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WO2000063392A1 - Nouvelle tyrosine phosphatase - Google Patents

Nouvelle tyrosine phosphatase Download PDF

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Publication number
WO2000063392A1
WO2000063392A1 PCT/JP2000/002455 JP0002455W WO0063392A1 WO 2000063392 A1 WO2000063392 A1 WO 2000063392A1 JP 0002455 W JP0002455 W JP 0002455W WO 0063392 A1 WO0063392 A1 WO 0063392A1
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dna
ptp
protein
seq
cells
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Japanese (ja)
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Kenji Shimizu
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KH Neochem Co Ltd
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Kyowa Hakko Kogyo Co Ltd
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Priority to AU36794/00A priority Critical patent/AU3679400A/en
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    • 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)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

Definitions

  • the present invention relates to a novel tyrosine phosphatase encoded on human chromosome 3p21, which is presumed to be a tumor suppressor gene.
  • Inactivation of tumor suppressor genes plays an important role in the development of cancer. Since there are two copies of a gene on the genome as an allele, inactivation of a tumor suppressor gene often results in loss of heterozygosity (hereinafter abbreviated as L0H) in one gene and one in the other. It is said that this is due to a mutation in the gene. Therefore, it is considered that there is a high possibility that a tumor suppressor gene is present at the site where L0H is frequently observed in cancer cells. In human lung cancer, sites where L0H occurs frequently have been reported. Among them, the short arm of chromosome 3 (3p) accounts for nearly 100% of small cell lung cancer and 60% of non-small cell lung cancer.
  • the deletion is found in%, it is considered to be the site of a gene involved in the development of lung cancer [0ncogene, 7, 445 (1992)].
  • L0H is observed in various regions of 3p, and among them, 3pl4, 3p21, and 3p25 are commonly found in many lung cancers, and these regions are presumed to contain tumor suppressor genes. [0ncogene, 7, 445 (1992)]. Deletions in these regions are seen in the early stages of lung cancer development, but reports indicate that the range of deletions increases with malignancy COncogene, 11, 2591 (1995)], and non-small cell cancers, especially lung glands Some reports are poor prognostic factors for cancer.
  • hybrid fusion cell clones of human chromosome 3 and mouse A9 cells two types of hybrid fusion cell clones that show tumorigenesis when transplanted into SCID mice are commonly deleted.
  • the deletion was 3p21 on human chromosome 3.
  • the region size is up to 1.6 cM (centimorgan) (between chromosome markers D3S1029 and D3S643). This deletion is thought to contain a tumor suppressor gene directly involved in tumorigenesis, but the gene has not been identified. '' Disclosure of the invention
  • the present inventors have conducted intensive studies on the tumor suppressor gene encoded at the 3p21 site of human chromosome where deletions are frequently observed, and have developed a novel ostium synphosphatase HD-PTP (histidine domain) involved in tumor suppression.
  • HD-PTP -cDNA encoding protein tyrosine phosphatase
  • the present invention provides the following (i) to (20).
  • a protein comprising the amino acid sequence of SEQ ID NO: 2.
  • a protein comprising an amino acid sequence in which at least one amino acid is deleted, substituted or added in the amino acid sequence of the protein of SEQ ID NO: 2, and having tyrosine phosphatase activity.
  • the protein comprising an amino acid sequence in which one or more amino acids are deleted, substituted or added in the amino acid sequence of the protein of SEQ ID NO: 2 in the above (2), and which has an oral synphosphatase activity, Molecular Cloning, A
  • the number of amino acids to be deleted, substituted or added is not particularly limited, it is a number that can be deleted, substituted or added by a well-known method such as the site-directed mutagenesis described above. It is preferably 1 to 20, more preferably 1 to 10, and still more preferably 1 to 5.
  • polypeptide of the present invention in order for the polypeptide of the present invention to have lipophilic synphosphatase activity, the amino acid sequence of SEQ ID NO: 2 and BLAST CJ. Mol. Biol., 215, 403
  • proteins having an amino acid sequence to which a tag peptide sequence is added are added.
  • DNA that hybridizes under stringent conditions and encodes a protein having tyrosinphosphatase activity refers to the nucleotide sequence represented by SEQ ID NO: 1, 3, 40 or 41.
  • DNA that can be obtained by using colony-'hybridization method, plaque 'hybridization method or Southern blot hybridization method as a probe with DNA After the hybridization at 65 ° C in the presence of 0.7 mol: 1.0 mol / L NaCl using the filter immobilized with DNA derived from colonies or plaques at 65 ° C, 0.1 x 2 x SSC ( saline-sodium citrate) solution [lx SSC solution (150 mmol / L NaCl, 15 mol / L sodium citrate); nx indicates an n-fold concentration solution. ], And DNA that can be identified by washing the filter under 65 ° C conditions can be given.
  • DNA abbreviation 1 Hybridization is described in Molecular 'Cloning 2nd Edition, Current'Protocols' in 'Molecular' Biology, DNA Cloning 1: Core Techniques, A Practical Approach, Second Edition, Oxford University Press (1995) (hereafter, DNA abbreviation 1) can be performed according to the method described in the experiment book. Specifically, as DNA that can be hybridized, Examples include DNAs having at least 80% homology with the nucleotide sequence represented by SEQ ID NO: 1, 3, 40 or 41, and preferably DNAs having 95% or more homology.
  • the transformant according to (7) is cultured in a medium, the protein according to (1) or (2) is produced and accumulated in the culture, and the protein is collected from the culture.
  • a therapeutic drug for cancer comprising the protein according to (1) or (2) as an active ingredient.
  • a therapeutic agent for cancer comprising the MA according to any one of the above (3) to (5) as an active ingredient.
  • a vector for treating a cancer gene comprising the DNA according to any one of the above (3) to (5).
  • oligonucleotide having a continuous nucleotide sequence of 10 to 60 residues, or a sequence complementary to the oligonucleotide, in the nucleotide sequence of the DNA according to any one of (3) to (5) above. And oligonucleotide derivatives that are derivatives of these oligonucleotides.
  • a diagnostic agent for cancer comprising the oligonucleotide according to (12) or (13).
  • a diagnostic agent for cancer comprising the antibody according to the above () 6) as an active ingredient.
  • the cDNA encoding the HD-PTP of the present invention is obtained by screening a cDNA library using, as a probe, a fragment of a gene that is a candidate for a cancer-related gene, obtained by the following transformation assay. can do.
  • Human cancer cells have acquired indefinite proliferation (immortalization), lost contact inhibition phenomenon during cell proliferation, anchorage-independent proliferation, decreased serum requirement for proliferation, decreased cell adhesion, nude mice Since it has different properties from normal cells, such as the ability to form tumors when transplanted, it is considered that there is a mutation in the chromosomal DNA of human cancer cells that causes the above properties.
  • One of the methods for screening such a causative gene is a transformation atsay method.
  • the Transformation Atsay method shows properties similar to those of normal cells or those of the above-mentioned cancer cells, which have no cancer traits other than infinite proliferation (tumorigenicity, growth ability in soft agar, etc.)
  • a DNA fragment derived from the chromosomal DNA of a human cancer cell is introduced into a host cell and expressed, and a cell clone is selected based on a change from a normal trait to a trait indicated by a human cancer cell as an indicator, and transformation is performed from the cell.
  • This is a method of isolating the chromosomal DNA fragment (causal gene) that caused the above.
  • the human cancer cell any cell can be used as long as it can extract chromosomal DNA.
  • KAL-1 [; Cancer Res., 51, 5392 (1991)] which is a cell line of human B-cell lymphoma is used as human cancer cells.
  • proteinase KZ phenol monoclonal form extraction method (Molecular Cloning, 2nd edition) can be mentioned.
  • a plasmid having a drug resistance gene serving as a marker in order to select the introduced cell.
  • the plasmid any plasmid can be used as long as it has a drug resistance functioning as a marker. Specific examples include pHyg having a hygromycin resistance gene [Mol. Cel. Biol., 5, 410 (1985)].
  • any method for introducing DNA into a host cell can be used.
  • electroporation (Cytotechnology, 3, 133 (1990) Proc. Natl. Acad. Sci. USA, 84, 7413 (1987)] and the like.
  • the traits of the cells will not change and will grow indefinitely, and other traits will be non-human animal cells that can be distinguished from cancer cells.
  • a cell into which is introduced is preferable.
  • Traits that can be distinguished from cancer cells include the above-described properties that are opposite to those specific to human cancer cells, such as anchorage-dependent growth, contact inhibition during cell growth, and serum requirements during growth. it can.
  • non-human animal cells A foreign human gene is expressed in a host cell to obtain a transformed cell having the same function as the cell containing the gene.
  • cells of animals which are not so far from humans in classification preferably mammalian cells other than human.
  • NIH3T3 RIB Cell Development Bank RCB0150
  • Rat-2 rat fibroblast cell line Rat-2 [Virology, 113, 408 (1981), ATCC CRL-1764].
  • transformed cells that is, cells into which a causative gene derived from a cancer cell has been introduced, can form colonies in a medium such as a soft agar medium.
  • a human cancer cell line is obtained.
  • the cells that have acquired the anchorage-independent trait possessed by form colonies in soft agar. Using the colony formation as an indicator, transformed cells can be easily isolated.
  • the human oncogene can be identified and obtained by performing DNA analysis using the colony forming cells isolated above in accordance with a conventional method. That is, after chromosomal DNA of the cells is cut with an appropriate restriction enzyme such as £ RI, the human oncogene is identified and obtained by Southern blot analysis (molecular cloning second edition, DNA cloning 1). Can be.
  • BLUR8 Pro Natl. Acad. Sci. USA, 77, 1398 (1980)
  • chromosomal DNA can be isolated from any human cell, and a DNA fragment labeled with a radioisotope, an enzyme, or the like can also be used.
  • the Alu sequence is a sequence that is specifically found in the introns of the human genome, and has a vast majority of 300,000 copies in human genomic DNA compared to other nucleotide sequences. Considered an array. Therefore, the labeled chromosomal DNA fragment can be used as a probe for identifying an intron in an oncogene. As a result of Southern blot analysis, colonies with bands of the same length were detected.
  • the chromosomal DNA is isolated from the colony, and the above-described operations from introduction into host cells to Southern blot are repeated using a DNA fragment derived from the chromosomal DNA.
  • a chromosomal DNA fragment derived from the selected colony causes transformation and that a band of the same length as the first band is detected by Southern blot analysis, a cancer trait is induced by the DNA fragment. You can see that it is triggered.
  • After isolating chromosomal DNA from the transformed cells it is partially cut into several kb to 20 kb with an appropriate restriction enzyme such as. The DNA fragment is inserted into a cloning vector and introduced into a host cell to prepare a DNA library.
  • any phage vector, plasmid vector, etc. can be used as long as it can autonomously replicate in E. coli K12 strain.
  • any microorganism that belongs to Escherichia coli can be used. Specifically, Escherichia coli XLl-Blue MRF '(Stratagene, Strategies, 5, 81 (1992)), Escherichia coli C600 (Genetics, 39, 440 (1954)), Escherichia coli Y1088 (Science, 222, 778) (1983)), Escherichia coli Y1090 CScience, 222, 778 (1983)), Escherichia coli NM522 CJ. Mol. Biol., 166, 1 (1983)), Escherichia coli K802 (J. Mol. Biol., 16, 118 (1966)), Escherichia coli JM105 (Gene, 38, 275 (1985)) and Escherichia coli LE392 (Molecular Cloning 2nd Edition) can be used.
  • the DNA library prepared as described above is subjected to colony hybridization or plaque hybridization (Molecular 'Cloning 2nd Edition) using the above-mentioned probe to obtain the DNA library. A more desired DNA clone can be obtained.
  • DNA fragment containing the oncogene derived from human cancer cells obtained from the clone is derived from chromosomal DNA, it is considered that the DNA fragment also contains introns.
  • cDNA of the gene is obtained by screening a cDNA library.
  • RNA or mRNA from appropriate cells or tissues.
  • cells include human gastric cancer cell line MKN45 [Jpn. J. Cancer Res. 77, 849 (1986), RIKEN Cell Development Bank RCB1001].
  • guanidine thiocyanate-cesium trifluoroacetate method As a method for preparing total RNA, guanidine thiocyanate-cesium trifluoroacetate method from human cells (Qlethods in Enzymol., 154, 3 (1987)), guanidine acid thionate / phenol-microform method (Analytical Biochemistry, 162) , 156 (1987)].
  • oligo (dT) cell mouth method (Molecular 'Cloning 2nd Edition) can be used.
  • mRNA can be prepared directly from human cells using a kit such as the Fast Track mRNA Isolation Kit (Invitrogen) or the Quick Prep mRNA Purification Kit (Pharmacia).
  • a cDNA library is prepared by preparing a cDNA, incorporating the cDNA into an appropriate vector, and introducing the cDNA into a host cell.
  • Specific methods for preparing a cDNA library include the method described in Molecular Cloning Second Edition, the method of Gubler and Hoffman [Gene, 25, 263 (1983)], or a commercially available kit such as Superscript Plasmid System for cDNA Synthesis and Plasmid Cloning (manufactured by Life Technologies) or ZAP-cDNA Synthesis Kit (manufactured by Staratagene) can be used. It can be performed according to
  • a chromosomal DNA fragment containing the oncogene obtained in (1) can be used.
  • RNA or mRNA is isolated from the clone obtained in the above (1) by the above method. Northern blot hybridization was performed using the isolated DM fragment and the RNA, and a fragment with a clear band was selected as a fragment containing exon, and used as a probe.
  • the nucleotide sequence of the cDNA of the novel human oncogene present in the cDNA clone thus obtained can be determined by the method described in (3) below. If it is considered that a full-length cDNA has not been obtained from the determined base sequence, a full-length cDNA can be obtained by the following method. That is, at both ends of the cDNA obtained by the above method Add Adabu Yuichi and perform PCR with primers based on the base sequence of this Adabu Yuichi and the obtained cDNA base sequence. 5, -MCE (rapid amplification of cDNA ends) and 3, -RACE CProc. Natl. Acad. Sci.
  • the obtained cDNA may be used as it is, or after the cDNA portion is digested with an appropriate restriction enzyme, subcloned into an appropriate cloning vector such as PUC118, and then used in a conventional nucleotide sequence analysis method, for example, the dideoxy method of Sanger et al. (Proc. Natl. Acad. Sci. USA, 74, 5463 (1977)] or the base sequence can be determined by analyzing using a base sequence analyzer such as 373A DNA Sequencer (Perkin Elmer). .
  • the amino acid sequence of the protein encoded by the cDNA can be known.
  • nucleotide sequence database such as GenBanks EMBL and DDBJ using a homology search program such as BLAST.
  • the nucleotide sequence determined by the above method includes the nucleotide sequence shown in SEQ ID NO: 1 of the cDNA encoding HD-PTP.
  • the genomic DNA is designed based on the base sequence of the cDNA obtained in (3) above, using human genomic DNA as type III. PCR CPCR using DNA that can be synthesized as a primer, A practical Approach, Oxford University Press ( 1991)]. Alternatively, a genomic DNA clone can be obtained from a human genomic DNA library that can be purchased from Clonetech, using the cDNA obtained in (3) as a probe. Cut.
  • HD-PTP DNA DNA encoding HD-PTP
  • HD-PTP DNA can also be prepared by chemical synthesis using a DM synthesizer based on the determined DNA base sequence.
  • a DNA synthesizer examples include a DNA synthesizer model 392 manufactured by Perkin Elmer utilizing the phosphoramidite method.
  • an oligonucleotide having a partial sequence of the DNA of the present invention or a complementary nucleotide sequence by a conventional method or the aforementioned DNA synthesizer.
  • Oligonucleotides (hereinafter referred to as HD-PTP oligonucleotides) can be prepared.
  • the HD-PTP oligonucleotide specifically, a DNA having the same sequence as 10 to 60 consecutive bases in the nucleotide sequence represented by SEQ ID NO: 1, 3, 40 or 41 or the MA Examples include DNAs having a complementary sequence.
  • the above-mentioned oligonucleotides in which the melting temperature and the number of bases of both are not extremely changed are preferable.
  • the antisense primer include MA having the base sequence represented by SEQ ID NO: 24, 25, 28 or 29.
  • oligonucleotide derivatives can also be mentioned as the oligonucleotide of the present invention.
  • the oligonucleotide derivative include: an oligonucleotide derivative in which a phosphodiester bond in an oligonucleotide is converted to a phosphorothioate bond; and a phosphodiester in an oligonucleotide.
  • HD-PTP is obtained using the method described in Molecular Cloning 2nd Edition or Current Protocols in Molecular Biology, etc., for example, by the following method, and by the method described in [1] above.
  • the prepared HD-PTP DNA can be expressed in host cells and prepared.
  • a recombinant DNA in which HD-PTP DNA is inserted downstream of the promoter in an appropriate expression vector is constructed.
  • a transformant expressing the HD-PTP protein can be obtained.
  • any cell that can express the target gene such as bacteria, yeast, animal cells, insect cells, and plant cells, can be used.
  • the expression vector those which can be autonomously replicated in the above-mentioned host cells or can be integrated into a chromosome, and which contain a promoter at a position where HD-PTP DNA can be transcribed, are used.
  • a prokaryote such as a bacterium
  • the recombinant DNA is capable of autonomous replication in the prokaryote and, at the same time, comprises a promoter, a ribosome binding sequence, HD-PTP DNA, and a transcription termination sequence.
  • the gene controlling the promotion may be included.
  • expression vectors examples include PKK233-2 (Pharmacia), pSE280 (Invitrogen), pGEMEX-1 (Promega), pQE-8 (QIAGEN), pKYPIO (Tokushosho 58-110600), p YP200 Agricultural Biological Chemistry, 48, 669 (1984)), pLSAl (Agric. Biol. Chem., 53, 277 (1989)), pGELl (Proc. Natl. Acad. Sci. USA, 82, 4306 (1985)), pBluescript II SK (-) (Stratagene), pGEX (Pharmacia), pET-3 (Novagen) and the like.
  • Any promoter can be used as long as it can be expressed in host cells such as Escherichia coli and Bacillus subtilis.
  • ⁇ promoter Isseki one P trp
  • ⁇ Promo one evening one P L promoter evening one
  • P R promoter evening one one such T7 promoter evening
  • promoters derived from Escherichia coli or phages such as it can.
  • two sets of P trp are connected in series, such as Promo Yuichi (P trp x 2), Promo Promo Yuichi, lacT7 Promo Yuichi, letl Promo Yoichiichi, etc. Can also be used.
  • a transcription termination sequence is not necessarily required for expression of the DNA of the present invention, but it is preferable to arrange a transcription termination sequence immediately below a structural gene.
  • Examples of the host cell include microorganisms belonging to the genus Escherichia, Bacillus, Corynebacterium, Brevibacterium, Pseudomonas, Serratia, etc., for example, Escherichia coli L1-Blue, Escherichia coli -XL2-Blue, Escherichia coli DHls Escherichia coli MC1000, Escherichia coli KY3276, Escherichia coli W1485, Escherichia coli JM109, Escherichia coli HB101, Escherichia coli No.49, Escherichia coli W3110s Escherichia col i NY49, Serratia ficaria, Serratia fonticola. Serratia liquefaciens Serratia marcescens, Bacillus subtil is s Bacillus amyloliquefaciens>
  • Brevibacterium immariophilum ATCC14068 Brevibacterium saccharolyticum ATCC14066, Corynebacterium glutajnicum ATCC13032, Corynebacterium glutamicum ATCC14067, Corynebacterium glutamicum ATCC13869,
  • Any method for introducing the recombinant DNA can be used as long as it is a method for introducing the DNA into the host cell.
  • the calcium chloride method [Proc. Natl. Acad. Sci. USA, 69, 2110 (1972) )]
  • Protoplast method JP-A-63-248394
  • electroporation method [Gene, 17, 107 (1982), Molecular & General Genetics, 168, 111 (1979)].
  • YEpl3 ATCC37115
  • YEp24 ATCC37051
  • YCp50 ATCC37419
  • pHS19 pHS15 and the like
  • Any promoter can be used as long as it can be expressed in a yeast strain.
  • Examples include a PH05 promoter, a PGK promoter, a GAP promoter, an ADH bromo promoter, a gal promoter, and a gal promoter.
  • Promoters include 10 promoters, heat shock polypeptide promoters, MF 1 promoters, CUP 1 promoters, and so on.
  • any method for introducing the recombinant DNA any method can be used as long as it is a method for introducing DNA into yeast. For example, an electroporation method [Methods in
  • an expression vector containing a promoter for transcription in the host animal cell, HD-PTP DNA, and a signal sequence for termination of transcription and polyadenylation of the transcript is used.
  • a promoter for transcription in the host animal cell HD-PTP DNA
  • a signal sequence for termination of transcription and polyadenylation of the transcript is used.
  • Any promoter can be used as long as it can perform transcription in animal cells, but it can be used for any of the early promoters of SV40 and the immediate early (IE) gene of human cytomegalovirus (CMV).
  • Promoter Yuichi Rous sarcoma virus (RSV) ⁇ ⁇ ⁇ Long-lasting retroviruses such as human immunodeficiency virus (HIV) and Moroni mouse leukemia virus (MMLV) ⁇
  • RSV Rous sarcoma virus
  • HSV human immunodeficiency virus
  • MMLV Moroni mouse leukemia virus
  • Promoters with virus-derived sequences such as repeats (long terminal repeats; LTRs), early promoters of SV40 SR artificial promoters with human T cell leukemia virus I LTRs artificially connected to them -, Or meta-oral thionein gene, e-actin gene, elongation factor (
  • Elongation factor examples include promoters of genes derived from animal cells such as one gene.
  • the enhancer of the IE gene of human CMV may be used together with the promoter.
  • an expression vector for example, PAGE107 (JP-A-3-22979, Cytotechnology, 3, 133, (1990)), pAS3-3 (JP-A-2-227075), pCDM8 CNature , 329, 840, (1987)], pcDNAI / Amp (manufactured by Invitrogen), pREP4 (manufactured by Invitrogen), pAGE103 GJournal of Biochemistry, m, 1307 (1987) ], PCDL81 and the like are used.
  • pCDL81 integrates the SR Hypromo overnight into the vector EBO-pcD Mol. Cell.
  • Xhol- This is a vector prepared by inserting DNA having a base sequence of I-Xbal-Kpnl-BajnHI) and Poly A signal.
  • Host cells include mouse myeloma cells, rat myeloma cells, mouse hybridoma cells, NIH3T3 cells which are mouse fibroblasts, Rat-2 cells which are rat fibroblasts, and Namalva which is a human cell.
  • Cells human fetal kidney cells, human leukemia cells, African green monkey kidney cells, Chinese hamster cells, CH0 cells, HBT5637 (JP-A-63-299), and the like.
  • Mouse / myeloma cells SP2 / 0, NS0, etc .
  • Rat 'myeloma cells YB2 / 0, etc .
  • human Namalwa cells Namalwa KJM-1 cells
  • human embryonic kidney cells HEK293 ATCC: CRL-1573
  • BALL-1 as human leukemia cells
  • COS-1 and C0S-7 African green monkey kidney cells.
  • any method for introducing the recombinant DNA any method can be used as long as it is a method for introducing DNA into animal cells.
  • an electro- volatilization method [Cytotechnology, 3, 133 (1990)]
  • a calcium phosphate method particularly Kaihei 2-227075
  • lipofection method Proc. Natl. Acad. Sci. USA, 84, 7413 (1987)] and the like.Constant HD-PTP with HD-PTP DNA integrated into host chromosomal DNA.
  • An expression cell can be selected by introducing an HD-PTP expression vector containing a sequence capable of expressing a resistance gene to a drug such as G418 or hygromycin into a host cell and culturing it in the presence of the drug.
  • a drug such as G418 or hygromycin
  • the expression of the dihydrofolate reductase (dhfr) gene was An HD-PTP constitutive expression vector containing such a sequence is introduced into a host cell, and cultivation is performed while increasing the concentration of methotrexate, a dhfr inhibitor, step by step. It can also increase the copy number of HD-PTP DNA.
  • a host cell for performing gene amplification using the dhfr gene a cell in which the dhfr gene does not function, for example, CHO / dhfr- (ATCC: CRL-9096) is used.
  • Baculovirus Expression Vectors A Laboratory Manual, WH Freeman and Company, New York (1992) (hereinafter, paculovirus, expression, vectors, laboratories,
  • the polypeptide can be expressed by the method described in Bio / Technology, 6, 47 (1988), Current 'Protocols in' Molecular Biology, and the like.
  • the recombinant gene transfer vector and the baculovirus are co-transfected into insect cells to obtain recombinant virus in the culture supernatant of insect cells, and then the recombinant virus is infected into insect cells.
  • the protein can be expressed.
  • the gene transfer vector used in the method include, for example, pVL1392, pVL1393, pBlueBacIII (all manufactured by Invitrogen) and the like.
  • the baculovirus for example, autographa californica nuclear polyhedrosis virus, which is a virus that infects night moth family insects, can be used.
  • the insect cells include Sf9 and Sf21 (baculovirus, expression vectors, vectors, laboratory, manual 1), ovary cells of Spodoptera frugiperda, and High 5 (manufactured by Invitrogen) which are ovarian cells of Trichoplusia ni. Etc. can be used.
  • the vector for introducing the recombinant gene into insect cells As a method of co-introducing Yuichi and the above baculovirus, for example, calcium phosphate method (Japanese Patent Laid-Open No. 2-227075), lipofection method [Proc. Natl. Acad. Sci. I can give it.
  • the protein of the present invention can be produced by culturing the transformant obtained as described above in a medium, producing and accumulating the protein of the present invention in the culture, and collecting from the culture.
  • the protein of the present invention is returned to the body of the patient by returning the cells to the body. It can also be expressed.
  • the method for culturing the transformant of the present invention in a medium can be performed according to a usual method used for culturing a host.
  • a culture medium for culturing a transformant obtained using a prokaryote such as Escherichia coli or a eukaryote such as yeast as a host contains a carbon source, a nitrogen source, inorganic salts, and the like which can be used by the organism. Either a natural medium or a synthetic medium may be used as long as the medium can efficiently culture C.
  • the carbon source may be any one that can be assimilated by the organism; glucose, fructose, sucrose, molasses containing these, carbohydrates such as starch or hydrolyzed starch, organic acids such as acetic acid, propionic acid, and the like. Alcohols such as ethanol and propanol can be used.
  • Nitrogen sources include ammonia, inorganic or organic acids such as ammonia, ammonium chloride, ammonium sulfate, ammonium acetate, and ammonium phosphate.
  • potassium potassium phosphate potassium potassium phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate, calcium carbonate, and the like can be used.
  • the culture is usually performed under aerobic conditions such as shaking culture or deep aeration stirring culture.
  • the culture temperature is preferably 15-40 ° C, and the culture time is usually 16-96 hours.
  • the pH is maintained at 3.0 to 9.0.
  • the pH is adjusted using inorganic or organic acids, alkaline solutions, urea, calcium carbonate, ammonia and the like.
  • an antibiotic such as ampicillin or tetracycline may be added to the medium during the culture.
  • an inducer may be added to the medium, if necessary.
  • an inducer may be added to the medium, if necessary.
  • isopropyl-I-D-thiogalactobilanoside is transformed with an expression vector using ⁇ promoter.
  • indoleacrylic acid or the like may be added to the medium.
  • Culturing is carried out under conditions such as normal 5% C0 2 presence.
  • the culture temperature is preferably 35-37 ° C, and the culture time is usually 3-7 days. If necessary, antibiotics such as kanamycin and penicillin may be added to the medium during the culture.
  • TNM-FH medium manufactured by Pharmingefi
  • Sf-900 II SFM medium As a medium for culturing a transformant obtained by using an insect cell as a host, commonly used TNM-FH medium (manufactured by Pharmingefi), Sf-900 II SFM medium (manufactured by Pharmingefi), Sf-900 II SFM medium (manufactured by Pharmingefi), Sf-900 II SFM medium (manufactured by Pharmingefi), Sf-900 II SFM medium (
  • ExCell400 LifeTechnologies
  • ExCe 11405 all manufactured by JRH Biosciences
  • the culture temperature is preferably 25-30 ° C, and the culture time is usually 1-4 days.
  • an antibiotic such as gentamicin may be added to the medium during the culturing.
  • a normal protein isolation and purification method may be used.
  • the cells when the protein of the present invention is expressed in a dissolved state in cells, the cells are collected by centrifugation after suspension of culture, suspended in an aqueous buffer, and then sonicated with a crusher, French press, Menton. The cells are disrupted using a Gaulin homogenizer, Dynomill, etc. to obtain a cell-free extract.
  • a normal protein isolation and purification method that is, a solvent extraction method, a salting out method using ammonium sulfate, a desalting method, a precipitation method using an organic solvent , Getylaminoethyl (DEAE)-Sepharose, DIAI0N HPA-75 (Mitsubishi Chemical) and other anion exchange chromatography methods, and S-Sepharose FF (Pharmacia) and other resins Cation exchange chromatography method used, hydrophobic chromatography method using resins such as butyl sepharose and phenylsepharose, gel filtration method using molecular sieve, affinity chromatography method, chromatofocusing method, etc.
  • a purified sample can be obtained by using techniques such as electrophoresis such as electrofocusing alone or in combination.
  • the cells When the protein is expressed in an insoluble form in the cells, the cells are similarly recovered, crushed, and centrifuged to obtain a precipitate fraction of the protein. After collecting the insoluble matter, the insoluble matter of the protein is solubilized with a protein denaturant. After diluting or solubilizing the solubilized solution to a solution containing no protein denaturing agent or diluting the concentration of the protein denaturing agent so that the protein is not denatured, and then restoring the protein to a normal three-dimensional structure.
  • a purified sample can be obtained by the same isolation and purification method as described above.
  • the protein of the present invention or its derivative such as a modified sugar is secreted extracellularly
  • the protein or its derivative such as a sugar chain adduct can be recovered in the culture supernatant. That is, a culture supernatant is obtained by treating the culture by a method such as centrifugation as described above, and a purification standard is obtained from the culture supernatant by using the same isolation and purification method as described above. Goods can be obtained.
  • the protein of the present invention can also be produced by a chemical synthesis method such as the Fmoc method (fluorenylmethyloxycarbonyl method) and the tBoc method (t-butyloxycarbonyl method). Chemical synthesis can also be carried out using a peptide synthesizer from Advanced ChemTech, Perkin Elmer, Pharmacia, Protein Technology Instrument, Synthecel Vega, PerSeptive, Shimadzu, etc.
  • Structural analysis of the purified protein of the present invention is carried out by a method usually used in protein chemistry, for example, a method described in Protein Structural Analysis for Gene Cloning (Hisashi Hirano, Tokyo Chemical Dojin, 1993). It is possible.
  • the method of immunization may be to administer the antigen directly to the animal subcutaneously, intravenously, or intraperitoneally, but to administer the antibody with a highly antigenic carrier bound thereto, or to administer the antigen with an appropriate adjuvant. Is preferred.
  • carrier proteins keyhole limpet and keyhole limbe Het hemosinin, bovine serum albumin, bovine thyroglobulin, and the like.
  • adjuvants include Complete Freund's Adjuvant, aluminum hydroxide gel, and pertussis vaccine.
  • Examples of the immunized animal include non-human mammals such as egrets, goats, mice aged 30 to 20 weeks, rats, and Hamichi Yuichi.
  • the antigen is administered 3 to 10 times every 1 to 2 weeks after the first administration.
  • the dose of the antigen is preferably 50 to 100 g per animal.
  • blood is collected from the fundus venous plexus or tail vein of the immunized animal, and the reactivity of the serum with the antigen is determined by enzyme immunoassay [enzyme immunoassay (ELISA): published by Medical Shoin. (1976 years)].
  • a non-human mammal whose serum shows a sufficient antibody titer is used as a source of serum or antibody-producing cells.
  • a polyclonal antibody can be prepared by separating and purifying the serum.
  • Monoclonal antibodies are prepared by fusing the antibody-producing cells with myeloma cells derived from non-human mammals to produce hybridomas, and culturing the hybridomas or administering the hybridomas to animals to cause the cells to develop ascites carcinoma. It can be prepared by separating and purifying the culture solution or ascites.
  • Antibody-producing cells are collected from antigen-administered non-human mammalian splenocytes, lymph nodes, peripheral blood, and the like.
  • mice 8-azaguanine-resistant mice (derived from BALB / c) myeloma cell line P3-X63Ag8-U1 (P3-U1) [Europ. J. Immunol. 6, 511 (1976)), SP2 / 0-Agl4 (SP-2) CNature, 276, 269 (1978)), P3-X63-Ag8653 (653) (J. Immunol., 123, 1548 (1979)), Any myeloma cells that can grow in vitro, such as P3-X63-Ag8 (X63) [Nature, 256, 495 (1975)], may be used.
  • Antibodies-A Laboratory Manual For culture and passage of these cell lines In accordance with the Antibodies-A Laboratory Manual (Cold Spring Harbor Laboratory, (1988), hereinafter abbreviated as Antibodies-A Laboratory Manual), until the time of cell fusion. Ensure a cell count of at least 2 x 10 7 cells.
  • a cell-aggregating medium such as polyethylene glycol 1000 (PEG-1000) is added, and the cells are fused and suspended in a medium.
  • PEG-1000 polyethylene glycol 1000
  • MEM medium or PBS 1.83 g of sodium phosphate, 0.21 g of phosphate monophosphate, 7.65 g of salt, 1 liter of distilled water, pH 7.2
  • HAT medium normal medium (1.5 ml ol / L glutamine, 5 xl (T3 ⁇ 4ol / L) in RPMI-1640 medium) so that only the desired fused cells can be selectively obtained.
  • a part of the culture supernatant is removed, and a sample that reacts with the antigen protein but does not react with the non-antigen protein is selected by enzyme immunoassay. Then, the clone is performed by the limiting dilution method, and those with a stable and high antibody titer determined by the enzyme immunoassay are selected as monoclonal antibody-producing hybridoma strains.
  • the antigen protein or cells expressing the antigen protein are coated on a 96-well plate, and the hybridoma culture supernatant or the purified antibody obtained by the above method is reacted as the primary antibody.
  • the plate After the first antibody reaction, the plate is washed and the second antibody is added.
  • the second antibody is an antibody obtained by labeling an antibody capable of recognizing the immunoglobulin of the first antibody with biotin, an enzyme, a chemiluminescent substance, a radiation compound, or the like. Specifically, if mice were used for hybridoma production, as the second antibody, An antibody that can recognize prolobulin is used.
  • a reaction is performed according to the substance labeled with the second antibody, and the antibody is selected as a hybridoma that produces a monoclonal antibody that specifically reacts with the antigen.
  • Monoclonal antibodies can be cultured in a culture solution obtained by culturing Hypridoma cells, or treated with pristane [0.5 ml of 2,6,10,14-tetramethylpenecane (Pristane) 0.5 ml intraperitoneally and reared for 2 weeks
  • pristane 0.5 ml of 2,6,10,14-tetramethylpenecane (Pristane) 0.5 ml intraperitoneally and reared for 2 weeks
  • the monoclonal antibody-producing hybridoma cells are intraperitoneally administered to the 8- or 10-week-old mouse or nude mouse to separate and purify the ascites from ascites cancer.
  • Methods for isolating and purifying monoclonal antibodies include centrifugation, salting out with 40-50% ammonium sulfate, caprylic acid precipitation, DEAE-Sepharose column, anion exchange column, protein A or G-column or There is a method in which chromatography using a gel filtration column or the like is performed alone or in combination. By this method, an IgG or IgM fraction can be collected to obtain a purified monoclonal antibody.
  • the subclass of the purified monoclonal antibody can be determined using a monoclonal antibody typing kit or the like.
  • the protein content can be calculated by the Lowry method or the absorbance at 280 nm.
  • the antibody subclass is an isotype within the class, and includes IgGl, IgG2a, IgG2b, and IgG3 in mice, and IgGK IgG2, IgG3, and IgG4 in humans.
  • Methods for detecting abnormalities such as deletion of the HD-PTP gene, changes in copy number, and chromosomal translocation include the Southern hybridization method.
  • the HD-PTP MA as a probe and Southern 'hybridization of chromosomal DNA cut with an appropriate restriction enzyme, abnormalities such as deletion of the HD-PTP gene, change in copy number, and chromosomal translocation were confirmed. can do.
  • Methods for detecting mutations such as substitution, deletion, and addition of the base sequence of the HD-PTP gene include Southern hybridization, PCR, and single-strand conformation polymoprphism (SSCP) (Pro Natl. Acad. Sci. USA, 86, 2766 (1989)].
  • SSCP single-strand conformation polymoprphism
  • cancer can be diagnosed by Southern hybridization analysis of chromosomal DNA with an oligonucleotide probe that hybridizes with the mutation site.
  • the hybrid method is a method in which a large number of panels with human chromosome fragments such as Gene-Bridge 4 are analyzed using a chromosome marker to determine which chromosome fragments are included. Performs polymerase chain reaction (PCR), which specifically amplifies the PTP gene, and identifies the detailed chromosomal location by analyzing the amplification results. It is a method.
  • PCR polymerase chain reaction
  • a signal hybridized to a human chromosome sample using HD-PTP DNA as a probe is detected, and the position of the signal on the sample is identified.
  • Probes are labeled with the radioactive isotope 3 H or biotin, 3 ⁇ 4-labeled by autoradiography, and biotin-labeled by avidin labeled with the fluorescent dye FITC (fluorescein isothiocyanate). Can be detected.
  • HD-PTP can be produced and obtained by the method described in [2].
  • the abnormalities of the HD-PTP gene are involved in carcinogenesis, and it is thought that the protein encoded by the abnormal gene is caused by carcinogenesis. It can be used as a cancer treatment.
  • the medicament containing the HD-PTP of the present invention can be administered alone as a therapeutic agent, but is usually mixed with one or more pharmacologically acceptable carriers to obtain a pharmaceutical preparation. It is desirable to provide it as a pharmaceutical preparation produced by any method well known in the art.
  • intravenous administration can be preferably mentioned.
  • Dosage forms include sprays, capsules, tablets, granules, syrups, emulsions, suppositories, injections, ointments, tapes and the like.
  • Formulations suitable for oral administration include emulsions, syrups, capsules, tablets, powders, and granules.
  • Liquid preparations such as emulsions and syrups can be prepared with water, sucrose, sorbitol, Sugars such as fructose, glycols such as polyethylene glycol and propylene glycol, oils such as sesame oil, olive oil, soybean oil, preservatives such as P-hydroxybenzoic acid ester, flavors such as strawberry flavor, peppermint, etc. Can be produced as an additive.
  • Capsules, tablets, powders, granules, etc. are excipients such as lactose, glucose, sucrose, mannitol, disintegrants such as starch and sodium alginate, lubricants such as magnesium stearate, talc, polyvinyl alcohol It can be produced using a binder such as hydroxypropylcellulose and gelatin, a surfactant such as fatty acid ester, and a plasticizer such as glycerin as additives.
  • Formulations suitable for parenteral administration include injections, suppositories, sprays and the like.
  • the injection is prepared using a carrier comprising a salt solution, a glucose solution, or a mixture of both.
  • Suppositories are prepared using carriers such as cocoa butter, hydrogenated fats or carboxylic acids. Sprays are prepared using the compound itself or a carrier which does not irritate the oral and respiratory tract mucosa of the recipient and disperses the compound as fine particles to facilitate absorption.
  • the carrier include lactose and glycerin.
  • Formulations such as aerosols and dry powders are possible depending on the properties of the compound and the carrier used.
  • the components exemplified as additives for oral preparations can be added.
  • the dose or frequency of administration varies depending on the desired therapeutic effect, administration method, treatment period, age, body weight, etc., but is usually 1 Ozg / kg Smg / kg per adult per day.
  • HD-PTP can be detected using the antibody described in [3].
  • Specific examples include an ELISA method using a microplate and a detection method using a fluorescent antibody method, a western blot method, and immunohistological staining.
  • HD-PTP can be quantified using the antibody described in [3]. Specifically, among the antibodies that react with HD-PTP in a liquid phase, Sanditsuchi ELISA method Ebito one flop using two different monochromatic one monoclonal antibody, labeled with a radioisotope such as 125 1 HD- The radioimmunoassay method using an antibody that recognizes PTP and HD-PTP can be used. BRIEF DESCRIPTION OF THE FIGURES
  • FIG. 1 shows a restriction enzyme map of the inserted DM fragment of chromosome DM clone SA2-26 of cell line KALI.
  • the numbers inside the figure represent the length of each ⁇ RI fragment in kb, and the gray fragment indicates the fragment hybridized with the Alul probe.
  • the lower line in the figure shows the positions of the ten types of fragments used for the probe of the KALI mRNA Northern blot 'hybridization.
  • the fragments marked with * are fragments that are hybridized with KALI mRNA and detected a band of about 6 kb, which are thought to contain exons.
  • FIG. 2 is a diagram showing the structure of a phage vector APSLl used for the preparation of a cDNA library of MKN45 and the cDNA insertion position.
  • H ⁇ dII I
  • N restriction enzyme site
  • AJ indicates a group of genes located in genes A to J of ⁇ phage.
  • KH54 and nin5 are genetic markers used in the input phage vector.
  • the structure of the plasmid vector pCDL81 used to construct PSL1 is shown below.
  • SR SR-ichi Promo
  • PolyA polyA signal
  • hph hygromycin resistance gene
  • Ap ampicillin Indicates a resistance gene.
  • FIG. 3 is a diagram showing a comparison of the amino acid sequences of the tyrosine phosphatase domains of HD-PTP and another protein tyrosine phosphatase (PTP).
  • PTP-1B P18031 (35-279)
  • PTP-HI P26045 (665-903)
  • MEG1 P29074
  • PTP-HI P18433 (260-503)
  • PTP-? P23467 (1722-- 1965
  • PTP- ⁇ ⁇ 23468
  • PTP-e ⁇ 23469
  • LAR P10586 (1360 to 1599)
  • PTP-P P23470 (869 to 1121)
  • PTP-S P23471 (1744 to 1993)
  • PTP-2C Q06124
  • FIG. 4 is a view showing a comparison of amino acid sequences between HD-PTP and rat PTP-TD14.
  • the upper row shows the amino acid sequence of HD-PTP, and the lower row shows amino acids that match in the amino acid sequence of rat PTP-TD14 by *, and the amino acids that do not match are shown.
  • the numbers indicate the position from the N-terminus of each protein.
  • FIG. 5 is a schematic diagram showing the structure of HD-PTP protein.
  • A shows the primary structural characteristics of the HD-PTP protein, where N is the N-terminal side and C is the C-terminal side.
  • the numbers below indicate the amino acid sequence numbers.
  • Y is the position of a tyrosine residue that can be phosphorylated by tyrosine kinase;
  • S is the position of an amino acid residue that can be phosphorylated by MAP kinase, which is a serine threonine kinase;
  • SHB can bind to SH3 Motifs Histidine
  • the line in the main shows the repeating structure for taking the Zn-leaf-like structure.
  • FIG. 6 is a view showing a comparison between the amino acid sequences of HD-PTP and BR01. The upper row shows the amino acid sequence of HD-PTP, and the lower row shows the amino acid sequence of BR01. The corresponding amino acids are indicated by I, and similar amino acids are indicated by. The numbers indicate the position from the N-terminus of each protein.
  • human chromosomal DNA was isolated by proteinase KZ phenol-monoclonal form extraction method (Molecular 'Cloning 2nd Edition). The DNA was purified by ethanol precipitation (Molecular Cloning, 2nd edition).
  • Rat fibroblast-based cell line Rat-2 CVirology, 113, 408 (1981), ATCC CRL-1764] was cultured on three 10-cm dishes in Dulbecco's modified MEM medium containing 5% pup serum. did.
  • the cells were cultured for 32 hours, and the obtained cultured cells per dish were applied to eight culture dishes (24 in total). After culturing the cells for 16 hours, The culture was continued with the addition of hygromycin at a concentration of 250 g / ml. Cells into which the DNA has been introduced become hygromycin-resistant and grow in a hygromycin-containing medium to form colonies. As a result of the culture, about 400 colonies were formed.
  • Chromosomal DNA was extracted from the isolated cells using the method described above. After 10 g of the DNA was cleaved with ⁇ RI, agarose gel electrophoresis was performed. After the electrophoresis, the DNA in the gel is transcribed at the same time, and the KAL-1 chromosomal DNA labeled with 32 P by the random primer method is a probe corresponding to the human Alu sequence (hereinafter referred to as the Alu sequence probe). ) was used to perform Southern hybridization, and human DNA contained in the colonies was analyzed.
  • the DNA was co-transfected into plasmid pHyg and Rat-2 cells, and hygromycin-resistant colonies were selected.
  • chromosomal DNA was extracted from the obtained colonies, the extracted DNA was digested with ⁇ RI, agarose gel electrophoresis was performed, and the DNA in the gel was transcribed in a filter, followed by Southern Alu sequence probe. Hybridization was performed to select six independent colonies with a 7 kb ⁇ RI fragment that hybridized in the same manner as the DNA from the first four colonies.
  • the chromosomal DNA from the colony is partially cut with ⁇ RI, and the DNA fragment and human DNA —Ligation with the ⁇ RI-cut arm of DASH II (stratagene), which is a divector, and after in vitro packaging, transfer to E. coli strain LE392 [Molecula 1-cloning 2nd edition] Was prepared.
  • the 6 ⁇ 10 5 plaque was screened by plaque hybridization using the same human Alu sequence probe as described above, and two positive clones having an inserted DNA of about 20 kb were isolated.
  • the inserted DM was cut into 6 fragments of 7.2 kb, 7.0 kb, 3.1 kb, 1.5 kb, l. Ok 0.2 kb with ⁇ RI, and 3 fragments (7.2 kb, 7.0 kb, 0.2 kb) Hybridized with the Alu sequence probe, and the remaining three fragments (3.1 kb, 1.0 kb, 1.5 kb) did not hybridize with the Alu sequence probe.
  • Fig. 1 was created for one of these positive clones (human SA2-26).
  • the DNA fragments cut with each restriction enzyme were subjected to Southern blot hybridization with an Alu sequence probe.
  • the following 10 types of fragments did not hybridize with the Alu sequence probe and were determined to contain no Alu sequence.
  • PSL1 As a vector, PSL1 was used. PSL1 was constructed by the following method.
  • PCDL81 described later in Example 7 was digested with the restriction enzyme ⁇ dlI. Two copies of the obtained DNA fragment of about 7.9 kb were ligated and the MA was inserted between the ⁇ dlI sites of DASH II (Stratagene) to construct a vector PSL1.
  • the structure of the input PSL1 is shown in FIG. Furthermore, as shown in FIG. 2, the PSL1 was cut with l, the cDNA was inserted into the l-cut DNA fragment from which pCDL81 had been removed by one equivalent, and the plasmid was inserted into the host E. coli strain LE392 to insert the cDNA. A library was prepared. This method eliminates short phage clones since little phage growth is observed when 0.5 to 13 kb of DNA is not inserted into the vector.
  • plaque hybridization was performed on the above-mentioned cDNA library according to a conventional method (molecular cloning, second edition). As a result, two positive clones [cKALll (4 kb) and cKAL16 (5.4 kb)] were obtained.
  • the nucleotide sequence of CKAL16 is shown in SEQ ID NO: 4.
  • An open 'reading' frame (0RF) encoding 1253 amino acids was present at base numbers 1-3759 of the base sequence.
  • cKALll had a structure in which an intron was inserted into the base sequence of cKAL16. That is, the nucleotide numbers 141 to 529, 676 to 960, 1273-1347, 1655- of SEQ ID NO: 5: 1740, 3680-3773, 3959-4084, 4190-4273, 413-4504, 4619 The ⁇ 4696th intron had been inserted.
  • cKALll is considered to be a cDNA clone derived from incompletely spliced mRNA, with the same gene as CKAL16, but with introns remaining.
  • the region from 829 to 1071 on the C-terminal side of the amino acid sequence of 0RF in the cDNA of CKAL16 contains a region homologous to the phosphatase region of another PTP, as shown in Fig. 3. It was considered the encoding cDNA.
  • the CKAL16 0RF starts from the 5 'end of the cDNA and has no 5' untranslated region, and its amino acid sequence shows high homology with the amino acid sequence of the PTP-TD14 from the 240th position onwards. Is not a full-length cDNA clone, and it was considered that the full-length cDNA was further extended to the 5 'end.
  • a cDNA fragment further extended to the 5 'end was amplified by the 5, -RACE method [Proc. Natl. Acad. Sci. USA, 85, 8998 (1988)]. Cloned. The nucleotide sequence of the cDNA fragment was determined, and the nucleotide sequence linked to the nucleotide sequence of CKAL16 was used as the nucleotide sequence of the full-length cDNA, and is shown in SEQ ID NO: 1.
  • nucleotide sequence of SEQ ID NO: 1 By comparing the nucleotide sequence of SEQ ID NO: 1 with the nucleotide sequence of cKALll, it was found that the 1st to 74th nucleotides of the nucleotide sequence of cKALll were also intron sequences. At positions 64 to 4971 in the nucleotide sequence of SEQ ID NO: 1, there is 0RF consisting of 1636 amino acids. This region was designated as a region encoding a novel human PTP, and the amino acid sequence is shown in SEQ ID NO: 2. When the amino acid sequence of this new human PTP is compared with the rat PTP-TD14 described above, the N-terminus is 180 amino acids longer than that of rat PTP-TD14. Although there was an input deletion, it had homology. The homology of the amino acid sequences of the 181st position and thereafter was 83%.
  • a phosphatase region having homology to another PTP was present at the C-terminal 1212 to 1454 positions of the amino acid sequence of the novel human PTP.
  • the conserved amino acid sequence (Val His Cys Ser Ala Gly (Val / Ile) Gly Arg (Thr / Ser) Gly ⁇ J. Biol. Chem. , 267, 140 (1992)] had been converted to serine.
  • At the 770th to 1128th there was a region consisting of histidine or cysteine at the head, and a structure consisting of a proline-rich 20 to 50 amino acid repeated 15 times.
  • the repeat region is named a histidine domain (His Domain; HD), and the new PTP is called HD-PTP.
  • His Domain His Domain
  • the N-terminal 778 amino acids also have homology to BR01 CMol. Cell. Biol., 16, 2585 (1996), a protein involved in the yeast MAP kinase signaling pathway.
  • BR01, rat PTP-TD14 having homology with BR01
  • the R10E12.1 gene (based on nucleotide sequence analysis of chromosome 3 of C. elegans)
  • tyrosine residues at positions 414, 665, 679, 922, 924, 998, 990, and 1243 of SEQ ID NO: 2 have a tyrosine residue that may be phosphorylated by tick synthase.
  • HD-PTP interacts with other proteins and has the potential to be involved in intracellular signal transduction.
  • the HD-PTP gene was found to be located between the markers indicating the location on the chromosome of Whitehead Research Institute-WI-11814 and WI-16174. This position was between the markers D3S3888 (telomere side) and D3S3334 (centromere side) located at 3p21.3 on the short arm of chromosome 3 (3p).
  • the 3p21.3 chromosome position coincided with the location where L0H is frequently found in lung cancer, anatomical cancer, and colon cancer. This position is 1.6 cM (centimorgan) on human chromosome 3 that is commonly deleted in hybrid fusion cells of human chromosome 3 and mouse A9 cells, such as those that are neoplastic in SCID mice.
  • the gene was considered to be a tumor suppressor gene present in 3p21.3, which was located in the region [Genes, Chromosome & Cancer, 20, 329 (1997)].
  • cKALll obtained in Example 1 was a cDNA clone in which introns remained, it was thought that most of the sequences matched the nucleotide sequence of genomic DNA, but some introns may have been spliced. There is also. Therefore, human genomic DNA is converted into type II, a fragment containing each of the possible exons of cKALll is amplified by PCR, and the nucleotide sequence is compared with the nucleotide sequence of cKALl1, whereby an intron is formed in the exon. Checked whether there is no.
  • the nucleotide sequence of SEQ ID No. 4 is 1 intron consisting of 9 in the exon corresponding to positions 1348 to 1654. It was found that one intron consisting of 93 bp was further present in the exons corresponding to positions 1741 to 3679. Further, the nucleotide sequence of the HD-PTP genomic DNA, which is further on the 5th side from the HD-PTP genomic DNA obtained by the above-described method, was compared with the primer using the nucleotide sequence of the HD-PTP cDNA obtained in Example 1. Analyzed by walking.
  • the cDNA having the nucleotide sequence of SEQ ID NO: 1 was composed of 25 exons, and that 24 introns were inserted between the exons.
  • the first intron between the first and second exons is about 5.5 kb
  • the second intron between the second and third exons is about 8 kb, which is as long as the first to twenty-fifth exons. Up to about 22kb.
  • SEQ ID NO: 40 is the region at the 5 'end of the first exon and the adjacent first intron
  • SEQ ID NO: 41 is the third exon and the third and second ends of the adjacent first intron.
  • the nucleotide sequence of the region at the 5 'end of the intron, SEQ ID NO: 3, shows the 3' end of the second intron and the regions from the third exon to the 25th exon adjacent thereto.
  • L0H on chromosome 3p21.3 in lung cancer patients Based on the method described in the literature [Cancer Res., 57, 1344 (1997)], microsatellite D3S3564, D3S3559, D3S3582, and D3S1568, which are microsatellite markers present in 3p21.3 of the human chromosome, were determined by PCR. By analyzing polymorphisms, 30 pairs of cancer tissues and normal tissues of lung cancer patients were examined for the presence or absence of L0H around 3 ⁇ 21.3. As a result, about 40% of the cancer tissues examined had deletions of one or more markers.
  • small cell lung cancer cell lines (Lu-130 (RIKEN Cell Bank RCB0465), Lu-135 (RIKEN Cell Bank RCB0468), NCI- H69 (ATCC HTB-119), NCI-H82 (ATCC HTB-175), NCI-N417 (ATCC CRL-5809), RERF-LC-MA (JCRB / HSRRB cell bank JCRB0812), SBC-5 (JCRB / HSRRB cells) Bank JCRB0819), NCI-H526 (ATCC CRL-5811), NCI-H209 (ATCC HTB-172), NCI-H841 (ATCC CRL-5845), NCI-H774 (ATCC CRL-5842), MS-18) PCR was performed using DNA as type III. After PCR, polyacrylamide gel electrophoresis was performed under non-denaturing conditions using the obtained reaction sample. After swimming, the gel was stained with silver.
  • the nucleotide sequence was determined using PCR primers.
  • Northern blot was performed using 20 ⁇ g of total RNA or 2 11 of 0 11 as a probe and using cKAL16 obtained in Example 1 as a probe.
  • cKAL16 obtained in Example 1 as a probe.
  • cell lines MKN74 and KAT0III no band was observed and it was considered that the HD-PTP gene was not expressed.
  • RNA was extracted from the adrenal gland by a conventional method, and Northern blot'hybridization was performed according to the method described in Example 5, using CKAL16 obtained in Example 1 as a probe.
  • HD-PTP is an intracellular phosphatase and is considered to be ubiquitous in all organs, it is likely that HD-PTP plays an important role in intracellular signal transduction .
  • SR T promoter a promoter derived from the LTR of human T-cell leukemia virus I (Mol. Cell. Biol., 8, 466 (1988)]
  • pCDL81 a vector for animal cell expression.
  • the multiple cloning site was prepared by synthesizing the nucleotide sequences shown in SEQ ID NOs: 38 and 39 using a DNA synthesizer.
  • the HD-PTP cDNA of CKAL16 cloned in Example 1 was inserted into PCDL81 to prepare an HD-PTP expression plasmid pDKL4f.
  • the transformant Escherichia coli DH5 / pDKL4f containing the HD-PTP expression plasmid pDKL4f was purchased from the National Institute of Advanced Industrial Science and Technology, Tsukuba East, Ibaraki, Japan on September 11, 1998. Deposited at Postal Code 3 0 5—8 5 5 6) Chome 1-3 as FERM BP-6499.
  • the expression plasmid DNA was introduced into the mouse cell line NIH3T3 or the rat cell line Rat-2 using the calcium phosphate method, and transformed cells were selected for hygromycin resistance. DNA is extracted from the selected transformed cells by a conventional method, subjected to agarose gel electrophoresis, and the gel is transferred to a filter.Then, cKAL16 cDNA is used as a probe to perform Southern blot hybridization, and the transformed cells are treated. It was confirmed that the introduced human HD-PTP cDNA was retained. RNA was extracted from the transformed cells by a conventional method, and Northern 'plot' hybridization was performed in the same manner as in Example 4 to detect the expression of the introduced human HD-PTP gene.
  • the transformed cells in which the expression of the human HD-PTP gene was observed showed colony formation in soft agar as shown in Example 1. This colony forming ability was found to be stronger as the expression level of the HD-PTP gene was higher according to the Northern plot. 2 ⁇ 10 7 transformed cells were injected subcutaneously into irradiated athymic nude mice and observed for tumor formation for 60 days. None of the mice formed tumors after injection. Therefore, this colony-forming ability was considered to be independent of tumor-forming ability.
  • HD-PTP cDNA was inserted into the ⁇ I site of pEGFP-Nl (Clonetech), a fusion protein expression vector with GFP (Green fluorescent protein), and the GFP was fused to the C-terminal side of HD-PTP (HD -PTP / GFP fusion protein) was prepared.
  • the plasmid was introduced into a human cell line 293tsA1609neo [Mol. Cell. Biol., 7, 379 (1987)] to express the HD-PTP / GFP fusion protein.
  • the transformed cells were observed with a fluorescence microscope, and the intracellular localization of the HD-PTP / GFP fusion protein was examined by detecting the fluorescence of GFP.
  • the HD-PTP / GFP fusion protein was present in the cell membrane, particularly in the cytoplasm near the nucleus, in a plurality of clusters, but not in the cell membrane or nucleus. This suggested that it might be located in a specific intracellular organ.
  • the molecular weight estimated from the amino acid sequence of HD-PTP was about 150 kDa, and the HD-PTP / GFP fusion protein was detected at a position of 150 kDa close to the expected molecular weight by molecular weight measurement by SDS-PAGE.
  • the fusion protein was detected at 70 kDa on non-denaturing PAGE. It was concluded that HD-PTP had a very compact globular structure in its native, undenatured state.
  • novel lip synthase of the present invention and its gene enables the diagnosis and treatment of cancer.
  • SEQ ID NO: 6 Description of artificial sequence: Sense primer for amplification of HD-PTP gene
  • SEQ ID NO: 8 Description of Artificial Sequence: sense primer for amplification of positions 3692-3939 in SEQ ID NO: 3 of HD-PTP gene including exon 14
  • SEQ ID NO: 9 Description of artificial sequence: Antisense primer for amplification of position 3692-3939 in SEQ ID NO: 3 of HD-PTP gene including exon 14
  • SEQ ID NO: 10 Delivery of artificial sequence: Sense primer for amplification of positions 4159-4421 in SEQ ID NO: 3 of HD-PTP gene including exon 15
  • SEQ ID NO: 11 Description of artificial sequence: Antisense primer for amplification of positions 4159-4421 in SEQ ID NO: 3 of HD-PTP gene including exon 15
  • SEQ ID NO: 12 Description of Artificial Sequence: Sense primer for amplification of positions 4625-5002 in SEQ ID NO: 3 of HD-PTP gene including exon 16
  • SEQ ID NO: 13 Description of Artificial Sequence: Antisense primer for amplification of positions 4625-5002 in SEQ ID NO: 3 of HD-PTP gene including exon 16
  • SEQ ID NO: 14 Description of Artificial Sequence: sense primer for amplification of positions 5014-5473 in SEQ ID NO: 3 of HD-PTP gene including exons 17 and 18
  • SEQ ID NO: 15 Description of Artificial Sequence: Antisense Primer for Amplification of Positions 5014-5473 in SEQ ID NO: 3 of HD-PTP Gene Containing Exons 17 and 18
  • SEQ ID NO: 16 Description of Artificial Sequence: Sense Primer for Amplification of Positions 5499-5984 in SEQ ID NO: 3 of HD-PTP Gene Containing Part of Exons 19 and 20
  • SEQ ID NO: 17 Description of Artificial Sequence: Antisense Primer for Amplification of Positions 5499-5984 in SEQ ID NO: 3 of HD-PTP Gene Containing Part of Exons 19 and 20
  • SEQ ID NO: 18 Description of Artificial Sequence: Exon 20 Primer for amplification of positions 5943-6281 in SEQ ID NO: 3 of the HD-PTP gene containing a part of
  • SEQ ID NO: 19 Description of artificial sequence: Antisense primer for amplification of positions 5943-6281 in SEQ ID NO: 3 of HD-PTP gene containing part of exon 20
  • SEQ ID NO: 20 Delivery of Artificial Sequence: Sense primer for amplification of positions 6191-6579 in SEQ ID NO: 3 of HD-PTP gene containing part of exon 20
  • SEQ ID NO: 21 Delivery of Artificial Sequence: Antisense primer for amplification of positions 6191-6579 in SEQ ID NO: 3 of the HD-PTP gene containing part of exon 20
  • SEQ ID NO: 22 Description of Artificial Sequence: Sense primer for amplification of positions 6478-6908 in SEQ ID NO: 3 of the HD-PTP gene containing part of exon 20
  • SEQ ID NO: 23 Description of Artificial Sequence: Antisense primer for amplification of positions 6478-6908 in SEQ ID NO: 3 of HD-PTP gene containing part of exon 20
  • SEQ ID NO: 24 Description of Artificial Sequence: Sense primer for amplification of positions 6866-7290 in SEQ ID NO: 3 of HD-PTP gene containing part of exon 20
  • SEQ ID NO: 25 Delivery of Artificial Sequence: Antisense primer for amplification of positions 6866-7290 in SEQ ID NO: 3 of the HD-PTP gene containing part of exon 20
  • SEQ ID NO: 26 Description of Artificial Sequence: Sense primer for amplification of positions 7244-7639 in SEQ ID NO: 3 of the HD-PTP gene containing part of exon 20
  • SEQ ID NO: 27 Description of Artificial Sequence: Antisense primer for amplification of positions 7244-7639 in SEQ ID NO: 3 of the HD-PTP gene containing part of exon 20
  • SEQ ID NO: 28 Description of Artificial Sequence: Sense primer for amplification of positions 7628-7888 in SEQ ID NO: 3 of HD-PTP gene including exon 21
  • SEQ ID NO: 29 Description of Artificial Sequence: Antisense primer for amplification of positions 7628-7888 in SEQ ID NO: 3 of HD-PTP gene including exon 21
  • SEQ ID NO: 30 Description of artificial sequence: Sense primer for amplification of positions 7897-8131 in SEQ ID NO: 3 of HD-PTP gene including exon 22
  • SEQ ID NO: 31 Description of Artificial Sequence: Antisense primer for amplification of positions 7897-8131 in SEQ ID NO: 3 of HD-PTP gene including exon 22
  • SEQ ID NO: 32 Description of Artificial Sequence: Sense primer for amplifying positions 8091-8379 in SEQ ID NO: 3 of HD-PTP gene including exon 23
  • SEQ ID NO: 33 Description of artificial sequence: Antisense primer for amplification of positions 8091-8379 in SEQ ID NO: 3 of HD-PTP gene including exon 23
  • SEQ ID NO: 34 Description of Artificial Sequence: Sense primer for amplification of positions 8317-8562 in SEQ ID NO: 3 of HD-PTP gene including exon 24
  • SEQ ID NO: 35_Description of artificial sequence Antisense primer for amplification of positions 8317-8562 in SEQ ID NO: 3 of HD-PTP gene including exon 24
  • SEQ ID NO: 36 Description of Artificial Sequence: Sense Primer for Amplification of Positions 8492-8905 in SEQ ID NO: 3 of HD-PTP Gene Containing Part of Exon 25
  • SEQ ID NO: 37 Description of Artificial Sequence: An antisense primer for amplification of positions 8492-8905 in SEQ ID NO: 3 of the HD-PTP gene containing part of exon 25
  • SEQ ID NO: 38 Description of artificial sequence: Multicloning site ( ⁇ I Not 1 Xbal pnl
  • SEQ ID NO: 39 Description of artificial sequence: Multicloning site (Xhol Notl Xbal Kpnl BamHI) Synthetic DNA for linker

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Abstract

L'invention concerne une nouvelle tyrosine phosphatase codée par le site 3p21 du chromosome humain, dont la délétion est fréquemment observée dans le cancer des poumons chez l'homme, ce qui fait supposer que cette nouvelle tyrosine phosphatase renferme un anti-oncogène. L'utilisation de cette tyrosine phosphatase et de son gène permet donc de diagnostiquer et de traiter le cancer.
PCT/JP2000/002455 1999-04-16 2000-04-14 Nouvelle tyrosine phosphatase Ceased WO2000063392A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU36794/00A AU3679400A (en) 1999-04-16 2000-04-14 Novel tyrosine phosphatase

Applications Claiming Priority (2)

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JP10884299 1999-04-16
JP11/108842 1999-04-16

Publications (1)

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WO2000063392A1 true WO2000063392A1 (fr) 2000-10-26

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PCT/JP2000/002455 Ceased WO2000063392A1 (fr) 1999-04-16 2000-04-14 Nouvelle tyrosine phosphatase

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002046384A3 (fr) * 2000-12-06 2003-10-23 Incyte Genomics Inc Kinases et phosphatases

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998049317A2 (fr) * 1997-04-28 1998-11-05 Sugen, Inc. Diagnostic et traitement de troubles lies a la phosphatase ou a la kinase

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998049317A2 (fr) * 1997-04-28 1998-11-05 Sugen, Inc. Diagnostic et traitement de troubles lies a la phosphatase ou a la kinase

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
LINGUANG CAO ET AL.: "A novel putative protein-tyrosine phosphatase contains a BR01-like domain and suppresses Ha-ras-mediated transformation", THE JOURNAL OF BIOLOGICAL CHEMISTRY,, vol. 273, no. 33, August 1998 (1998-08-01), pages 21077 - 21083, XP002929073 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002046384A3 (fr) * 2000-12-06 2003-10-23 Incyte Genomics Inc Kinases et phosphatases

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