WO2001083738A1 - Facteur de regulation de differenciation et de determination du sexe - Google Patents

Facteur de regulation de differenciation et de determination du sexe Download PDF

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Publication number
WO2001083738A1
WO2001083738A1 PCT/JP2001/003728 JP0103728W WO0183738A1 WO 2001083738 A1 WO2001083738 A1 WO 2001083738A1 JP 0103728 W JP0103728 W JP 0103728W WO 0183738 A1 WO0183738 A1 WO 0183738A1
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Prior art keywords
protein
present
dna
seq
antibody
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PCT/JP2001/003728
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English (en)
Japanese (ja)
Inventor
Takashi Sugihara
Renu Wadhwa
Sunil C. Kaul
Osamu Ohara
Takahiro Nagase
Nobuo Nomura
Original Assignee
Chugai Seiyaku Kabushiki Kaisha
Kazusa Dna Research Institute
National Institute Of Advanced Industrial Science And Technology
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Priority to AU2001252651A priority Critical patent/AU2001252651A1/en
Publication of WO2001083738A1 publication Critical patent/WO2001083738A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • 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
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • 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
    • C12Q1/6879—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for sex determination
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
    • G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
    • G01N2333/4701—Details
    • G01N2333/4703—Regulators; Modulating activity

Definitions

  • the present invention relates to genes and proteins involved in the mechanism of sex determination, and their production and use. Background art
  • Fem-1A gene is present on chromosome mouse # 17 and is highly expressed in skeletal muscle cells.
  • Fem-1B is located on mouse chromosome 9 and is expressed in mature, testis cells.
  • human Fem-1B having 99% homology at the amino acid level with mouse Fem-1B has been cloned (Ventura-Holman T, et al. Biochem Biophys Res Co. un. 2000 Jan. 7; 267 (l): 317-20).
  • the present inventors examined the expression of protein in testis tissue using the antibody of unknown protein p33 (Wadhwa R, et al. (1991) Mutat Res 256: 243-254) that causes immortalization. was found to stain sperm cells, the final differentiation point in the testis (Fig. 1). When the gene of the protein stained with this antibody was tried using the immunoscreening method, multiple testis-specific genes were isolated (Fig. 2).
  • the present inventors proceeded with analysis of a gene fank having a novel sequence among these isolated genes.
  • a database search was performed using the nucleotide sequence information of the fank gene, no gene having a nucleotide sequence showing significant homology was found on the database.
  • a database was searched using the amino acid sequence information of the protein encoded by the fank gene, a part of the structure of the protein was identified by F. elegans FEM- l (sex determination factor-1) (27% homology to mouse FEM-la protein, 25% homology to FEM-lb protein) .
  • the present invention relates to the fank gene and the protein encoded by the gene, molecules related to these molecules, and their production and use.
  • the present invention provides
  • a polynucleotide comprising at least 15 nucleotides complementary to a DNA consisting of the nucleotide sequence of SEQ ID NO: 1 or a complementary strand thereof, and
  • a method for examining abnormal DNA expression according to (1) comprising: (a) preparing a sample from a testis or ovary of a subject;
  • step (c) comparing the measured value obtained in the step (b) with the measured value in a healthy subject.
  • the present invention provides a gene “fank” encoding a novel protein that is thought to regulate the differentiation of spermatogonia into spermatocytes.
  • the nucleotide sequence of human-derived fank cDNA is shown in SEQ ID NO: 1, and the amino acid sequence of the protein encoded by the cDNA is shown in SEQ ID NO: 2.
  • human-derived fank cDNA has 0RF encoding a protein consisting of 617 amino acids.
  • fank Since the gene expression of fank is low in infertile mice, it can be applied to the prevention and treatment of infertility diseases caused by impaired cell differentiation such as testicular and ovarian dysgenesis in males and females.
  • fank is considered to have a similar structure to the sex determinant in e7e a / 2s. By enhancing the function, it is expected to be applied to production management such as gender separation.
  • the present invention also includes a protein that is functionally equivalent to the fank protein (a protein consisting of the amino acid sequence of SEQ ID NO: 2).
  • Such proteins include, for example, mutants of the fank protein, homologs of organisms other than mice, and the like.
  • “functionally equivalent” means that the target protein has a biological function equivalent to that of the fank protein.
  • the biological function of the fank protein includes, for example, a function that controls sex determination.
  • a protein functionally equivalent to the protein can be prepared.
  • Amino acid mutations can also occur in nature.
  • a protein comprising one or more amino acids in the amino acid sequence of the fank protein which is altered and functionally equivalent to the protein is also included in the present invention.
  • the number of amino acids to be mutated in such a mutant is usually within 30 amino acids, preferably within 15 amino acids, and more preferably within 5 amino acids (eg, within 3 amino acids).
  • the amino acid residue to be mutated is desirably mutated to another amino acid that preserves the properties of the amino acid side chain.
  • the properties of amino acid side chains include hydrophobic amino acids (A, I, L, M, F, P, W, Y, V), and hydrophilic amino acids (R, D, ⁇ , C, ⁇ , Q, G, H, K, S, T), amino acids with aliphatic side chains (G, A, V, L, I, P), amino acids with hydroxyl-containing side chains (S, ⁇ , ⁇ )
  • Proteins in which a plurality of amino acid residues are added to the amino acid sequence of the fank protein include fusion proteins containing these proteins.
  • the fusion protein is a fusion of these proteins and another peptide or protein, and is included in the present invention.
  • the frame encoding the DNA encoding the fank protein (the protein consisting of the amino acid sequence of SEQ ID NO: 2) and the DNA encoding the other peptide or protein should match. This can be introduced into an expression vector, and expressed in a host.
  • Other peptides or proteins to be fused with the protein of the present invention are not particularly limited.
  • peptides to be fused with the protein of the present invention include, for example, FLAG (Ho ⁇ , ⁇ . ⁇ . et ah, BioTechnology (1988) 6, 1204-1210), and 6 His (histidine).
  • HA agglutinin
  • proteins to be fused with the protein of the present invention include, for example, GST (Glutathione-S-transferase), HA (Influenza agglutinin), immunoglobulin constant region, ⁇ —Galactosidase, MBP (maltose binding protein) and the like.
  • a fusion protein can be prepared by fusing a commercially available DNA encoding the peptide or protein with a DNA encoding the protein of the present invention and expressing the fusion DNA prepared thereby. .
  • the present invention includes a protein that encodes a DNA that hybridizes with the DNA encoding the fank protein and is functionally equivalent to the fank protein.
  • proteins include, for example, homologs derived from human and mouse and other mammals (for example, rat, magpie, magpie, etc.).
  • the conditions of hybridization for isolating MA encoding a protein functionally equivalent to the fank protein can be appropriately selected by those skilled in the art.
  • the conditions for high predication include, for example, low stringency conditions.
  • Low stringency conditions are the conditions of 10% formamide, 5 x SSPE, lx dend ol solution, and IX salmon sperm MA.
  • the conditions are 25% formamide, 5 ⁇ SSPE IX Denhardt's solution, and IX salmon sperm DNA.
  • More preferable conditions for high predication include high stringency conditions.
  • High stringency conditions include, for example, 50% formamide, 5 x SSPE, lx den Hart solution, lx salmon sperm MA conditions.
  • a gene amplification method using a primer synthesized based on the sequence information of DNA (SEQ ID NO: 1) encoding the fank protein such as the polymerase chain reaction (PCR) method, is used to synthesize the fank protein with the fank protein. It is also possible to isolate DNA encoding functionally equivalent proteins.
  • the proteins functionally equivalent to the fank protein which are encoded by the DNA isolated by these hybridization techniques and gene amplification techniques, are usually a fank protein (a protein consisting of the amino acid sequence of SEQ ID NO: 2) and an amino acid sequence. Has high homology.
  • the protein of the present invention also includes a protein that is functionally equivalent to the fank protein and has high homology to the amino acid sequence of the protein. High homology usually means at least 50% or more identity, preferably 75% or more identity, more preferably 85% or more identity, and even more preferably 95% or more identity at the amino acid level. .
  • the amino acid sequence and nucleotide sequence identity can be determined by the algorithm described in the literature (Wilbur, WJ and Lip, DJ Proc. Natl. Acad.
  • the protein of the present invention may differ in amino acid sequence, molecular weight, isoelectric point, presence / absence and form of sugar chains, etc., depending on the cell, host, or purification method for producing the protein described below. However, as long as the obtained protein has the same function as the fank protein, it is included in the present invention. For example, when the protein of the present invention is expressed in a prokaryotic cell, for example, Escherichia coli, a methionine residue is added to the N-terminal of the amino acid sequence of the original protein. ⁇ The protein of the present invention also includes such a protein. .
  • the protein of the present invention can be prepared as a recombinant protein or a natural protein by methods known to those skilled in the art. If it is a recombinant protein, a DNA encoding the protein of the present invention (for example, a DNA having the nucleotide sequence of SEQ ID NO: 1) is inserted into an appropriate expression vector, and this is introduced into an appropriate host cell. After recovering the transformant and obtaining an extract, the extract is subjected to chromatography such as ion exchange, reverse phase, gel filtration, or affinity chromatography in which an antibody against the protein of the present invention is immobilized on a column. It is possible to purify and prepare by applying or by combining a plurality of these columns.
  • chromatography such as ion exchange, reverse phase, gel filtration, or affinity chromatography
  • the protein of the present invention is expressed in a host cell (for example, an animal cell or Escherichia coli) as a fusion protein with glutathione S-transferase protein or as a recombinant protein to which a plurality of histidines are added.
  • a host cell for example, an animal cell or Escherichia coli
  • the expressed recombinant protein can be purified using a glutathione column or a nickel column. After purification of the fusion protein, if necessary, regions of the fusion protein other than the target protein can be cleaved with thrombin or Factor Xa and removed.
  • the protein is a natural protein
  • a method known to those skilled in the art for example, an antibody that binds to the protein of the present invention (described below) against an extract of a tissue or a cell expressing the protein of the present invention (for example, testis) may be used. Purification by the action of the bound affinity column Can be isolated.
  • the antibodies may be polyclonal or monoclonal.
  • the present invention also includes partial peptides of the protein of the present invention.
  • the partial peptide of the present invention can be used, for example, for preparing an antibody against the protein of the present invention, screening for a compound that binds to the protein of the present invention, or screening for an accelerator or inhibitor of the protein of the present invention. In addition, it can be an antagonist of the protein of the present invention or a competitive inhibitor.
  • the partial peptide of the present invention When used as an immunogen, the partial peptide of the present invention has an amino acid sequence of at least 7 amino acids or more, preferably 8 amino acids or more, and more preferably 9 amino acids or more. When used as a competitive inhibitor of the protein of the present invention, at least 100 amino acids or more, preferably 200 amino acids or more, more preferably 300 amino acids or more
  • the partial peptide of the present invention can be produced by genetic engineering techniques, known peptide synthesis methods, or by cleaving the protein of the present invention with an appropriate peptidase.
  • the peptide may be synthesized by, for example, either a solid phase synthesis method or a liquid phase synthesis method.
  • the DNA encoding the protein of the present invention is used for the production of the protein of the present invention in iflvi TO and J'i3iiro as described above, and is also caused by, for example, an abnormality in the gene encoding the protein of the present invention.
  • Application to gene therapy of a disease or a disease that can be treated by the protein of the present invention is also considered.
  • the DNA of the present invention may be in any form as long as it can encode the protein of the present invention. That is, it does not matter whether it is cDNA synthesized from mRNA, genomic DNA, or chemically synthesized DNA. Further, as long as it can encode the protein of the present invention, a DNA having an arbitrary nucleotide sequence based on the degeneracy of the genetic code is included.
  • the DNA of the present invention can be prepared by a method known to those skilled in the art.
  • a cDNA library is prepared from cells expressing the protein of the present invention, It can be prepared by using a part of the DNA sequence (for example, SEQ ID NO: 1) as a probe and performing hybridization.
  • the cDNA library may be prepared, for example, by the method described in the literature (Sam brooK, J. et al., Molecular Cloning, Cold Spring Harbor Laboratory Press (1989)), or using a commercially available MA library. Good.
  • RNA is prepared from cells expressing the protein of the present invention
  • cDNA is synthesized using reverse transcriptase
  • oligo DNA is synthesized based on the sequence of the DNA of the present invention (for example, SEQ ID NO: 1).
  • it can be prepared by performing a PCR reaction using this as a primer and amplifying cDNA encoding the protein of the present invention.
  • Genomic DM can be isolated by screening the genomic DNA library using the obtained cDNA as a probe.
  • mMA is isolated from cells, tissues, and organs (for example, testis) that express the protein of the present invention.
  • mRNA can be isolated by a known method, for example, guanidine ultracentrifugation (Chirgwin, J.M. et al., Biochemistry (1979) 18,
  • the total RNA was prepared by the AGPC method (Chomczynski, P. and Sacchi J., Anal. Biochem. (1987) 162, 156-159), etc., and the total RNA was prepared using the mRNA Purification Kit (Pharmacia). Purify mRNA from RNA. Also, QuickPrep mRNA Purification
  • MRM can also be prepared directly by using Kit (Pharmacia).
  • CDNA is synthesized from the obtained mRNA using reverse transcriptase.
  • cMA synthesis AMV
  • Reverse transcriptase First-strand cDNA Synthesis Kit (Shodaigaku Kogyo) can be used.
  • the 5-RACE method Frohman, MA et al.
  • the 5 Ampli FINDER RACE Kit manufactured by Clontech
  • the polymerase chain reaction polymerase chain reaction; PCR.
  • cMA can be synthesized and amplified.
  • the resulting DNA fragment of interest is prepared from the PCR products, and c is connected to the vector one DNA, than this to prepare a recombinant vector, the desired recombinant vector was selected colonies and then is introduced into E. coli etc. Prepare.
  • the nucleotide sequence of the desired MA can be confirmed by a known method, for example, the dideoxynucleotide chain-initiation method.
  • a nucleotide sequence having higher expression efficiency can be designed in consideration of the codon usage of the host used for expression (Grantham, R. et a 1., Nucelic Acids Research ( 1981) 9, r43-74).
  • the DNA of the present invention can be modified by a commercially available kit or a known method. Modifications include, for example, digestion with restriction enzymes, insertion of synthetic oligonucleotides—appropriate DNA fragments, addition of a linker, insertion of an initiation codon (ATG) and / or termination codon (TM, TGA, or TAG). And the like.
  • the DNA of the present invention includes MA consisting of a base sequence from base a at position 91 to base a at position 1941 in the base sequence of SEQ ID NO: 1, or a DNA containing the base sequence.
  • the MA of the present invention is a DNA that hybridizes with a DNA consisting of the nucleotide sequence shown in SEQ ID NO: 1 and includes a DNA that encodes a protein functionally equivalent to the above-described protein of the present invention.
  • the conditions in the hybridization can be appropriately selected by those skilled in the art, and specifically, the conditions described above can be used. Under these conditions, DNA with higher homology can be obtained as the temperature is increased.
  • the MA is preferably a naturally occurring DNA, such as a cDNA or chromosomal DNA.
  • the present invention also provides a vector into which the DNA of the present invention has been inserted.
  • the vector of the present invention is useful for retaining the DNA of the present invention in a host cell or expressing the protein of the present invention.
  • the vector for example, when E. coli is used as a host, the vector is amplified in E. coli (e.g., JM109, 5 dishes, HB101, XLlBlue), etc.
  • the restriction is not particularly limited as long as the gene has an “ori” of the above and has a transformed gene selected from Escherichia coli (eg, a drug-resistant gene that can be identified by any drug (ampicillin, tetracycline, kanamycin, chloramphenicol)). Absent.
  • vectors examples include M13-based vectors, pUC-based vectors, pBR322, pBluescript ⁇ pCR-Script, and the like.
  • pGEM-Ts pDIRECT, pT7 and the like can be mentioned in addition to the above vector.
  • an expression vector is particularly useful.
  • the host in addition to having the above characteristics such that the vector is amplified in Escherichia coli, the host may be used in a host such as E.
  • coli such as JM109, DH5 or HB101 ⁇ XLlBlue.
  • a promoter that can be efficiently expressed in E. coli for example, lacZ promoter overnight (Ward et al., Nature (1989) 341, 544-546; FASEB J. (1992) 6, 2422- 2427), having the araB promoter (Better et al., Science (1988) 240, 1041-1043), or the T7 promoter.
  • Such vectors include pGEX-5 ⁇ -1 (Pharmacia), “QIAexpress systemj (Qiagen)”, pEGFP, or pET (in this case, the host is T7 RNA polymerase) in addition to the vector described above.
  • BL21 which expresses zeolites is preferred).
  • the vector may also include a signal sequence for polypeptide secretion.
  • a signal sequence for protein secretion the pelB signal sequence (Lei 3 SP et al J. Bacteriol. (1987) 169, 4379) may be used for production in E. coli periplasm.
  • the introduction of the vector into the host cell can be performed using, for example, a calcium chloride method or an electroporation method.
  • a mammalian expression vector for example, pcDNA3 (manufactured by Invitrogen), pEG F-BOS (Nucleic Acids. Res.
  • insect cell-derived expression vector eg, Factory Bac-to-BAC baculovairus expression system j (manufactured by Gibco BRL), pBacPAK8), plant-derived expression vector (Eg, ⁇ 1, pMH2), animal vector-derived expression vectors (eg, HSV pMV, pAdexLcw), retrovirus-derived expression vectors (eg, pZIPneo), yeast-derived expression vectors (eg, rpicia Expression Kitj (manufactured by Invitrogen), pNVll, SP-Q01), and an expression vector derived from Bacillus subtilis (for example, pPL608, PTH50).
  • insect cell-derived expression vector eg, Factory Bac-to-BAC baculovairus expression system j (manufactured by Gibco BRL), pBacPAK8)
  • plant-derived expression vector Eg, ⁇ 1, pMH2
  • animal vector-derived expression vectors eg, HSV
  • the promoter required for expression in cells such as the SV40 promoter (Mulligigan et al., Nature (1979)) 277, 108), MMLV-LTR Promoter Yuichi, EFla Promo Promoter Yuichi (Mzushima et al., Nucleic Acids Res. (1990) 18, 5322), CMV promoter, etc. It is more preferable to have a gene (for example, a drug resistance gene that can be distinguished by a drug (neomycin, G418, etc.)) for selecting transformation of E. coli. Examples of vectors having such properties include pMAM, pDR2, pBK-RSV, pBK-CMV, pOPRSV, pOP13, and the like.
  • a vector having a DHFR gene complementing the nucleic acid synthesis pathway-deficient CH0 cell is used.
  • pCHOI a vector having a DHFR gene complementing the nucleic acid synthesis pathway-deficient CH0 cell
  • MTX methotrexate
  • transient expression of a gene is intended, express the SV40 T antigen
  • COS cells having the gene on the chromosome are used to transform the cells into a vector having a replication origin of SV40 (such as pcD).
  • a replication origin those derived from poliovirus, adenovirus, sipapirovirus (BPV) and the like can also be used.
  • the expression vector should be used as a selection marker, such as aminoglycoside transferase (APH) gene, thymidine kinase (TK) gene, Escherichia coli xanthinguanine phosphoribosyltransferase (Ecogpt) gene, dihydro It can contain the folate reductase (dhfr) gene and the like.
  • APH aminoglycoside transferase
  • TK thymidine kinase
  • dhfr folate reductase
  • the DNA of the present invention is incorporated into an appropriate vector, for example, by a retrovirus method, a ribosome method, a cationic ribosome method, an adenovirus method, or the like.
  • a retrovirus method for example, a retrovirus method, a ribosome method, a cationic ribosome method, an adenovirus method, or the like.
  • a method for introduction into a living body This makes it possible to perform gene therapy for a disease caused by a mutation in the fank gene of the present invention.
  • the vector used include, but are not limited to, an adenovirus vector (eg, pAdexlcw) and a retrovirus vector (eg, pZIPneo).
  • General genetic manipulation such as insertion of the DNA of the present invention into a vector can be performed according to a conventional method (Molecular Cloning, 5.61-5.63).
  • Administration into a living body may be performed by the ex'ra method or the inw'ra method.
  • the present invention also provides a host cell into which the vector of the present invention has been introduced.
  • the host cell into which the vector of the present invention is introduced is not particularly limited, and for example, Escherichia coli and various animal cells can be used.
  • the host cell of the present invention can be used, for example, as a production system for producing or expressing the protein of the present invention.
  • Production systems for protein production include in vitro and j'n'ra production systems. In vitro production systems include production systems using eukaryotic cells and production systems using prokaryotic cells.
  • Animal cells include mammalian cells such as CHO (J. Exp. Med. (1995) 108, 945), COS C3T3, Kazuma Mie, BHK (baby hamster kidney), HeLa, Vero, amphibian cells, For example, African omega oocytes (Valle, et al., Nature (1981) 291, 358-340) or insect cells such as Sf9, Sf21, and Tn5 Are known.
  • CHO cells in particular, dhfr-CHO (Proc. Natl. Acad. Sci. USA (1980) 77, 4216-4220) and CHO K-1 (Proc.
  • the vector can be introduced into a host cell by, for example, a calcium phosphate method, a DEAE dextran method, a method using Cationic ribosome D0TAP (manufactured by Boehringer Mannheim), an electoral poration method, or a ribofusion method. You.
  • Nicotiana tabacum As plant cells, for example, cells derived from Nicotiana tabacum (icotiam tabacum) are known as a protein production system, which may be callus cultured.
  • Fungal cells include yeast, for example, Saccharomyces ”) genus, for example, Saccharomyces cerevisiae, filamentous fungi, for example, Aspergillus genus, for example, Aspergillus niger (spergi llus niger). ing.
  • prokaryotic cells When prokaryotic cells are used, there are production systems using bacterial cells.
  • the bacterial cells include E. coli, for example, JM109, DH5 and HB101, and Bacillus subtilis.
  • the protein is obtained by transforming these cells with the desired DNA and culturing the transformed cells in vitro.
  • the culturing can be performed according to a known method.
  • a culture solution of animal cells for example, DMEM, MEMs RPMI1640, IMDM can be used.
  • a serum replacement solution such as fetal calf serum (FCS) may be used together, or serum-free culture may be performed.
  • FCS fetal calf serum
  • the pH during culturing is preferably about 6-8. Culture is usually performed at about 30 to 40 ° C for about 15 to 200 hours, and the medium is replaced, aerated, and agitated as necessary.
  • examples of a system for producing a protein in in> c include a production system using animals and a production system using plants. Intended for these animals or plants The DNA is introduced and the protein is produced and recovered in the body of the animal or plant.
  • the “host” in the present invention includes these animals and plants.
  • mice When using animals, there are production systems using mammals and insects. As mammals, goats, horses, pigs, mice, and mice can be used (Vicki Glasser, SPECTRUM Biotechnology Applications, 1993). When a mammal is used, a transgenic animal can be used.
  • the desired MA is prepared as a fusion gene with a gene encoding a protein specifically produced in milk, such as goat casein.
  • the DNA fragment containing the fusion gene is injected into a goat embryo, and the embryo is transplanted into a female goat.
  • the target protein can be obtained from milk produced by the transgenic goat born from the goat that has received the embryo or its progeny. Hormones may be used in transgenic goats as appropriate to increase the amount of milk containing proteins produced by transgenic goats (Ebert, KM et al., Bio / Technology (1994) 12, 699-7 02).
  • silkworms can be used as insects, for example.
  • the target protein can be obtained from the body fluid of the silkworm by infecting the silkworm with a baculovirus into which DNA encoding the target protein has been inserted (Sus drawing, M. et al., Nature (1985) 315, 592-594).
  • tobacco when using a plant, for example, tobacco can be used.
  • MA encoding the protein of interest is introduced into a plant expression vector, for example, MON530, and this vector is introduced into a bacterium such as Agrobacterium ⁇ bacterium tumefaciens.
  • the bacteria are infected with tobacco, for example, Nicotiana tabacum, and the desired polypeptide can be obtained from the leaves of this tobacco (Julian K. -C. Ma et a I., Eur. J. Immunol. (1994) 24, 131-138).
  • the protein of the present invention thus obtained can be isolated from the inside or outside of the host cell (such as a medium) and purified as a substantially pure and homogeneous protein.
  • the separation and purification of the protein may be carried out by using the separation and purification methods used in ordinary protein purification, and is not limited. For example, chromatographic columns, filters, ultrafiltration, salting out, solvent precipitation, solvent extraction, distillation, immunoprecipitation, SDS-polyacrylamide gel electrophoresis, isoelectric focusing, dialysis, recrystallization, etc. When combined, proteins can be separated and purified.
  • chromatography examples include affinity chromatography, ion exchange chromatography, hydrophobic chromatography, gel filtration, reverse phase chromatography, and adsorption chromatography (Strategies for Protein Purification and Characterization: A Laboratory). Course Manual. Ed D aniel R. Marshak et al., Cold Spring Harbor Laboratory Press, 1996). These types of chromatography can be performed using liquid phase chromatography, for example, liquid phase chromatography such as HPLC and FPLC. The present invention also encompasses highly purified proteins using these purification methods.
  • the protein can be arbitrarily modified or partially removed by reacting the protein with an appropriate protein modifying enzyme before or after purification.
  • protein-modifying enzyme include trypsin, chymotrypsin, lysylendopeptidase, protein kinase, and glucosidase.
  • the present invention also provides an antibody that binds to the protein of the present invention.
  • the form of the antibody of the present invention is not particularly limited, and includes a monoclonal antibody as well as a polyclonal antibody. It also includes antisera obtained by immunizing immunized animals such as rabbits with the protein of the present invention, polyclonal antibodies and monoclonal antibodies of all classes, as well as human antibodies and humanized antibodies obtained by genetic recombination.
  • the protein of the present invention used as a sensitizing antigen for obtaining an antibody is not limited to the animal species from which it is derived, but is preferably a protein derived from a mammal, for example, a human, mouse or rat. Particularly, human-derived proteins are particularly preferable. Human-derived proteins can be obtained using the gene sequences or amino acid sequences disclosed herein.
  • the protein used as the sensitizing antigen may be a complete protein or a partial peptide of the protein.
  • the partial peptide of the protein include an amino group (N) terminal fragment and a carboxy (C) terminal fragment of the protein.
  • antibody refers to an antibody that reacts with the full length or fragment of a protein.
  • a gene encoding the protein of the present invention or a fragment thereof is inserted into a known expression vector system, and the vector is used to transform a host cell described in the present specification. Fragments may be obtained by a known method, and these may be used as a sensitizing antigen. Further, a cell expressing the protein, a lysate thereof, or a chemically synthesized protein of the present invention may be used as the sensitizing antigen. It is preferable that the short peptide is appropriately bound to a carrier protein such as protein phosphate hemocyanin, pepsin albumin, and ovalbumin to form an antigen.
  • a carrier protein such as protein phosphate hemocyanin, pepsin albumin, and ovalbumin to form an antigen.
  • the mammal to be immunized with the sensitizing antigen is not particularly limited, but is preferably selected in consideration of compatibility with the parent cell used for cell fusion. In general, rodents are used. Eyes, egrets, and primates are used.
  • mice for example, mice, rats, hamsters and the like are used.
  • a heronoid animal for example, a heron is used.
  • monkeys are used as primates.
  • monkeys of the lower nose for example, cynomolgus monkeys, rhesus monkeys, baboons, chimpanzees, etc. are used.
  • Immunization of an animal with a sensitizing antigen is performed according to a known method.
  • a sensitizing antigen is injected intraperitoneally or subcutaneously into a mammal.
  • the sensitizing antigen is diluted and suspended with PBS (Phosphate-Buffered Saline), physiological saline, or the like in an appropriate amount.
  • PBS Phosphate-Buffered Saline
  • physiological saline or the like in an appropriate amount.
  • a normal adjuvant for example, Freund's complete buffer
  • An appropriate amount of adjuvant is mixed, emulsified, and administered to mammals. Thereafter, it is preferable to administer the sensitizing antigen mixed with an appropriate amount of incomplete Freund's adjuvant several times every 4 to 21 days.
  • an appropriate carrier can be used at the time of immunization with the sensitizing antigen.
  • C Immunization is performed in this way, and it is confirmed by a conventional method that the level of the desired antibody in the serum increases.
  • the blood of a mammal sensitized with the antigen is removed after confirming that the desired antibody level in the serum has increased.
  • the serum is separated from the blood by a known method.
  • a serum containing the polyclonal antibody may be used.
  • a fraction containing the polyclonal antibody may be further isolated from the serum and used.
  • a fraction that recognizes only the protein of the present invention is obtained using an affinity column to which the protein of the present invention is coupled, and the fraction is further purified using a protein A or protein G column.
  • immunoglobulin G or M can be prepared.
  • the immune cells may be removed from the mammal and subjected to cell fusion.
  • preferred immune cells used for cell fusion include spleen cells.
  • the other parent cell to be fused with the immunocyte is preferably a mammalian myeloid cell, more preferably a myeloid cell that has acquired characteristics for selecting fused cells by a drug.
  • the cell fusion of the immune cells and myeloma cells is basically performed according to a known method, for example, the method of Milstein et al. (Galfre, G. and Milstein, Methods Enzymol. (1981) 73, 3-46). be able to.
  • the hybridoma obtained by cell fusion is selected by culturing it in a normal selective culture medium, for example, a HAT culture medium (a culture medium containing hypoxanthine, aminopterin and thymidine). Cultivation in the HAT culture medium is the desired hybrid. It is performed for a period of time sufficient to kill cells other than one cell (non-fused cells), usually for several days to several weeks. Next, a conventional limiting dilution method is performed to screen and close the hybridoma producing the desired antibody.
  • a normal selective culture medium for example, a HAT culture medium (a culture medium containing hypoxanthine, aminopterin and thymidine). Cultivation in the HAT culture medium is the desired hybrid. It is performed for a period of time sufficient to kill cells other than one cell (non-fused cells), usually for several days to several weeks. Next, a conventional limiting dilution method is performed to screen and close the hybridoma producing the desired antibody.
  • human lymphocytes for example, human lymphocytes infected with EB virus
  • a sensitized lymphocyte with a human-derived myeloma cell having permanent division ability, for example, U266, to obtain a hybridoma that produces a desired human antibody having a protein binding activity (Japanese Patent Application Laid-Open No. 63_16888). Gazette).
  • the obtained hybridoma is transplanted intraperitoneally into a mouse, ascites is collected from the mouse, and the obtained monoclonal antibody is subjected to, for example, ammonium sulfate precipitation, protein II, protein G column, DEAE ion exchange chromatography, the present invention. It can be prepared by purifying the above protein using a coupled affinity column or the like.
  • the antibody of the present invention is used for purification and detection of the protein of the present invention, and is also a candidate for an agonist and an angoniist of the protein of the present invention. It is also conceivable to apply this antibody to antibody therapy for diseases involving the protein of the present invention.
  • a human antibody or a humanized antibody is preferable in order to reduce immunogenicity.
  • a transgenic animal having a repertoire of human antibody genes is immunized with a protein serving as an antigen, a protein-expressing cell or a lysate thereof to obtain antibody-producing cells, and a hybridoma obtained by fusing this with myeloma cells is used.
  • a human antibody against the protein see International Publication Nos. W092-03918, W093-2227, W094-26022, W094-25585, W096-33735 and W096-34096).
  • cells in which immune cells such as sensitized lymphocytes that produce antibodies are immortalized by oncogenes may be used.
  • the monoclonal antibody thus obtained can also be obtained as a recombinant antibody produced using genetic recombination technology (for example, Borrebaeck, AK and Larrick 3 JW 5 THERAPEUTIC MONOCLONAL ANTIBODIES, Published in the Unit ed Kingdom by MCMILLAN PUBLISHERS LTD, 1990).
  • Recombinant antibodies are produced by cloning DNA encoding them from hybridomas or immunized cells such as sensitized lymphocytes that produce the antibodies, incorporating the DNA into an appropriate vector, and introducing this into a host.
  • the present invention includes this recombinant antibody.
  • the antibody of the present invention may be a modified antibody fragment thereof as long as it binds to the protein of the present invention.
  • Fab, F (ab ') 2, Fv or a single chain Fv (scFv) in which an Fv of an H chain and an L chain are linked by an appropriate linker Huston, JS et al., Proc. .Atl. Acad. Sci. USA (1988) 85, 5879-5883.
  • the antibody is treated with an enzyme, for example, papain or pepsin, to generate an antibody fragment, or a gene encoding these antibody fragment is constructed and introduced into an expression vector. Expressed in cells (eg, Co, MS et al., J.
  • modified antibody an antibody bound to various molecules such as polyethylene glycol (PEG) can be used.
  • PEG polyethylene glycol
  • the “antibody” of the present invention also includes these modified antibodies.
  • Such a modified antibody can be obtained by subjecting the obtained antibody to chemical modification. These methods are already established in this field.
  • the antibody of the present invention can be prepared using a known technique by using a chimeric antibody composed of a variable region derived from a non-human antibody and a constant region derived from a human antibody or a CDR derived from a non-human antibody (complementarity determination). Constant region), a human antibody-derived FR (framework region), and a constant region.
  • the antibody obtained as described above can be purified to homogeneity.
  • the separation and purification of the antibody used in the present invention may be performed by the separation and purification methods used for ordinary proteins. For example, if appropriate selection and combination of chromatographic columns such as affinity chromatography, filtration, ultrafiltration, salting out, dialysis, SDS polyacrylamide gel electrophoresis, isoelectric focusing, etc.
  • Antibodies can be separated and purified (Antibodies: A Laboratory Manual. Ed Harlow and David Lane, Cold Spring Harbor Laboratory, 1988), but are not limited thereto.
  • the concentration of the antibody obtained above can be measured by measuring absorbance or by enzyme-linked immunosorbent assay (ELISA).
  • Columns used for affinity chromatography include protein A column and protein G column.
  • columns using Protein A include Hyper D, POROS, Sepharose F.F. (Pharmacia) and the like.
  • chromatography other than affinity chromatography examples include ion exchange chromatography, hydrophobic chromatography, gel filtration, reverse phase chromatography, and adsorption chromatography (Strategies for Protein). Purification and Characterization: A Laboratory Course Manual. Ed Daniel R. Marshak et al., Cold Spring Harbor Laboratory Press, 1996) 0 These chromatography can be performed using liquid phase chromatography such as HPL FPLC.
  • Methods for measuring the antigen-binding activity of the antibody of the present invention include, for example, measurement of absorbance, enzyme-linked immunosorbent assay (ELISA), EIA (enzyme-linked immunosorbent assay), and RIA (radioimmunoassay). Measurement method) or a fluorescent antibody method can be used.
  • ELISA enzyme-linked immunosorbent assay
  • EIA enzyme-linked immunosorbent assay
  • RIA radioimmunoassay
  • a secondary antibody that recognizes an enzyme for example, an antibody labeled with alkaline phosphatase
  • an enzyme substrate such as P-nitrophenyl phosphate
  • Antigen binding activity can be evaluated.
  • a protein fragment for example, a fragment consisting of its C-terminal may be used.
  • BIAcore Pharmacia
  • the antibody of the present invention is brought into contact with the sample contained in the sample which is expected to contain the protein of the present invention, and an immune complex of the antibody and the protein is detected or measured.
  • the method for detecting or measuring a protein of the present invention can be carried out.
  • INDUSTRIAL APPLICABILITY The method for detecting or measuring a protein of the present invention can specifically detect or measure a protein, and thus is useful for various experiments and the like using proteins.
  • the present invention also provides a polynucleotide comprising at least 15 nucleotides complementary to the DNA encoding the fank protein (SEQ ID NO: 1) or its complementary strand.
  • the “complementary strand” refers to one strand of a double-stranded nucleic acid composed of A: T ( ⁇ ⁇ ⁇ in the case of RNA) and G: C base pairs with respect to the other strand.
  • “complementary” is not limited to a sequence that is completely complementary to at least 15 contiguous nucleotide regions, but is at least 70, preferably at least 80, more preferably 90, and even more preferably 95 ° / It is only necessary to have the above homology on the base sequence.
  • the algorithm described in this specification may be used as an algorithm for determining homology.
  • nucleic acids include a probe primer used for detection and amplification of MA encoding the protein of the present invention, a probe primer for detecting the expression of the DNA, and expression of the protein of the present invention.
  • a nucleotide derivative eg, an antisense oligonucleotide or ribozyme, or a DNA encoding the same
  • Such a nucleic acid can also be used for producing a DNA chip. When used as a ply, the region on the 3 'side is complementary, and a restriction enzyme recognition sequence, evening DNA, etc. can be added to the 5' side.
  • the antisense oligonucleotide includes, for example, an antisense oligonucleotide that hybridizes at any position in the nucleotide sequence of SEQ ID NO: 1.
  • This antisense oligonucleotide is preferably an antisense oligonucleotide for at least 15 or more consecutive nucleotides in the base sequence of SEQ ID NO: 1. More preferably, at least 15 or more consecutive nucleotides are an antisense oligonucleotide containing a translation initiation codon.
  • the antisense oligonucleotide derivatives and modifications thereof can be used. Examples of the modified form include a modified lower alkylphosphonate such as a methylphosphonate type or an ethylphosphonate type, a phosphorothiote modified form or a phosphoroamidate modified form.
  • Antisense oligonucleotides include not only those whose nucleotides corresponding to nucleotides constituting a predetermined region of DNA or mRNA are all complementary sequences, but also those having nucleotides represented by SEQ ID NO: 1 in which DNA or mRNA and the oligonucleotide are represented by SEQ ID NO: 1. As long as it can be hybridized specifically, it includes those in which a mismatch of one or more nucleotides is present.
  • the antisense oligonucleotide derivative of the present invention acts on cells producing the protein of the present invention to bind to DNA or mRNA encoding the protein, thereby inhibiting the transcription or translation of the protein or degrading the mRNA. Or by suppressing the expression of the protein of the present invention, thereby effectively suppressing the action of the protein of the present invention.
  • the antisense oligonucleotide derivative of the present invention can be used as an external preparation such as a coating agent or a poultice by mixing with a suitable base material which is inactive against the derivatives.
  • excipients may be added to tablets, powders, granules, capsules, ribosome capsules, It can be a lyophilized agent such as a propellant, a liquid, a nasal drop and the like. These can be prepared according to a conventional method.
  • the antisense oligonucleotide derivative of the present invention is applied directly to the affected area of the patient, or is applied to the patient so as to be able to reach the affected area as a result of intravenous administration or the like.
  • an antisense-encapsulated material that enhances durability and membrane permeability can be used.
  • ribosome, poly-L-lysine, lipid, cholesterol, lipofectin or derivatives thereof can be mentioned.
  • the dose of the antisense oligonucleotide derivative of the present invention can be appropriately adjusted according to the condition of the patient, and a preferred amount can be used. For example, it can be administered in the range of 0.1 to 100 mg / kg, preferably 0.1 to 50 mg / kg.
  • the antisense oligonucleotide of the present invention inhibits the expression of the protein of the present invention and is therefore useful in suppressing the biological activity of the protein of the present invention. Further, an expression inhibitor containing the antisense oligonucleotide of the present invention is useful in that it can suppress the biological activity of the protein of the present invention.
  • the present invention also provides a method for screening for a compound that binds to the protein of the present invention.
  • the protein of the present invention is brought into contact with a test sample expected to contain a compound binding thereto, the binding activity between the protein and the test sample is detected, and the protein binds to the protein of the present invention. Selecting a compound that has activity.
  • the protein of the present invention used for the screening may be a recombinant protein or a protein of natural origin. It may be a partial peptide. It may also be a form expressed on the cell surface or a form as a membrane fraction.
  • the test sample is not particularly limited and includes, for example, a cell extract, a cell culture supernatant, a fermentation microorganism product, a marine organism extract, a plant extract, a purified or crude protein, a peptide, a non-peptidic compound, and a synthesis. Low molecular weight compounds and natural compounds.
  • the protein of the present invention, which is brought into contact with a test sample may be, for example, a purified protein, a soluble protein, or a carrier. As a fusion protein with another protein, as a form expressed on a cell membrane, or as a membrane fraction, and contacted with a test sample.
  • Such screening can be performed by, for example, immunoprecipitation. Specifically, it can be performed as follows.
  • the gene encoding the protein of the present invention is inserted into a vector for expressing a foreign gene such as pSV2neo, pcDNAI, pCD8, etc., so that the gene is expressed in animal cells or the like.
  • the promoters used for expression include the SV40 early promoter (Rigby In Williamson (ed.), Genetic Engineering, Vol. 3.Academic Press, London, p. 83-141 (1982)) and the EF-1 promoter (Kim Gene 91, P-2 17-223 (1990)), CAG promoter (Niwa et al. Gene 108, p.193-200 (1991)),
  • RSV LTR promoter (Cullen Methods in Enzymology 152, p.684-704 (1987)), SR a promoter (Takebe et al. Mol. Cell. Biol. 8, p. 66 (1988)), CMV itsuru ed iate early promoter (Seed and Arffo Proc. At 1.Acad.Sci. USA 84 3 P.3365-3369 (1987)), SV40 late promoter (Gheysen and Fiers J. ol.Appl.Genet. 15 p.385-394 (1982)), Adenovirus late promoter (Kaufman et al. Mol. Cell. Biol. 9, p. 946 (1989)), HSV TK promoter, and other commonly used promoters. Is also good.
  • the protein of the present invention is expressed as a fusion protein having a monoclonal antibody recognition site by introducing a recognition site (epitope) of the monoclonal antibody of which specificity is known into the N-terminal or C-terminal of the protein of the present invention. be able to.
  • a commercially available epitobu antibody system can be used (Experimental Medicine, 85-90 (1995)).
  • a vector that can express a fusion protein with galactosidase, maltose binding protein, glutathione S-transferase, green fluorescent protein (GiT), etc. via a multicloning site is commercially available. .
  • polyhistidine His-tag
  • influenza agglutinin HA human c-myc
  • FLAG Vesicular sto matrtis virus glycoprotein
  • VSV-GP Vesicular sto matrtis virus glycoprotein
  • T7-tag human simple herpes virus sugar Epitopes such as proteins (HSV-tag) and E-tags (epitopes on monoclonal phages) and monoclonal antibodies recognizing them
  • HSV-tag Vesicular sto matrtis virus glycoprotein
  • T7-tag human simple herpes virus sugar Epitopes such as proteins (HSV-tag) and E-tags (epitopes on monoclonal phages) and monoclonal antibodies recognizing them
  • HSV-tag proteins
  • E-tags epitopepitopes on monoclonal phages
  • monoclonal antibodies recognizing them can be used for screening of proteins that bind to the protein of the present invention. It can be used as
  • an immune complex is formed by adding these antibodies to a cell lysate prepared using an appropriate surfactant.
  • This immune complex comprises the protein of the present invention, a protein capable of binding thereto, and an antibody.
  • immunoprecipitation can also be performed using antibodies against the protein of the present invention.
  • Antibodies against the protein of the present invention include, for example, a gene encoding the protein of the present invention introduced into an appropriate E. coli expression vector, expressed in E. coli, and the expressed protein is purified. It can be prepared by immunizing goats and chickens. In addition, the synthesized It can also be prepared by immunizing the above animal with a partial peptide of the protein.
  • the immune complex can be precipitated using Protein A Sepharose or Protein G Sepharose.
  • the protein of the present invention is prepared, for example, as a fusion protein with an epitope such as GST, the present invention can be carried out by utilizing a substance that specifically binds to these epitopes, such as glutathione-Sepharose 4B.
  • An immune complex can be formed in the same manner as in the case where an antibody of the above protein is used.
  • SDS-PAGE is generally used for analysis of immunoprecipitated proteins.
  • the bound proteins can be analyzed by the molecular weight of the protein.
  • the protein of interest can be purified directly from the SDS-polyacrylamide gel and its sequence determined.
  • Examples of a method for isolating a protein binding to the protein using the protein of the present invention include, for example, a West Western blotting method (Skolnik, EY et al., Cell (1991) 65, 83- 90). Specifically, a cDNA library using a phage vector (eg, gtll, ZAP, etc.) is prepared from cells, tissues, and organs (eg, testis) that are expected to express the protein that binds to the protein of the present invention. The protein expressed on LB-agarose and expressed on the filter is immobilized, and the purified and labeled protein of the present invention is reacted with the filter on the filter to produce the protein of the present invention.
  • a West Western blotting method Skolnik, EY et al., Cell (1991) 65, 83- 90.
  • a cDNA library using a phage vector eg, gtll, ZAP, etc.
  • Plaques expressing the protein bound to the light protein may be detected by labeling.
  • the method for labeling the protein of the present invention include a method using the binding property of biotin and avidin, and a method specific for the protein of the present invention or a peptide or polypeptide fused to the protein of the present invention (for example, GST).
  • a method using an antibody that binds to the DNA a method using a radioisotope, a method using fluorescence, and the like.
  • the protein of the present invention or a partial peptide thereof is expressed in yeast cells by fusion with an SRF DNA binding region or a GAL4 DNA binding region, and a protein that binds to the protein of the present invention.
  • CDNA library that is expressed in a form fused with the VP16 or GAL4 transcriptional activation region from cells that are expected to express E. coli, is introduced into the above yeast cells, and the positive clones detected Live from The rally-derived cDNA is isolated (when the protein that binds to the protein of the present invention is expressed in the yeast cell, the binding of both activates the repo all-in-one gene, and a positive clone can be confirmed).
  • a protein encoded by the cDNA By introducing the expressed cDNA into Escherichia coli and expressing it, a protein encoded by the cDNA can be obtained, whereby a protein that binds to the protein of the present invention or its gene can be prepared.
  • the repo-inhibitory genes used in the lid system include, for example, the HIS3 gene, the Ade2 gene, the LacZ gene, the CAT gene, the resiferase gene, the PAI-1 (plasminogen activator inhibitor t ypel) gene and the like, but are not limited thereto. Screening by the two-hybrid method can be performed using mammalian cells in addition to yeast cells. Screening for a compound that binds to the protein of the present invention can also be performed using affinity chromatography.
  • the protein of the present invention is immobilized on a carrier of an affinity column, and a test sample which is expected to express a protein that binds to the protein of the present invention is applied here.
  • the test sample includes, for example, a cell extract, a cell lysate, and the like. After applying the test sample, the column is washed, and the protein bound to the protein of the present invention can be prepared.
  • the obtained protein is analyzed for its amino acid sequence, an oligo DNA is synthesized based on the amino acid sequence, and a DNA encoding the protein is obtained by screening a cDNA library using the DNA as a probe. Can be.
  • the method for isolating not only proteins but also compounds that bind to the protein of the present invention includes, for example, synthetic proteins, natural product banks, immobilized proteins of the present invention, Alternatively, a method of screening a molecule that binds to the protein of the present invention by using a random phage peptide display library, or a screening method using high throughput by combinatorial chemistry technology (Wrighton NC; Farrell FX; Chang R; Kashyap AK; Barnbone FP; Mulcahy LS; Johnson DL; Barrett RW; Jolliffe LK; Dower WJ., Sma 11 eptides as potent mimetics of the protein hormone erythropoietin, Science (UNITED STATES) Jul 26 1996, 273 p458-64, Verdine GL., The combina torial chemistry of nature.Nature (ENGLAND) Nov 7 1996, 384 pll-13, Hog an JC Jr., Direct
  • a biosensor utilizing the surface plasmon resonance phenomenon can be used as a means for detecting or measuring the bound compound.
  • a biosensor utilizing the surface plasmon resonance phenomenon is used to determine the phase between the protein of the present invention and a test compound.
  • the interaction can be observed in real time as a surface plasmon resonance signal using a trace amount of protein and without labeling (for example, BIAcore, manufactured by Pharmacia). Therefore, it is possible to evaluate the binding between the protein of the present invention and the test compound by using a biosensor such as BIAcore.
  • the present invention also provides a method for screening a compound that regulates the activity of the protein of the present invention.
  • This method comprises contacting the spermatogonia with the protein or peptide of the present invention and a test sample, or contacting the test sample with a transformed spermatogonia expressing the protein or peptide of the present invention, Detecting the spermatogonia differentiated into cells and selecting a compound that increases or decreases the number of spermatogonia differentiated into spermatocytes as compared to the case where the test sample is not contacted (control). .
  • the test sample is not particularly limited, and examples thereof include a cell extract, a cell culture supernatant, a fermented microorganism product, a marine organism extract, a plant extract, a purified or crude protein, a peptide, a non-peptide compound, Synthetic low molecular compounds, natural compounds and the like can be used.
  • the protein or peptide of the present invention to be used may be one prepared by a gene recombination technique, one derived from nature, or one synthesized.
  • Diseases to be treated or prevented include, for example, infertility.
  • the protein of the present invention or a compound that can be isolated by the screening of the present invention can be used for humans or other animals, such as mice, rats, guinea pigs, egrets, chicks, cats, dogs, sheep, bush, foxes, monkeys, When used as a medicament for baboons and chimpanzees, it can be administered as a pharmaceutical composition formulated by known pharmaceutical methods, in addition to directly administering the protein or the isolated compound itself to patients. It is. For example, tablets, capsules, elixirs, and microcapsules, which are sugar-coated as required orally, or aseptic solution or suspension in water or other pharmaceutically acceptable liquids It can be used parenterally in the form of injections.
  • pharmacologically acceptable carriers or vehicles specifically, sterile water, saline, plants Appropriate combination with oil, emulsifier, suspending agent, surfactant, stabilizer, flavor, excipient, vehicle, preservative, binder, etc.
  • Can be formulated by mixing with The amount of the active ingredient in these preparations is such that an appropriate dose in the specified range can be obtained.
  • Excipients that can be incorporated into tablets and capsules include, for example, binders such as gelatin, corn starch, tragacanth gum, acacia, excipients such as crystalline cellulose, corn starch, gelatin, and alginic acid.
  • Swelling agents such as magnesium stearate, sweeteners such as sucrose, lactose or saccharin, and flavoring agents such as peppermint, coconut oil or cellulose are used. May further contain a liquid carrier such as an oil or fat in addition to the above materials.
  • Sterile compositions for injection can be formulated according to normal pharmaceutical practice using a vehicle such as distilled water for injection.
  • Aqueous injection solutions include, for example, saline, isotonic solutions containing glucose and other adjuvants, such as D-sorbitol, D-mannose, D-mannitol, sodium chloride, and suitable solubilizing agents.
  • glucose and other adjuvants such as D-sorbitol, D-mannose, D-mannitol, sodium chloride, and suitable solubilizing agents.
  • alcohols specifically, ethanol, polyalcohols, for example, propylene glycol, polyethylene glycol, nonionic surfactants, for example, polysorbate 80 (TM) , or HC0-50 may be used in combination.
  • the oily liquid includes sesame oil and soybean oil, and may be used in combination with benzyl benzoate or benzyl alcohol as a solubilizer. It may also be combined with a buffer, for example, a phosphate buffer, a sodium acetate buffer, a soothing agent, for example, proforce hydrochloride, a stabilizer, for example, benzyl alcohol, phenol, or an antioxidant.
  • a buffer for example, a phosphate buffer, a sodium acetate buffer, a soothing agent, for example, proforce hydrochloride, a stabilizer, for example, benzyl alcohol, phenol, or an antioxidant.
  • the prepared injection solution is usually filled into an appropriate ampoule.
  • Administration to patients can be performed, for example, by intraarterial injection, intravenous injection, subcutaneous injection, etc., or intranasally, transbronchially, intramuscularly, transdermally, or orally by a method known to those skilled in the art. It can do better.
  • the dosage varies depending on the patient's weight, age, administration method, etc. Those skilled in the art can appropriately select an appropriate dose.
  • the compound can be encoded by DNA, the DNA may be incorporated into a gene therapy vector to perform gene therapy.
  • the dose and the administration method vary depending on the patient's body weight, age, symptoms and the like, but can be appropriately selected by those skilled in the art.
  • the dose of the protein of the present invention varies depending on the administration subject, target organ, symptoms and administration method. For example, in the case of an injection, usually 1 dose is used for an adult (assuming a body weight of 60 kg). It is considered to be about 100 to 20 mg per serving. ⁇
  • the dose of the compound that binds to the protein of the present invention or the compound that modulates the activity of the protein of the present invention varies depending on the symptoms. However, in the case of oral administration, in general, in adults (with a body weight of 60 kg), 1 It is believed to be about 0.1 to 100 mg per day, preferably about 1.0 to 50 mg, more preferably about 1.0 to 20 mg.
  • the single dose varies depending on the subject of administration, target organ, symptoms and administration method.
  • the dose in the case of injection, usually in adults (with a body weight of 60 kg), It may be convenient to administer about 0.01 to 30 mg, preferably about 0.1 to 20 mg, more preferably about 0.1 to 10 mg per day by intravenous injection.
  • the dose can be administered in terms of the amount converted per 60 kg body weight or the amount converted per body surface area.
  • the DNA of the present invention is incorporated into a vector that guarantees its expression in vivo as described above. It may be introduced into a living body by a method such as the catonic ribosome method or the adenovirus method. As a result, it is possible to perform gene therapy for a disease caused by mutation of the fank gene of the present invention. In vivo administration can be performed by the J'O method or the ⁇ W'6> method.
  • the present invention also provides a method for examining abnormal expression of the DNA of the present invention.
  • the test method of the present invention is a method characterized by detecting the expression of the DNA of the present invention in the sexual tissue of a subject and comparing the expression with a healthy person. More specifically, (a) the testis or testis of the subject Is a step of preparing a sample from the ovary, (b) a step of measuring the amount of a transcription product or a translation product of the DNA of the present invention in the sample, and (c) a step of measuring the value obtained in step (b) in a healthy subject. Comparing with a measured value.
  • abnormal expression of the DNA of the present invention includes both abnormal transcription levels and abnormal translation levels of the DNA of the present invention.
  • the DNA of the present invention showed reduced expression in infertile mice (Example 3). Therefore, for example, a test for infertility can be performed using the abnormal expression of the DNA of the present invention as an index.
  • the detection of a transcription product and a translation product of the DNA of the present invention in a testis sample or an ovary sample prepared from a subject can be performed by a method known to those skilled in the art.
  • Suitable methods for detecting the transcription product of the DNA of the present invention include, but are not limited to, Northern blotting, RT-PCR, and the like.
  • Suitable methods for detecting the translation product of the DNA of the present invention include, but are not limited to, Western blotting and immunoprecipitation.
  • the subject will be able to express the DNA of the present invention. It is determined to be abnormal. For example, in a test for infertility, if the amount of the transcript or translation product of the DNA of the present invention in the test subject is significantly lower than that in a healthy subject, the test subject is suspected of having It can be determined that there is a predisposition to the disease.
  • FIG. 1 is a photograph showing the results of detecting the expression of p33 in testis tissue.
  • FIG. 2 is a diagram showing a list of testis-specific genes isolated by the immunoscreening method.
  • FIG. 3 is a diagram showing an AM repetition structure in a fank.
  • FIG. 4 is a diagram showing the arrangement of the fank on the chromosome. BEST MODE FOR CARRYING OUT THE INVENTION
  • the present inventors were examining the expression of a gene encoding an unknown protein p33 that causes immortalization by the immunoscreening method. Accordingingly, a gene fragment that is highly expressed in the testis and ovary of a mouse coincidentally got a fank. The sequence of this gene fragment was a novel sequence in which no homology could be found in one bank of DNA sequence data. In addition, this gene fragment was cloned from a mouse testis library using a probe labeled with a 32 P isotope. As a result, a clone having a length of about l. 1 kb was obtained. The sequence of the cDNA clone was determined by the dideoxy termination method and analyzed by the ABI377 automatic sequencer.
  • EST clones were searched by combi-evening analysis to determine the full-length human sequence. Furthermore, based on the human EST clones, it was checked against an in-house database, and full-length cDNA was obtained from Kazusa Full Clone Library.
  • the entire base sequence of the obtained fank cDNA is shown in SEQ ID NO: 1. No homology was found in the obtained full-length cDNA sequence in the DNA sequence overnight.
  • the resulting cDNA encodes a protein consisting of 617 amino acids (pi-7.44), and no significant match was found in the published protein database. However, although no DNA sequence match was found, BLASTP analysis of the amino acid sequence revealed that the mouse and human; fank amino acid sequences were partially established in c.egBfls and were established as sex-determining genes. It was a protein with a similar structure to the Fem-1 family. It was also confirmed that the Fem-1 family had an ANK repeat structure, which is a common structure (Fig. 3). [Example 2] Chromosome arrangement
  • a novel protein and a gene thereof which are closely involved in testis cell differentiation and have a structure similar to the Fem-family are provided. Since the fank protein and its gene of the present invention are considered to have a structure similar to that of the sex determinant in Wea a / is, their application to production control of livestock and the like is also expected.

Abstract

Selon l'invention, un gène « fank » est isolé par dosage immunologique au moyen d'un anticorps dirigé contre une protéine p33 inconnue, induisant une immortalisation. Une partie de la structure de la protéine codée par ce gène est similaire à la structure de FEM-1, connu sous le nom de facteur de détermination du sexe.
PCT/JP2001/003728 2000-04-28 2001-04-27 Facteur de regulation de differenciation et de determination du sexe WO2001083738A1 (fr)

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AU2001252651A AU2001252651A1 (en) 2000-04-28 2001-04-27 Sex determinative differentiation regulatory factor

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JP2000-134294 2000-04-28

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Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
NAGASE T. ET AL.: "Prediction of the coding sequences of unidentified human genes. XX. The complete sequences of 100 new cDNA clones from brain which code for large proteins in vitro", DNA RES., vol. 8, no. 2, 27 April 2001 (2001-04-27), pages 85 - 95, XP002943489 *
SPENCE A.M. ET AL.: "The product of fem-1, a nematode sex-determining gene, contains a motif found in cell cycle control proteins and receptors for cell-cell interactions", CELL, vol. 60, no. 6, 1990, pages 981 - 990, XP002943488 *

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