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WO2001066716A1 - Polypeptide ayant une activite de phosphodiesterase - Google Patents

Polypeptide ayant une activite de phosphodiesterase Download PDF

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Publication number
WO2001066716A1
WO2001066716A1 PCT/JP2001/001720 JP0101720W WO0166716A1 WO 2001066716 A1 WO2001066716 A1 WO 2001066716A1 JP 0101720 W JP0101720 W JP 0101720W WO 0166716 A1 WO0166716 A1 WO 0166716A1
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polypeptide
oligonucleotide
dna
gene
cells
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PCT/JP2001/001720
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English (en)
Japanese (ja)
Inventor
Hiromasa Miyaji
Motoko Haruoka
Toshio Ota
Ayako Kawabata
Sumio Sugano
Yusuke Nakamura
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Kyowa Hakko Kogyo Co.,Ltd.
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Priority to AU2001236101A priority Critical patent/AU2001236101A1/en
Publication of WO2001066716A1 publication Critical patent/WO2001066716A1/fr

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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)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/04—Drugs for disorders of the alimentary tract or the digestive system for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/18—Drugs for disorders of the alimentary tract or the digestive system for pancreatic disorders, e.g. pancreatic enzymes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A61P11/06—Antiasthmatics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/12—Drugs for disorders of the urinary system of the kidneys
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
    • A61P19/10—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease for osteoporosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
    • A61P25/16—Anti-Parkinson drugs
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/18—Antipsychotics, i.e. neuroleptics; Drugs for mania or schizophrenia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/22—Anxiolytics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/24—Antidepressants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/08—Antiallergic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/12—Antihypertensives
    • 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

Definitions

  • the present invention relates to a novel phosphodiesterase polypeptide, a DNA encoding the polypeptide, a vector containing the DNA, a transformant transformed with the vector, and a method for producing the phosphodiesterase polypeptide. .
  • the present invention also relates to a method for using the polypeptide, for example, a method for screening for a compound having agonistic or angonist activity using the polypeptide or an antibody thereof, and a medicament containing the polypeptide or an antibody thereof.
  • Linear nucleotides are known to mediate cellular responses to many extracellular stimuli, including stimuli from G protein-coupled receptors (GPCRs).
  • Cyclic nucleotide phosphodiesterase (PDE) hydrolyzes 3,, 5, and monocyclic nucleotides such as 3,5,5 monocyclic adenosine monophosphate (cAMP) and 3,5,5-cyclic guanosine monophosphate (cGMP).
  • PDE Cyclic nucleotide phosphodiesterase
  • cAMP 3,5,5 monocyclic adenosine monophosphate
  • cGMP 3,5,5-cyclic guanosine monophosphate
  • PDE is, c AMP and cGMP concentration
  • the further regulate the magnitude and duration of signals through cyclic nucleotides number of experimental results from PDE activity other signals at steady state intracellular It has been shown to be controlled by a variety of information from the transduction system, occupying an important position in the "cross-talk" between signal transduction pathways and intracellular control networks [Trend. Pharmacol. Sci., 2i, 217 (1997), Physiol. Rev., 75, 725 (1995), Arch.Biochem. Biopys. 3 322, 1 (1995), and Endocrinol. Rev., 16, 370 (1995)] .
  • Vertebrate PDEs have structure, localization, regulation, selective PDEP and sensitivity to harmful agents, etc.
  • cAMP PDE cGMP PDE inhibited by cAMP PDE 10
  • PDE 10 Many families consist of subtypes encoded by different genes and their splice variants (isoforms) [Kidney International, 55, 29 (1999), Cyclic Nucleotide Phosphodiesterases: Structure, Regulation and Drug Action, John Wiley & Sons, New York (1990), and Biochem. Biophys. Res. Commun., 261, 551 (1999)]. Tissue and cell-specific expression has been reported for several PDE isoforms [Trend. Pharmacol. Sci., 22, 217 (1997), Physiol. Rev., 75, 725 (1995), and Arch. Biochem. Biophys., 32, 1 (1995)]. To date, more than 30 species of PDE have been reported, but more unknown PDEs are expected.
  • PDEs usually have a structure consisting of three functional domains (Trend.
  • the region consisting of about 270 amino acids (catalytic core domain) existing from the center to the C-terminal side is conserved in all vertebrate-derived PDEs and constitutes the catalytic domain. This region is well conserved between genes within individual PDE families, with over 80% identity in the amino acid sequence. In addition, amino acid sequences of about 25% to 40% are identical between different PDE gene families. Although two Zn 2+ binding motifs exist in the catalytic core domain (J. Biol.
  • the catalytic core domain of one of the PDE3 families contains other PDE families.
  • the first Zn 2+ binding motif has been destroyed due to the insertion of a 44 amino acid that is not present in the cell.
  • a conserved motif of HDXXHXXXXN amino acid residues are represented by one letter; H; histidine, D; aspartic acid, N; asparagin, X; any amino acid residue) is found in the catalytic domain of all PDEs. Have been.
  • the region containing the catalytic core domain of PDE 4 was expressed in E. coli and was shown to have PDE activity [J. Biol. Chem., ML, 18929 (1992)].
  • the conserved catalytic domain contains sequences that define the specificity of the PDE family for substrate affinity and sensitivity to inhibitors. CJ. Biol. Chem., 274, 4839 (1999)]. While the catalytic domain is well conserved, the ⁇ ⁇ terminal regulatory domain is PD ⁇ The structure and size differ between subtypes and isoforms [Cyclic Nucleotide Phosphodiesterases: Structure, Regulation and Drug Action, John Wiley & Sons, New York (1990)].
  • This domain contains ligand binding sites, phosphorylation sites, membrane binding sites, sequences involved in protein-protein interactions, and the like. Fully clear the functional significance of relatively small C-terminal domains It has not been. It has been reported that the serine residue at position 487 of the PDE4B2B isoform is phosphorylated by mitogen-activated protein kinase (Biochem. J., 316, 751 (1996)).
  • PDE subtype or isoform-specific agonists or antagonists are expected to have a prophylactic or therapeutic effect on a variety of diseases involving variations in intracellular cyclic nucleotide levels. For example, many functions related to the immune and inflammatory responses are inhibited by agents that increase intracellular cAMP [Mol. Pharmacol., 47, 1164 (1995)]. On the other hand, cGMP is involved in the functions of smooth muscle, lung and brain cells. CPharmac. Ther., 51, 13 (1991)]. Non-specific PDE inhibitors and inhibitors that are selective for PDE subtypes have been synthesized, and some of them have been studied experimentally or clinically for their therapeutic potential [ Trends Pharmacol.
  • PDE 3 inhibitors have been developed as antithrombotic agents, antihypertensive agents, and inotropic agents useful in treating depressive heart failure. Recently, it has been reported that PDE 3B is involved in the inhibition of insulin secretion by lebutin [J. Clin. Invest., ⁇ , 869 (1998)]. As a PDE 4 inhibitor, rolipram has been used as a therapeutic agent for depression. TNF-Hi
  • HIV-1 replication may be inhibited by suppressing TNF-production by lipopolysaccharide stimulation [AIDS, 9, 1137 (1995)].
  • it has activity to suppress the production of cytokines such as TNF- and inuichi-feron-a, so it can be used for encephalomyelitis (encephalomyelitis ⁇ multiple sclerosis (MS), tardive dyskinesia, etc.)
  • PDE 4 inhibitors have anti-inflammatory effects and are expected to be used as therapeutic agents for rheumatism, etc.
  • they have anti-inflammatory effects as well as bronchodilator effects, and as therapeutic agents for asthma.
  • PDE PDE 3 inhibitor
  • mesangial cells a PDE 3 inhibitor
  • mesangial cells a PDE 3 inhibitor
  • Inhibits the production of reactive oxygen metabolites J. Biol. Chem., 272, 9854 (1997).
  • PDE inhibitors have been shown to suppress the development of proteinuria in anti-Thy-1 nephritis rats [J. Clin. Invest., 98, 262 (1996)].
  • Non-specific drugs are less desirable because their effects on cAMP or cGMP-mediated signaling in tissues and cells other than the target tissue and cells are problematic.
  • An object of the present invention is to provide a novel PDE polypeptide and a DNA encoding the PDE polypeptide.
  • the present invention also provides diabetes, ischemic heart disease, hypertension, nephritis, nephritis, ulcer, allergy, asthma, rheumatism, osteoporosis, pain, anxiety using the PDE polypeptide or an antibody recognizing the polypeptide. It is an object of the present invention to provide a medicament for preventing and / or treating symptom, fibrosis, manic depression, Parkinson's disease, dementia, infectious disease or malignant tumor.
  • the present inventors prepared a cDNA library from HepG2 cells and randomly analyzed a base sequence. Based on the gene sequence information of human PDE10A (GenBank: AB020593), the human cDNA sequence thus obtained was analyzed using BLAST search homology search software, and the catalytic sites of human PDE10A and human PDE5A were analyzed. A partial sequence having homology with was found. By analyzing these sequences, expressing the polypeptide and detecting the PDE activity, the present invention has been completed.
  • the present invention relates to the following inventions (1) to (33).
  • a polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 15.
  • transformant is a transformant selected from the group consisting of microorganisms, animal cells, plant cells, and insect cells.
  • the transformant according to any one of (5) to (8) is cultured in a medium, the polypeptide described in (1) is produced and accumulated in the culture, and the polypeptide is isolated from the culture.
  • Oligonucleotide derivative is an oligonucleotide derivative in which the phosphoric ester bond in the oligonucleotide is converted to a phosphorothioate bond, and the phosphodiester bond in the oligonucleotide is N3, -P5 'phosphoramidite.
  • Oligonucleotide derivative converted to date bond Oligonucleotide derivative in which ribose and phosphodiester bond in oligonucleotide are converted to peptide nucleic acid bond
  • Peracyl in oligonucleotide is C-15 provinylperacyl
  • Oligonucleotide derivative in which oligosaccharide in oligonucleotide is substituted with C-15 thiazoleperacyl Oligonucleotide derivative in which cytosine in oligonucleotide is substituted with C-5 propynylcytosine, oligo In nucleotides Tosin is phenoxazine-modified cytosine (
  • Oligonucleotide derivatives substituted with phenoxazine-modified cytosine Oligonucleotide derivatives substituted with phenoxazine-modified cytosine
  • oligoribonucleotide derivatives in which the ribose in DNA is substituted with 2,10-propylribose oligoribonucleotide derivatives in which the ribose in DNA is substituted with 2,10-propylribose
  • ribose in the oligonucleotide with 2'-methoxyethoxy ribose The oligonucleotide according to (10), which is an oligonucleotide derivative selected from the group consisting of substituted oligonucleotide derivatives.
  • a method for detecting mRNA encoding the polypeptide according to (1) comprising using the oligonucleotide according to (10) or (11).
  • the method for suppressing the expression of the polypeptide according to (1) comprising using the oligonucleotide according to (10) or (11.).
  • a method for screening a compound that varies the phosphodiesterase activity of the polypeptide which comprises contacting the polypeptide according to (1) with a test sample.
  • a method for screening a compound that changes the expression of a gene encoding the polypeptide comprising contacting a cell that expresses the polypeptide according to (1) with a test sample.
  • test sample is contacted with a transformant having the promoter DNA described in (24) and a plasmid containing the repo overnight gene linked downstream of the promoter DNA. Measuring the translation product content of the reporter gene. A method for screening a compound which varies the efficiency of transcription by said promoter.
  • the repo overnight gene is the chloramphenicone acetyltransferase gene, the galactosidase gene, the luciferase gene, the ' ⁇ -glucuronidase gene, the equorin gene, and the green fluorescent protein.
  • a medicament comprising the compound according to (19), (23) or (27). : (32) Diabetes, ischemic heart disease, hypertension, nephritis, Tengitis, ulcer, allergy, asthma, rheumatism, osteoporosis, pain, anxiety, schizophrenia, manic depression, Parkinson's disease, dementia, infectious disease or malignancy From (28), which is a medicament for the prevention or treatment of tumors
  • Figure 1 shows a comparison between the amino acid sequence of the new human PDE (upper row: amino acid numbers are indicated by numbers + and) and the amino acid sequence of human PDE5A (GenBank: CAA06170) (lower row: amino acid numbers are indicated by numbers + ").
  • An asterisk indicates a matching amino acid residue, and a period indicates a homologous amino acid residue.
  • Figure 2 shows the amino acid sequence of the new human PDE (upper row: the amino acid number is indicated by a number +,) and the amino acid sequence of the hPDE10A (GenBank: BAA78034) (lower row: the amino acid number is a number)
  • An asterisk indicates a matching amino acid residue, and a period indicates a homologous amino acid residue. (Aminoic acid residues are indicated by one letter.) The underline indicates the HDXXHXXXXN motif.
  • FIG. 3 shows the construction process of plasmid P200-EB and a restriction enzyme map.
  • Fig. 4 shows a poly (A) + MA file of human heart, brain, placenta, lung, liver, skeletal muscle, kidney, and knee using a partial sequence (about 0.2 kb) of the novel human PDE homolog cDNA as a probe. -Filtration of CHurnan Multiple Tissue Northern Blots (Clontech)] and poly (A) + RNA filter of human heart, brain, liver, kidney, placenta and lung [Human Normal Tissue mENA blot I (Normalized) filter]
  • FIG. 7 is a diagram showing the results of performing Northern Hybridization on Yuichi (Toyobo Co., Ltd.).
  • FIG. 5 is a diagram showing a construction process of a plasmid pGST-PDE and a restriction enzyme map.
  • FIG. 6 shows the results of PDE activity measurement in the expressed E. coli soluble fraction.
  • GST indicates Escherichia coli transfected with only black and white
  • GST-PDE indicates Escherichia coli expressing the GST-PDE fusion protein.
  • FIG. 7 is a diagram showing the construction process of plasmids p23-2k and p23-lk and a restriction enzyme map.
  • FIG. 8 is a diagram showing a comparison between the amino acid sequence of the novel human PDE (upper) and the amino acid sequence of human PDE5A (GenBank: CAA06170) (lower). Asterisks indicate identical amino acid residues, and periods indicate homologous amino acid residues. (Amino acid residues are indicated by single letter notation.) Underline indicates HDXXHXXXXN motif.
  • Figure 9 shows the ⁇ (3 ⁇ 4 primer designed based on the sequence information of the novel human PDE homolog cDNA, and the cDNA prepared from mMA of human tissues of the kidney, lung, mammary gland, prostate, skeletal muscle, and testis. PCR was performed and the results of agarose electrophoresis of the amplified product are shown.
  • kb indicates kilobase pairs
  • Ap indicates an ampicillin resistance gene
  • T7 indicates a T7 promoter
  • GST indicates glutransthione S trans.
  • polypeptide of the present invention is characterized by comprising any one of the following amino acid sequences.
  • a polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or Rooster S, which has at least one amino acid deleted, substituted or added in SEQ ID NO: 15, and having phosphodiesterase (PDE) activity Molecular Cloning, A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1989) (hereinafter abbreviated as Molecular Cloning 2nd Edition), Current Protocols in Molecular Biology, Supplement 1-38, John Wiley & Sons (1987 -1997) (hereinafter abbreviated as current 'protocol' in 'molekiura' biology), Nucleic Acids Research, 10, 6487 (1982), Proc. Natl. Acad. Sci.
  • PDE phosphodiesterase
  • 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 above-described site-directed mutagenesis method, and is one to several tens. The number is preferably 1 to 20, more preferably 1 to 10, and still more preferably 1 to 5. Further, in order for the polypeptide of the present invention to have phosphodiesterase activity, the homology with the amino acid sequence of SEQ ID NO: 1 or 15 is determined by BLAST [
  • the present invention also provides a DNA encoding the above-mentioned polypeptide of the present invention.
  • Specific examples of the DNA encoding the polypeptide of the present invention include DNA having any one of the following nucleotide sequences.
  • base sequence encoding a polypeptide that hybridizes under stringent conditions and has phosphodiesterase activity refers to a DNA having the base sequence of SEQ ID NO: 2 or SEQ ID NO: 16 as a probe.
  • Colony-hybridization method plaque; DNA obtained by using the hybridization method or Southern blot hybridization method, specifically, DNA derived from a colony or plaque.
  • the SSC 0.1- to 2-fold concentration
  • saline-sodium citrate solution (the composition of a 1x concentration SSC solution is composed of 15 Ommo 1 / L sodium chloride and 15 mmo 1 / L sodium citrate), and fill at 65 ° C. Can be identified by washing the DNA.
  • DNA that can be hybridized is calculated using analysis software such as BLAST (J. Mol. BioL, 215, 403, 1990) and FASTA (Methods in Enzymology, 183, 63-69).
  • BLAST J. Mol. BioL, 215, 403, 1990
  • FASTA Methods in Enzymology, 183, 63-69
  • the public database such as GenBank and Swiss_Plot can be used as the database.
  • the origin of the DNA of the present invention is not particularly limited, but is preferably a mammal, and particularly preferably a human.
  • RNA Total RNA from appropriate cells or tissues to create a DNA library Alternatively, prepare mRNA.
  • RNA guanidine thiocyanate-cesium trifluoroacetate method [Methods in Enzymology, 154, 3 (1987)], acid guanidine thiochanetone 'phenol' Cloguchiform (AGPC) method [AnalyticaI Biochemistry, 162 5 156 (1987), and Experimental Medicine 1937 (1991)].
  • Methods for preparing mRNA as poly (A) + RNA from total RNA include the Oligo (dT) immobilized cellulose column method (Molecular 'Cloning 2nd Edition') and a method using oligo dT latex. it can.
  • Kits such as Fast Track mRNA Isolation Kit (Fast Track mRNA Isolation Kit; manufactured by Iiwitrogen) and Quick Prep mRNA purification kit (Quick Prep mRNA Purification Kit; manufactured by Pharmacia) It can also be used to prepare mRNA directly from tissues or cells.
  • a cDNA library is prepared by an ordinary method.
  • Large intestine is used as a cloning vector for preparing a cDNA library.
  • Any phage vector, plasmid vector, etc. can be used as long as they can replicate autonomously in the bacterial K12 strain.
  • any microorganism belonging to Escherichia coli can be used. Specifically, Escherichia coli XL Bok Blue MRF, [Stora evening Gene Co., Strategies, 5 3 81 (1992 ) ], Escherichia coli C6QQ [Genetics,
  • Examples of commercially available cDNA libraries include cDNA libraries of various organs derived from humans, mice, rats, rats, and egrets, such as Clothtec and Lifetech Oriental.
  • the DNA of the present invention or a cDNA clone containing a part thereof was isolated or labeled with a fluorescent probe.
  • a primer prepared based on the nucleotide sequence can be used as the primer.
  • An adapter is added to both ends of the cDNA, and PCR is performed with a primer based on the base sequence partially identifiable with the base sequence of this adapter.5'-RACE (rapid amplification of cDNA ends) and 3,-RACE [Proc. Natl. Acad. Sci. USA, 85 3 8998 (1988)] to obtain cDNA fragments at the 5 'end and the 3' end from the sequence used for the primer. be able to.
  • the full-length DNA of the present invention can be obtained.
  • a cDNA encoding the full-length polypeptide may be obtained as follows. it can.
  • a single-stranded cDNA library derived from various organs or cells can be prepared according to a conventional method or a standard kit. For example, it can be prepared by the following method.
  • RNA is extracted from various organs or cells by the guanidium thiosinate phenol chloroform method [Anal. Biochem., 162, 156 (1987)]. If necessary, treat all RNAs with Deoxyribonuclease I (Life Technologies) to degrade chromosomal DNA that may be contaminated. For each of the obtained total RNAs, a single-stranded cDNA library can be prepared by using the SUPERSCRIPT TM Preamplification System for First Strand cDNA System (manufactured by Life Technologies) using oligo (dT) primers or random primers.
  • SUPERSCRIPT TM Preamplification System for First Strand cDNA System manufactured by Life Technologies
  • oligo (dT) primers or random primers random primers.
  • the nucleotide sequence of DNA was obtained by digesting the DNA fragment as it is or by using an appropriate restriction enzyme or the like, and incorporating the fragment into a vector by a conventional method. Sanger) et al. [Proc. Natl. Acad. Sci. USA, 74, 5463 (1977)] or Perkin Elmer: 373A DNA Sequencer, Pharmacia, Lycoa It can be determined by analysis using a base sequence analyzer such as (LI-C0R).
  • Examples of the plasmid containing the DNA of the present invention and a part thereof obtained by the above method include, for example, plasmid hepl0314 having a part of the DNA consisting of the base sequence represented by SEQ ID NO: 2, and SEQ ID NO: 16 And plasmids p23-lk and p23-2k having a part of the DNA consisting of the base sequence represented by
  • Escherichia coli JM109 / hepl0314 containing plasmid hepl0314 was designated as FERM BP-6976 as of December 22, 1999 by the Institute of Life Science and Industrial Technology, National Institute of Advanced Industrial Science and Technology. Deposited with No. 3 (zip code 305-8566).
  • the target DNA can also be prepared by chemical synthesis using a DNA synthesizer.
  • the DNA synthesizer include a DNA synthesizer manufactured by Shimadzu Corporation using the thiophosphite method, and a DNA synthesizer mode 1392 manufactured by Perkin Elmer Inc. using the phosphoramidite method. it can.
  • nucleotide sequence database such as GenBank, EMBL and DDBJ using a homology search program such as BLAST.
  • the homology search programs such as FASTA and FrameSearch (FrameSearch) are used to search for amino acid sequences such as GenPept, PIR and Swiss-Prot. By searching, existing genes with homology can be searched.
  • an antisense oligonucleotide having a partial sequence of the DNA of the present invention or a sense Oligonucleotides such as nucleotides can be prepared.
  • oligonucleotide examples include a DNA having the same sequence as the consecutive 5 to 60 bases in the base sequence of the DNA or a DNA having a sequence complementary to the DNA.
  • the above-mentioned oligonucleotides whose melting temperature (T m) and number of bases do not extremely change are preferred.
  • I oligonucleotides include the oligonucleotides represented by SEQ ID NOS: 10, 11, 17, or 18.
  • oligonucleotides can also be used as the oligonucleotide of the present invention.
  • oligonucleotide derivatives include an oligonucleotide derivative wherein the phosphoric acid Jiesu ether bond in the oligonucleotide is converted to Hosuhorochioe Bok bond, a phosphodiester bond N 3 in O oligo nucleotides, - the P 5 5 Hosufoamide one preparative bond Converted oligonucleotide derivative, oligonucleotide derivative in which ribose and phosphodiester bond in oligonucleotide are converted to peptide nucleic acid bond, peracyl in oligonucleotide substituted with C-15 propynyl peracyl Oligonucleotide derivatives, Oligonucleotide derivatives in which peracyl in the oligonucleotide is substituted by C-15thiazo'-peruracil, Oligonucleotide derivatives in which cytosine in the oligonucleotide is substitute
  • Oligonucleotide derivatives substituted with phenoxazine-modified cytosine Oligonucleotide derivatives substituted with 2,10-propylribose, or ribose in the oligonucleotide has been substituted with 2′-methoxetoxyribose Oligonucleotide derivatives, etc. [Cell Engineering, 16, 1463 (1997)].
  • a recombinant vector expressing the polypeptide of the present invention is obtained by constructing a recombinant vector in which the DNA of the present invention is inserted downstream of an appropriate expression vector, and introducing the vector into a host cell.
  • the polypeptide of the present invention can be produced by obtaining and culturing the transformant.
  • the host cells any microorganisms (bacteria, yeast, etc.), animal cells, insect cells, plant cells, etc., can be used as long as they can express the gene of interest.
  • the expression vector a vector capable of autonomously replicating in the above-mentioned host cell, capable of being integrated into a chromosome, and containing a promoter at a position capable of transcribing the DNA of the present invention is used.
  • the polypeptide gene expression vector of the present invention is capable of autonomous replication in a prokaryote and has a promoter, a ribosome binding sequence, and a DNA of the present invention. And a recombination vector comprising a transcription termination sequence. A gene that controls the promoter overnight may be included.
  • expression vectors include pB.Trp2, pBTAc Is pBTac2 (both commercially available from Boehringer Mannheim), PKK233-2 (Pharmacia), PSE280 (Invitrogen), pGEEX- ⁇ [Promega ( Promega), QE-8 (QIAGEN), pKYPIO (JP-A-58-U0600), pKYP200 (Agric. Biol. Chem., 8, 669 (1984)), pLSAl CAgric. Biol. Chem. , 53, 277 (1989)), pGELl [Pro Natl. Acad. Sci.
  • the promoter may be any promoter that can be expressed in host cells such as Escherichia coli and Bacillus subtilis.
  • promoters derived from Escherichia coli or phage such as ii ⁇ promoter overnight (Pii ⁇ ), lac promoter overnight (Plac), P L promoter, promoter overnight, T7 promoter overnight, etc. , SP01 Promoter, SP02 Promo Overnight, penP Promo Overnight, etc.
  • the liposome binding sequence it is preferable to use a plasmid in which the distance between the Shine-Dalgarno sequence and the initiation codon is adjusted to an appropriate distance (for example, 6 to 18 bases).
  • transcription termination sequence is not necessarily required for the expression of the DNA of the present invention, it is preferable to arrange the transcription termination sequence immediately below the structural gene.
  • the host cell Eshierihia, Serratia, Bacillus, Burebibakute genus, Corynebacterium, the genus Microbacterium, microorganisms belonging to Shiyudomonasu genus like, for example, Escherichia coli XL1-Blue s Escherichia coli XL2-Blue, Escherichia coli Keio ⁇ Escherichia coli MC1000 S Escherichia coli KY3276, Escherichia coli W1485, Escherichia coli JM109, Escherichia coli HB101, Escherichia coli No.49, Escherichia coli W3110, Escherichia coli NY49, Serratia ficaria ⁇ Serratia fonticola, Serratia Iiguefaciens s Serratia marc6sc6ns , Bacillus subtil is, Bacillus amyloliquefaciens ⁇ Brevibacterium ammoniage
  • Corynebacterium glutamicmn ATCC14Q67 S Corynebacterium glutamicum ATCC13869, Corynebacterium acetoacidophilum ATGC1387Q N Microbacterium ammoniaphilum ATCC15354, Pseudomonas sp. D- 0110 , or the like can be mentioned.
  • Any method for introducing a recombinant vector can be used as long as it is a method for introducing DNA into the above host cells, for example, a method using lucidium ion [Proc. Natl. Acad. Sci. USA, 69, 2110 ( 1972)], protoplast method (JP-A-63-248394), electroporation method [Gene, 17, 107 (1982), Molecular & General Genetics, 168, ill (1979)].
  • YEpl3 ATCC37115
  • YEp24 ATCC37051
  • YCp50 ATCC37419
  • pHS19 pHS15, etc.
  • Any promoter can be used as long as it can be expressed in yeast strains, such as PH05 Promo, PGK Promo, GAP Promo, ADH Promo, Promoters such as gal 1 promoter, gal 10 promoter, heat shock polypeptide promoter, MF 1 promoter, CUP 1 promoter, etc.
  • any method can be used as long as it is a method for introducing DNA into yeast, and examples thereof include an electoporation method, Qiethods in Enzymology, 194, 182 (1990)) and a spheroplast method [ Proc. Natl. Acad. Sci. USA, 81, 4889 (1984)] and the lithium acetate method Journal of Bacteriology, 153, 163 (1983)].
  • expression vectors such as pcDNA I / Amp (manufactured by Invitrogen), pcDNAI, pCDM8 (Nature, 329, 840 (1987)), pAGE107 [ Kaihei 3-22979, Cytotechnology, 3, 133 (1990)], pREP4 (Invitrogen), pAGE 103 (Journal of Biochemistry, 101, 1307 (1987)), pAMo, pAMoA, pAS3-3 (Special Kaihei 2) -227075) is used.
  • any promoter can be used as long as it can be expressed in animal cells.
  • the promoter of the IE (i-ediate early) gene of cytomegalovirus (CMV) can be used.
  • CMV cytomegalovirus
  • SV40 early promoter or metallothinin promoter can be used.
  • retrovirus promoter can be used.
  • heat shock promoter can be used.
  • animal cells examples include mouse and mouse cells, rat's mouse cells, mouse 'hybrid cells, and human cells such as Namalwa cells or femalwa KJM-1 cells CCytotechnology, 1 3 151 (1988)], can be mentioned the human embryonic kidney cells, human leukemia cells, Afurikami Dorizaru kidney cells, CH0 cells is a cell of Chinese hamster, and HBT5637 (JP 63- 299) or the like.
  • Mouse / myeloid cells include SP2 / 0, NS0, etc., Rat'myeloid cells, YB2 / 0, etc., and human embryonic kidney cells, such as HEK293 (ATCC: CRL-1573), human Examples of leukemia cells include BALL-1 and examples of African green monkey kidney cells include COS-1 and COS-7.
  • any method for introducing DNA into animal cells can be used.
  • the recombinant gene transfer vector and the baculovirus were co-transfected into bizoa cells to obtain the recombinant virus in the insect cell culture supernatant, and then the recombinant virus was infected to the insect cells, and Peptides can be expressed. '
  • Examples of the gene transfer vector used in the method include pVL1392, pVL1393, pBlueBacIII (all manufactured by Invitrogen) and the like.
  • Examples of baculoviruses are viruses that infect night roth moths, such as Atographa's force, Reformi force, Nuclea, polyhedrosis, and virus.
  • Ru can Autographa caiiforaica nuclear polyhedrosis virus
  • ovary cells of Spodoptera frugiperda ovary cells of Trichoplusia ni, cultured cells derived from silkworm ovary, and the like can be used.
  • Spodoptera frugiperda ovary cells include Sf9 and Sf21 (baculovirus' Expression Vectors' Laboratory 'manual), etc. Bombyx mori M and the like can be mentioned as cultured cells derived from silkworm ovaries.
  • Co-transfection of the above-described recombinant gene transfer vector and the above baculovirus into insect cells for preparing a recombinant virus includes, for example, a calcium phosphate method.
  • a method for expressing a gene in addition to direct expression, secretory production, fusion protein expression, and the like can be performed according to the method described in Molecular 'Cloning, 2nd edition.
  • sugar or sugar chain-added polypeptide When expressed by yeast, animal cells or insect cells, a sugar or sugar chain-added polypeptide can be obtained.
  • the polypeptide of the present invention is produced by culturing the transformant obtained as described above in a medium, producing and accumulating the polypeptide of the present invention in a culture, and collecting the polypeptide from the culture. be able to.
  • the cells are returned to the living body, thereby obtaining the polypeptide of the present invention.
  • the peptide can also be expressed in a patient's body.
  • the method of culturing the transformant of the present invention in a culture medium can be performed by a conventional method for culturing a host It can be done according to the method.
  • a culture medium for culturing a transformant obtained by 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 can be used as long as the medium can be efficiently cultured.
  • the carbon source may be any one that can be assimilated by the organism, such as glucose, fructose, sucrose, molasses containing these, carbohydrates such as starch or starch hydrolysate, and organic acids such as acetic acid and propionic acid. Acids, alcohols such as ethanol and propanol and the like can be used.
  • Nitrogen sources include ammonia, ammonium chloride, ammonium sulfate, ammonium acetate, ammonium phosphate, and other ammonium or inorganic salts of organic acids, other nitrogen-containing compounds, and peptone, meat extract, yeast extract, and corn starch. 1. Casein hydrolyzate, soybean meal and soybean meal hydrolyzate, various fermented cells, digested products thereof, and the like can be used.
  • potassium phosphate monobasic, potassium phosphate dibasic, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate, calcium carbonate and the like can be used.
  • the cultivation is usually carried out under aerobic conditions such as shaking culture or deep aeration stirring culture.
  • the culturing temperature is preferably 15 to 40 ° C, and the culturing time is usually 16 to 96 hours.
  • the pH is maintained at 3.0 to 9.0.
  • the pH is adjusted using an inorganic or organic acid, an alkaline solution, urea, calcium carbonate, ammonia, or the like.
  • an antibiotic such as ampicillin or tetracycline may be added to the medium during the culture.
  • an indicator When culturing a microorganism transformed in an expression vector using an inducible promoter as the promoter, an indicator may be added to the medium, if necessary.
  • an indicator For example, when culturing a microorganism transformed with an expression vector using a promoter, isopropyl-1 /?-D-thiogalactopyranoside or the like is used; —When culturing a microorganism transformed with an expression vector using Yuzuichi, indoleacrylic acid or the like may be added to the medium.
  • a medium for culturing the transformant obtained using animal cells as a host commonly used RPMI1640 medium [The Journal of the American Medical Association, 199, 519 (1967)], Eagle's MEM medium [Science, 122, 501 (1952)), DMEM medium (Virology, ⁇ , 396 (1959)), 199 medium (Proceeding of the Society for the Biological Medicine, 73, 1 (1950)), or fetal calf serum etc.
  • a used medium or the like can be used.
  • Culture is usually p H 6 ⁇ 8, 3 0 - performing 4 0 ° C 5% C 0 2 under the conditions such as the presence 1-7 days.
  • an antibiotic such as kanamycin, penicillin, streptomycin or the like may be added to the medium during the culture.
  • TNM-II medium Pharmingen
  • Sf-900 II SFM medium Sf-900 II SFM medium
  • ExCell400 ExCell405 (all manufactured by JRH Biosciences)
  • Grace's Insect Medium Neture, 195, 788 (1962)
  • the cultivation is usually performed under conditions of pH 6 to 7, 25 to 30 ° C. for 1 to 5 days.
  • an antibiotic such as genyumycin may be added to the medium during the culture.
  • an ordinary enzyme isolation and purification method may be used.
  • the polypeptide of the present invention when expressed in a dissolved state in a cell, after completion of the culture, the cell is collected by centrifugation, suspended in an aqueous buffer, and then subjected to an ultrasonic homogenizer, a French press, The cells are disrupted with a Manton Gaurin homogenizer, Dynomill, etc. to obtain a cell-free extract.
  • an ordinary enzyme isolation and purification method that is, a solvent extraction method, a salting out method using ammonium sulfate, a desalting method, and a precipitation method using an organic solvent , Getylaminoethyl (DEAE)-Sepharose, anion exchange chromatography using a resin such as DIAION HPA-75 (manufactured by Mitsubishi Chemical Corporation), and a positive electrode using a resin such as S-Sepharose FF (manufactured by Pharmacia) Ion exchange chromatography, hydrophobic chromatography using resins such as butyl sepharose and phenylsepharose, gel filtration using molecular sieves, affinity chromatography, chromatofocusing, etc.
  • a purified sample can be obtained by using techniques such as electrophoresis such as electrofocusing alone or in combination.
  • the cells When the polypeptide is expressed by forming an insoluble substance in the cells, the cells are similarly recovered, crushed, and centrifuged. After recovering the polypeptide, the insoluble form of the polypeptide is solubilized with a protein denaturant.
  • the solubilized solution was diluted or dialyzed into a solution containing no protein denaturing agent or a solution in which the concentration of the protein denaturing agent was so low that the protein was not denatured, to form the polypeptide into a normal three-dimensional structure. Thereafter, a purified sample can be obtained by the same isolation and purification method as described above.
  • the polypeptide of the present invention or a derivative such as a modified sugar chain thereof is secreted extracellularly, the polypeptide or the derivative such as a glycosylated product thereof can be recovered in the culture supernatant.
  • a soluble fraction is obtained by treating the culture by a method such as centrifugation as described above, and a purified sample is obtained from the soluble fraction by using the same isolation and purification method as described above. Can be obtained.
  • the polypeptide of the present invention can be produced as a fusion protein with another protein, and purified using affinity chromatography using a substance having affinity for the fused protein.
  • affinity chromatography using a substance having affinity for the fused protein.
  • the polypeptide of the present invention can be produced as a fusion protein with protein A according to the method described in 05-336963, W094 / 23021, and can be purified by affinity chromatography using immunoglobulin G.
  • polypeptide of the present invention can be produced as a fusion protein with an F1ag peptide and purified by affinity chromatography using an anti-F1ag antibody [Proc. Natl. Acad. Sci. USA, 86, 8227 (1989), Genes & Dev., A, 1288 (1990)]. Furthermore, it can be purified by affinity chromatography using an antibody against the polypeptide.
  • polypeptide of the present invention can also be produced by chemical synthesis methods such as the Fmoc method (fluorenylmethyloxycarbonyl method) and the tB0c method (t-butyloxycarbonyl method). be able to.
  • Structural analysis of the purified polypeptide of the present invention is described in a method commonly used in protein chemistry, for example, protein structural analysis for gene cloning (Hisashi Hirano, published by Tokyo Kagaku Dojin, 1993). The method can be implemented by the following method. ,
  • a polyclonal antibody can be produced by using the purified full-length or partial fragment of the polypeptide of the present invention obtained by the method described in the above [2] as an antigen and administering it to an animal.
  • the dose of the antigen is preferably 50 to 100 zg per animal.
  • a peptide it is preferable to use, as an antigen, a peptide obtained by covalently bonding the peptide to a carrier protein such as keyhole limpet haemocyanin or bovine tilogin purine.
  • a peptide serving as an antigen can be synthesized by a peptide synthesizer.
  • the administration of the antigen is performed 3 to 10 times every 1 to 2 weeks after the first administration.
  • Blood is collected from the fundus venous plexus 3 to 7 days after each administration, and the reaction of the serum with the antigen used for immunization is determined by enzyme-linked immunosorbent assay [Enzyme-linked immunosorbent assay (ELISA): published by Medical Shoin 1976 Antibodies-A Laboratory Manual, Cold Spring Harbor Laboratory Press (1988)
  • a polyclonal antibody can be obtained by obtaining serum from a non-human mammal whose serum shows a sufficient antibody titer against the antigen used for immunization, and separating and purifying the serum.
  • Separation and purification methods include centrifugation, salting out with 40-50% saturated ammonium sulfate, force prillic acid precipitation (Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, (1988)), or DEAE-Cephalone.
  • a rat whose serum shows a sufficient antibody titer against the antigen used for immunization is used as a source of antibody-producing cells.
  • the spleen is removed 3 to 7 days after the final administration of the antigenic substance to the rat showing the antibody titer.
  • the spleen is shredded in MEM medium (manufactured by Nissui Pharmaceutical), loosened with forceps, centrifuged at 1,200 rpm for 5 minutes, and the supernatant is discarded.
  • MEM medium manufactured by Nissui Pharmaceutical
  • the resulting precipitate fraction spleen cells were treated with Tris ammonium chloride buffer (pH 7.6.5) for 1 to 2 minutes to remove red blood cells, and then washed three times with MEM medium. Splenocytes are used as antibody producing cells.
  • myeloma cells cell lines obtained from mice or rats are used.
  • 8-azaguanine resistant mouse derived from BALB / c myeloma cell line
  • P3-X63Ag8-Ul CCurr.Topics Microbiol. Immunol., 81, 1 (1978), Eur.J.I maraudal nol., 6, 511 (1976)], SP2 / 0-Agl4 (SP- 2) (Nature, 276, 269 (1978)), P3-X63-Ag8653 (653) [J. Immunol., 123, 1548 (1979)] ⁇ P3-X63-Ag8 (X63) [Nature, 495 (1975) ] and the like can be used.
  • these cell lines include 8-Azaguanin medium [RPMI-1640 medium glutamine (1.
  • the resulting precipitated fraction of cell groups may Hoku, and, in the group of cells, with stirring, at 37 ° C, 10 8 antibody-producing cells per polyethylene glycol one Roux 1000 (PEG- 1000) 2 g MEM Add 0.2 to 1 mL of a mixed solution of 2 mL and 0.7 mL of dimethyl sulfoxide (DMS 0), and add 1 to 2 mL of MEM medium several times every 1 to 2 minutes.
  • PEG- 1000 polyethylene glycol one Roux 1000
  • DMS 0 dimethyl sulfoxide
  • the suspension was dispensed by 100 ⁇ L / well to the plate for a 96-well culture, 5% Rei_0 2 in I Nkyube Isseki one, at 37 ° C? Incubate for ⁇ 14 days.
  • a purified full-length or partial fragment of the polypeptide of the present invention used as an antigen is coated on an appropriate plate, and the obtained supernatant is obtained from a hybridoma culture supernatant or (2-4) described later.
  • the purified antibody is reacted as the first antibody, and the second antibody is reacted with an anti-rat immunoglobulin antibody labeled with biotin, an enzyme, a chemiluminescent substance, a radiation compound, or the like, followed by a reaction according to the labeling substance.
  • Those which specifically react with the polypeptide of the present invention are selected as hybridomas producing monoclonal antibodies against the polypeptide of the present invention.
  • Pristane-treated [2,6,10,14-tetramethyl-pen-decane (Pristane) -0.5 mL was intraperitoneally administered and bred for 2 weeks] to 8-10 week old mice or nude mice, (2 -Intraperitoneal injection of 5 to 20 x 10 6 hybridoma cells producing a monoclonal antibody against the polypeptide of the present invention obtained in 3) I do.
  • Hypridoma develops ascites cancer in 10 to 21 days.
  • the ascites is collected from the mouse with ascites tumor and centrifuged at 3,000 rpm for 5 minutes to remove solids. 'From the obtained supernatant, a monoclonal antibody can be purified and obtained by the same method as that used for the polyclonal antibody.
  • the antibody subclass is determined using a mouse monoclonal antibody evening kit or a rat monoclonal antibody typing kit.
  • the protein amount is calculated by the oral method or the absorbance at 28 Onm.
  • the polypeptide of the present invention is expressed in Escherichia coli, yeast, insect cells, animal cells, etc. as a host, or prepared in DNA or in vitro in African omega frog oocytes.
  • Expression of the cRNA obtained by the microinjection method (Methods in Enzymology, Z07, 225 (1992), Methods in Enzymology, 254, 458 (1995)), and in v: itro translation products were measured for PDE activity.
  • PDE activity is measured using a cyclic nucleotide (eg, [ 3 H] cAMP or [ 3 H] cGMP) labeled with a detectable reagent (eg, a radioactive, fluorescent, or colorimetric reagent).
  • a detectable reagent eg, a radioactive, fluorescent, or colorimetric reagent.
  • the polypeptide of the present invention is expressed in a yeast (e.g., Saccharomyces cerevisiae strain PP5 (ATCC number 96135), Saccharomyces cerevisiae strain 10DAB (ATCC number 74049)), and recovery of heat shock sensitivity, sensitivity to nitrogen deficiency, etc. is used as an index. ; PDE activity can also be detected [Proc. Natl. Acad. Sci. USA, 90 5 11970 (1993), J. Biol. Chem., 274, 4839 (1999)].
  • Test samples include synthetic compounds, naturally occurring proteins, artificially synthesized proteins, peptides, carbohydrates, lipids, modified forms and derivatives thereof, and mammals (for example, mice, rats, guinea pigs, hamsters).
  • the agonist or aminogonist of the polypeptide of the present invention obtained by the above method can be used alone as a therapeutic agent, but is usually one or more pharmacologically acceptable. It is desirable to mix it with a carrier and use it as a pharmaceutical preparation produced by any method well known in the pharmaceutical art.
  • oral administration or parenteral administration such as oral, respiratory, rectal, subcutaneous, intramuscular, and intravenous administration.
  • parenteral administration such as oral, respiratory, rectal, subcutaneous, intramuscular, and intravenous administration.
  • Examples of the dosage form of the therapeutic agent include ointments, sprays, capsules, tablets, granules, syrups, emulsions, suppositories, injections, tapes and the like.
  • Formulations suitable for oral administration include emulsions, syrups, capsules, tablets, powders, granules and the like.
  • Liquid preparations such as emulsions and syrups are prepared from sugars such as water, sucrose, sorbitol, fructose, glycols such as polyethylene glycol, propylene glycol, sesame oil, olive oil, soybean oil, etc. It can be produced using preservatives such as oils, p-hydroxybenzoic acid esters, etc., and flavors such as bery flavor, peppermint and the like as additives.
  • 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 chloride Binders such as alcohol, hydroxypropyl cellulose and gelatin, surfactants such as fatty acid esters, and plasticizers such as glycerin can be used as additives.
  • Formulations suitable for parenteral administration include injections, suppositories, sprays and the like.
  • An injection can be prepared using, for example, a carrier composed of a salt solution, a glucose solution, or a mixture of both.
  • Suppositories can be prepared using carriers such as cocoa butter, hydrogenated fats and carboxylic acids.
  • the agonist or angonist obtained above can be used directly as a propellant, but it does not irritate the oral cavity and the respiratory tract mucosa of the recipient, and the conjugate is used as fine particles. Sprays prepared using a carrier or the like that disperses and facilitates absorption are preferred.
  • the carrier include lactose and glycerin.
  • Formulations such as aerosols and dry powders can be prepared depending on the properties of the agonist or angelic gonist obtained above and the carrier.
  • the dose or frequency of administration varies depending on the desired therapeutic effect, administration method, treatment period, age, body weight, etc., but is usually 10 zg / kg to 8 mg / kg per adult per day.
  • the compound for regulating the expression of the polypeptide of the present invention can be searched and identified by using the antibody of the present invention.
  • any cell, cell line, or tissue expressing the polypeptide of the present invention can be used.
  • a cell, cell line, or tissue in which the expression of the polypeptide has been observed can be used by the method of immunologically detecting with the antibody described in [7] or the method of detecting m: RNA.
  • Suitable cell lines include, for example, Hep (3 ⁇ 4 cells).
  • test sample those described in the test sample in the above [5] can be used.
  • the cells expressing the polypeptide of the present invention are suspended in a medium capable of growing the cells, a test sample is added to the medium, and the cells are contacted with the cells.
  • Quantify the expressed polypeptide content of the cells As a method for quantification, for example, a method using the following immune cell staining can be mentioned.
  • Cultured cells can be used as they are for suspension cells.
  • the cells are washed with a PBS buffer and suspended in a fixative.
  • the fixative include a PBS buffer containing 3.7% formaldehyde.
  • An example of the reaction solution that is permeable to the moon is a PBS buffer solution containing 0.1% Triton X-100.
  • the treated cells are suspended in a buffer for staining of immune cells (PBS containing 1% BSA, 0.02% EDTA, 0.05% sodium azide), etc., and 1 to 20 ⁇ 10 5 cells are suspended. Dispense into a round bottom 96-well plate.
  • the monoclonal antibody of the present invention is dispensed on the plate.
  • Examples of the monoclonal antibody include the culture supernatant of a hybridoma producing the monoclonal antibody of the present invention obtained in [3] (2-3), and the purified monoclonal obtained in [3] (2-4). Antibodies can be mentioned. Furthermore, an antibody labeled with the monoclonal antibody can also be used.
  • an antibody labeled with a monoclonal antibody for example, a biotin-labeled antibody can be mentioned.
  • a biotin-labeled antibody can be prepared by a known method (enzyme antibody method: interdisciplinary project, 1985).
  • the above antibody is diluted to a concentration of 0.1 to 50 ⁇ g / mL using an immune cell staining buffer or an immune cell staining buffer containing 10% animal serum.
  • a buffer for immunocell staining is added to the plate, and the cells are washed.
  • FITC fluorescein isothiocyanate
  • strebutavidin labeled with a fluorescent dye such as FITC or phycoerythrin is dispensed into the above plate in an amount of 5-0 to 500 ⁇ L / well, and the plate is iced. Leave in the light for 30 minutes under cooling.
  • Expression-modulating compounds can be identified by searching for a test sample that increases or decreases the polypeptide content of the present invention as compared to a system to which no test sample is added.
  • the cell expressing the polypeptide or the mRNA of the present invention for example, the cell line described in the above [6] (1) can be used, and as the test sample, the one described in the above [5] can be used.
  • Cells expressing the polypeptide of the present invention or mRNA encoding the polypeptide are suspended in a medium capable of growing the cells, a test sample is added to the medium, and the cells are contacted. After that, the content of the mRNA expressed in the cells is quantified by a conventional Northern hybridization method, an RNA dot-protocol hybridization method, and an RT-PCR method. I do.
  • a gene fragment encoding the polypeptide of the present invention can be mentioned as a probe that can be used for the hybridization method or the like and a primer that can be used for the HT-PCR method or the like.
  • an oligonucleotide having the same sequence as the contiguous 5 to 6 bases in the nucleotide sequence of SEQ ID NO: 2 or SEQ ID NO: 16 an oligonucleotide having a sequence complementary to the oligonucleotide, It can be suitably used.
  • An expression-regulating compound can be identified by searching for a test sample that increases or decreases the mRNA content of the polypeptide of the present invention as compared to a system to which no test sample is added.
  • Plasmid containing DNA linked to a repo-all-one gene downstream of a region that regulates transcription of a gene encoding the polypeptide of the present invention (hereinafter, abbreviated as a transcription control region). After contacting the transformant transformed with the test sample with the test sample, the expression controlling compound can be searched and identified by quantifying the expression level of the polypeptide encoded by the reporter gene.
  • a transcription control region is usually contained 5 ′ upstream of a gene.
  • the 5, upstream region of the gene encoding the polypeptide of the present invention can be prepared using, for example, Genome Walker kits (Clontech).
  • a fragment obtained by cutting this region using an appropriate restriction enzyme to an appropriate length can be used as a transcription control region.
  • the reporter gene any gene can be used as long as the translation product of the gene is stable in cells and the amount of the translation product can be easily quantified.
  • Examples of peptides include chloramphenic acid luciferase transferase (CAT), 5-galactosidase (5-gal), luciferase (luc), glucuronidase, equorin, green fluorescent protein (GFP) ) Etc. can be given.
  • Any cell may be used as a host cell into which the repo overnight plasmid containing the transcription control region is introduced, but preferably, the polypeptide of the present invention described in [6] (1) is used.
  • a cell line in which expression of the polypeptide mRNA has been confirmed can be used.
  • test sample described in [5] above can be used.
  • the repo overnight gene is ligated to the downstream of the transcription control region by an ordinary method, and the resulting plasmid is used to transform host cells by an ordinary method. .
  • gene selection for positive selection such as G418 resistance gene
  • negative selection marker for simple herpesvirus thymidine kinase diphtheria toxin A fragment gene, etc.
  • Vector and a cell line in which a part of the chromosomal gene encoding the polypeptide of the present invention is replaced with a repo overnight gene
  • the transformant is suspended, for example, in a medium in which the cells can grow, a test sample is added to the medium, and the cells are brought into contact with each other.
  • the amount of the encoded polypeptide is detected and determined by a method appropriate for the polypeptide.
  • Expression-controlling compounds can be identified by searching for a test sample that increases or decreases the content of the polypeptide encoded by the repo overnight gene as compared to a system in which the test sample is not added.
  • the DNA of the present invention is obtained by using the DNA as a probe for RNA extracted from tissues and cells of non-human mammals such as humans and mice in the same manner as in [1] (1).
  • the mRNA of the polypeptide gene of the present invention in the tissue or cell can be detected or quantified.
  • tissue expression distribution of the polypeptide of the present invention can be known.
  • the oligonucleotide of the present invention was used as a specific primer of the DNA of the present invention and extracted from tissues and cells of non-human mammals such as humans and mice in the same manner as [1] (1).
  • RT-PCR reverse transcription PCR; PCE Pr-otocols (1990)
  • mRNA encoding the polypeptide of the present invention can be detected and quantified.
  • the method for quantifying the mRNA can be used for diagnosis of a disease state associated with the present gene. it can.
  • a drug can be evaluated by comparing the expression amount of the mRNA with or without the drug.
  • the information obtained by these methods on the tissue and cells in which the polypeptide of the present invention is expressed and the information on what kind of stimulus the cells receive when the expression level changes are as follows. It is useful for analyzing the involvement of the polypeptide of the present invention in physiological functions and disease states.
  • the mutation of the gene encoding the polypeptide of the present invention is carried out by performing Southern hybridization (second edition) on genomic DNA using the DNA as a probe. Can be detected.
  • Detection of the mutation may cause the mutation in the gene, for example, diabetes, ischemic heart disease, hypertension, nephritis, knee inflammation, ulcer, allergy, asthma, rheumatism, osteoporosis, Diagnosis of diseases such as pain, anxiety, schizophrenia, manic depression, Parkinson's disease, dementia or malignancy can be made.
  • a single nucleotide polymorphism (single nucleotide polymorphism) can be obtained by amplifying the gene encoding the polypeptide of the present invention using PCR or the like and analyzing the base sequence, or by performing analysis using a DNA chip or the like.
  • polymorphisms SNP
  • the polymorphism of the gene may be related, for example, diabetes, ischemic heart disease, hypertension, nephritis, tengitis, ulcer, allergy, asthma, rheumatism, osteoporosis , Pain, anxiety, schizophrenia, manic depression, par
  • RNA antisense 'oligonucleotide (RNA, DNA or a derivative thereof) of the present invention to suppress the transcription of the gene encoding the polypeptide of the present invention or the translation of mRNA, 46, 681 (1991), Bio / Technology, 9, 358 (1992)]
  • RNA antisense 'oligonucleotide
  • such genes may be involved in the pathogenesis, such as diabetes, ischemic heart disease, hypertension, nephritis, tengitis, ulcer, It can be used for the prevention and treatment of diseases such as allergy, asthma, rheumatism, osteoporosis, pain, anxiety, schizophrenia, manic depression, Parkinson's disease, dementia, infectious disease and malignant tumor.
  • diseases such as allergy, asthma, rheumatism, osteoporosis, pain, anxiety, schizophrenia, manic depression, Parkinson's disease, dementia, infectious disease and malignant tumor.
  • the above-mentioned antisense 'oligonucleotide is preferably an oligonucleotide having a sequence complementary to 5 to 60 consecutive nucleotides in the nucleotide sequence of the DNA encoding the polypeptide of the present invention, preferably the polynucleotide of the present invention. It is designed and prepared based on the nucleotide sequence complementary to 5 to 60 bases in the translation initiation region of the DNA encoding the peptide, and is then administered to a living body.
  • the medicament containing the DNA of the present invention can be prepared by the same method as the above-mentioned [5], which is the same as the method for preparing a pharmaceutical preparation of an agonist or an agonist of the polypeptide of the present invention.
  • the prepared pharmaceutical preparation can be administered in the same manner as in the above [5].
  • polypeptide of the present invention can be obtained by the method described in [2] using the DNA of the present invention.
  • polypeptides of the present invention include diabetes, ischemic heart disease, hypertension, nephritis, tengitis, ulcer, allergy, asthma, rheumatism, osteoporosis, pain, anxiety, schizophrenia, E depression, Parkinson's disease, Remedies or prophylactics for diseases such as dementia, infectious diseases or malignancies are contemplated.
  • the medicament containing the polypeptide of the present invention can be prepared and prepared by using the same method as the above-mentioned [5] for preparing the pharmaceutical preparation of the agonist or gonist of the polypeptide of the present invention.
  • the pharmaceutical preparation can be administered in the same manner as in the above [5].
  • the oligonucleotide of the present invention is a single-stranded or double-stranded retrovirus, It can be incorporated into viral vectors such as adenovirus and adeno-associated virus, and other vectors, and used as gene therapy vectors for gene therapy.
  • An antibody against the polypeptide of the present invention can be produced by the method described in [3] using the polypeptide of the present invention as an antigen.
  • the polypeptide of the present invention can be detected or quantified immunologically.
  • Specific examples include the ELISA method using a microtiter plate, immunohistochemical staining using an enzyme-labeled antibody method or a fluorescent antibody method, and a detection method using a western blot method.
  • sandwich ELISA using two types of monoclonal antibodies that recognize different polypeptides among antibodies that react with the polypeptide of the present invention in the liquid phase, labeling with radioisotopes such as ⁇ ⁇ And a radioimmunoassay method using the polypeptide of the present invention and an antibody that recognizes the polypeptide of the present invention.
  • the antibody of the present invention can also be used for immunological and tissue staining using pathological tissue sections. Using the antibody of the present invention, the polypeptide of the present invention present in cells or tissues of healthy subjects and subjects is immunologically detected or quantified, and the amount is compared between healthy subjects and subjects.
  • the subject's diabetes By examining whether it has changed, the subject's diabetes, ischemic heart disease, hypertension, nephritis, Tengitis, ulcer, allergy, asthma, rheumatism, osteoporosis, pain, anxiety, schizophrenia, manic depression, It can be used for diagnosis of pathological conditions such as Parkinson's disease, dementia, infectious disease and malignant tumor.
  • the antibody of the present invention to immunologically detect or quantify the polypeptide present in tissues and cells of various pathological model animals, and comparing with a normal animal, the polypeptide in the pathological state can be obtained. The importance of peptides can be clarified. Furthermore, a drug can be evaluated by comparing the expression level of the polypeptide depending on the presence or absence of the drug.
  • the medicament containing the antibody of the present invention can be prepared using the same method as the above-mentioned [5], which is the same as the method for preparing a pharmaceutical preparation of the polypeptide of the present invention or an agonist of the present invention.
  • the pharmaceutical preparation can be administered in the same manner as in the above [5].
  • the compounds that regulate the expression of the agonist, angiogonist of the present invention and the polypeptide gene of the present invention include diabetes, ischemic heart disease, hypertension, nephritis, nephritis, ulcer, allergy, asthma, rheumatism. It is expected to be used for the treatment or prevention of diseases such as osteoporosis, pain, anxiety, schizophrenia, manic depression, Parkinson's disease, dementia, infectious disease and malignancy.
  • Patent application for application filed on March 7, 2000 which forms the basis of the priority claimed in the present application 200 0 — 6 1 4 6 4 and Japanese application for patent application, filed on July 10, 2000 2 All the contents described in 0 0 0—2 0 8 6 10 shall be cited as disclosure in this specification.
  • Example 1 Cloning of a human cDNA fragment encoding a protein having PDE activity (1) Preparation of a cDNA library derived from HepG2 cells
  • Plasmid DNA was obtained from each Escherichia coli clone of the cDNA library prepared in (1) above according to a conventional method, and the nucleotide sequence of the terminal of cDNA contained in each plasmid was determined.
  • the nucleotide sequence was determined using a kit (BigDye Terminator Cycle Sequencing FS Ready Reaction Kit, manufactured by PE Biosystems) and a DNA sequencer (ABI PRISM 377, manufactured by PE Biosystems).
  • the primer the synthetic DNA shown in SEQ ID NOs: 7 and 8 was used.
  • plasmid hepl0314 contained a cDNA of about 2.Ikb from base number 406 of the nucleotide sequence described in SEQ ID NO: 2, and was described in SEQ ID NO: 1. 339 a in new polypeptides
  • a DNA polymerase described in SEQ ID NO: 9 and SEQ ID NO: 10 was designed, and the following method was used using the Human Pancreas Marathon-Ready cDNA kit (manufactured by Klontech) by the method shown below.
  • the PCB was amplified.
  • TaKaRa PCR Thermal Cycler 480 heat at 95 ° C for 10 minutes, perform 30 cycles at 94 ° C for 30 seconds and 60 ° C for 2 minutes as one cycle, and further heat at 72 ° C for 8 minutes did.
  • primers of SEQ ID NO: 10 and AP2 were each 0.2 mol / L, and each component was 200 mol / L dNTP ( dATP s dGTP, dCTP, dTTP) mixed solution, Taq Gold polymerase (manufactured by Pakin Elmer) 2.5 units, and 1 1 & 9 01 (reaction solution 5 () ⁇ containing 1 buffer solution) PCR was carried out in the same manner.
  • the obtained PCR reaction solution was fractionated, and after confirming that an approximately 0.7 kb MA fragment was amplified by agarose gel electrophoresis, the DNA fragment was separated using a QIAEX II Gel Extraction Kit (manufactured by QIAGEN) according to the manual. Purified.
  • Example 2 Shown in 5 5 end side MA primer first and SEQ ID NO: 1 0 shown in the Northern High Priestess die peptidase one Chillon sequence based on the information of nucleotide sequence determined by expression analysis Example 1 of mRNA by No. 1 1: Example 2 A 3 'end DNA primer was designed and synthesized. 0.2 mol / L of each of the two types of obtained primers, plasmid of Example 1
  • each component 200 ⁇ mol / L dNTP (dATP, dGTP, dCTP, dTTP) mixture
  • PCR was performed under the following conditions using a reaction solution (50 / L) containing 2.5 units of ExTaq polymerase (Takara Shuzo) and IXExTaq buffer.
  • BAP (Hereinafter abbreviated as BAP).
  • C75 (Takara Shuzo Co., Ltd.) was added, and a dephosphorylation reaction was performed at 60 ° C for 30 minutes.
  • the reaction solution was subjected to agarose electrophoresis, and pBluescript II KS (-)-derived! was used using a QIAEX II Gel Extraction Kit (manufactured by QIAGEN).
  • the RV-BAP treated fragment (3.0 kb) was purified.
  • a buffer solution consisting of 50 ⁇ l / L Tris-HCl (pH 7.5), 10 orchid ol / L magnesium chloride, 1 orchid ol / L DTT, and 100 hol / L sodium chloride 50 ⁇ l of plasmid p200 ⁇ L, dissolved in 10 units of! ⁇ RI (Takara Shuzo) and (Takara Shuzo), and digested at 37 ° C for 3 hours. After phenol extraction and ethanol precipitation, the DNA fragment was subjected to a binding reaction using a DNA Blunting Kit (Takara Shuzo) according to the manual.
  • Escherichia coli JM109 strain was transformed using the recombinant plasmid DNA obtained by the reaction, and a plasmid p200-EB for preparing a Northern analysis probe was prepared by an ordinary method.
  • Figure 3 shows the construction process and restriction map of the plasmid.
  • a buffer solution consisting of 10 ⁇ g of the prepared plasmid p200-EB at 10 ⁇ l / L Tris-hydrochloric acid (pH 7.5), 10 mmol / L magnesium chloride, 50 orchids ol / L sodium chloride, and 1 ol / L DTT 50
  • the mixture was dissolved in L, 30 units of IIIi (Takara Shuzo) was added, and the digestion reaction was performed at 37 ° C for 6 hours. Using the reaction mixture, phenol-mouth opening extraction and ethanol precipitation were performed to recover DNA fragments. ''
  • Lg of the DNA fragment was converted into 40 orchid ol / L Tris-hydrochloride (pHS.O), 6 ol / L magnesium chloride, 2 ol / L spermidine, 10 mfflol / L DTT ⁇ 1 ol / L ATP, lmmol / L CTP.
  • 1 ol / L GTPs 0.65 ⁇ ol / L UTP, 0.35 ol / L digoxigenin-; - ⁇ -dissolved in 50 zL of buffer, 40 units of T7 RNA polymerase (Peringa-Mannheim ) And in vitro transcription reaction was performed at 37 ° C for 2 hours.
  • the hybrid was subjected to 50% formamide, 5 times concentration of SSC (1 times concentration of SSC was 150 ol / L sodium chloride and 15 ol / L sodium citrate), 0.5% sodium dodecyl sulfate (hereinafter abbreviated as SDS), 2% blocking reagent (manufactured by Behringer Mannheim), O.lmg / mL salmon sperm DNA was immersed in a buffer solution (hereinafter abbreviated as hybridization buffer), and prehybridization was performed at 70 ° C
  • the film was immersed in a hybridization buffer in which the above-mentioned digoxigenin-labeled cMA probe was dissolved at a concentration of 1 g / mL, and hybridization was performed at 70 ° C. for 15 hours.
  • the film is immersed once in a buffer consisting of 2 times concentration SSC and 0.1% SDS at 70 ° C for 10 minutes, and then 70 ° C in a buffer consisting of 0.2 times concentration SSC and 0.1% SDS once. C, washed three times under conditions of immersion for 30 minutes.
  • the film is immersed for 15 minutes at room temperature in a buffer solution (hereinafter abbreviated as DIG I buffer solution) consisting of 100 t ol / L maleic acid (pH 7.5) and 150 ol / L sodium chloride. Washing was performed twice under the conditions to remove SDS.
  • DIG I buffer solution consisting of 100 t ol / L maleic acid (pH 7.5) and 150 ol / L sodium chloride.
  • the filter was immersed in a buffer solution consisting of 100 mmol / L maleic acid (pH 7.5), 150 t ol / L sodium chloride, and 1% blocking reagent (hereinafter abbreviated as DIGI I buffer solution), and left at room temperature for 1 hour. Proking was performed.
  • the filter was immersed in a solution of anti-digoxigenin antibody Fab fragment (manufactured by Boehringer-Mannheim) diluted 10,000 times with DIGII buffer, and the antigen-antibody reaction was performed at room temperature for 30 minutes. went.
  • the filter was washed three times with DIG I buffer at room temperature for 30 minutes to remove excess antibody, and then 100 mmol / L Tris-HCl (pH 9.0), 100 mL / L chloride were added. It was immersed in a buffer solution containing sodium and 50 t ol / L magnesium chloride (hereinafter abbreviated as DIGIII buffer solution) for 5 minutes to equilibrate.
  • DIGIII buffer solution a buffer solution containing sodium and 50 t ol / L magnesium chloride
  • Fig. 4 shows the results. Approximately 2. s was found in the kidney
  • Example 1 E. coli expressing a glutathione S transferase (hereinafter abbreviated as GST) fusion protein was used. Produced.
  • GST glutathione S transferase
  • DNA-end DNA primer shown in SEQ ID NO: 13 were designed and synthesized.
  • the obtained PCR reaction solution was fractionated, and it was confirmed by agarose electrophoresis that a DNA fragment of about l.Okb was amplified.
  • 10 t ol / L magnesium chloride 1 hi ol / L DTT
  • 100 t ol / L chloride After dissolving in 50 ⁇ L of a buffer solution containing lithium, 10 units of iHI (manufactured by Takara Shuzo) were added, and digestion reaction was performed at 37 ° C. for 3 hours.
  • the DNA fragment was reconstituted with 10 l / l Tris-HCl (pH 7.5), 10 l / l magnesium chloride, limol / L DTT, 50 l / l sodium chloride, 0.01 l / l Dissolve in 50 ⁇ L of buffer solution consisting of 10% serum albumin, add 10 units of 1 (Takara Shuzo) and incubate the digestion reaction at 37 ° C for 3 hours. went.
  • the reaction solution was subjected to agarose electrophoresis, and the I-I fragment (1. Okb) was purified using a QIAEX II Gel Extraction Kit (manufactured by QIAGEN). '
  • digestion reaction was performed at 37 ° C for 3 hours.
  • the reaction solution was subjected to agarose electrophoresis, and an HI-I fragment (9.6 kb) was purified using a QIAEX II Gel Extraction Kit (manufactured by QIAGEN).
  • plasmid pPDE-1393 was obtained by a conventional method. 2 ⁇ g of the plasmid pPDE-1393 was added to 50 t ol / L Tris-hydrochloride (pH 7.5), 10 t ol / L magnesium chloride, 1 t ol / L DTT, 100 t ol / L sodium chloride, 0.01% The solution was dissolved in a buffer solution consisting of serum albumin and O.
  • Plasmid pT-1 obtained in Example 1 was prepared from 50 ol / L Tris-monohydrochloride (pH 7.5), 10 t ol / L magnesium chloride, 1 t ol / L DTT, and 100 t ol / L sodium chloride.
  • the resulting solution was dissolved in 50 L of a buffer solution, and 10 units of l £ V and £ 1 were added thereto, and the digestion reaction was carried out at 37 ° C for 3 hours.
  • the reaction solution was subjected to agarose electrophoresis, and V- £ i ⁇ I (0.3 kb) was purified using QIAEX II Gel Extraction K (manufactured by QIAGEN).
  • plasmid pGEX-5X-1 (Pharmacia) was added to 33 ⁇ l / L Tris-HCl (pH 7.9), dissolved in 50 L of a buffer consisting of 10 ol / L magnesium acetate, 0.5 L ol / L DTT, and 66 L ol / L potassium acetate, added 10 units of l, and added at 30 ° C. A time digestion reaction was performed.
  • the DNA fragment was used in 50 ol / L tris monohydrochloride (pH 7.5), 10 ol / L magnesium chloride, 1 ol / L DTT ⁇ 100 ol / L sodium chloride was dissolved in 50 ⁇ L of a buffer solution containing 0.01% Pseudoserum albumin and 0.01% Tron X-100, and 10 units of l was added to carry out a digestion reaction at 37 ° C. for 3 hours. The reaction solution was subjected to agarose electrophoresis, and an I-MI fragment (5. Okb) was purified using a QIAEX II Gel Extraction Kit (manufactured by QIAGEN).
  • a 50 ng (5.0 kb) binding reaction was carried out using DNA Ligation Kit Ver.2 (Takara Shuzo) according to the manual.
  • the recombinant plasmid DNA obtained by the reaction was used to transform E. coli JM109 strain, and Escherichia coli JM109 / pGST-PDE strain containing plasmid pGST-PDE was obtained by a conventional method.
  • Figure 5 shows the construction process and restriction map of the plasmid.
  • the cells were recovered by centrifugation (3,000 rpm for 10 minutes), and the extract (20 orchids ol / L Tris-acetic acid (pH7.5), 2 ol / L magnesium chloride, 1 ol / L DTT, 250 units) / mL aprotinin, 40 ⁇ g / mL phenylmethylsulfonyl fluoride, and l / g / mL peptide A), and disrupt Escherichia coli using an ultrasonic crusher (T0MY model UR-200R). did.
  • the extract was centrifuged at 10,000 rpm for 30 minutes, and the supernatant was used for PDE activity measurement.
  • PDE activity was determined by the method of Kincaid, RL and Manganiello, VC Ofethod. Enzymol., 159, 457-470 (1988)).
  • [3 ⁇ 4] -cAMP or [3 ⁇ 4] -cGMP as a substrate 300 L reaction solution [50 ol / L of ⁇ , ⁇ ⁇ -bis (2-hydroxyethyl) -2_aminoethanesulfonate ( ⁇ 7. 2), 1 mol / L magnesium chloride, 0.1 lotol / L ethylene glycol bis (?-Aminoethyl ether) -NJ, N, J, -disodium tetraacetate].
  • the substrate concentration was; mol / L.
  • [3 ⁇ 4] -cAMP and [3 ⁇ 4] -cGMP degradation amount The amount of degradation by the Escherichia coli soluble fraction was determined by subtracting the amount of non-catalytic hydrolysis from the total amount of degradation. The protein amount was quantified using the Bio Rad Protein Assay method. As a control, an E. coli soluble fraction into which only plasmid pGEX-5X-1 was introduced was used. Fig. 6 shows the results. -As described above, it has been clarified that the polypeptide of the present invention has PDE activity for hydrolyzing cAMP and cGMP.
  • Example 4 Cloning of a full-length human cDNA fragment encoding a protein having PDE activity
  • the solution was dissolved in 50 / L, 40 units of I and gEl (Takara Shuzo) were added, and the digestion reaction was performed at 37 ° C for 4 hours.
  • the reaction solution was subjected to agarose electrophoresis, and a DNA fragment of about 1 kb was recovered.
  • the DNA fragment was purified using QIAEX II Gel Extraction Kit (manufactured by QIAGEN) according to the manual.
  • the purified DNA fragment is labeled with horseradish peroxidase using an ECL direct nucleic acid labeling / detection system (Amersham). And used as a probe.
  • Plaque hybridization was performed using 2 ⁇ 10 5 clones of a cDNA library derived from human fetal kidney (manufactured by Clontech, trade name: Human Fetal Kidney 5′-STRETCH PLUS cDNA Library) using the probe. Two independent phage clones (vector: human gt10) hybridizing were obtained (clones 6-1 and 6-1).
  • the operation of the hybrid is all performed by ECL direct nucleic acid labeling.
  • the cDNA fragment contained in clone 23-1 of the phage clone was recombined from the phage vector to the plasmid vector.
  • 20 ⁇ g of the DNA of the phageclone 23-1 was added to 50 ol / L tris-hydrochloride (pH 7.5), 10 orchids ol / L magnesium chloride, 100 thigh ol / L sodium chloride, 1 liter ol / L After dissolving in 30 zL of buffer solution containing LDTT, 15 units of!
  • ⁇ RI (manufactured by Takara Shuzo Co., Ltd.) was added, and the digestion reaction was performed at 37 ° C for 4 hours.
  • the reaction solution was subjected to agarose electrophoresis, and DNA fragments of about 2 kb and 1 kb were recovered.
  • the DNA fragment was purified using a QIAEX II Gel Extraction Kit (manufactured by QIAGEN) according to the manual.
  • the DNA fragment was dissolved in 30 zL of a buffer containing 50 t / ol / L Tris-monohydrochloride ( ⁇ 90) and 1 t / ol / L magnesium chloride, and 0.5 unit of BAP (E. coli C75) (Takara Shuzo) ) was added and a dephosphorylation reaction was performed at 60 ° C. for 30 minutes.
  • the reaction solution was subjected to agarose electrophoresis, and a DNA fragment of about 3.0 kb was recovered.
  • the MA fragment was purified using a QIAEX II Gel Extraction Kit (QIAGEN) according to the manual.
  • Escherichia coli JM109 strain was transformed using the recombinant plasmid DNA obtained by the binding reaction, and plasmids p23-Ik and p23-2k were obtained by a conventional method.
  • the inserted DNA fragments of plasmids p23-lk and p23-2k can be ligated at the ⁇ M site, and the approximately 3.0 kb cDNA described in SEQ ID NO: 16 And a novel polypeptide consisting of 576 amino acids described in SEQ ID NO: 15 was encoded in the cDM.
  • the structures of p23-lk and p23-2k are shown in FIG.
  • FIG. 8 shows an alignment between the amino acid sequence and the human PDE5A amino acid sequence (GenBank: CM06170).
  • Two types of primers SEQ ID NO: 17 and SEQ ID NO: 18 each 1.0 ⁇ 1 / human tissue
  • 2 ⁇ L cDNA library made from nRNA, 200 ⁇ mol / L dNTP dATP, dGTP, PCR was carried out under the following conditions using a reaction solution containing a mixture of dCTP and dTTP), 2.5 units of Taq Gold polymerase (manufactured by Pakinkin Elmer) and lxTaq Gold buffer (Mg plus).
  • the DNA of the novel PDE polypeptide obtained according to the present invention it is possible to use the DNA of $ karauria, ischemic heart disease, hypertension, nephritis, rheumatism, ulcer, allergy, asthma, rheumatism, osteoporosis, pain, anxiety, Diagnosis, prevention, and treatment of diseases such as schizophrenia, manic depression, Parkinson's disease, dementia, infectious diseases, and malignant tumors are possible.

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Abstract

La présente invention concerne : un polypeptide ayant une activité de phosphodiestérase (PDE) ; un procédé permettant de produire ce polypeptide ; l'ADN codant pour ce polypeptide ; un vecteur de recombinaison obtenu par intégration de ce polypeptide ; un transformant portant ce vecteur de recombinaison ; un anticorps reconnaissant le polypeptide mentionné ci-dessus ; un procédé permettant de quantifier le polypeptide et un procédé de coloration immunologique dans lequel est utilisé ledit anticorps ; un procédé de criblage d'une substance modifiant l'expression d'un gène codant pour le polypeptide mentionné ci-dessus ; un procédé de criblage d'une substance modifiant l'activité du polypeptide ; et des médicaments, etc., permettant le diagnostic, la prévention ou le traitement du diabète, des troubles cérébraux, des troubles rénaux, du cancer, etc., grâce à l'utilisation de l'ADN mentionné ci-dessus ou de l'anticorps mentionné ci-dessus. L'utilisation de l'ADN du polypeptide PDE de l'invention permet le diagnostic, la prévention et le traitement du diabète, des cardiopathies ischémiques, de l'hypertension, de la néphrite, de la pancréatite, de l'ulcère, de l'allergie, de l'asthme, du rhumatisme, de l'ostéoporose, de la douleur, de l'anxiété, de la schizophrénie, de la psychose maniaco-dépressive, de la maladie de Parkinson, de la démence, des maladies infectieuses, de la tumeur maligne, etc.
PCT/JP2001/001720 2000-03-07 2001-03-06 Polypeptide ayant une activite de phosphodiesterase WO2001066716A1 (fr)

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Publication number Priority date Publication date Assignee Title
WO2002000854A3 (fr) * 2000-06-26 2002-07-11 Bayer Ag Regulation de l'enzyme humaine de type phosphodiesterase
WO2004029617A2 (fr) * 2002-09-24 2004-04-08 Bayer Healthcare Ag Diagnostic et therapeutique de maladies associees a la phosphodiesterase humaine 11a (pde11a)
WO2004042387A2 (fr) * 2002-11-08 2004-05-21 Bayer Healthcare Ag Diagnostics et traitements de maladies associees a la phosphodiesterase 11a1 humaine (pde11a1)
WO2004042386A2 (fr) * 2002-11-08 2004-05-21 Bayer Healthcare Ag Moyens diagnostiques et therapeutiques destines a des maladies associees a la phosphodiesterase 11a3 (pde11a3) humaine

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WO2000040733A1 (fr) * 1999-01-07 2000-07-13 Incyte Pharmaceuticals, Inc. Phosphodiesterases de nucleotides cycliques humaines

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WO2000040733A1 (fr) * 1999-01-07 2000-07-13 Incyte Pharmaceuticals, Inc. Phosphodiesterases de nucleotides cycliques humaines

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CHING-SHWUN LIN ET AL.: "Identification of three alternative first exons and an intronic promoter of human PDE5A gene", BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, vol. 268, no. 2, February 2000 (2000-02-01), pages 596 - 602, XP002941709 *
DAVID R. REPASKE ET AL.: "A polymerase chain reaction strategy to identify and clone cyclic nucleotide phosphodiesterase cDNAs", THE JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 267, no. 26, 1992, pages 18683 - 18688, XP002941713 *
KEIZO YUASA ET AL.: "Isolation and characterization of two novel phosphodiesterase PDE11A variants showing unique structure and tissue-specific expression", THE JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 275, no. 40, October 2000 (2000-10-01), pages 31469 - 31479, XP002941710 *
LINDSAY FAWCETT ET AL.: "Molecular cloning and characterization of a distinct human phosphodiesterase gene family: PDE11A", PROC. NATL. ACAD. SCI. USA, vol. 97, no. 7, March 2000 (2000-03-01), pages 3702 - 3707, XP002941711 *
PETER STACEY ET AL.: "Molecular cloning and expression of human cGMP-binding cGMP-specific phosphodiesterase(PDE5)", BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, vol. 247, no. 2, 1998, pages 249 - 254, XP002941712 *

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002000854A3 (fr) * 2000-06-26 2002-07-11 Bayer Ag Regulation de l'enzyme humaine de type phosphodiesterase
WO2004029617A2 (fr) * 2002-09-24 2004-04-08 Bayer Healthcare Ag Diagnostic et therapeutique de maladies associees a la phosphodiesterase humaine 11a (pde11a)
WO2004029617A3 (fr) * 2002-09-24 2004-07-22 Bayer Healthcare Ag Diagnostic et therapeutique de maladies associees a la phosphodiesterase humaine 11a (pde11a)
WO2004042387A2 (fr) * 2002-11-08 2004-05-21 Bayer Healthcare Ag Diagnostics et traitements de maladies associees a la phosphodiesterase 11a1 humaine (pde11a1)
WO2004042386A2 (fr) * 2002-11-08 2004-05-21 Bayer Healthcare Ag Moyens diagnostiques et therapeutiques destines a des maladies associees a la phosphodiesterase 11a3 (pde11a3) humaine
WO2004042387A3 (fr) * 2002-11-08 2004-07-15 Bayer Healthcare Ag Diagnostics et traitements de maladies associees a la phosphodiesterase 11a1 humaine (pde11a1)
WO2004042386A3 (fr) * 2002-11-08 2004-07-15 Bayer Healthcare Ag Moyens diagnostiques et therapeutiques destines a des maladies associees a la phosphodiesterase 11a3 (pde11a3) humaine

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