+

WO1999031238A1 - Nouvelle proteine - Google Patents

Nouvelle proteine Download PDF

Info

Publication number
WO1999031238A1
WO1999031238A1 PCT/JP1998/005612 JP9805612W WO9931238A1 WO 1999031238 A1 WO1999031238 A1 WO 1999031238A1 JP 9805612 W JP9805612 W JP 9805612W WO 9931238 A1 WO9931238 A1 WO 9931238A1
Authority
WO
WIPO (PCT)
Prior art keywords
protein
kdr
dna
cdna
domain
Prior art date
Application number
PCT/JP1998/005612
Other languages
English (en)
Japanese (ja)
Inventor
Masabumi Shibuya
Naoyuki Yabana
Original Assignee
Kyowa Hakko Kogyo 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 Kyowa Hakko Kogyo Co., Ltd. filed Critical Kyowa Hakko Kogyo Co., Ltd.
Priority to AU15065/99A priority Critical patent/AU1506599A/en
Publication of WO1999031238A1 publication Critical patent/WO1999031238A1/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/71—Receptors; Cell surface antigens; Cell surface determinants for growth factors; for growth regulators

Definitions

  • the present invention relates to a protein that induces KDR in the vascular endothelial thread fflSS3 ⁇ 43 ⁇ 4a ⁇ (VEGF) receptor-KDR (kinase insert domain-containing receptor; hereinafter, referred to as KDR).
  • VEGF vascular endothelial thread fflSS3 ⁇ 43 ⁇ 4a ⁇
  • KDR kinase insert domain-containing receptor
  • VPF Vascular permeability fector
  • VEGF Vascular endothelial growth factor
  • OVLShibuya Advances in Cancer Research, 161, 281h 995
  • VPF / VEGF is a protein consisting of homodimers and having a molecular weight of about 40,000.
  • VEGF is known to be a high-level, high-sensitivity probe for tube sex [14. N. Ferrara et al .; Biochem.
  • Blocking VEGF can also prevent water and water.
  • Rt-l farnesoiase insert domain-containing receptor
  • mice Homologs of the mouse VEGF receptor KDR in mice are Plk-1 CWMatthews et al .; Proc. Natl. Acad. ScL USA, S8, 9026 991), A-Ullich et al .; W094 11499 Priolity Nov. 13 (1992), BJMillauer 12, 835 (1993)].
  • the fflW domain consists of seven imglobulin domains, and the domain within the thread has a tyrosinki ⁇ " zedomin.
  • a quantity of 180-200-kilodaltons ( ⁇ volume ⁇ ⁇ belongs to the tyrosinkinase family! VEGF specifically binds to nt-1 and KDR / Flk-1 at KD ifi ⁇ 20pM and 75pM, respectively Flt-1 and KDR / Flk-1 ⁇ ⁇ specifically expressed on vascular endothelial cells Natl. Acad. Sd. USA Q, 7533 (1993), RLKendaU et al., Proc. Natl. Acad. ScL USA 2Q, 8915 (1993)].
  • KDR When KDR is expressed in the blood vessels of the porcine artery, it becomes VEGF and proliferates and proliferates in the vein of the porcine artery.
  • lk tube ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ J J J J J J J J J J J J J J J J J J J J J J J J J J J J J J J J J J J J J J J J J J J J J J J J J J J J J J J J J J J J J J J J J J J J J J J J J J J J J J J J J J J J J J J J J J J J J J J J J J J J J J J J J J J J J J J J J J J J J J J J J J J J J J J J J
  • F ⁇ fSt of this tyrosine chitose-type receptor pig is ligated with its ligand; It is thought that the tyrosine is released by re-entering the tyrosine, and this protein is initiated by the receptor self-redirecting TVr through another protein via the SH2 domain. It is thought that ff ⁇ c fSS ⁇ is performed by inducing a dripping.
  • the age of KDR also causes the TVr 3 ⁇ 4S to be subjected to GF ⁇ by VEGF in vascular endosperm [J Waltenberger et al., J. Biol.
  • the SH2 domain is also white, and PLC-, GAP (ras G Pase activating protein), PI3-kinase vphosphatidylinositol 3-kinase), and Nck are affected by the intravascular intravenous play, which has been diluted with VEGF. (D. Guo et al., J. Biol. Chem., 22Q, 6729 (1995)]. However, it has been reported as a domain within the KDR thread. Not done.
  • ⁇ induces ⁇ ff «3 ⁇ 4 of KDR by ⁇ to VEGF receptor KDR, and causes abnormalities such as HffM ⁇ im, fibril formation, and ⁇ ⁇ transportation, diabetes mellitus i4WM, m, dryness, etc. in old rheumatism.
  • abnormalities such as HffM ⁇ im, fibril formation, and ⁇ ⁇ transportation, diabetes mellitus i4WM, m, dryness, etc. in old rheumatism.
  • the inventors of the present invention have been eager to elaborate and, as a result, have isolated the cDNA encoding the protein that induces (7 ⁇ ⁇ ) as the domain within the KDR thread from the ⁇ cDNA library. , The Rooster E ⁇ ij decision and led to the present invention
  • a fflM receptor (VEGF) receptor consisting of one or several amino ⁇ deletions or added amino acids in the amino acid sequence of the protein (a)
  • KDR KDR insert domain-containing receptor
  • amino deletions or additions can be identified by the method of the specialty experiment, which is a preoperative technique, and one or several amino acids can be defined as the specialty modification.
  • the amino acid that can be deleted or added by the method is is ( ⁇ .
  • Proteins consisting of amino acids J with one or several amino acids deleted or added, and having the properties of the KDL domain of KDR, are described in J. Sambrook et al .; Molecular Cloning, A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1989), FMFrederick et al; Current Protocols in Molecular Biology, Supplement 1-38, John Wiley & Sons (1987-1997), MJ.ZoUer and M.Snith; Nucleic Acids Research, IQ , 6487 (1982),
  • the protein of the present invention may be referred to as a KDR resator " ⁇ protein.
  • the second (7) description of the present invention is a DNA encoding the above-mentioned protein, a DNA having a rooster seed No. 1, 2 and 27 A protein that hybridizes under the stringent cow with DNA having ⁇ Sfi ⁇ j read at the seeding number of 2 and 27, and induces KDR as a domain within the thread of VEGF receptor KDR It is a DNA that is a DNA character.
  • the DNA obtained by using a filter is prepared by using a filter immobilized with plaque-derived DNA, using a filter with immobilized DNA, 0.7 ⁇ ; under L0M Nad, 65 ° C 0.1 X to 2 X SSC (saline-sodium citrate) [1 SSC descendant night (150 mM NaCl, 15 mM que ⁇ "trim); n X is n-density n times
  • SSC saline-sodium citrate
  • the DNA of Tori Ban No. 1, 2 and 27 can be selected from the following: Can include DNA having a homolog of 95% Rii.
  • a third aspect of the present invention is an Itoh vector containing the above DNA layer.
  • the fifth invention of the present invention relates to a method for producing a protein, comprising feeding the above-mentioned S-form in a culture medium, allowing the above-mentioned protein to be added during a period of difficulty, and listening to the protein from the outside. ! T3 ⁇ 43 ⁇ 4
  • the sixth (7) description of the present invention relates to the DNA
  • the oligonucleotide having the sequence-as the oligonucleotide for example, Two and twenty-seven strengths, an oligonucleotide having the same or different as the male or female: Can be
  • a seventh aspect of the present invention is an anti-C that recognizes the above protein.
  • the eighth (7) description of the present invention is the above-mentioned [white matter ⁇ ⁇ extraction method, in which a body is used.
  • the ninth aspect of the present invention relates to the above-mentioned ligoneucle; ⁇ or iiH3 ⁇ 4t body as an extreme component, abnormal blood ⁇
  • the 10th ⁇ i above of the present invention is a disease that is caused by abnormal vascular growth or a disease caused by abnormal ff $ giSt of KDR, using a lignonucleotide or ⁇ a a body ⁇ as a mechanical component. is there.
  • a method for screening the quality of KDR which comprises using the DNA of the eleventh (Mi) of the protein of the present invention.
  • the present invention will be described in detail below.
  • the cDNA of the protein of the present invention can be prepared by a twchhy rid method.
  • Examples of the one-hybrid method include, for example, the following: the one-brid method [S. Fields et al .; Nature, Q245 (1989)].
  • the Teng-to-Brid method is a method for producing a yeast protein in which a DNA domain and a transcription domain are separated, for example, a protein capable of forming a complex with a target protein using GAL4 and tH:
  • a vector capable of expressing a protein with two domains of GAL4, that is, protein X (in the present invention, the KDR domain) is used as a DNA DNA ⁇ domain (GAL domain) and a protein.
  • a vector to be expressed by ⁇ and a protein ′ (the protein S encoded by each cDNA in the cDNA library according to the present invention) and a ffi ⁇ protein (GAL4 S14f human domain)
  • the yeast clones that interact with protein X and protein X to form multiple copies are transcribed.
  • the dangling domain takes on the form of a DNA promoter, which is transcribed, and is expressed as a reporter. Then, using the reporter HI expression as a key, the cDNA ability of the protein that interacts with the domain within the thread; screening the contained form and extracting the plasmid from the transformed form to obtain the desired cDNA Clone itf.
  • a KDR cDNA clone can be obtained from a cDNA library by performing the following steps:
  • RNA from the KDR-expressing DNA or human, for example, human moon is shown.
  • Methods for steaming all EA include the thiosian guanidine-trifluorene g ⁇ t shim method [H. Okayama et al., Methods in En2ymology J, 397], the guanidine dithiocyanate phenol. Clonal form (AGPC) method [P. Chomczynski and N. Sacchi; Analytical Biochemista, J £ 2, 156 (1987)]. Use this total RNA
  • ⁇ mENA can be transferred after using a kit such as Fast Track mRNA Isolation Kit [Invitrogen Co., Ltd., Quick Prep mENA Purification Kit [Pharmacia Co., Ltd. Liu].
  • This mENA is converted into cDNA, and a suitable vector is introduced into Sukutadashi to produce a cDNA library.
  • Encouraging cDNA Libraries ⁇ See J. Sambrook et al .; Molecular Cloning, A
  • a phage vector, a plasmid vector or the like can be used as long as it can be used in the MK12 strain.
  • ZAP Express [Stratagene II, Strategies, 5, 58 (1992)]
  • pBluescriptn SK (+) [NucleicAcids Research, 12, 9494 (1989)]
  • Lambda ZAP II (Stratagene Bye, gtlO, gtll [DNA Cloning, APracticalApproach, 1, 49 (1985)]
  • XTViplEx clone-tech crane, XExCeU (Fano! ⁇ Shea recitation, pT7T318U (Fano-shear crane, pcD2
  • Ruko ⁇ a can: Specifically, Escherichia mli XLl-Blue certificate F [Stratagene II, Strategies, 5, 81 (1992)], Escherichia coH CeOO [R Appleyard; Genetics, 22, 440 (1954)],
  • Sacoharomvffis visiae ability and suitable trait has a marker, for example, TRP1, LEU2, etc., and the promoter U expressed at» is a promoter of the bulcono hydrogenase (ADH) promoter, such as GAL. It is a vector that can express a DNA domain. For insertion of this ⁇ , KDR cDNA, there is an appropriate control site in the portion corresponding to the C-terminal side of the DNA binding domain, and it can also be formed with w ⁇ , -m bacteria such as fflM of vector DNA. ⁇ I 1 or the like; ⁇ traits Ma in ⁇ - is desired arbitrary one with the force scratch.
  • ADH bulcono hydrogenase
  • vectors examples include pGBT9 (Clontech ⁇ ), pASl [T. Durfee et al., Genes & Development,?, 555/993], pAS2-l (Clontech ⁇ ), and the like.
  • the domain fragment in the thread of the KDR cDNA obtained in (1-1) was purified to obtain the DNA binding domain. Insert the protein into the control site at the end so that the protein hood fits.
  • a fragment of the KDR cDNA thin domain can be cut out using an appropriate control such as Asp 8C) 6 (77i at the EamHI site based on i, or a primer from the Rooster IJ that only ⁇ ⁇ ⁇ 3 ⁇ 4 ⁇ ⁇ in the intracellular domain.
  • PCR [MJMcPherson et al .; PCR, A practical Approach, Oxford University Press (1991)] to fight “f” (2) ⁇
  • a protein library for inverting D4f human domains and a cDNA library The yeast can be made from yeast Saccha drawing vcfis occidental visiae and has a suitable marker H »5 ⁇ , such as RP1, LEU2, etc., and a promoter expressed in gfS, such as ADH promoter Yanagi, GAL4, etc. It is a vector that can express the ⁇ 14 ⁇ domain. There is a suitable site on the C-terminal side of the ⁇ , inverted ⁇ 3 ⁇ 4ttf domain, and ⁇ ⁇ ⁇ and large ⁇ bacteria, such as; ⁇ , in vector DNA, and fungi such as ampicillin ftH4 » In ⁇ what has the marker power s desired ,.
  • Such vectors include pGAD [CT, Chien et al .; Proc NatLAca ScL USASa SSTS iiggiW, pGAD424 (Clontech tt) and pACT [T. Durfee et al .; Genes & Development, 2, 555 (1993)], pACT2-l ( Black
  • KDR The protein interacting with the Dm domain is considered to be expressed in the same fflSM position as KDR. Therefore, (1-1) and! Transfer cDNA from ⁇ and insert it into the control site at the C-terminus of the! ⁇ Protein fragment conversion domain described above to save the cDNA library. Due to the orientation of the domain and the fume, the # ⁇ represents the age protein between the transgenic domain and the protein encoded by the cDNA. Zhao's library that can be used on shelves, for example, the MATCHMAKER cDNA library (Clontech) can also be used.
  • HIS3 was used as a reporter " ⁇ ", and was used as a reporter for lacZ as a reporter, when it was fed on a minimal medium without His, and lacZ was used as a reporter. Look at all the colonies.
  • the colony contains two kinds of plasmids, KDR ( ⁇ plasmid intracellular domain protein expression plasmid and protein-discovered cDNA plasmid).
  • KDR ⁇ plasmid intracellular domain protein expression plasmid and protein-discovered cDNA plasmid.
  • the method is as follows. Then, the cDNA clone is obtained by ⁇ R of the transformant based on the marker of the protein 3 ⁇ 4 cDNA plasmid, and the plasmid DNA is obtained from the obtained 3 ⁇ fungus [D. Rickwood and BDHames; DNA Cloning 2, Expression Systems, A Practical Approach, Second Edition, Oxford University Press 995), C.-T. Chien et al .; Proc. Natl. Acad. ScL, S8, 9578 999)]).
  • KDR yarn! Use the DNA domain of the inner domain for expression of the age protein, and in the vector, the domain of the KDR thread MS When both cDNA clones are introduced into the inn, It is possible to bear that the protein encoded by the obtained cDNA specifically binds to the intracellular domain of KDR.
  • the obtained cDNA clone is left as is, and the cDNA portion is cut out with an appropriate IW3 ⁇ 4 and pUCU8 etc. (3 ⁇ 41 After subcloning into a suitable cloning vector, the salt is used for normal SIE ⁇ Zhe method, for example, Sanger Natl Acad. Sd. ⁇ JS. 24, 5463 (1977)] Alternatively, the DNA sequence is determined by using a DNA sequence 1 ⁇ from Perkin Elmer or Fanare Masia.
  • cDNA ⁇ is a new rooster
  • a homology ⁇ program such as WLAST, BLAST, etc.
  • a database such as GenBank, EMBL and DDBJ
  • the obvious homology, which is considered to be [?] In the source, can be obtained by the ⁇ f ⁇ S system.
  • Examples of the cDNA encoding the novel protein serving as an intracellular domain of KDR obtained as in m include a cDNA clone having a rooster 5 or 6.
  • KDR receptor protein DNA DNA encoding the protein that functions as the intracellular domain of KDR.
  • the cDNA of the protein of the present invention obtained in (3), and the mEA plasmid obtained in Northern blotting (4), The ⁇ which is not expected to be obtained can be used to obtain the cDNA of the protein of the present invention by the following method.
  • the novel amino translation of the protein of the present invention can be confirmed by examining amino translation databases such as GenPept, PIR and Swiss-Prot using a homology search program such as FASTA or Framesearch.
  • a homology search program such as FASTA or Framesearch.
  • a cDNA clone having a suitable roto-ban ffi «EffiE ⁇ from rooster seed Nos. 1, 2 and 27 can be mentioned.
  • the cDNA of the present invention is considered to be a protein, and the cDNA is substituted in the same manner as in (1-1), and adapters are added to both ends of the cDNA. Salts of the cDNA clones obtained in (3) PCR Perform PCR with primers in the row P. 5'-RACE and 3'-RACE
  • a primer based on fflE ⁇ of the cDNA was used to express the protein of the present invention.
  • ⁇ is considered to be the same as (1-1) and the cDNA converted to [ ⁇ ] is to be subjected to PCR [MJMcPherson et al .; PCR ⁇ ApracticalApproac Oxford University Press (1991)] as a cDNA library.
  • PCR MJMcPherson et al .; PCR ⁇ ApracticalApproac Oxford University Press (1991)] as a cDNA library.
  • DNA encoding the target protein of the present invention can be linked.
  • the DNA encoding the target protein of the present invention can be obtained by chemically synthesizing the DNA with DNA.
  • a DNA strand is used to synthesize an oligonucleotide such as an oligonucleotide and a sense oligonucleotide having a part of the rooster IJ of the DNA of the present invention using a DNA fiber.
  • an oligonucleotide such as an oligonucleotide and a sense oligonucleotide having a part of the rooster IJ of the DNA of the present invention using a DNA fiber.
  • oligonucleotide examples include a DNA having the same rooster as that of the fibrous 10 to 50 in the DNA contained in the DNA or a DNA having a rooster with the DNA. No. 1, 2 and 27
  • the rooster selected by the ⁇ ⁇ 1 1 if fi fi fi fi DNA 1 ⁇ ⁇ ⁇ DNA ⁇ ⁇ ⁇ ⁇ ⁇ DNA ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA
  • the oligonucleotide is also an oligonucleotide of the present invention.
  • oligonucleotides can also be mentioned as the oligonucleotide of the present invention.
  • Words ⁇ Conductive DNA Derivatives in which diesteno phosphate in DNA is converted to phosphoric acid carbonate ⁇ DNA, Diesteno phosphate in DNA is converted to N 3 '-P 5' phosphoamidate Derivative DNA, Derivative DNA in which ribose and riiesteno in DNA are converted to peptides, Derivative DNA in which peracyl in DNA is fiberized with C-5 propynyl peracyl, Peracyl in DNA is C_5 thiazolyl peracyl Fiber-derivative DNA, derivative DNA in which cytosine in DNA is replaced with C-5 propynylcytosine, derivative DNA in which cytosine in DNA is replaced with phenoxazine-modified cytosine, ribose in DNA is 2 '— O-propylribose-derived DNA, or 2'-me
  • Any lodging can be used as long as it can express the target ⁇ ⁇ , such as m.
  • an expression vector that can be used in the above-mentioned inns, or that can be used in the body, and that has a promoter at a position where the DNA of the present invention can be transcribed is used.
  • ⁇ is a protein expression vector according to the present invention.
  • Source is capable of self-organization in a nucleus, as well as a promoter and a ribosome; i ⁇ fE a DNA of the present invention; It is preferable that the vector is composed of wm & xiao ⁇ vector:
  • the promoter is ⁇ ⁇ - ⁇ .
  • expression vectors include ⁇ 233-2 (Pharmacia ft), pSE280 (Invitrogen ⁇ , pGEMEX-l [Promega tt], pQE-8 (QIAGEN) tt, pKYPIO
  • the promoter may be any promoter that can be expressed in a host such as a fungus.
  • a promoter one P ⁇ rp
  • lac_ promoter P L promoter one
  • P R promoter one coater P S E promoter, such as T7 promoter
  • promoter one terpolymer derived from ⁇ Ya fur ⁇ , SPO l promoter , SP02 promoter pen P promoter and the like.
  • artificially modified promoters such as a promoter (P ⁇ rp XS), a tail promoter, a lacT7 promoter, an let promoter, and the like, which are obtained by combining two promoters, can also be used.
  • the ribosome, ⁇ g ij may be any as long as it can be expressed in the spores of the fungus, etc., but it is necessary to divide the Shine-Dalgarno rooster E ⁇ J and the initiation codon.
  • a suitable ⁇ for example, 6 to 18 fibers of plasmid; preferred).
  • transcription is not necessarily required for expression of the DNA of the present invention, and transcription sia is distributed to sr of « ⁇ 3 ⁇ 4. Les ,.
  • the original organisms include those belonging to the genus Escherichia, Serratia, Corynebacterium, Brevipacterium, Pseudomonas, and Batinoles, such as Escherichia mliXLl-Blu Escherichia coli XL2-B1UR. Escherichia coli DHL Escherichia MC Escherichia coli KY3276, Escherichia coli W1485, Esdierichia coli JM109, Escherichia coli HBim. Escherichia ooli No.49. Escherichia coli W3110. Escherichia coli NY49, Bacillus sbilis.
  • ATCC13870 Micmhac ⁇ rium ammoniaphilum ATCC1 and the like can be mentioned.
  • DNA can be introduced into the above-mentioned host cells: Method using calcium ion [S. Cohen et al .; Proc. Natl. Acad ScL US ⁇ £ 2, 2110 (1972)], arotoplast method (63-2483942), electrotrophic method, or Gene, II, 107 (1982 and Molecular & General Genetics, J £ S, Ulhi 979 ) And the like.
  • the promoter may be any one that can be expressed in a bacterial strain, or may be one that is out of alignment.
  • a PH05 promoter, a PGK promoter, a GAP promoter, an ADH promoter, a gal promoter, or a gal promoter may be used.
  • promoters such as heat shock polypeptide promoter, MFal promoter, and CUP1 promoter.
  • S ⁇ S strains include Saocharomyces cerevisae. Schizasaccharomyces pomhe,
  • Klyveromyces lactis Trichosporon ullulans. Sdiwanniomyces aUuvius. I can do it.
  • the method As a method for introducing a delicate vector, if the method is to introduce DNA into the accessory, it can be used to adjust the displacement.
  • the electroporation I ⁇ method (DJVl. Beckerand L. Guarente; Methods. Enzymol. , 124, 182 (1990)]
  • the spheroplast method [D. Shortle et al., Proc. NatLAcai ScL USA SI. 4889 (1984)]
  • the lithium method H. Ito et al., Journal of Bacteriology 153, 163 (1983)]. Natl. Acad. Sci. US 15, 1929 (1978).
  • a spore as a host, as an expression vector, for example, pAGE107 [JP-A-3-22979; H. Miyaji et al., Cytotechnology, 3, 133, (1990)], pAS3-3 (Paragraph 2- 227075), pCDM8 [B. Seed; Nature, 229, 840, (1987)], pcDNAI / Amp (Invitrogenenne, pREP4 (Invitrogen ⁇ , pAGE103 [T Mizukami et al .; Journal of Biochemistry, 101, 1307 (1987) )] And so on.
  • any of the promoters that can be expressed in ft «shelves can be used.
  • the promoter of the cytomegalovirus (CMV) IE immediate early
  • ⁇ ⁇ the promoter of the cytomegalovirus
  • the SV40 promoter One example is a metallotionin motor, a retrovirus promoter, a heat shock promoter, an SR promoter, and the like.
  • the vesicles are human ⁇ , Luba (Namalwa), and monkey thread MS, COS. «, CHO thread of Chinese hamster! ⁇ , HBT5637 (Showa 63-299).
  • a method for introducing DNA into Soitoshima can be used.
  • the 7-rhd method (Parahei 2-227075), the lipofection method [P.LFelgner et al., Proc. Natl. Acad. Sci. USA S4, 7413h 987)]
  • ⁇ includes, for example, DR O'Reilly et al .; Baculovirus Expression Vectors, A Laboratory Manual, WH Freeman and Company, New York (1992), Current Protocols in Molecular Biology, Supplement 1-38, (1987- 1997), VALuckow and
  • Proteins can be expressed by the method described in M.D. Summers; Bio Ibchnology S, 47 (1988).
  • transfection vector used in the method for example, pVL1392, pVL1393, pBlueBacHI (in vitro gene) and the like can be mentioned.
  • Autographa californica nuclear polyhedrosis virus which is an insect virus, such as Autographa californica nuclei polyhedrosis virus, can be used.
  • the protein of the present invention can be obtained by culturing the mia body obtained in the manner described in m: above in a culture medium, accumulating the protein of the invention in the culture medium, and recovering the medium.
  • the method of culturing the present invention in a culture medium can be performed according to a usual method used for culturing a host.
  • a culture medium for culturing a form obtained by using a prokaryote such as ⁇ iigfW, which is a difficult organism, as a host is a culture medium that contains charcoal, «3 ⁇ 4,» ⁇ , etc. It is a medium that can be used for any purpose.
  • the charcoal 3 ⁇ 43 ⁇ 4f may be any as long as the substance can be assimilated, such as gnorecose, fructose, sucrose, molasses, starch or starch ⁇ ] ⁇ ⁇ ⁇ ⁇ Wt ⁇ ), ⁇ , propio: / ⁇ , Ethanol, propanono anorecono, etc. can be used.
  • ⁇ such as ammonia, ammonium chloride, ammonium, ammonium, l> m ammonium, etc. or ammonia, soya (7 ⁇ 3 ⁇ 43 ⁇ 4 ⁇ ⁇ ), along with peptone, meat extract, alongside extract, Corn steep liquor, casey ⁇ 3 ⁇ 41 ⁇ advice, lees and soybeans ⁇ ⁇ sou, each cell, and other types of bacteria can be used.
  • Examples thereof include lithium potassium, potassium dipotassium, potassium bamboosium, » ⁇ gnesium, sodium chloride, sulfuric acid ⁇ , manganese sulfate, W, calcium carbonate, and the like.
  • Culture is carried out under normal ⁇ ⁇ culture or under 3 ⁇ 4 »3 ⁇ 4 ⁇ # ⁇ * ⁇ .
  • the cultivation is preferably 15-40 ° C, and the cultivation 0 ⁇ is usually 16-96 B # ⁇ .
  • During fermentation pH should be 3.0 ⁇ 9.0.
  • the reaction is carried out using acid or alkaline, urea, calcium, ammonia, etc.
  • a substance such as ampicillin tracycline may be added to the culture medium during culture.
  • the indu When culturing the transformant using an inducible ferromotor or an expression vector, the indu may be added to the culture medium as necessary: for example, ia ⁇ ⁇ When culturing a product formed using an expression vector using a promoter, cultivate a product prepared using an expression vector using a promoter such as isobromopropyl- ⁇ -D-thiogalactopyranoside. In this case, the medium may be indolacrino.
  • the culture medium for culturing the mi ⁇ body obtained using the spores as a host is reduced to "" ⁇ .
  • RPMI1640 medium [The Journal of the American Medical Association, I22, 519 (1967)], Ea ⁇ e MEM medium [Science, 122, 501 (1952)], DMEM medium [Virology, S, 396 (1959)], 199 Culture medium [Prooeeding of the Society for Biological Medicine, 23, 1950]] or a medium in which bovine wombs are stimulated can be used for these mediums.
  • Culture is usually at pH 6-8, 30-40. C, 5% C0 2 ⁇ such as ⁇ (under cow:! Intends to 7 days.
  • a medium such as lysine or syrin may be added to the medium during the culture.
  • a TNM-FH medium called “Pharmingen”, a so-called Sf-900II SFM medium (Life Technology 3 ⁇ 4f3 ⁇ 4) And ExCell400, ExCeU405 [both JRH Bay: “Yen ⁇ ” (JRH Biosciences)) B, Grace's Insect Medium [Nature, 125, 788 (1962)] and the like can be used.
  • a substance such as gentamicin may be added to the medium during the culture.
  • the protein of the present invention in order for the protein of the present invention to be expressed in a transgenic form in a filament, after completion of the culture, as was recovered from a distance and the aqueous system JS-screened, sonication, French press, Menton gaurine homogenase ⁇ ⁇ ", Thread 3 ⁇ 4 is crushed by Dynomino to obtain a thread-free liquid. Salting out method using ammonium sulfate, etc., 3 ⁇ 4 ⁇ method using a shelf, acetylethyl (DEAE)-Sepharose, DIAIONHPA-75 ( ⁇ it ⁇ ) using resin, etc.!
  • ⁇ T-on chromatographic method S -Sepharose FF ( ⁇ T-on chromatographic method using a resin such as Fano ⁇ Chine, ⁇ ⁇ -chromatographic method using a resin such as Butinole Faros and Phenylsepharose, a gel using a sieve Filtration method, affinity mouth chromatography One method, chromatography Focusing method, etc.
  • ⁇ Swim in the swimming island Kr & ⁇ can be used to obtain insects.
  • 3 ⁇ 4 ⁇ which was expressed as a result of expression of the protein in Itoshima, was obtained by recovering the protein by a conventional method from the amount obtained by crushing and recovering the ⁇ and performing distant ⁇ The body of the protein is soluble in the protein.
  • the word ⁇ ! Melts into a sickle that does not contain protein difficulties or ugliness or that does not change the protein, but the protein does not change, or the protein is composed into a normal standing iW After this, ⁇ 12 and! I ⁇ t® to remove more! Goods can be obtained.
  • the protein in the culture supernatant can recover the derivative of the protein with a weight of 0%.
  • an article can be obtained by using the above and [ ⁇ ] from the word 141 minutes. .
  • the protein of the present invention can also be obtained by the Fmoc method (fluoreninolemethyloxycarbonyl method), the tBoc method (t-butyloxycarboninole method), etc. Also, Advanced ChemTfech, Nokinkin Eno! ⁇ 1, Fanocia, Protein Technology Instrument (Synthecell-Vega), and Protein Tfechnology Instrument. The company, PerSeptive, can also combine and combine peptides from the island's abdominal crop ff ⁇ .
  • the antigen is administered 3 to 10 times every 1 to 2 weeks after the first administration. 3 ⁇ 7 days after each administration, it is better to recommend it from ⁇ » ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ 1 1 1 ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ 1976 ⁇ E. Harlow and D. Lane: Antibodies-A W 99/31
  • the serum used in rabbits, goats, mice, rats, or hamsters showed sufficient antibody titers against the serum used for destruction.
  • Purification can be carried out using a conventional method of chromatography using ammonium salt, cabril removal, DEAE-Sepharose column, protein column or gel filtration column.
  • a mouse or rat As a medullary movement, a mouse or rat is used.
  • 8-azaguanine ⁇ us derived from BALB / c
  • 3 ⁇ 4S B »P3-X63Ag8-Ul hereinafter abbreviated as P 3—U 1 [Curr.TbpicsMiciObiol.Immunol., Ai, 1 (1978)], [Europ. J. Immunol., 511 976)], SP2 / 0-Agl4 (SP-2) [Nature, 226, 269 978)], P3-X63-Ag8653 (653) [J.
  • Nopridoma strain that contains a monoclonal antibody which is a protein of the present invention.
  • a monoclonal antibody against the protein of the present invention which was obtained in (2-3), was conjugated to a monoclonal antibody.
  • Tesshi the spoon was Mausuka Agata, 3, 0 0 0 r P a solid content of 5 minutes to at m [ ⁇ to.
  • the obtained supernatant is subjected to salting out by 40 ⁇ 50% « ⁇ Chromatography using DAE-Sepharose column, Protein A-column, or column chromatography using Cell mouth Fine GSL2000 (Biochemical Chemistry Earth). I do.
  • the subclass of the antibody is determined using a mouse monoclonal antibody typing kit or a rat monoclonal antibody typing kit.
  • the protein content is calculated by the absorbance at 280 nm.
  • the DNA of the present invention can be obtained by performing Northern blot hybridization on RNA extracted as [1 ⁇ (1-1) and ( ⁇ ) from human-derived yarn using this as a probe.
  • the level of detection of the mEA of the protein of the present invention can be determined; frT can be determined. Can be.
  • the oligonucleotide of the present invention is used as a specific primer of the DNA of the present invention to reduce RNA extracted from human ([1] (1-1) and I ⁇ from human).
  • the mRNA of the present invention can also be detected by RT-PCR using the same DNA as that of the present invention; By using this as a probe and performing in situ hybridization on human fragments, it is possible to know a finer distribution of the oligonucleotide of the present invention, such as identification of the protein of the present invention. Can be.
  • the oligonucleotide (RAZDNA) of the present invention can be used by tranferring the transcription or mRNA of the protein of the present invention (Murakami; Chemistry, 46, 681 991), PS Universal, Bio Ifechnology. , 2, 358 (1992)], which is thought to be a potential source of VEGF-KDR ⁇ ⁇ 3 ⁇ 4 ⁇ 3 ⁇ 4 ⁇ ij ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ It is possible to treat diseases that progress due to abnormal blood such as fresh blood.
  • the protein of the present invention can be obtained by using the DNA of the present invention and [2] the method of freezing.
  • screening for inhibiting the binding of the KDR intradomain to the protein of the present invention can be performed.
  • [1] use the cDNA of the KDR cell domain obtained in (1-1), and [2] use the method of language to select the domain protein in the ffl ⁇ of the KDR thread and use the protein of the present invention.
  • KDR Itoshima domain proteins in vitro After that, polyacrylamide gel electrophoresis is performed to detect a rise in the amount.
  • a cDNA clone of the obtained protein of the present invention inverted domain
  • a protein of the present invention ⁇ ⁇ ⁇ ⁇ ⁇ protein poor expression plasmid
  • the amount of 3 ⁇ 4 ⁇ can be aged by adding it, and it is possible to screen for destruction, and such destruction is considered to include fff ⁇ Sl of VEGF-KDR system. Therefore, it can be used to treat diseases that progress due to solid culture, inflammation in masticatory rheumatism, diabetes mellitus msm, ⁇ m, and dry abnormal blood.
  • an antibody against the protein of the present invention can be obtained by the method of [3] language.
  • the protein of the present invention can be detected by using an antibody against the protein of the present invention.
  • an antibody that inhibits the age of the protein of the present invention and KDR an antibody that inhibits the interaction between the protein of the present invention and the protein of the present invention, WTOfTOi ⁇ . It is thought that VEGF-KDR system is inhibited, so administration of such an antibody will result in solid phase ® ⁇ c occupation, »formation, littMfi rheumatism ( ⁇ ⁇ ⁇ if ⁇ , diabetes t4 S ⁇ S, ⁇ dry). Abnormal blood, etc. ⁇ It is possible to cure illness that progresses due to raw disease.
  • Figure 1 shows the KDR cDNA clone pBluescriptn KDR, KDR thread Domain 1 in MS?
  • the plasmid pAS-KDR for the no-brid method and the plasmid pAS-KDR and the cDNA library used for the two / brid method were used.
  • FIG. 2 shows the results of a northern blot for the distribution of the expression of the KDR receptor H ⁇ protein DNA1 in humans.
  • FIG. 3 shows the results of a Northern blot for investigating the distribution of KDR receptor protein DNA2 in humans.
  • FIG. 4 is a diagram showing the deletion of the K868M mutant KDR cDNA plasmid pBluescriptH KDR M-2.
  • FIG. 5 shows the result of a tuno-brid using plasmid clone # 37 and; KDR (pAS-KDR) or T ⁇ r mutant KDR.
  • Fig. 6 shows the use of plasmid clone # 2 and wild-type MKDR (pAS-KDR) or T ⁇ r mutant KDR. .
  • the fragment to be amplified in (A) is 810 bp, and since the BamHI site is located near the 5 'end and the site is located at the 3' end in the SIJ of this fragment, the DNA is cut with gi ⁇ EamHI and The DNA fragment of about 740 bp is used, and the fragment that flank in (B) is 1059 bp, and the primer is 7 ⁇ 5fe) haI site in 33 ⁇ 43 ⁇ 4.
  • ⁇ cytoca ⁇ ⁇ ⁇ near the 5 'end works in IW ⁇ Aial and hal, and a fragment of about 990bp is recovered.
  • the plasmid obtained by subcloning the KDR cDNA fragment of pBluescript ⁇ SK into pBluescript ⁇ SK was cut with BamHI and Xhal, and ligation was performed with the amHI- ⁇ al fragment of (A) and the ⁇ al-Xhal fragment of (B).
  • Primer Represents the entry made by KDR-2.
  • GAL4BD DNA ⁇ domain of GAL4
  • GAL4AD »GAL4 remains ⁇
  • KDRC KDR cDNA thread domain fragment
  • the JME ⁇ J of the obtained KDR cDNA was determined by the 'oxy method, and was determined as follows: »[BITbrman et al .; Biochem. Biophys. Res. Commun., 1S2, 1579 (1992)] (1)
  • the unknown two amino acids at the N-terminus are Mel ⁇ ATG) and Gln 2 (CAG), and the 272 bp 5 '"amino acid.
  • Val 297 (QTA) ⁇ He (ATA), Thr 7 '72 (ACG) - Ala (QCG), Gly 787 (QGG) - ⁇ Arg (CGG), Asn 835 (AAQ ⁇ I ⁇ s (AAG), Glu 848 (GAG) ⁇ Val (GTG), Thr 134 (ACG)- ⁇ SerCTCG) [S. Sawano et al .; Cell Growth & DiffercntiationJ, 213 (1996)].
  • this mutation creates one Aii l site in the fragment of the (B) increase
  • the plasmid pUC-KDRC is transformed into the Affi-primer KDR-2 (salt sequence P No. 15 ⁇ ⁇ )) K K K K K K K K K K K K K K K K K K ⁇ K ⁇ Pro Pro ⁇ 15 15 15 ⁇ ⁇ ⁇ 15 Immediately after the BamHI site ⁇ ; ⁇ Introduce one SC; cut this modified pUC-KDRC with W ⁇ EamHI and Sail; cut about 1.7 kb of the KDR cDNA fragment; cut this EamHI-Sall fragment mouth Nte Tsuno ⁇ hybrid 3 ⁇ 4 of Kkusha ⁇ 7 was subcloned between EamHI / Sall site of Kuta ⁇ pAS2-l, a plasmid pAS2-KDR and I Lou P AS2-KDR ⁇ !
  • Fig. 1 Screening method is performed according to the manual of Clonetech, which is sized for the library.
  • pAS2-KDR Sa chammvops oprpvisiae strain CG1945 (Introduced into Clonetech using the ⁇ lithium method: GAL4 binds to chromosome in CG1945 strain Downstream of EJIJ, it has histidine HIS3 and / 3 galactosidase lacZ as reporter Hfi ⁇ ", and is a strain requiring tributophan, leucine, and histidine.
  • Plasmid PAS2-KDR and KDR yarn E ⁇ The Sl form that expresses each protein of the cDNA clone of the protein that corresponds to the domain is GAL4 DNA ⁇ domain and GAL4 transgenic by the binding protein that encodes the KDR thread domain and cDNA.
  • the I4f-Dori domain is transcribed, so the HIS3 and lacZ transcripts downstream of the GAL4 DNA domain are transcribed, resulting in histidine tropism and / 3-galactosidase 3 ⁇ 43 ⁇ 41 «.
  • the ⁇ S ⁇ body was cultured in a minimal medium containing leucine and became leucine ⁇ ⁇
  • 3 ⁇ 4 ⁇ cDNA plasmid is recovered from the body, and the protein encoded by the cDNA in the recovered plasmid is DR
  • Clones # 37 and clones were identified as the clones:
  • Clones 37 forces; including cDNA salt »Row C
  • Plasmid clone # 37 which is the clone identified in (2) above, is cut with EffiRI and Egin, and a 1.9 kb fragment of cDNA 3 ⁇ 4r ⁇ * is cut into a fiber, subcloned between the HincIlZEamHI sites of the vector "pUC118" and nested deletion.
  • JME ⁇ J is the rooster No.5 ⁇ .
  • ⁇ 3 ⁇ 4H ⁇ is 1985 bp, which corresponds to the 283rd and later of the salt ij of the E ⁇ E1.
  • the homology of this ⁇ to the GenBank database was determined by BLAST search. As a result, no ⁇ 7 ⁇ MH ⁇ rooster U was found.
  • the cDNA containing clone # 37 is considered to be a novel cDNA that is known as a KDR receptor, and is named KDR receptor Hi ⁇ protein DNA1.
  • Plasmid clone # 2 which has been cloned in (2) (> ⁇ ), is cut with ECQRI and Xhol, and the cDNA 3 ⁇ 4rtj # 3 ⁇ 4 1.6 kb fragment is excised, and after ossification of * ⁇ , the vector “pUC118 Subcloning into the Hindi site of pDNA2-RX1 and pUK2-RXl, based on the nested deletion method, based on the series of cDNA deletions, and the plasmid before deletion.
  • the sequence Sl ⁇ was performed using a sequence kit from one company, DNA Inc., and DNA sequence 1 ⁇ ⁇ ABI310. ⁇ ] make a decision ⁇
  • Rooster No. 6 i The obtained; ⁇ ⁇ is 1610 bp, which corresponds to the 27th and following characters of Rooster No.1. This:
  • Homologous search was performed by BLAST search using GenBank.
  • the cDNA containing Clone # 2 is also considered to be a novel cDNA involved in the identification of KDR receptor 1 and this is named KDR receptor "protein DNA2".
  • CMSS Example 2 KDR receptor protein DNA release, cloth
  • KDR receptor protein DNA1 current distribution
  • the release of the KDR receptor protein DNA1 in humans was compared to the Multiple Tissue Northern Blot (Clontech tt), a mENA blot of human fibers ('brain, lunar disjunction, lunar, skeletal muscle, M).
  • a Northern blot By performing a Northern blot
  • the mENA of the KDR receptor “protein DNA1” has two views of 2.7 kb and 2.0 kb, and that the mRNA is not sewn; (It is considered that ⁇ 16
  • Human ⁇ ( ⁇ ⁇ (KDR receptor protein DNA2 is distributed to human fibers (I: mold, brain, lung, moon, skeletal muscle, haze); II. M thymus, anterior, testis, speaking, small intestine, colon , White rt) mRNA blots using Multiple Tissue Northern Blot I and II (Clonetech II).
  • mENA of KDR resorbu ⁇ ⁇ protein DNA2 is found to have two views, 3 kb and 6 kb. In the small intestine and colon, the crumbs were crushed, while the mRNA of 6 kb was found in the thymus, testis, and leukemia of the thymus.
  • a 2.2 kb target ECQRI fragment was a vector "" ECQR of pUCU9 [Subcloned into the site and obtained plasmid pUK137-Rl and pOKl37-Rl by EmRt and ⁇ 7: ®f ⁇ Fight U3 ⁇ 453 ⁇ 40.61 * wr and subcloning between pucii9 * ⁇ ⁇ Do ⁇
  • Escherichi cdiDH5ii which is a bacterium containing PUK137-R1) UKl37-Rl November 1997
  • the KDR receptor "" ⁇ protein DNA1 is linked to # 3 ⁇ 41 ⁇ 2, so KDR C 3 ⁇ 4 3 ⁇ 4 ⁇ ⁇ ⁇ 3 ⁇ 4 j j j ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇
  • the KDR receptor obtained in Step 1 is the cDNA of protein DNA2. Since the mENA was 0 ft, which became evident in Step 2, it does not encode cDNA i ⁇ length. Since it has Hft ⁇ ', the cDNA rfeftiD clone Make it! ⁇
  • a clone containing the longest cDNA of about 3.0 kb was selected from the positive clones obtained from Bracno and Ibridice. This clone was subjected to ECQRI, subcloned into the ECQEI site of the vector pUC119 ( ⁇ ⁇ -), and the plasmid pUK102-Rl was subcloned with the cDNA (I ECQR [fragment 3.0 kb]. The 3kb 1111 ⁇ (13 ⁇ 4 was considered to be the full-length cDNA clone corresponding to the above.
  • the KDR receptor protein DNA2 of pUK102-Rl was subjected to 5 ⁇ ⁇ 3 ⁇ 4 ⁇ , and the # 2 KDR receptor pig obtained in 3 ⁇ 4 ⁇ 1 was obtained.
  • ⁇ Synthetic protein DNA2 cDNA CD3 ⁇ 43 ⁇ 4S ⁇ J is 5bp 27 bp longer than 5 ⁇ »J KDR receptor of P UK102-R1"
  • Protein DNA2 cDNA is about 1.4kb longer than # 2 cDNA, but 5 ' It is considered to be a clone, not 3 ' ⁇ , but it is considered to be a clone.
  • the novelty of this amino translation is achieved by performing homology t ⁇ on the amino translation database Genpept :, PIR, Swiss-Prot, PRF, and PDB by Blast search.
  • a human leukocyte cDNA library (Clontech; vector, gtlO) was screened for a DNA2 clone from the human isolated cDNA library. Screening was performed in the same manner as the ning.
  • the human leukocyte cDNA library was again screened with plaqueno and hybridization, and the obtained clone: 1.8 kb from the eve clone cDNA ⁇ tf clone This clone was subjected to ECQRI and corresponds to cDNA.
  • Lys 868 which neutralizes the kinase activity of KDR, has difficulty in Met (hereinafter referred to as the K868M mutation).
  • ⁇ was introduced into the DRcDNA clone used in Zongyin 1 to encode KDR.
  • A Primer K14N «1 Ban No. 16) and Primer KMR-2 ® ⁇ No. 17
  • B Primer KMN-2 (Tori ⁇ I Ban No. 18) and Primer K3R 3 ⁇ 4 1 PCR No. 19) Perform PCR with two sets of primers ⁇ :
  • KNR-2 and KMN-2 are mutually exclusive primers with K868M variation 3 ⁇ 4K ⁇ . ⁇ IX the two combined DNA fragments and mix them.3 ⁇ 4 Next, use primers 2 and 5 used in Example 1 for PCR! The 0.8 kb cDNA fragment encoding KDR was used (Fig. 4).
  • the fragment is restricted with gg ⁇ EamHI and: Ikil
  • EamHI-fragment K868M mutation 3 ⁇ 4 ⁇ ,
  • an approximately 5.2 kb oil-EamHI fragment obtained by ligating pBluescriptll KDR with EamHI and 1, and pBluescriptn KDR was digested with: Ikgl and oil About 1.5 kb: Ikj fctl fragment with ligation ® ⁇ , ⁇ 868 ⁇ mutant KDR cDNA ⁇ l ⁇ t plasmid pBluescriptn KDR M-2
  • the language is 2% glucose and 5mM 3-aminotriazole ⁇ ⁇ , leucine, histidine, tryptophan 3 ⁇ 4 ⁇ manale, combined / ⁇ 3 ⁇ 4 Lb, 30 days at 30 C C, histidine dwarfism
  • Figure 5 shows the results of using plasmid claw # 37.
  • KDR and KDR receptor pig H ⁇ protein DNA1 and KDR receptor protein DNA2 have Lys 868 , which means that tyrosine chitase activity of KDR is necessary.
  • KDR receptor "KDR site which is ⁇ protein"
  • KDR resator " ⁇ In order to examine the power of a protein to KDR, we used a mutant of KDR and carried out the yeast two-hybrid method: A modified version of rnm l (l) with one base inserted immediately below the EamHI site! pUC-KDRC (7 "Main strand 3 ⁇ 4 ⁇ Using male primer 1 (1) and ⁇ The Tr on the C «side of the kinase domain of KDR was replaced with Phe by mutation introduction. The primer used was YF12 [Tyr (1136 YF13 [Tyi 1175)-Phe], YF14 with iffi IJ of Toriban No.
  • the warrior TVr mutant 7 was combined with the mutation ⁇ :, as multiple mutants of T r were mutated, YF12 + 14 [Tyr (1136, 1214) ⁇ Phe], YF12 + 13 + 14 [1 ⁇ 1136, 1175, 1214) ⁇ Phe], YF14 + 15 [1Vr (1214, 1223) ⁇ Phe], YF14 + 16 [1 ⁇ (1214, 1305) ⁇ Phe], YF1G -17 [ TVr (1305, 1309) ⁇ Phe] and YF12 + 14 + 16 [1Vr (1136, 1214, 1305) ⁇ Phe]: ⁇ The introduction of each mutation
  • the Hi Tr mutant KDR plasmid thus obtained was digested with EamHI and Sail, inserted between EamHlZSaJI of PAS2-1, and a plasmid in which Tr-transformed KDR was inserted into these pAS2-l. Introduced into strain HCG1945, cultured in Leu, His, rp 3 ⁇ 4r ⁇ Mana ⁇ ⁇ with 2% glucose and 80 mM 3-aminotriazole for 4 days at 30 ° C for 4 days, Check whether or not His
  • the iyr mutant plasmid of Arthropod magna 7 views in the TO ⁇ KI ⁇ body, KDR TVr (1305) was changed to Phe (YF16) On the other hand, a decrease in growth in a medium containing His was observed. On the other hand, the TVr mutation ⁇ ⁇ ⁇ 'in the other 6 views was a completely unrecognized force under the union ⁇ A mutant in which multiple Tyrs were transferred to Phe Nyaray, even a mutant of TVr (1305)
  • the abnormal blood t ⁇ f such as »flame, m, ⁇ m.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Biochemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Toxicology (AREA)
  • Zoology (AREA)
  • Biophysics (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Immunology (AREA)
  • Cell Biology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

On a tenté de clarifier une substance qui active, par liaison au récepteur KDR du facteur de croissance cellulaire de l'endothélium vasculaire (VEGF), la transduction du signal de KDR, et provoque ainsi des néovascularisations anormales, telles que la prolifération de tumeurs solides, la formation de métastases, l'arthrite de la polyarthrite rhumatoïde, la rétinopathie diabétique, la rétinopathie de la prématurité et le psoriasis. L'invention concerne en particulier une protéine capable de se lier au domaine intracellulaire du récepteur KDR de VEGF pour activer la transduction du signal de KDR, un ADN codant cette protéine, un ADN recombinant obtenu par incorporation de l'ADN dans un vecteur, un transformant portant l'ADN recombinant, un procédé de production de ladite protéine au moyen dudit transformant, et un anticorps pouvant reconnaître cette protéine. L'utilisation de l'ADN protéique du récepteur KDR telle que décrite ci-dessus permet de clarifier le mécanisme de transduction du signal du récepteur KDR de VEGF et de traiter des maladies comportant une évolution des symptômes due à des néovascularisations anormales, telles que la prolifération de tumeurs solides, la formation de métastases, l'arthrite de la polyarthrite rhumatoïde, la rétinopathie diabétique, la rétinopathie de la prématurité et le psoriasis.
PCT/JP1998/005612 1997-12-12 1998-12-11 Nouvelle proteine WO1999031238A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU15065/99A AU1506599A (en) 1997-12-12 1998-12-11 Novel protein

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP34347497 1997-12-12
JP9/343474 1997-12-12

Publications (1)

Publication Number Publication Date
WO1999031238A1 true WO1999031238A1 (fr) 1999-06-24

Family

ID=18361812

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP1998/005612 WO1999031238A1 (fr) 1997-12-12 1998-12-11 Nouvelle proteine

Country Status (2)

Country Link
AU (1) AU1506599A (fr)
WO (1) WO1999031238A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005093055A1 (fr) * 2004-03-26 2005-10-06 Frontier Science Co. Ltd. Levures transformees pour detecter et mesurer les dioxines et procedes de detection, de mesure et de depistage pour dioxines employant lesdites levures

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996031625A1 (fr) * 1995-04-07 1996-10-10 Cytogen Corporation Polypeptides presentant un domaine fonctionnel important, et leurs procedes d'identification et d'utilisation

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996031625A1 (fr) * 1995-04-07 1996-10-10 Cytogen Corporation Polypeptides presentant un domaine fonctionnel important, et leurs procedes d'identification et d'utilisation

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
LEHMANN J. M., RIETHMUELLER G., JOHNSON J. P.: "NCK, A MELANOMA CDNA ENCODING A CYTOPLASMIC PROTEIN CONSISTING OF THE SRC HOMOLOGY UNITS SH2 AND SH3.", NUCLEIC ACIDS RESEARCH, INFORMATION RETRIEVAL LTD., GB, vol. 18., no. 04., 1 January 1990 (1990-01-01), GB, pages 1048., XP002915759, ISSN: 0305-1048 *
MARGOLIS B., ET AL.: "HIGH-EFFICIENCY EXPRESSION/CLONING OF EPIDERMAL GROWTH FACTOR- RECEPTOR-BINDING PROTEINS WITH SRC HOMOLOGY 2 DOMAINS.", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES, NATIONAL ACADEMY OF SCIENCES, US, vol. 89., 1 October 1992 (1992-10-01), US, pages 8894 - 8898., XP002915762, ISSN: 0027-8424, DOI: 10.1073/pnas.89.19.8894 *
MEISENHELDER J., HUNTER T.: "THE SH2/SH3 DOMAIN-CONTAINING PROTEIN NCK IS RECOGNIZED BY CERTAIN ANTI-PHOSPHOLIPASE C-ZL MONOCLONAL ANTIBODIES, AND ITS PHOSPHORYLATION ON TYROSINE IS STIMULATED BY PLATELET-DERIVED GROWTH FACTOR AND EPIDERMAL GROWTH FACTOR TREATMENT.", MOLECULAR AND CELLULAR BIOLOGY., AMERICAN SOCIETY FOR MICROBIOLOGY, WASHINGTON., US, vol. 12., no. 12., 1 December 1992 (1992-12-01), US, pages 5843 - 5856., XP002915760, ISSN: 0270-7306 *
PARK D., RHEE S. G.: "PHOSPHORYLATION OF NCK IN RESPONSE TO A VARIETY OF RECEPTORS, PHORBOL MYRISTATE ACETATE, AND CYCLIC AMP.", MOLECULAR AND CELLULAR BIOLOGY., AMERICAN SOCIETY FOR MICROBIOLOGY, WASHINGTON., US, vol. 12., no. 12., 1 December 1992 (1992-12-01), US, pages 5816 - 5823., XP002915761, ISSN: 0270-7306 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005093055A1 (fr) * 2004-03-26 2005-10-06 Frontier Science Co. Ltd. Levures transformees pour detecter et mesurer les dioxines et procedes de detection, de mesure et de depistage pour dioxines employant lesdites levures

Also Published As

Publication number Publication date
AU1506599A (en) 1999-07-05

Similar Documents

Publication Publication Date Title
US7858091B2 (en) Antibodies to insulin-like growth factor binding protein and uses thereof
JP2002514928A (ja) 脊椎動物テロメラーゼ遺伝子およびタンパク質ならびにその使用
KR20170138410A (ko) 비-천연 세마포린 3 및 이의 의학적 용도
WO2006073052A1 (fr) Nouvel inhibiteur d'angiogenèse
JP2002501738A (ja) ６７個のヒト分泌タンパク質
JP2002506627A (ja) ９５個のヒト分泌タンパク質
JP2002505871A (ja) ３１個のヒト分泌タンパク質
JPWO2002033094A1 (ja) Ｖｐｌｆの活性を阻害する抗体
WO1999031238A1 (fr) Nouvelle proteine
US20040081972A1 (en) Novel physiologically active peptide and use thereof
WO2010007701A1 (fr) Agent thérapeutique et agent de test pour maladie à nécrose myocardiale
CN1952129B (zh) Angptl4缺失突变体及其应用
JP2001017188A (ja) 新規なｖｅｇｆ／ｐｄｇｆ様因子
US7250488B1 (en) Transcriptional activation inhibitory protein
JP2000060571A (ja) 新規な哺乳類のペプチド及びそれをコードするポリヌクレオチド
JPH119288A (ja) 新規ｈａｓ２スプライシング変種ｈｏｅｆｃ１１：慢性腎不全、炎症性疾患および心筋虚血における標的
US6908765B1 (en) Polypeptide—human SR splicing factor 52 and a polynucleotide encoding the same
CN108329387A (zh) 癌症相关的肿瘤特异转录本lin28b-tst及其用途
WO2001060855A1 (fr) Nouvelle proteine humaine associee a la regulation du cycle cellulaire et sa sequence de codage
CN100443501C (zh) 人骨髓基质细胞来源的线粒体转运蛋白分子及其编码序列和用途
JP2005505252A (ja) 低酸素症誘導因子及び脈管形成を誘導するため及び筋肉機能を改善するためのそれらの使用
US20040038210A1 (en) Novel polypeptide-type II fibronectin 10 and a polynucleotide encoding the same
CN100381466C (zh) 一种人Pif1基因、其编码蛋白及其应用
CN102212126B (zh) 具有抑制内皮细胞生长活性的重组EDI-8t蛋白
JP5099535B2 (ja) Ｋａｎｋ４遺伝子の癌治療及び癌検出並びに創薬への利用

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AU BG BR CA CN CZ HU IL JP KR MX NO NZ PL RO SG SI SK UA US VN

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): 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

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
NENP Non-entry into the national phase

Ref country code: KR

122 Ep: pct application non-entry in european phase
NENP Non-entry into the national phase

Ref country code: CA

点击 这是indexloc提供的php浏览器服务，不要输入任何密码和下载