Tes を特異的に検出 ·測定するサンドィツチ EIA系 力く構成できる。 EIA系は 1 ステップ法、 2 ステップ法のいずれも可能であり、 標 識抗体は Fab， -HRPに限定されない。 各反応緩衝液の «や反応条件は測定の目的 に応じて、 短縮、 延長など調整できる。 また、 標準品となるヒト N- Tes は、 糸職 ±咅養上清、 糸瑚包培養上清または実施例 2記載あるいはそれ以外の方法で発現した 組換え体から精製すること力できる。 精製にはイオン交換、 ゲルろ過、 抗ヒト N- Tes モノクローナル抗体を用いたァフィ二ティーク口マトグラフィ一またそれ以 外の各種ァフィニティーク口マトグラフィ一の組み合わせによつて達成される。According to the following method, 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 In addition, 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.
(a) 標識抗体の調製 (a) Preparation of labeled antibody
抗ヒト N - Tes モノクローナル抗体を 0. 1M NaCl を含む 0. 1雌酸緩衝液、 H4. 2 に抗体量の 2%(W/W) のペプシンを加え、 37°C、 24時間消化する。 消ィ匕物に 3M Tri s-HCl 、 pH7. 5 を添加し、 反応を停止する。 0. 1Mリン酸緩衝液、 H7. 0で 新化 したウルトロゲル AcA54 カラムによるゲルろ過で F(ab' )2 画分を分取する。 この F(ab' ) 2 画分に最終濃度 0. 01M となるようにシステアミン塩酸塩を添加し、 37°C 、 1. 5 時間還元し、 5m EDTA含有 0. 1Mリン酸緩衝液、 H6. 0で 奐 Ϊ化したウルト 口ゲル AcA54 力ラムによるゲルろ過で Fab'画分を分取する。 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.
上記の操作とは別に HRPを 0. 1Mリン酸緩衝液、 H7. 0 に溶解、 HRPの 25倍モル 量の EMCSを DMF溜夜として加え、 30°C、 30分間反応させる。 これを 0. 1Mリン酸緩 衝液、 H6. 0 で 射化した NICK- 5カラム （Pharmacia)でゲルろ過しマレイミ ド標 識 HRP画分を分取する。  Separately from the above procedure, dissolve HRP in 0.1 M phosphate buffer, H7.0, add 25 times the molar amount of HRP to EMCS as DMF overnight, and react at 30 ° C for 30 minutes. This is subjected to gel filtration on a NICK-5 column (Pharmacia) sensitized with a 0.1 M phosphate buffer and H6.0, and the maleimide-labeled HRP fraction is collected.
Fab'画分とマレイミ ド標識 HRPを等モルとなるように両画分を混合し 4°C、 20 時間反応させた後、 Fab'の 10倍モノ I*の N-ェチルマレイミ ドで未反応のチオール 基をプロックする。 これを 0. 1Mリン酸緩衝液、 H6. 5で 化したウノレト口ゲル AcA54 カラムでゲルろ過し、 Fab' -HRP標識抗体を分取する。 これに 0. 1% BSAおよ び 0. 001%クロルへキシジンを添加して 4°Cで保存する。  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.
(b) モノクローナル抗体結合担体の調製 (b) Preparation of monoclonal antibody binding carrier
抗ヒト N- Tes モノクロ一ナル抗体を 0. 1Mリン酸緩衝液、 H7. 5 に溶解し、 50 / g/mLの濃度に調製する。 このモノクロ一ナル抗体激夜を 96穴マイク口プレー卜に ゥエルあたり 100 ； Lずつ加え、 4°C、 18時間静置する。 モノクローナル抗体溶 液を除去し、 生理 ^^液で 1 回、 0. 05% ¾een20, 0. 1M NaCl, 5mM CaCl2 含有 Tr is - HCl緩衝液、 pH8. 0で 3回洗浄後、 1% BSA, 0. 1M NaCl, 5m CaCl 2含有 Tris - H CI辑衝液、 H8. 0 を加えブロッキングする。 Dissolve the anti-human N-Tes monoclonal antibody in 0.1 M phosphate buffer, H7.5, and adjust to a concentration of 50 / g / mL. Add the intense night of this monoclonal antibody to a 96-well microphone plate at 100 L per well and let stand at 4 ° C for 18 hours. Removing the monoclonal antibodies solvent solution, once with physiological ^^ solution, 0. 05% ¾een20, 0. 1M NaCl, 5mM CaCl 2 containing Tr is -. HCl buffer, pH 8 after 0-3 washes, 1% BSA , 0.1 M NaCl, 5m CaCl 2 containing Tris-H Add CI buffer and H8.0 to block.
(c) 1ステップサンドィッチ EIA法  (c) One-step sandwich EIA method
精製したヒト N- Tes画分を標準抗原としてヒト N_Tes 標準曲線を作成す る。 1% BSA, 0. 05% Bri j35, 0. 05¾ Tween20, 0. 1M NaCl, 5mM CaCl 2 含有 Tris - H CI緩衝液、 Ηδ. Οで段階希釈した標準ヒト Ν- Tes を 60 zLずつ分注、 それぞれに 1% BSA, 0. 05% Bri j35, 0. 05% Tween 20, 0. 1M NaCl, 5mM CaCl 2^ Tri s-HCl 衝液、 pH8. 0で 100ng/50 zL に調製した標識抗体 Fab' -HRPを 60〃Lずつ?忝加し十 分混和する。 調製した抗体結合マイクロプレートを 0. 05% Tween20, 0. 1M NaCl, 5m CaCh 含有 Tris- HC1緩衝液、 PH8. 0で 3回洗浄し、 標準抗原と標識抗体混合 液を 100 zL/ゥエルずつ添加する。 室温で 1 時間反応した後 0. 05% Tween20, 0. 1 M NaCl, 5mM CaC 含有 Tris - HC1緩衝液、 H8. 0で 3回洗浄する。 次に、 6%ジメ チルホルムアミ ド、 0. 005%過酸化水素含有 0. 1 M酢酸緩衝液 （PH5. 5)に溶解した 0. 01% 3， 3'，5， 5' -テトラメチルベンジジンをゥエルあたり 100 〃L添加し、 室温 で 20分間反応後、 2N硫酸を 100 L添加し反応を停止する。 この反応混液の 450 nmをマイクロプレートリ一ダーを用いて測定し、 標準曲線を求める。 Prepare a standard human N_Tes curve using the purified human N-Tes fraction as the standard antigen. 1% BSA, 0. 05% Bri j35, 0. 05¾ Tween20, 0. 1M NaCl, 5mM CaCl 2 containing Tris -. H CI buffer, Itaderuta by 60 zL standard human Nyu- Tes serially diluted with Ο dispensing , Labeled antibody Fab prepared at 100 ng / 50 zL with 1% BSA, 0.05% Bri j35, 0.05% Tween 20, 0.1 M NaCl, 5 mM CaCl 2 ^ Tris-HCl buffer, pH 8.0 'Add the HRP by 60〃L each time, and mix thoroughly. 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 µL 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.
測定検体は、 ヒト血清、 脊髄液、 血漿、 関節液、 尿および唾液などヒ卜に由来 する体«分、 各種ヒト糸纖の抽出液、 ヒト由来あるいは組換え体など各種培養 細胞の細胞抽出液、 ±咅養上清などから調製される。 それぞれの測定検体は、 標準 ヒト N- Tes にかえて上記の 1 ステップサンドイッチ EIA に供し、 ヒト N- Tes と同時に反応を進行させる。測定検体から得られた標準曲線にあてはめ、 測定検 体に含まれるヒト N- Tes の量を算出する。 実施例 7 ： N-Tes のクローニング  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
(1) 一次スクリーニング (1) Primary screening
発現べクタ一 PEKA8で構築されたヒト胎児腎臓由来 cDNA発現ライブラリ一の 30 クローンの大腸菌を 1プールとして顯 I培地 (1M Tris-HCl, 1. 25% tryptone, 2 . 5% yeast extract, 125 ni NaCl, 0. 4% glycerol ; pH 7. 2)にて 37°Cで 16時間培 養し、 ±咅養大腸菌 2 mはりポリエチレングリコールを用いたアルカリ法 俊法） にてプラスミ ドを精製した。 96穴マイクロプレートに 1穴あたりおよそ 50， 000個の 293T細胞を 5 %牛胎]; ¾ώ 清を含む画腿で播き（0. 1 mL/穴） 、 24時間培養した。 MMP- 9， 固 P- 2， MT1-MMPの 全長 cDNAを pSG5(Stratagene)のクロ一ニングサイトに揷入した発現ベクターを作 成し、 1穴あたりプロ固 P- 9発現ブラスミ ド 3 ng, プロ匪 P- 2発現ブラスミ ド 20 ng, MT1-MMP発現プラスミ ド 30 ngを上記ライブラリー DNA 100 ngとともに Trans IT LT1 遺 fe?導入 I¾¾(Mirus) にてトランスフエクシヨンした。 37°Cで 48 時間培 、 培地を除き、 細胞を 1穴あたり 50マイクロリツトルの SDS- PAGEサン プルバッファ一 (50 mM Tris-HCl pH6. 8, 10¾ glycerol, 0. 1% ΒΡΒ, 2% SDS) で 懸濁し穏やかな超音波破砕にて可溶化した。 37°Cで 30分加显後、 5マイクロリッ トルをゼラチンザィモグラフィ一に供し、 潜在型、 中間体、 活性化型隠 P- 9、 固 P-2 を検出した。 一次スクリ一ニングによりライブラリ一より活性化型國 P - 2の 産生を抑制する遺伝子ブラスミ ドの集団を特定した。 Expression vector 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 導入 (Mirus). Incubate at 37 ° C for 48 hours, remove the medium, and remove cells from the wells with 50 μL / well SDS-PAGE sample buffer (50 mM Tris-HCl pH 6.8, 10 8, glycerol, 0.1% ΒΡΒ, 2% SDS ) And solubilized by gentle sonication. After heating at 37 ° C for 30 minutes, 5 microliters were subjected to gelatin zymography to detect latent, intermediate, activated hidden P-9 and solid P-2. A primary screening screen was used to identify a population of gene brasmids that suppress the production of activated P-2 from the library.
任意の 12検体にっレ、て、 ゼラチンザィモグラフィ一で角？!斤したところ、 すべて から 92 kDaのプロ固 P- 9， 68 kDaのプロ MMP- 2および 64 kDaの活' ,中間体匪 P - 2力認められた。 しカヽしな力くら、 検体 9において、 プロ画 P - 2の切断が抑制され 、 活性型中間体删 P - 2 カ'比較的少ないことカ壞察された。  Any 12 specimens, and the corner with gelatin zymography? When baked, all showed 92 kDa pro-P-9, 68 kDa pro-MMP-2 and 64 kDa active P--2 intermediate. In the specimen 9, the cleavage of the pro-form P-2 was suppressed and the activated intermediate 删 P-2 カ 'was relatively small.
(2) 二次スクリーニング (2) Secondary screening
一次スクリーニングの検体 9で用いた^！遺伝子プラスミ ドの靠団を大腸菌 XL -IBlue (Stratagene) に導入しアンピシリン含有 LBプレ一ト上で 24時間培養した o 大腸菌コロニー 50個を 1個づっ分離し、 删 I 培地にて 37°Cで 16時間培養し、 培 養大腸菌 2 mlよりプラスミ ドを精製した。 前項記載の一次スクリーニングと同様 にプロ MMP- 9，プロ應 P- 2， MT卜讀 P発現プラスミ ドと精製プラスミ ドを 293T細胞に 導入し、 ゼラチンザィモグラフィ一にて潜在型、 中間体、 活性化議 P- 9、 MMP- 2 を検出した。 活性化 §¾ΜΡ- 2 の産生を抑制した単一プラスミ ドを同定した。 任意の 12検体にっ ヽて、 ゼラチンザィモグラフィ一で角斜斤したところ、 検体 3 、 5および 6で 64 kDaの活' ftS中間体 ΜΡ- 2カく全く見られなかった。 これらのレ ーンに用いたプラスミ ドは 1. 4 kbの挿入配列を有しており、 LI- COR DNA シーク ェンサ一Model 4200L(S)- 2を して揷人された DNAの塩基配列を した (Acc ession No. AB056866)o この配列において、 最初の 5，側の 1038残基は、 Testican 3¾S配列 (Access ion No. 應 016950) の 311 - 319力く欠損している以外では Test i can 3と同一であつた。 1039残 ¾J¾降の配列はュ二一クであつた。 ォ一プンリ一 ディングフレームは 108 から 1046残基で見出され、 Testican 3の N-末端断片に相 当する 313 アミノ酸残基をコードしていた。 ただし、 Testican 3の 64から 66残基 部分を欠き、 また C-末端に変化が見られた。 この遺伝子が、 Testi can 3の N-末端 側をコードするものであったことから、 N-Tes と命名した。 N- Tes の 3'部分と Te st ican 3の cDNAi^S配列力く、 第 4染色体ゲノムデータベース（BAC clone RP11-44 0L30, Accession No. AC020599) 上に見出されたことから N- Tes は Testican 3の ^ Used for primary screening sample 9 The gene plasmid was transferred into 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 删 I medium. After culturing for 16 hours, plasmid was purified from 2 ml of cultured E. coli. In the same manner as the primary screening described in the previous section, 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 §¾ΜΡ-2 was identified. Square beveling with gelatin zymography for any of the 12 samples showed that no 64 kDa active ftS intermediate ΜΡ-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 3¾S sequence (Accession No. 016950). It was. The sequence of the remaining 1039 was ュ J¾. The open reading frame was found at residues 108 to 1046 and encoded 313 amino acid residues corresponding to the N-terminal fragment of Testican 3. However, residues 64 to 66 of Testican 3 were missing, and changes were found at the C-terminus. Since this gene encoded the N-terminal side of Testcan 3, it was named N-Tes. 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
N-Tes の N -末端側 21アミノ酸残基は、 疎水' f生のシグナルペプチド、 22-85 アミ ノ酸残基は、 Testi can 1、 Testican 2および Testican 3以外では見られないュニ —クな酉 リであった。 また、 86- 191アミノ酸残基は、 システィンリッチな fol l is tatin-l ike (FS) ドメイン、 87 - 311アミノ酸残基は、 extracel lular Ca2 +糸吉合 (E C)ドメインであった。 Testican 3は、 じ-末端側に1;1^1"0£101)111 （TY)様ドメイン と 2ケ所の糖鎖修飾部位を持つ。 実施例 8 ：組換え Testican 1, Testi can 2, Testican 3および BM- 40 のクロー二 ング 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
C-末端側に FLAGェピトープを付加した Testi can 1， Testi can 2， Test ican 3 および BM 40 を発現するプラスミ ドを以下の様にして作製した。 マルチクロ一二 ングサイ卜と FLAGェピトープ (Asp- Tyr- Lys- Asp- Asp- Asp- Asp- Lys 〔配列番号： 12〕 ) をコードした DNA断片を pFLAG- CTC plasmid (IBI FLAG Biosystems, NY) からプライマー N26 と C24 (IBI FLAG Biosystems, NY) による PCRで調製した。 この断片を Hind 111と8£1 I Iで切断した pSG5 (Clonetech) クロ一ニングサイト に挿入し PSG- FLAGと称するプラスミ ドを作製した。 cDNAは、 ヒトあるいはマウス 胎盤由来の total RNA より 8 mer ランダムプライマーと ReverTra Ace reverse t ranscriptase (T0Y0B0, Japan)で合成した。 ヒト Testican 1、 ヒト Testi can 2、 ヒト Testican 3およびマウス BM- 40 に対するプライマ一を GeneBank™ accession numbers: CAA51999, CAA04774, 016950 およぴ 009242 に言己載の酉己列に基 づいて作製した。 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-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). This fragment was inserted into pSG5 (Clonetech) cloning site cut with Hind 111 and 8 £ 1 II to prepare a plasmid called PSG-FLAG. 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 ™ accession numbers: CAA51999, CAA04774, 016950 and 009242.
Tes ti can 1-5' GCAGATCTAGCTCGAGCAACTCGGACTAG 〔配列番号 13〕 Tes ti can 1-3， GGAGATCTCCATATGTACCCGACCTCATC ¾己列番号 14〕 Tes t i can 2-5' CAGGTCGAAnCAGACCACGATGCG 〔配列番号 15〕 Tes ti can 2-3' GGAGATCTCCAGATGTAGCCCCCGTCGTC 〔配列番号 16〕 Tes ti can 3-5' AAAGCAGCGAGnGGCAGAG ¾己列番号 17〕 Tes ti can 3-3' CATGMTTCMTGTATACATCATGGTC ¾己列番号 18〕 BM - 40- 5' ： GAGGGATCCCAGCATCATGAGGGC 〔配列番号 19〕 BM- 40 - 3' ： GCAGGATCCGTGAACmGATCAC 〔配列番号 20〕 Testi can 1-5 'GCAGATCTAGCTCGAGCAACTCGGACTAG [SEQ ID NO: 13] Testi can 1-3, GGAGATCTCCATATGTACCCGACCTCATC ¾ Own column number 14] Testi can 2-5' CAGGTCGAAnCAGACCACGATGCG [SEQ ID NO: 15] Tes ti can 2-3 'GGAGATCTCGATCCGCCCC No. 16] Tes ti can 3-5 'AAAGCAGCGAGnGGCAGAG ¾ Own column number 17) Tes ti can 3-3' CATGMTTCMTGTATACATCATGGTC ¾ Own column number 18] BM-40-5 ': GAGGGATCCCAGCATCATGAGGGC [SEQ ID NO: 19] BM-40-3' : GCAGGATCCGTGAACmGATCAC [SEQ ID NO: 20]
PCR にて増幅した各 cDNA断片は、 プライマ一中の BamHI あるいは Bgl IIサイト で切断し、 pSG- FLAGの Bgl 11サイトに揷入した。 Tes ti can 3 cDNA は、 pSG- FLAG の Sma I および Bgl IIサイトに揷入した。 各 cDNA断片は、 LI -COR DMシークェン サ一でその塩基酉己列を確認した。 それぞれの発現べクタ一を pSG- Test i can 1 - FLA G， pSG-Testican 2- FLAG, pSG - Testican 3- FLAGおよび pSG- BM - 40- FLAGと称した  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と pSG- Testican 3- FLAGを 293T細胞に導入し、 N- Tes と Testic an 3の分布を調べた。 N- Tes と Testican 3に付加した FLAGェピトープは、 遺伝子 導入した細胞のライゼートおよび培養上清に見出された。 分子量はそれぞれ 37 k Da、 60 kDaを示した。 一方、 Testican 3-FLAG は、 遺伝子導入した細胞の ECMか ら見出された力、 N- Tes- FLAGは検出されなかったことから、 N-Tes は可溶性のタ ンパク質であると判断された。 実施例 9 ：免疫沈降による N- Tes と ΜΤΊ- MPの結合 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. On the other hand, 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 ΜΤΊ-MP by immunoprecipitation
径 60 mmの培養ディッシュに 293T細胞を播き込み、 1 の pSG- N- Tes- FLAGお よび の pSG - ΜΉ-匪 P発現プラスミ ドを導入し、 48時間後に 100 zCi/ml の Pro-mix L-(35S) in vitro cell labeling mix (Amersham Pharmacia Biotech) で 4時間、 標識した。 細胞は、 RIPA緩衝液 (150 mM NaCl, 5 mM EDTA, 1% Trito n-X100, 0. 1% SDS含有 10 mM Tris-HCl buffer, pH 7. 4)で溶解し、 15, 000 rpm 1 0分間遠心して上清を得た。 400 ng irCMTl-MMP抗体 114- 1F2 (第一フアインケ ミカノレ） あるいは抗 FLAG抗体 M2 (S i ma) をこの上清に加え 4 °Cで 16時間ィンキュ ベ一卜した。 抗原一抗体の複合体は、 20 Lの Protein- G Sepharose4B (Amersh am Pharmacia Biotech) で回収し、 12% SDS - PAGEに供した。 ゲルを乾燥し放射活 性を Bio-image Analyzer BAS1000 (Fuj i Fi lm)で検出した。 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. 400 ng 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).
διΜΤΙ-ΜΜΡ モノクローナル抗体は、 ΜΉ- MMP遺伝子のみを導入した細胞から 50 kDaおよび 47kDa C MTl-ΜΜΡを沈殿させた。 N- Tes- FLAGと MT1-固 P遺伝子を共導 入した細胞力、ら 37 kDaの N - Tes - FLAGが御 T1 - MMP モノクローナル抗体によって MT 1-MMP と共に沈殿した力く、 N- Tes- FLAG遣 fe?のみを導入した細胞から、 この抗体 によって N-Tes- FLAGは沈殿しなかった。  The διΜΤΙ-ΜΜΡ monoclonal antibody precipitated 50 kDa and 47 kDa C MTl-ΜΜΡ from cells into which only the ΜΉ-MMP gene had been introduced. N-Tes-FLAG and MT1-fixed P gene co-introduced cell force, and 37 kDa N-Tes-FLAG was precipitated together with MT1-MMP by T1-MMP monoclonal antibody. N-Tes-FLAG was not precipitated by this antibody from cells into which only the FLAG transfection was introduced.
抗 FLAG抗体は、 N- Tes- FLAG遺伝子を導人した細胞から 35 kDaの N- Tes- FLAGを沈 殿させた。抗 FLAG抗体は、 MT1- MP遺伝子を導人した細胞から MT1-匿を沈殿さ せなかった力 N - Tes - FLAGと MT1-醒 P遺伝子を共導入した細胞から MT1 -丽 P力く共 沈した。 同様に、 MT3-删 P もまた、 N- Tes- FLAGと共沈が確認された。 これらの結 果から、 MT1 -匪 P と MT3- MMP は、 N - Tes - FLAGと安定な複合体を形成すること力く示 された。 UniO： MT- Ps の阻害活性を有する N- Tes ドメィンの決定  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. Similarly, it was confirmed that MT3- 删 P also co-precipitated with N-Tes-FLAG. These results strongly indicate that MT1-band P and MT3-MMP form a stable complex with N-Tes-FLAG. UniO: Determination of N-Tes domain with MT-Ps inhibitory activity
(1) N-Tes変異体の発現べクタ一の作製  (1) Construction of expression vector for N-Tes mutant
N-Tesの欠 ί!¾異体 85、 97および 122の cDNA断片を ρΕΑΚ8の 5'プライマーと以 下に記載したプライマーによる PCRで増幅した。  N-Tes deficient! CDNA fragments of variants 85, 97 and 122 were amplified by PCR with the 5 'primer of ρΕΑΚ8 and the primers described below.
85-3' ： TGAGATCTCnAGCTGGATCTAAAGCCTG 〔配列番号： 21〕 85-3 ': TGAGATCTCnAGCTGGATCTAAAGCCTG [SEQ ID NO: 21]
97-3' ： TGAGATCTTCCTGCTTCTTTCATCCTGTGTG 〔配列番号： 22〕 得られた DNA断片を Bgl I Iで切断し、 pSG- FLAGに挿入した。 97-3 ′: TGAGATCTTCCTGCTTCTTTCATCCTGTGTG [SEQ ID NO: 22] The obtained DNA fragment was cut with BglII and inserted into pSG-FLAG.
122C/S変異体 cDNAは、 以下のプライマ一による連続的な PCRにて 4ケ所のシス ティン残基をセリン残基に置換して作製した。下線は、変異を導入した塩基であ る。 C/Sl-2: AAGGATCCMGCnAAAGATGAAMGTAG 〔配列番号 23〕 C/S3 : CGCCATAAAGTMGCAnGCTCAAG 〔配列番号 24〕 C/S4: CAGACTGCAGTCAGCAHAGTCACC 〔配列番号 25〕 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]
(2) N-Tes/Testican 2キメラの作製 (2) Construction of N-Tes / Testican 2 chimera
N-Tes/Testican 2キメラ cDNA断片を作製した。 550番から始まる Test i can 2の C-末端側 cDM断片を Testican 2-3' プライマーおよび  An N-Tes / 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 ¾己列番号： 26) を用いて増幅した。 N- Tes cDNAの C-末端側断片 (359 #-1046番） を BamHI およ び Bgl I Iで切断除去し、 増幅した断片を挿入した。 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.
(3) Test i can 2/N - Tesキメラの作製  (3) Preparation of Test i can 2 / N-Tes chimera
Tes ti can 2/N - Tesキメラ cDNA断片を作製した。 Testican 2の N-末端側 cDNA ( 279番- 550番） の cDNA断片を Testican 2-5' プライマ一および 550番から始まる AGG COTGGTGGTATCCAGGGCTTCAT ¾ 番号： 27] を用いて増幅した。增幅した断片は、 N-Tes cDNAの N-末端側 (1番- 550番） と置換 し/  A 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].置換 Replace the widened fragment with the N-terminal side of N-Tes cDNA (No. 1-550) /
(4)結果 (4) Result
Testican 1および Testican 3の発現は、 ΜΏ-ΜΜΡおよび ¥Γ3- MMP によるプロ讀 Ρ-2活性化を阻害した力く、 Testican 2および BM - 40の発現では、 阻害活性は認め られなかった。 N- Tes の MT1-匪 Pおよ ti¾T3- MMP P且害活性ドメインを決定するた めに欠損変離を作製した。 N- Tesの N-末端側の 122アミノ酸残基（Δ122)は、 野生型 N - Tes と同等のプロ画 P - 2活性化阻害能を有していた。 N- 側の 97、 85 残基からなる欠損変難 （Δ97、 Δ85) は、 野生型より低いレベルで ffll -議 Ρお よび MT3 - MMPを阻害した。 Δ122の 4つのシスティン残基をセリン残基に置換し た変異体（M22C/S) は、 阻害活性を腿していた。 しかしながら、 N-Tesの 33 -84残基を欠損したものは、 P且害活†生を失った。  The expression of Testican 1 and Testican 3 strongly inhibited the activation of pro-reading II-2 by ΜΏ-ΜΜΡ and ¥ Γ3-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 ti¾T3-MMMP P and toxic domains. The 122 amino acid residue (Δ122) 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 (Δ97, Δ85) inhibited ffll--and MT3-MMP at lower levels than wild-type. The mutant in which four cysteine residues of Δ122 were substituted with serine residues (M22C / S) had a higher inhibitory activity. However, those lacking residues 33-84 of N-Tes lost P and no harmful activity.
N-Tesのシグナルとユニークドメインを Testican 2の FS、 EC、 TYおよび C-末端 ドメインカ、ら成るキメラ（N- Tes/Tes-2) は、 充分な阻害活性を示したが、 Testic an 2のシグナルとユニークドメインで置き替えられた N- Tes (Tes-2/N_Tes)は、 阻害活' f生を失った。 これらのこと力、ら、 N - Tes の N -末端のユニークドメイン力 \ MT1-MMPおよび ΙΓ3-删 P の阻害に必須であること力確認された。 実施例 11：半 的 RT- PCRによる N- Tes および Testican 3 mRNAの角靳 A chimera (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. These facts confirmed that the unique domain force at the N-terminal end of N-Tes was essential for inhibition of MT1-MMP and ΙΓ3- 删 P. Example 11: Quantification of N-Tes and Testican 3 mRNA by Semi-RT-PCR
(1) 臨床検体  (1) Clinical specimen
ヒトグリオ一マ糸纖は、 10人の患者より脳廳除去^ ¾に伴う正常脳糸職と共 に調製した。 Total RNA調製のため、 検体は摘出後、 液体窒素で急速冷凍し- 80 °Cでィ呆存した。 また、 糸&織検査のため 4%パラホルムアルデヒド固定液で 18から 24 時間 (4°C) で固定した。 切片をへマトキシリンおよびェォシンで染色し、 検鏡し た。 脳 S藤の^は、 Kleihues, P. , Burger, PC , Schei thauer, BW.， "Histol ogical Typing of Tumours of the Central Nervous System", Berl in, Springe r-Verlag, 1993， p 11-20. 記載の判定基準で行った。 すべて 織に対し て、 切除前に化学療法、 放射線治療は行われていない。  Human glioma fiber was prepared from 10 patients together with a normal brain thread associated with removal of the brain. For total RNA preparation, 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.
(2) 半 的 RT- PCRによる N- Tes および Testican 3 mRNAの角科斤  (2) Semi-assisted RT-PCR of N-Tes and Testican 3 mRNA
Nakada, M. , Nakamura, H. , Ikeda, Ε. , Fuj imoto, Ν. , Yamashi ta, J. , Sato ， Η. , Seiki, Μ.， and Okada, Y. , "Expression and tissue local ization of m embrane - type 1， 2, and 3 matrix metal loproteinases in human astrocyt ic t umors. ", Am J Pathol. 154: 417-28, 1999.記載の方法に従って、 半 的 RT - P CRによる N- Tes， Testican 3および GAPDHの mRNAの解析を行った。 プライマ一は 下記のものを用いた。  Nakada, M., Nakamura, H., Ikeda, Ε., Fuj imoto, Ν., Yamashi ta, J., Sato, Η., Seiki, Μ., And Okada, Y., "Expression and tissue localization of membrane-type 1, 2, and 3 matrix metal loproteinases in human astrocytic ticumors. ", Am J Pathol. 154: 417-28, 1999. Testican 3 and GAPDH mRNA were analyzed. The following primers were used.
RT-N-Tes-5' GAGTGGTGCTACTGCTTCCA ¾己列番号 6〕 RT - N - Tes- 3， GTGCHCnGGTGGCTCATA 〔配列番号 7〕 RT-Tes3-5' TGCTAnGGACCAGTCAGAGCTC 〔配列番号 28〕 RT-Tes3-3' TCCAACACTGCCATGACACTGTG 〔配列番号 29〕 (3) 結果 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
半 的 RT- PCRにより正常脳およびグリオ一マ組織の N - Tes および Testican 3 の mRNA レベルを調べた。正常脳において、 -Tes および Yestican 3のいずれ も力《検出された力く、 グリオ一マ糸纖において低下が認められた。 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.
グリオ一マ細) 3包株 T98 および U87 における N- Tes mRNA発現は正常脳と同等であ つた力 U251細胞では有為に低かった。 一方、 検出可能なレベルの Test i can 3を 発現するグリォーマ細胞株は存在しなかった。  The expression of N-Tes mRNA in the three strains T98 and U87 was significantly lower in U251 cells, which were comparable to normal brain. On the other hand, no glioma cell lines expressing detectable levels of Test Can 3 were present.
図 6に、 NB (normal brain; 正常脳組織， n=18)； LGA (low-grade astrocytom a ； ί£¾凰細胞腫， η=9)； ΑΑ (anaplastic astrocytoma; 未分ィ匕 ¾細胞腫， n=8) ； GB (gl ioblastoma; グリア芽細^！重， n=34)； Meta (metastatic brain tumor ; 脳へ転移した 癌， ΙΡ9)及び GCL (gl ioma cell l ine ;グリオ一マ細胞株， n= 4)の Tes ti can 3と N- Tes の発現レベルを半 的 RT- PCRで角? ) した結果を示した 悪 tt^が高くなるにつれて、 N-Tes の発現レベルの低下が認、められた。 歸 fe例 12 ： N- Tesあるいは Tes tican 3による癌の浸潤阻害  Figure 6 shows NB (normal brain tissue, n = 18); LGA (low-grade astrocytoma; ί £ ί phoenoma, η = 9); ΑΑ (anaplastic astrocytoma; undivided 分 cytoma) , N = 8); GB (glioblastoma; n = 34); Meta (metastatic brain tumor; cancer that has metastasized to the brain, ΙΡ9) and GCL (gliooma cell line; gloma cells) 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
(1) 糸且換え Testican 3、 N-Tes および " - Tes- del 発現細胞株の植泣  (1) Transplantation of cell lines expressing testosterone 3, N-Tes and "-Tes-del
pSG-Tes ti can 3- FLAG, pSG-N- Tes- FLAGおよび pSG-N- Tes- del- FLAGのそれぞれ を Xbalで切断し、 pSG5の early SV40プロモータ一、 ラビット /3- グロビンのイン トロン I I、 T7バクテリオファージプロモータ一、 それぞれの cDNAおよびポリアデ 二レーシヨンシグナルを含む断片を切り出し、 予め Xbalで切断した pCEP4(Invi tr ogen) に挿入した。 得られた発現ベクターを pCEP- Testi can 3 - FLAG、 pCEP-N-Tes -FLAGおよび pCEP- N- Tes- del - FLAG と称した。  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-Tes tican 3- FLAGおよび pCEP - N - Tes - FLAGを erbB2で癌化した MDCK細胞あ るいは U251グリオ一マ細胞に導入し、 400 〃g/mLのハイグロマイシン B 下で 選択した。  pCBP-Testican3-FLAG and pCEP-N-Tes-FLAG were introduced into MDCK cells or U251 glioma cells carcinomatized with erbB2 and selected under 400 μg / mL hygromycin B.
(2) コラ一ゲンゲルにおける i咅養  (2) i-culture in collagen gel
I 型コラーゲンゲル (Ni tta Gelatin) を調製した。 1 mLのコラーゲンをゲル化 した径 35 譲の培養ディッシュに 3， 000個の U251細胞を含む 6 /L のコラーゲン ゲルを置き、 さらに 1 mLのコラーゲンゲルでサンドイッチした。 ゲルに 2 niLの培 地を添加し、 5日間培養した。 MDCK-erb2細胞の浸潤アツセィには、 径 35 誦の 培養ディッシュにおよそ 3， 000個の細胞を含むコラーゲンをゲル化し 2 mLの培地 を添カロし、 7日間 i咅養した。 (3) 結果 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. For the infiltration of MDCK-erb2 cells, 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. (3) Result
安定的に N- Tes-FLAG あるいは Tes t i can 3-FLAG遺伝子を導人した U251で両遺 伝子の発現を抗 FLAG抗体によるウエスタンプロッティングで確認した。 N- Tes- FL AGおよび TesUcan 3-FLAG融合タンパク質は、 それぞれ 37 kDaおよび 55 kDaのバ ンドを示した。 野 βυ251, pCEP4, N- Tes- FLAGおよび TesUcan 3-FLAGを導人 した U251のコラーゲンゲルに ¾ ^る浸潤を比較した。 N- Tes- FLAGあるいは Test ic an 3-FLAG の導入は、 U251の浸潤能を明らかに低下させた。  The expression of both genes was confirmed by Western blotting using an anti-FLAG antibody in U251 stably carrying the N-Tes-FLAG or Testcan 3-FLAG gene. The N-Tes-FLAG and TesUcan 3-FLAG fusion proteins exhibited 37 kDa and 55 kDa bands, respectively. We compared the infiltration of collagen gel of U251, which was derived from wild βυ251, pCEP4, N-Tes-FLAG and TesUcan 3-FLAG. The introduction of N-Tes-FLAG or Testic an 3-FLAG clearly reduced the infiltration capacity of U251.
MT1- MP を発現し、 コラーゲンゲル中で浸潤生育する erbB2で癌化した MDCK細 胞 (MDCK- erbB2)に N- Tes- FLAGあるいは Testican 3-FLAG発現プラスミドを導入し 、 導入後の性質を比較した。 N- Tes- FLAGあるいは TesUcan 3-FLAG遺伝子の導人 によって、 形態学的変化は全く誘導されなかった。 親钿胞株あるいは空べクタ一 導 AMDCK - erbB2細胞株は、 コラ一ゲンゲル中で浸潤生育を示した力、 注目すべき ことに、 N- Tes FLAGあるいは Test i can 3-FLAG遺^の導入後、 浸潤生育能が低 下した。 頻 fe例 13：組換え Test i can 3、 N-Tes およびM- Tes-del の精製  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
(1) 組換え Test i can 3、 N-Tes および Tes- del の発現系  (1) Recombinant Test i can 3, N-Tes and Tes-del expression system
pCEP-Testican 3-FLAG, pCEP N- Tes- FLAGおよび pCEP-N- Tes- del- FLAGを 293E BNA細胞に導人し、 00 a g/mLのノ、ィグロマイシン B存在下で選択して Tes t i can 3-FLAG、 N- Tes FLAGおよび "N- Tes- de卜 FLAGを産生する細胞株を得た。  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.
(2) 組換え Test i can 3、 N-Tes および "N- Tes-del の発現  (2) Expression of recombinant Test can 3, N-Tes and "N-Tes-del
それぞれの発現株を口一ラーボトルに播き込み、 10% FCS含有 DMEMでコンフル ェン卜まで培養した。 ±き養液を FCS不含の DMEMに交換し 2日間培養を 繞した後 、 ±咅¾±清を回収した。 ±咅養上清 0. 5 mLを濃縮し、 抗 FLAG抗体によるウェスタン ブロッテイ ングで発現を確認した。  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, ± 咅 ¾ ± was recovered. 0.5 mL of the culture supernatant was concentrated, and expression was confirmed by Western blotting using an anti-FLAG antibody.
(3) 組換え TesUcan 3、 N-Tes および" N- Tes- del の精製  (3) Purification of recombinant TesUcan 3, N-Tes and "N-Tes-del
培 *±清を遠心し細胞残渣を除去した後、 抗 FLAG抗体ァフィ二ティ一ゲノレに供 し、 精製した。 抗 FLAG抗体によるウェスタンプロッティングおよび CBB染色で発 現と純度を検討した。 実施例 14：ポリクローナル抗体の作製 The culture was centrifuged to remove cell debris, and then subjected to anti-FLAG antibody affinity purification to purify. Expression and purity were examined by Western blotting and CBB staining with anti-FLAG antibody. Example 14: Preparation of polyclonal antibody
(1) 抗原ポリペプチドの調製  (1) Preparation of antigen polypeptide
N-Tes のアミノ酸配列中より、 N-Tes に特徵的な配列として次の配列を選択し た。  From the amino acid sequence of N-Tes, the following sequence was selected as a sequence specific to N-Tes.
CFQRQQGKR 〔配列番号： 11) このポリペプチドを Fmoc- bop法で合成した。 合成したペプチドは、 逆相 HPLCに より 80〜90%純度以上に精製した。  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.
(2) ポリペプチドと BSAの複合体の調製  (2) Preparation of complex of polypeptide and BSA
各ポリぺプチドをぺプチ FN末端に付加したシスティン残基を介してゥシ血清 アルブミン (BSA) と糸吉合させ、 抗原コンジュゲートを調製した。  Antigen conjugates were prepared by combining each polypeptide with cysteine serum albumin (BSA) via cysteine residues added to the peptide FN terminus.
(3) ゥサギ她清の調製  (3) Preparation of Egret
前項 (2) で調製した BSA-ポリぺプチドを完全フロイントアジュバントと共に日 本白色種メス家兎 2羽に皮下投与 （0. 15 mg/羽） し、 初回免疫した。 これらのゥ サギに 2週、 4週、 6週、 8週、 10週後に 0. 3 mg/羽の BSA -ポリペプチド複合体 を不^フロイントアジュバントと共に内皮に追加免疫し、 11週後に全採血を行 なった。 得られた血液を遠心分離し、 抗 BSA -ポリペプチド複合体ポリクローナル 抗体を含むゥサギ!¾&1清を得た。  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.
免疫した BSA-ポリべプチド複合体を固相化した 96穴プレートにて ELISAを行い 、 全採血して得た ML清の力価を測定して抗体の を確認した。 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.
表 1 吸光度 (0.D) Table 1 Absorbance (0.D)
希釈倍率  Dilution ratio
免疫前採血 全採血  Pre-immune blood sampling Whole blood sampling
1,000 0.086 1.883  1,000 0.086 1.883
2,000 0.070 1.832  2,000 0.070 1.832
4,000 0.067 1.678  4,000 0.067 1.678
8,000 0.058 1.195  8,000 0.058 1.195
16,000 0.058 0.783  16,000 0.058 0.783
32,000 0.052 0.392  32,000 0.052 0.392
64,000 0.051 0.226  64,000 0.051 0.226
128,000 0.047 0.148  128,000 0.047 0.148
(4) ポリクロ一ナル抗体の精製 (4) Purification of polyclonal antibodies
前項 (3) で得られたゥサキ 清をそれぞ ^40%飽和硫酸ァンモニゥムで塩折 し、 得られた沈殿を 60mM NaCl含有 40mM KH2P04- NaOH (pH8)で溶解後、 同緩衝液 で5 Pfi化した DEAE- S印 hacel (Amersham Pharmacia Biotech) ゲルカラム （直径 32腿、 長さ 35cm) に供した。 素通り画分を 50%飽和硫酸アンモニゥムで塩折し、 得られた沈殿を 0.1M リン酸緩衝液 (pH7.0) で溶解、 同緩衝液で透析しゥサギポ リク口一ナノレ抗体を得た。 実施例 15： irCN-Tes モノク口一ナル抗体の作製 The Usaki Qing obtained in the above (3) respectively were salted out with ^ 40% saturated sulfuric Anmoniumu, 60 mM and the resulting precipitate NaCl containing 40mM KH 2 P0 4 - was dissolved in NaOH (pH 8), with the same buffer 5 Pfi-formed DEAE-S mark hacel (Amersham Pharmacia Biotech) gel column (diameter 32 thigh, length 35 cm) was used. The flow-through fraction was salt-broken with 50% saturated ammonium sulfate, and the obtained precipitate was dissolved in a 0.1 M phosphate buffer (pH 7.0) and dialyzed with the same buffer to obtain a one-negative antibody of Sagipoly mouth. Example 15: Production of irCN-Tes monoclonal antibody
(1) 免疫抗原の調製 (1) Preparation of immunizing antigen
N-Tesの C-末端側に His タグを付加した融合タンパク質を発現する発現べクタ — pCTCN- TesHisを導人した大腸菌 JM109を 4mLの 50 g/mL Ampicillinを含む LB 培地に接種、 37°Cで 16時間培養し、 1次カルチャーを調製した。 これを 400mLの 50 g/mL Ampicillinを含む LB培地に再接種、 37°Cでおよそ 3時間、対数増殖期 中期まで培養した。 IPTG (最終濃度 0. lmM) を添加し、 さらに 4時間培養を継続 し、 組換え融合タンパク質の遞を誘導した。  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.
±咅養液を直ちに氷冷し、 遠心 (5，000rpm、 20min)で菌体を回収 20mLの lmg/mL lysozyme含有 20mM Tris- HCl(pH8.0)に懸濁し 15分間、 氷中に置いて細^ を破 壌した。菌体懸濁液を氷冷下で超音波破砕（30秒、 10回） 、遠心 (18， 000rpm、 10 min) して inclusion bodyを回収した。 Inclusion bodyを lmLの 0. 1 M NaCl含有 10 mM リン酸緩衝液 (pH 7. 0)で再懸濁し洗浄、 遠心 (15, 000rpm、 lOmin)して沈殿 を回収、 これを lmLの 8M urea, 0. 1 M NaCl含有 10慮 リン酸緩衝液 (pH 7. 0)に 溶解した。 Immediately cool the nutrient solution on ice, and collect the cells by centrifugation (5,000 rpm, 20 min). The cells broke down. Ultrasonic disruption of the cell suspension under ice cooling (30 seconds, 10 times), centrifugation (18,000 rpm, 10 min) to collect the inclusion body. The inclusion body was resuspended in 1 mL of 10 mM phosphate buffer (pH 7.0) containing 0.1 M NaCl, washed, centrifuged (15,000 rpm, 10 min), and the precipitate was collected. It was dissolved in a phosphate buffer (pH 7.0) containing 0.1 M NaCl.
上言己の糸且換え大 g昜菌 (400mL培養） より調製した不溶 'ί生封入体を 8 M urea, 0. 5 M NaCl, 10 mM imidazole含有 50mMリン酸緩衝液 (ρΗ7· 5) で可溶ィ匕し、 Ni++ C helating Sepharoseゲルカラムに供した。 洗浄後、 8 urea, 0. 5 M NaCl, 0. 5 imidazole含有 50 mM リン酸緩衝液 (pH7. 5) で溶出、 得られた精製画分に 1% SD S, 0. 5¾ 2-メルカプトエタノールを添加した。室温で 30分間置いた後、 2% D0Cを 添加して PBSで透析した。 2日間、 計 7回の夕 換を行った。 さらに組換え夕 ンパク質画分を PBSで 2. 5 mLに調製し、 PBSで ti化した PD- 10 にてバッファ一 交換した。 精製画分より 5 zL とり還元 SDS- PAGEに供し、 CBB にて染色し、 タン パク質 1. 4 mg、 糸艘 95% と見積もった。 The insoluble 'ί living inclusion bodies prepared from the above-mentioned thread-recombinant large-scale bacteria (400 mL culture) were treated with 50 mM phosphate buffer (ρΗ7.5) 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, 0.5¾ 2-mercaptoethanol was added to the purified fraction. Was added. After 30 minutes at room temperature, 2% D0C was added and dialyzed against PBS. A total of seven evenings were held over two days. Further, the recombinant protein fraction was adjusted to 2.5 mL with PBS, and the buffer was exchanged with PD-10 tied with PBS. 5 zL was taken from the purified fraction, subjected to reducing SDS-PAGE, stained with CBB, and estimated to be 1.4 mg of protein and 95% of the boat.
(2) 抗体の作製と選択 (2) Preparation and selection of antibodies
①免疫は以下のスケジュ—ル  ① Immunization schedule
6週齢 Balb/C雌、 2匹  6 week old Balb / C female, 2
lmg/ml N-Tes 0. 1M NaCl 含有 50 m リン酸緩衝液 (pH7. 0) 初回免疫： 45. 5 ^g/300 〃1/マウス （^フロイントアジュバント） 腹腔内 投与  lmg / ml N-Tes 0.1m NaCl-containing 50m phosphate buffer (pH 7.0) Initial immunization: 45.5 ^ g / 300 〃1 / mouse (^ Freund's adjuvant) Intraperitoneal administration
追加免疫： 20日後 41. 1 g/185 〃1/マウス 0空内投与  Booster boost: 20 days later 41.1 g / 185 〃1 / mouse 0 air administration
最終免疫： 55日後 36. 7 g/220 〃1/マウス 静脈内投与  Final immunization: 55 days later 36.7 g / 220 〃1 / mouse Intravenous administration
最終免疫の 3日後 （58日後） に細胞融合を行った。 Cell fusion was performed 3 days (58 days) after the final immunization.
②スクリーニング (ELISA法によるハイプリ ドーマの検索)  ②Screening (Search for high prioma by ELISA)
ELISA:固相 100 ng/ゥエルで免疫抗原をコート。 ELISA: Immobilized antigen coated with 100 ng / well of solid phase.
細胞融合の 11日後にスクリ一ニングを実施。 35株を選択。 Screening was performed 11 days after cell fusion. Select 35 shares.
③クローニング （限界希釈）  ③ Cloning (limited dilution)
限界希釈でクロ一ニング、 ELISAで抗体産生ハイプリ ド一マを選択。 9クロ一 ンを選抜。 ④サブクラスアツセィ Cloning by limiting dilution and antibody-producing hybridoma by ELISA. 9 clones selected. ④Subclass Atsushi
9クローンについてサブクラスを決定した 表 2 クローン No . サブクラス  Table 2 Clone No. subclass determined for 9 clones
288 - 9H3 y lb / κ  288-9H3 y lb / κ
288-12H5 y 2a / κ  288-12H5 y 2a / κ
288-14F8 2b / κ  288-14F8 2b / κ
288-17B9 y 2a / κ  288-17B9 y 2a / κ
288-21D11 y 2a / κ  288-21D11 y 2a / κ
288-25D10 7 2a /  288-25D10 7 2a /
288-27E11 I K  288-27E11 I K
288-28D11 y 2a / K  288-28D11 y 2a / K
288-29B6 7 1 / K  288-29B6 7 1 / K
⑤ィムノブロッティングによる選択 Selection by simno blotting
免疫抗原を 12% SDS- PAGEで展開、 ニトロセルロースメンブレンに転写、 各ハイ プリ ドーマ培^：清を 1次抗体として添加、 抗マウス Ig(G+A+M)- HRP を 2次抗体 として使用、 染色した。 いずれの培養上清も免疫抗原と強く反応した。  Immunogen developed on 12% SDS-PAGE, transferred to nitrocellulose membrane, each hybridoma culture medium added: primary antibody added, anti-mouse Ig (G + A + M) -HRP used as secondary antibody Stained. All culture supernatants reacted strongly with the immunizing antigen.
実施例 13に言己載の pCEP- Test i can 3- FLAGヽ pCEP- N- Tes- FLAGおよび pCEP- N- Tes -del -FLAGを導入した 293EBNA細胞より調製した細胞抽出画分を 12% SDS- PAGEで 展開、 ニトロセルロースメンブレンに転写、 サブクラス毎に任意に選択した 288 -9H3 ( r 2b Ικ ) 、 288- 12H5 (ァ 2a / κ ) および 288-29Β6 (ァ l/ ) の各ハイブ リ ドーマ培 清を 1次抗体として添加、 抗マウス Ig(G+A+M)- HRP を 2次抗体と して使用、 染色した。 いずれのクローンも組換え Test i can 3 - FLAGおよひ - Tes - FLAGと良く した力 検討した 3種のクローンには N- Tes-de卜 FLAGと反応する ものは含まれなかった。  The cell extract fraction prepared from 293EBNA cells transfected with pCEP-Testican3-FLAG ヽ pCEP-N-Tes-FLAG and pCEP-N-Tes-del-FLAG described in Example 13 was subjected to 12% SDS. -Developed on PAGE, transferred to nitrocellulose membrane, and hybridomas of 288-9H3 (r2bΙκ), 288-12H5 (α2a / κ) and 288-29Β6 (αl /) selected arbitrarily for each subclass 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.
実施例 13に言己載の pCEP- N- Tes- FLAGおよび pCEP - N - Tes- del- FLAGを導入した 29 3EBNA細胞培 ¾±清より精製した N- Tes- FLAGおよび "N- Tes - de卜 FLAGを ％ SDS-PA GEで展開、 ニトロセルロースメンブレンに転写した。 前項言己載の各ハイプリ ド一 マ培^:清を 1 次抗体として添加、 抗マウス Ig(G+A+M)- HRP を 2次抗体として使 用し染色した。 、ずれのクローンも組換え N- Tes - FLAGと反応することを確認した o また、 検討した 9種のクローンのうち 288- 21D11 は、 N- Tes- 1- FLAGと反応し た。 産業上の利用可能性 N-Tes-FLAG and "N-Tes-de The FLAG was developed with% SDS-PAGE and transferred to a nitrocellulose membrane. Use HRP as secondary antibody Used and stained. In addition, it was confirmed that the shifted clone also reacted with the recombinant N-Tes-FLAG. O Of the nine clones examined, 288-21D11 reacted with N-Tes-1-FLAG. Industrial applicability
MT1- MP及び T3-匪 Pなどの画 Psに対して阻害作用を有する N- Tes は、 癌の浸 潤 ·転移、血管新生などを抑制に有効であると期待されるし、 アミロイド前駆体 分解抑制によるアルツハイマー病の進行抑制にも有効であると期待される。 また その N- Tes に対する抗体は、 診断薬としても期待される。  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.
本発明は、 前述の説明及び難例に特に記載した以外も、 実行できることは明 らかである。上述の教示に鑑みて、 本発明の多くの改¾¾ぴ¾¾が可能であり、 従ってそれらも本件添付の請求の範囲の範囲内のものである。  Obviously, the present invention can be practiced other than as specifically described in the foregoing description and examples. Many modifications of the present invention are possible in light of the above teachings, and so are within the scope of the appended claims.

Claims

請 求 の 範 囲 The scope of the claims
1 . (A) ヒト由来の N - Tes ポリぺプチド若しくは 1. (A) N-Tes polypeptide of human origin or
(B) (i) 該 N - Tes のァミノ酸配列と少なくとも 60%の相同性を有し且つ  (B) (i) having at least 60% homology with the amino acid sequence of the N-Tes;
(i i) (a) Glycosaminoglycan attachment si teを欠いている、  (i i) (a) lacking the Glycosaminoglycan attachment site,
(b) TY domainや CWCV domainを欠いている、  (b) Lack of TY domain or CWCV domain,
(c) C末端が特徴的な配列 Gly- Lys - Argを有している、 及び (c) the C-terminal has a characteristic sequence Gly-Lys-Arg, and
(d) N- Tesのアミノ酸配列のうちの少なくとも 5〜313個の連続したァ ミノ酸残基を有する (d) having at least 5 to 313 consecutive amino acid residues in the amino acid sequence of N-Tes
から成る群から選ばれた榭敫を有しているもの Having 榭 敫 selected from the group consisting of
であることを特徵とするポリぺプチド又はその塩。 Or a salt thereof.
2. 配列表の配列番号： 2で表される了ミノ酸配列のうち、  2. Of the amino acid sequence represented by SEQ ID NO: 2 in the sequence listing,
(i) 少なくとも連続した 5〜70個のァミノ酸残基を有するもの、  (i) having at least 5 to 70 consecutive amino acid residues,
(i i) 少なくとも 続した 71〜150個のァミノ酸残基を有するもの、  (ii) having at least 71 to 150 amino acid residues consecutive,
(i i i) 少なくとも 铳した 1 〜313個のアミノ酸残基を有するもの、  (iii) having at least 1 to 313 amino acid residues,
(iv) 少なくとも第 22番目〜第 122番目のァミノ酸配列を有するもの、  (iv) having at least the 22nd to 122nd amino acid sequence,
(v) 第 22番目〜第 313番目のァミノ酸配列を有するもの、  (v) having the 22nd to 313rd amino acid sequence,
(vi) 第 1番目〜第 313番目のァミノ酸配列を有するもの、 及び  (vi) those having the 1st to 313rd amino acid sequences, and
(vi i) それらのいずれか一つと実質的に同等のアミノ酸配列を有するもの からなる群から選ばれたポリぺプチドであることを特徴とする請求項 1記載のポ リぺプチド又はその塩。  (vii) The polypeptide or the salt thereof according to claim 1, which is a polypeptide selected from the group consisting of those having an amino acid sequence substantially equivalent to any one of them.
3. 請求項 1または 2記載のポリぺプチドの部分べプチドまたはその塩。 3. A partial peptide of the polypeptide according to claim 1 or 2, or a salt thereof.
4. 下記の性質： 4. The following properties:
1) MT-讓 P類の MMP類活性化能を抑制する機能を有し、 且つ  1) It has the function of suppressing the ability of MT-Substitutes P to activate MMPs, and
2) (i) 配列表の配列番号： 2で表されるアミノ酸配列において、 Ala2 2 〜G1 y1 Z 2のアミノ酸配列のうち、 少なくとも 5〜101個の纖したアミノ酸残基を有 するもの、 及び 2) (i) sequence listing SEQ ID NO: in the amino acid sequence represented by 2, the amino acid sequence of Ala 2 2 ~G1 y 1 Z 2 , which have the amino acid residues at least 5 to 101 amino纖, as well as
(i i) N- Tesのァミノ酸酉己列のうちの少なくとも 5〜313個の速繞したァミ ノ酸残基を有するもの (ii) At least 5 to 313 of the amino acids of N-Tes Having no-acid residues
から成る群から選ばれたものである Selected from the group consisting of
ことを | ^とするポリぺプチド又はその塩。 | Or a salt thereof.
5. (i) MT-删 P類が ΜΉ-國 P又は MT3- MPである及び/又は（i i) M P類が MMP-2であることを樹敫とする請求項 4記載のポリぺプチド又はその塩。  5. The polypeptide according to claim 4, wherein (i) the MT-II Ps are ΜΉ-P or MT3-MP and / or (ii) the MPs are MMP-2. Its salt.
6. 請求項 1〜 5のレ、ずれか一記載のポリぺプチドをコ―ドする: 配列 を有することを樹敷とする核酸。  6. A polypeptide encoding the polypeptide according to any one of claims 1 to 5, wherein the nucleic acid has a sequence.
7. 配列表の配列番号： 1で表される塩基配列のうち、 オープンリーディ ングフレーム部分またはそれと実質的に同等な塩基配列を有することを特徴とす る請求項 6記載の核酸。  7. The nucleic acid according to claim 6, wherein the nucleic acid has an open reading frame portion or a nucleotide sequence substantially equivalent thereto among the nucleotide sequence represented by SEQ ID NO: 1 in the sequence listing.
8. 請求項 6または 7記載の核酸を含有することを特徽とするべクタ一。  8. A vector characterized by containing the nucleic acid according to claim 6 or 7.
9. 請求項 6又は 7記載の核酸ある 、は請求項 8記載のベクタ一を含有す ることを とする形質車云換体。  9. A transgenic vehicle comprising the nucleic acid according to claim 6 or 7, which contains the vector according to claim 8.
10. 宿主細胞が大腸菌、 酵母、 293T細胞、 CH0細胞および COS細胞からな る群から選ばれたものであることを特徴とする請求項 9記載の形質転換体。  10. The transformant according to claim 9, wherein the host cell is selected from the group consisting of Escherichia coli, yeast, 293T cells, CH0 cells and COS cells.
11. 請求項 10記載の形質転換体によって発現させて得たものであることを 特徴とする請求項 1〜 5のいずれか一記載のポリぺプチド。  11. The polypeptide according to any one of claims 1 to 5, which is obtained by being expressed by the transformant according to claim 10.
12. 配列表の配列番号： 1で表される塩基配列のうち、 オープンリ一ディ ングフレーム部分又はその一部あるいはそれと実質的に同等な塩基配列を PCR増 幅するに有効なブラィマ一。  12. A primer useful for amplifying the base sequence represented by SEQ ID NO: 1 in the open reading frame part or a part thereof or a base sequence substantially equivalent thereto among the base sequence represented by SEQ ID NO: 1.
13. 次のものに対する抗体：  13. Antibodies to:
(i) 請求項 1記載のポリぺプチドまたはその塩、  (i) the polypeptide of claim 1 or a salt thereof,
(i i) 請求項 2記載のボリぺプチドまたはその塩、  (i i) the polypeptide or a salt thereof according to claim 2,
(i i i) 請求項 3記載の部分ペプチドまたはその塩、  (iii) the partial peptide according to claim 3, or a salt thereof,
(iv) 請求項 4記載のポリペプチドまたはその塩、 あるいは  (iv) the polypeptide of claim 4, or a salt thereof, or
(V) 請求項 5記載のポリぺプチドまたはその塩。 (V) The polypeptide according to claim 5, or a salt thereof.
14. 請求項 1〜 5の 、ずれか一記載のポリぺプチドまたはその塩と特異的 に免疫反応する抗体を測定 として用いることを樹敫とする請求項 1〜 5のい ずれか一記載のポリぺプチドまたはその塩の免疫学的測定方法。 14. The method according to any one of claims 1 to 5, wherein an antibody that specifically immunoreacts with the polypeptide or the salt thereof according to any one of claims 1 to 5 is used as a measurement. An immunoassay for a polypeptide or a salt thereof.
15. 請求項 1〜 5の ヽずれか一記載のポリぺプチドまたはその塩と特異的 に免疫 する抗体を含むことを特徵とする請求項 1〜 5のいずれか一記載のポ リぺプチドまたはその塩の免疫学的彻 j定 «。 15. The polypeptide according to any one of claims 1 to 5, which comprises an antibody that specifically immunizes with the polypeptide according to any one of claims 1 to 5 or a salt thereof. Its salt immunological 彻 j determination «.
16. 請求項 1〜 5の 、ずれか一言己載のポリぺプチド、 その一部のぺプチド またはそれらの塩；あるいは請求項 6または 7記載の核酸；あるいは請求項 13記 載の抗体を含有していることを樹敷とする医薬。  16. The polypeptide according to claims 1 to 5, or a partial peptide thereof or a salt thereof; or the nucleic acid according to claim 6 or 7; or the antibody according to claim 13; Pharmaceuticals that contain as a barrier.
17. (i) MT1-MMP又は MT3-画 P活性阻害剤、  17. (i) MT1-MMP or MT3-fraction P activity inhibitor,
(i i) 癌の浸潤、 転移抑制及びズ又は阻害剤、  (ii) cancer invasion, metastasis suppression and inhibition or inhibitors,
(i i i)血管新生阻  (i i i) Angiogenesis inhibition
(iv) アルツハイマー治療剤、 及び  (iv) Alzheimer's therapeutic agent, and
(v) 関節破壌治麵  (v) Joint fracture repair
から成る群から選ばれたものであることを とする請求項 1 6記載の医薬。 17. The medicament according to claim 16, which is selected from the group consisting of:
18. 請求項 1〜 5の 、ずれか一記載のポリぺプチド、 その一部のぺプチド またはそれらの塩の生物学的活性を促進または阻害する化合物またはその塩を含 有していることを特徵とする医薬。  18. The composition according to claim 1, which contains the compound or a salt thereof which promotes or inhibits the biological activity of the polypeptide according to any one of claims 1 to 5, a part of the peptide or a salt thereof. Specialty pharmaceuticals.
19. 請求項 1〜 5の 、ずれか一記載のポリぺプチド、 その一部のぺプチド またはそれらの塩の生物学的活性を促進または阻害する化^)のスクリ一ニング 方法およびスクリーニングキット。  19. A method and a screening kit for screening the polypeptide according to any one of claims 1 to 5, which promotes or inhibits the biological activity of the polypeptide, a part of the polypeptide or a salt thereof.
20. 固 Psから成る群力、ら週ま'れたものの発現ブラスミ ドと検体遺伝子を宿 田胞に導入し、発現する ΜΜΡ についての検出を指標とすることを特徵とする隱 20. The secretion that specializes in introducing the expression strength of a group consisting of solid Ps, the expression plasmid and the sample gene of the one that has been released into Sukuda, and using the detection of the expressed と す る as an index.
Psから成る群から選ばれたものの活性調節に関与する遺伝子のスクリ—ニング方 法およびスクリ一ニングキット。 A screening method and a screening kit for a gene selected from the group consisting of Ps and involved in the activity control.
21. MMP-2、 MT1-MMP発現プラスミ ドと検体プラスミ ドを 293T細胞などの 宿主钿胞に導入し、 例えば潜在型、 中間体、 活性化議 P- 2を検出にかけること を とする請求項 2 0記載のスクリーニング方法およびスクリーニングキット o ·  21. A request to introduce MMP-2, MT1-MMP expression plasmid and specimen plasmid into host cells such as 293T cells, and to detect, for example, latent form, intermediate, and activation P-2 Item 20. The screening method and screening kit according to Item o.
22. 下記の指標：  22. The following indicators:
(a) N-Tesの発現量が正常部位に比べ低いこと、  (a) the expression level of N-Tes is lower than that in a normal site,
(b) N-Tes ポリペプチド又はそれから誘導されたペプチド断片、 (c) N-Tes遺伝子又はそれから誘導された mRNAなどの核酸、 及び (b) an N-Tes polypeptide or a peptide fragment derived therefrom, (c) N-Tes gene or nucleic acid such as mRNA derived therefrom, and
(d)御 - Tes抗体  (d) your-Tes antibody
から成る群から週ま'れたものを指標として使用することを糊敫とするグリォ一マ の検出方法。 A method for detecting gloma using glue from the group consisting of:
PCT/JP2001/011529 2000-12-27 2001-12-27 Novel peptides and activities thereof WO2002057448A1 (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
JP2002558500A JPWO2002057448A1 (en) 2000-12-27 2001-12-27 Novel peptide and its activity

Applications Claiming Priority (2)

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

Publications (1)

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

Family

ID=18863704

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP2001/011529 WO2002057448A1 (en) 2000-12-27 2001-12-27 Novel peptides and activities thereof

Country Status (2)

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

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998046757A2 (en) * 1997-04-15 1998-10-22 Genetics Institute, Inc. Secreted proteins and polynucleotides encoding them
WO1999023110A1 (en) * 1997-10-31 1999-05-14 Eli Lilly And Company Hptlg gene from human hypothalamus
WO1999046281A2 (en) * 1998-03-10 1999-09-16 Genentech, Inc. Novel polypeptides and nucleic acids encoding the same
WO2000032221A2 (en) * 1998-12-01 2000-06-08 Genentech, Inc. Promotion or inhibition of angiogenesis and cardiovascularization
WO2000053756A2 (en) * 1999-03-08 2000-09-14 Genentech, Inc. Secreted and transmembrane polypeptides and nucleic acids encoding the same

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998046757A2 (en) * 1997-04-15 1998-10-22 Genetics Institute, Inc. Secreted proteins and polynucleotides encoding them
WO1999023110A1 (en) * 1997-10-31 1999-05-14 Eli Lilly And Company Hptlg gene from human hypothalamus
WO1999046281A2 (en) * 1998-03-10 1999-09-16 Genentech, Inc. Novel polypeptides and nucleic acids encoding the same
WO2000032221A2 (en) * 1998-12-01 2000-06-08 Genentech, Inc. Promotion or inhibition of angiogenesis and cardiovascularization
WO2000053756A2 (en) * 1999-03-08 2000-09-14 Genentech, Inc. Secreted and transmembrane polypeptides and nucleic acids encoding the same

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 (en) 2004-05-20

Similar Documents

Publication Publication Date Title
JP3837494B2 (en) Soluble RAGE protein
US7358061B2 (en) Methods of diagnosing thyroid carcinoma
JP2008131944A (en) Family of immunomodulators designated leukocyte immunoglobulin-like receptors (lir)
CN102712687A (en) MASP isoforms as inhibitors of complement activation
JP2002543787A (en) A family of immunomodulators termed leukocyte immunoglobulin-like receptors (LIRs)
JPH10210982A (en) New protein
JP4295516B2 (en) Gastrokine and its derived peptides including inhibitors
JP3800175B2 (en) Novel aggrecanase
JP4841054B2 (en) Novel insulin / IGF / relaxin family polypeptide and DNA thereof
CA2343720A1 (en) Novel antibodies, drugs containing these antibodies and methods for screening compounds by using these antibodies
JP2003189874A (en) Agent for regulating galectin-9 activity
JP2000050885A (en) Antibody against cathepsin and its utilization
JPH05501951A (en) Recombinant human thyroid peroxidase
JP4171228B2 (en) Soluble type RAGE measurement method
JP2001231578A (en) Protein belonging to il-1 family
WO2007037245A1 (en) Polypeptide having anti-angiogenic activity
WO2002057448A1 (en) Novel peptides and activities thereof
JP2000270874A (en) New membrane-bound metalloprotease
JP2003321494A (en) Method for adjusting activation of prommp-7
US6800473B1 (en) Human cathepsin L2 protein, gene encoding said protein and use thereof
WO2004048565A9 (en) Apoptosis-associated protein and use thereof
JP2003000249A (en) ACTIVATION OF proMMP-2 WITH CLAUDINS THROUGH MT-MMPs
JP2006180813A (en) Aminopeptidase o and its use
WO2004040302A1 (en) REGULATION OF INTERACTION BETWEEN RAPL AND Rap1
JP2004329040A (en) Polycerase i and its utilization

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