WO2001066716A1 - Polypeptide having phosphodiesterase activity - Google Patents

Abstract

Novel polypeptide having a PDE activity; a process for producing this polypeptide; a DNA encoding this polypeptide; a recombinant vector obtained by integrating this polypeptide; a transformant carrying this recombinant vector; an antibody recognizing the above polypeptide; a method of quantifying the polypeptide and an immunological staining method by using this antibody; a method of screening a substance changing the expression of a gene encoding the above polypeptide; a method of screening a substance changing the activity of the polypeptide; and drugs, etc. for diagnosing, preventing or treating diabetes, brain diseases, kidney diseases, cancer, etc. by using the above DNA or the above antibody. Use of the DNA of the novel PDE polypeptide enables diagnosis, prevention and treatment of diabetes, ischemic heart diseases, hypertension, nephritis, pancreatitis, ulcer, allergy, asthma, rheumatism, osteoporosis, pain, anxiety, schizophrenia, manic-depressive psychosis, Parkinson's disease, dementia, infectious diseases, malignant tumor, etc.

Description

 Specification

 Polypeptides having phosphodiesterase activity

 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. Background art

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). In addition, they play an important role in regulating the concentration of these cyclic nucleotides in cells by producing the corresponding nucleoside 5, monophosphate. CPharma Ther., 51, 13 (1991)]. That, 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. Trend. Pharmacol. Sci., 11, 150 (1990), Physiol. Rev., 75, 725 (1995), Arch. Which form a large family of different subtypes and their isoforms. Biochem. Biophys.,, 1 (1995), Endocrinol. Rev. 16, 370 (1995), Kidney International, 55, 29 (1999), Cyclic Nucleotide Phosphodiesterases: Structure, Regulation and Drug Action, John Wiley & Sons, New York (1990), and Phosphodiesterase Inhibitors, Academic Press, New York (1996)]. To date, the PDE superfamily has been characterized by its biochemical and enzymatic properties, as well as the comparison of amino acid sequences by cloning of the corresponding cDNAs. ~ PDE 10) (Kidney International, 55, 29 (1999), J. Biol. Chem., 274, 18438 (1999), Gene, 234, 109 (1999), and Proc, Natl. Acad. Sci. USA, 96, Brief (1999)].

1. Ca ²⁺ / calmodulin-dependent PDE (PDE 1)

 2. c GMP stimulated PDE (PDE 2)

 3. PDEs inhibited by cGMP (PDE 3)

 4. cAMP-specific PDE (PDE 4)

 5. cGMP-specific PDE (PDE 5)

 6. cGMP-specific photoreceptor Yuichi PDE (PDE 6)

 7. High affinity, cAMP specific PDE (PDE 7)

 8. High affinity, cAMP specific PDE (PDE 8)

 9. cGMP-specific PDE (PDE 9)

10. c AMP PDE cGMP PDE inhibited by cAMP (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.

Pharmacol. Sci., 22, 217 (1997), Physiol. Rev., 75, 725 (1995), Arch. Biochem. Biophys., 322, 1 (1995), and Endocrinol. Rev., 16, 370 (1995). ]. 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. Chem., 69, 22477 (1994)), 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 制 御 terminal regulatory domain is PD ΕThe 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. Sci., 22, 217 (1997), Physiol., Rev., 75, 725 (1995), Phosphodiesterase Inhibitors, Academic Press, New York (1996)]. For example, 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., ΙΟ, 869 (1998)]. As a PDE 4 inhibitor, rolipram has been used as a therapeutic agent for depression. TNF-Hi

 Promotes replication of HIV-1. rol ipram

It has been pointed out that HIV-1 replication may be inhibited by suppressing TNF-production by lipopolysaccharide stimulation [AIDS, 9, 1137 (1995)]. In addition, 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. In addition, they have anti-inflammatory effects as well as bronchodilator effects, and as therapeutic agents for asthma. Exp. Immunol., Fiber, 126 (1995), Am. J. Respir. Grit. Care Med., 157, 351 (1998), whose potential has been evaluated.] PDE is involved in the proliferation of many cells Of the treatment of malignant tumors It is also of interest as an evening gate [Proc. Natl. Acad. Sci. USA, 91, 5330 (1994)]. In addition, in a system using rat mesangial cells, a PDE 3 inhibitor has a mitogenic effect on mesangial cells [J. Clin. Invest., 96, 401 (1995)]. Inhibits the production of reactive oxygen metabolites (J. Biol. Chem., 272, 9854 (1997)). Furthermore, PDE inhibitors have been shown to suppress the development of proteinuria in anti-Thy-1 nephritis rats [J. Clin. Invest., 98, 262 (1996)].

 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 In order to prevent or treat diseases in which abnormalities in signal transduction via cyclic nucleotides may be involved in the onset and progression of disease states, select PDE subtypes involved in the disease and further select isoforms There is a demand for the development of an effective agonist and an evening gonist. 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.

 In the case of PDEs, the minimal amount of enzyme present and catalysis of basically the same reaction, and the overlap of substrate specificities, etc. results in purification and isolation of isoforms from tissues or cells. Separation is not easy. Isolation of novel isoforms using classical enzymological techniques is hampered by the limitations of currently used purification methods and the difficulty in confirming that a single purified enzyme preparation has been obtained. It is rare. Other approaches have been to use isoform-selective assay conditions and to separate and identify subtype isoforms using immunological techniques. These approaches require establishing the criteria needed to identify subtypes and isoforms, which can be time consuming, labor intensive, and technically challenging. As a result, many studies have been performed with partially purified PDE preparations containing multiple subtypes or isoforms.

New tissue or cell specific; PDE isoforms using recombinant DNA technology If it can be prepared in large quantities, it is expected that these difficulties can be overcome and more specific and safe agonist and angelic gonist development will be possible. Disclosure of the invention

 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.

 That is, the present invention relates to the following inventions (1) to (33).

 (1) A polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 15.

 (2) DNA encoding the polypeptide according to (1).

 (3) a DNA having the nucleotide sequence of SEQ ID NO: 2 or SEQ ID NO: 16;

 (4) A recombinant vector containing the DNA of (2) or (3).

 (5) A transformant having the recombinant vector according to (4).

 (6) The transformant according to (5), wherein the transformant is a transformant selected from the group consisting of microorganisms, animal cells, plant cells, and insect cells.

(7) The transformant according to (6), wherein the microorganism is a microorganism belonging to the genus Escherichia. (8) The transformant according to (7), having a deposit number of FERM BP-6976.

 (9) 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. The method for producing a polypeptide according to (1), wherein the polypeptide is collected.

 (10) A sense oligonucleotide having the same sequence as the contiguous 5 to 60 bases in the nucleotide sequence of DNA according to (2) or (3), and an antisense oligonucleotide having a sequence complementary to the sense oligonucleotide Oligonucleotides selected from the group consisting of nucleotides and oligonucleotide derivatives of these sense or antisense oligonucleotides.

 (11) 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), oligoribonucleotide derivatives in which the ribose in DNA is substituted with 2,10-propylribose, and 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.

(12) A method for detecting mRNA encoding the polypeptide according to (1), comprising using the oligonucleotide according to (10) or (11). (13) The method for suppressing the expression of the polypeptide according to (1), comprising using the oligonucleotide according to (10) or (11.).

 (14) An antibody that recognizes the polypeptide of (1).

 (15) The method for immunologically detecting a polypeptide according to (1), comprising using the antibody according to (14).

 (16) The method for immunohistochemical staining of a polypeptide according to (1), wherein the antibody according to (14) is used.

 (17) An immunohistochemical, comprising the antibody according to (14).

 (18) 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.

 (19) A compound obtained by the method according to (18).

 (20) 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. .

 (21) A method according to (12), wherein the mRNA encoding the polypeptide according to (1) is detected to detect a change in the expression of a gene encoding the polypeptide. The screening method according to (20).

(22) A method according to (15), wherein the polypeptide according to (1) is detected to detect a change in the expression of a gene encoding the polypeptide. ).

 (23) A compound obtained by the method according to any one of (20) to (22).

(24) A promoter DNA that controls transcription of a gene encoding the polypeptide according to (1).

(25) The 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.

 (26) The repo overnight gene is the chloramphenicone acetyltransferase gene, the galactosidase gene, the luciferase gene, the 'β-glucuronidase gene, the equorin gene, and the green fluorescent protein. The screening method according to (25), which is a gene selected from the group consisting of tin genes.

 (27) A compound obtained by the method according to (25) or (26).

 (28) A medicament comprising the polypeptide according to (1).

 (29) A physician comprising the oligonucleotide according to (10) or (11).

 (30) A medicament comprising the antibody according to (14).

 (31) 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

The medicament according to any one of (31).

 (33) 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 The medicament according to any one of (28) to (31), which is a medicament for diagnosis of: BRIEF DESCRIPTION OF THE FIGURES

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. Show. :) Underline indicates HDXXHXXXXN motivation.

 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)

+ Asterisk). 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, and 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 Ρ (¾ 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.

In the above figure, kb indicates kilobase pairs, Ap indicates an ampicillin resistance gene, T7 indicates a T7 promoter, and GST indicates glutransthione S trans. Show Ferrarize. BEST MODE FOR CARRYING OUT THE INVENTION

 The polypeptide of the present invention is characterized by comprising any one of the following amino acid sequences.

 (A) the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 15, or

 (B) an amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 15 wherein one or more amino acids are deleted, substituted and / or added, and encodes a polypeptide having phosphodiesterase activity Amino acid sequence.

 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. USA, 79, 6409 (1982) ヽ Gene , 34, 315 (1985), Nucleic Acids Research, 13, 4431 (1985), Proc. Natl. Acad. Sci, USA, 82, 488 (1985), Proc. Natl. Acad. Sci. USA, 81, 5662 (1985). 1984), Science, 224, 1431 (1984), International Publication W085 / 00817, and Nature, 316, 601 (1985), etc. Using the site-directed mutagenesis method described, for example, by introducing a site-directed mutagenesis into a DNA encoding a polypeptide having an amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 15. Can be.

Although 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 [

J. Mol. Biol., 215 ₅ 403 (1990)] or FASTA (Methods in Enzymology, 183, 63 (1990)), etc. It is preferably at least 70%, more preferably at least 80%, further preferably at least 90%, particularly preferably at least 95%, most preferably at least 98%. Does not include polypeptides.

 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.

 (A) the nucleotide sequence of SEQ ID NO: 2 or SEQ ID NO: 16:

 (B) One nucleotide sequence that hybridizes with the nucleotide sequence described in (A) above under stringent conditions and encodes a polypeptide having phosphodiesterase activity.

 The above “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. After performing hybridization at 65 ° C in the presence of 0.7-1.0mo1 / L NaC1 using the immobilized filter, the SSC (0.1- to 2-fold concentration) was used. 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.

Hydride is described in Mole Yura's' Clothing Second Edition, Current Protocol 'in' Molequira. Biology ,; DN A Clonin 1: Core Techniques. A Practical Approach, Second Edition, Oxford University Press (1995), etc. can be performed according to the method described in the experiment book.

 Specifically, 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). A DNA having at least 80% or more, preferably 90% or more, more preferably 95% or more, and particularly preferably 98% or more homology with the nucleotide sequence represented by SEQ ID NO: 2 or SEQ ID NO: 16. Can be.

 However, the known DNAs are not included in the DNAs of the present invention.

 Hereinafter, a method for implementing and using the present invention will be described in detail.

[1] Acquisition of DNA of the present invention and preparation of oligonucleotide

 Genes with homology to the amino acid sequence of human PDE10A (GenBank: AB020593) were searched for from gene data bases and protein databases by using the Blast, Smith-Waterman method, etc. (Compugen)] Search using homology search software.

 The public database such as GenBank and Swiss_Plot can be used as the database.

 In addition, random and large-scale nucleotide sequencing of the cDNAs of the clones in the privately owned cDNA library was performed, and the obtained sequence data was integrated and the created private data base was used. You can also.

 When the obtained gene having homology to human PDE10A (GenBank: AB020593) is only a partial base sequence of the gene, such as EST (Expressed Sequence Tag), The full length can be obtained, and the DNA of the present invention can be obtained from the cDNA.

 The origin of the DNA of the present invention is not particularly limited, but is preferably a mammal, and particularly preferably a human.

 (1) Preparation of cDNA library

c Total RNA from appropriate cells or tissues to create a DNA library Alternatively, prepare mRNA.

Total: As methods for preparing 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.

 Appropriate cells or tissues were found from the database: Escherichia coli was searched for one type of cDNA library that contained EST, etc., and used to construct the library. It is preferable to use cell lines or the like derived from such cells. Using the obtained total RNA or mRNA, a cDNA library is prepared by an ordinary method.

 Methods for preparing a cDNA library are described in Molecular Cloning 2nd Edition, the current 'protocol' in Molecular Kiura Biology, DNA Clonin 1: Core Techniques, A Practical Approach, Second Eaition, Oxford University Press (1995), etc. Methods or commercially available kits, such as Superscript Plasmid System Plasmid cDNA Synthesis and Plasmid-^ 7 pn ^ SuperScript Plasmid System for cDNA Synthesis and Plasmid Cloning; Gibco BRL ) Co., Ltd.) or a method using Zappoo cDNA 'Synthesis Kit [ZAP-cDNA Synthesis Kit, Straugene Co., Ltd.].

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.

 Specifically, ZAP Express (Stratagene, Inc., Strategies, 5, 58 (1992)), Bluescript II SK (+) (Nucleic Acids Research, 17, 9494 (1989)), Lambda ZAP II (Strata GtlO, Agtll [DNA Cloning, A Practical Approach, 1, 49 (1985)], ATriplEx (Clontech), ExCell (Pharmacia), pT7T318U (Pharmacia) ), PcD2 (Mol. Cell. Biol., 3, 280 (1983)), pUC18 [Gene, 33, 103 (1985)], pAMo [J. Biol. Cem., 268, Z2782-22787 (1993) ), Also known as pAMoPRC3Sc (Japanese Patent Laid-Open No. 05-336963)].

As the host microorganism, 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,

39, 440 (1954)), Escherichia coli Y1088 (Science, 222, 778 (1983)),

Escherichia coli Y109Q (Science, 222, 778 (1983)), Escherichia coli NM522

[J. Mol. Biol., 166, 1 (1983)], Escherichia coli K802 [J. Mol. BioL, 16,

118 (1966)], Escherichia coli JM105 [Gene, 38 ₅ 275 (1985)], Escherichia coli S0LRTM Strain (manufactured by Stratagene), Escherichia coli LE392 (Molecular Cloning _2nd Edition), etc. can be used. . In addition to the cDNA library prepared by the above method, a commercially available cDNA library can also be used.

 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.

 (-2) Acquisition of the DNA of the present invention

From the cDNA library prepared in (1) above, the DNA of the present invention or a cDNA clone containing a part thereof was isolated or labeled with a fluorescent probe. Can be selected by the colony hybridization method or the plaque hybridization method [Molecular cloning 2nd edition] using E. coli.

 As a probe, a fragment obtained by partially amplifying a part of cDNA by a method using PCR CP CE. Protocols, Academic Press (1990)], Oligonucleotides based on known nucleotide sequences can be used.

 When the nucleotide sequence of both the 5, 5 and 3 terminal ends of the full-length cDNA is known by EST, etc., 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.

 By linking the obtained cDNA fragments, the full-length DNA of the present invention can be obtained.

 If the cDNA obtained from the cDNA library does not encode a full-length polypeptide, a cDNA encoding the full-length polypeptide may be obtained as follows. it can.

PCR using a single-stranded cDNA library prepared from various organs or cells or a cDNA library prepared by the above-described method as a gun and using a primer set specific to the cDNA. Thus, an organ or cell that expresses a gene corresponding to the cDNA is identified, and a cDNA library as a probe is used for a cDNA library derived from the identified organ or cell using the cDNA hybridization method (molecular By carrying out the 'cloning second edition', cDNA containing the full length of the cDNA can be newly selected from the cDNA library, 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.

 Total 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 ™ Preamplification System for First Strand cDNA System (manufactured by Life Technologies) using oligo (dT) primers or 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).

-Also, by selecting DNA that hybridizes under stringent conditions with the DNA obtained by the above method, the desired DNA derived from other tissues or other animals, such as humans and mice, is obtained. can do. Based on the nucleotide sequence information obtained by the above method, the target DNA can also be prepared by chemical synthesis using a DNA synthesizer. Examples of 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.

 The novelty of the obtained nucleotide sequence can be confirmed by searching a nucleotide sequence database such as GenBank, EMBL and DDBJ using a homology search program such as BLAST.

 After converting the new nucleotide sequence to an amino acid sequence, 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.

 (3) Preparation of oligonucleotide of the present invention

 Using the DNA or DNA fragment of the present invention obtained by the above-mentioned method, an antisense oligonucleotide having a partial sequence of the DNA of the present invention or a sense Oligonucleotides such as nucleotides can be prepared.

 Examples of the oligonucleotide 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. A DNA having the same sequence as 5 to 60 consecutive nucleotides in the nucleotide sequence represented by SEQ ID NO: 2 or SEQ ID NO: 16 or a DNA having a sequence complementary to the DNA. When used as a sense primer and an antisense primer, the above-mentioned oligonucleotides whose melting temperature (T m) and number of bases do not extremely change are preferred.

 Examples of I oligonucleotides include the oligonucleotides represented by SEQ ID NOS: 10, 11, 17, or 18.

Further, derivatives of these oligonucleotides (herein, oligonucleotides Can also be used as the oligonucleotide of the present invention.

Examples of the 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 substituted by C-5 provinylcytosine, Oligonucleo Cytosine in soil Fuenokisajin ί Takazari cytosine (

Oligonucleotide derivatives substituted with phenoxazine-modified cytosine), oligonucleotide derivatives in which the ribose in the oligonucleotide has been substituted with 2,10-propylribose, or ribose in the oligonucleotide has been substituted with 2′-methoxetoxyribose Oligonucleotide derivatives, etc. [Cell Engineering, 16, 1463 (1997)].

 [2] Preparation of polypeptide of the present invention

 (1) Preparation of transformant

 In order to express the DNA of the present invention obtained by the method described in the above [1] in a host cell and produce the polypeptide of the present invention, Molecular Cloning Second Edition, Current Protocol The method described in “Molecular biology” can be used.

That is, 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. As 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. As 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.

When a prokaryote such as a bacterium is used as a host cell, 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. Examples of expression vectors include pB.Trp2, pBTAc Is pBTac2 (both commercially available from Boehringer Mannheim), PKK233-2 (Pharmacia), PSE280 (Invitrogen), pGEEX-Ι [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. USA, 82, 4306 (1985)), pBluescript II SK (-) (Stratagene), pTrs32 (FERM BP-5408) , PGHA2 (FER M BP-400) _N pGKA2 (FERM BP-6798) _s pT e rm2 (JP-A-3-22979, US4686191.US4939094 _S US51 60735), pGEX (Pharmacia), pET-3 (Novagen) ), PSupex, pUB110, pTP5pC194, pTrxFus (Inv Zeron), pMAL-c2 (New England Biolabs), and the like.

The promoter may be any promoter that can be expressed in host cells such as Escherichia coli and Bacillus subtilis. For example, promoters derived from Escherichia coli or phage, such as ii 卫 promoter overnight (Pii 卫), lac promoter overnight (Plac), P _L promoter, promoter overnight, T7 promoter overnight, etc. , SP01 Promoter, SP02 Promo Overnight, penP Promo Overnight, etc. In addition, the promotion of two Ptrps in series (Pi | 卫 x2), the promotion of tac promotion, the promotion of lacT7 promotion, let I It is also possible to use an artificially designed and modified Promo One Night, such as the One Night Promo.

 As 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).

 Although the 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ヽ_{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ヽ_{Serratia fonticola, Serratia Iiguefaciens s Serratia marc6sc6ns} , Bacillus subtil is, Bacillus amyloliquefaciens ^ Brevibacterium ammoniagenesess Brevibacterium immariophilum ATCC14068, Brevibacterium saccharolyticum AT.CC14066, Corynebacterium glutamicmn ATCC13032,

_{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)].

When a yeast strain is used as a host cell, for example, YEpl3 (ATCC37115), YEp24 (ATCC37051), YCp50 (ATCC37419), pHS19, pHS15, etc. are used as expression vectors. Can be.

 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.

As the host cell, Saccharomyces, Schizosaccharomyces support steward Russia My genus, Kuruibe opening Mrs. genus Trichosporon, can this匕力^s raise the yeast strain belonging to the genus Shiwaniomisesu such as, specifically, Saccharomyces cerevisiae _s Schizosaccharomyces pombe, Kluyveromyces iactis _s Trichosporon puliulans, Schwaimiomyces alluvius _s Pichia pastor is, and the like.

 As a method for introducing a recombinant vector, 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)].

 When an animal cell is used as a host, 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. For example, the promoter of the IE (i-ediate early) gene of cytomegalovirus (CMV) can be used. , SV40 early promoter or metallothinin promoter, retrovirus promoter, heat shock promoter, SR hypromo, etc. Also, Enhansa of the IE gene of CMV One may be used with the Promo All Night.

Examples of animal cells 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.

 As a method for introducing a recombinant vector, any method for introducing DNA into animal cells can be used.

Cytotechnology, 3, 133 (assigned)], calcium phosphate method (Japanese Patent Publication No. 2-227075), lipofection method CProc. Natl. Acad. Sci. USA, 84, 7413 (1987)), Virology, 52, 456 (1973) And the like.

When insect cells are used as hosts, for example, Baculovirus Expression Vectors, A Laboratory Manual, .H. Freeman and Company, New York (1992) , Molecular 'Baioroji one' § 'Laboratory' manual (Molecular Biology, a Laboratory manual) , is described in the current 'pro Toco one Luz' in Molecular ■ Bruno Lee Oroji _{one, Bio / Technology, 6 5 47} (1988) , etc. the methods, it is possible to express Poribe peptide ₉

 That is, 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) Rereru that the force ^s use and the like.

 As the insect cells, 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.

(Japanese Unexamined Patent Publication (Kokai) No. 2-227075), Lipofxion method CProc. Natl. Acad. Sci. USA, 8, 7413 (1987)].

 As 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.

 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.

 In addition, after introducing an appropriate expression vector for expressing the polypeptide of the present invention into cells collected from a patient's living body, 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.

 (2) Culture of transformants

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.

 As the inorganic salt, 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. During the culture, 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.

 If necessary, an antibiotic such as ampicillin or tetracycline may be added to the medium during the culture.

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. 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.

 As 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.

 If necessary, an antibiotic such as kanamycin, penicillin, streptomycin or the like may be added to the medium during the culture.

 As a medium for culturing the transformant obtained using insect cells as a host, commonly used TNM-II medium (Pharmingen), Sf-900 II SFM medium (Life Technologies Inc.) ExCell400, ExCell405 (all manufactured by JRH Biosciences), Grace's Insect Medium (Nature, 195, 788 (1962)) and the like.

 The cultivation is usually performed under conditions of pH 6 to 7, 25 to 30 ° C. for 1 to 5 days.

 If necessary, an antibiotic such as genyumycin may be added to the medium during the culture.

 (3) Isolation and purification of expressed polypeptide

 In order to isolate and purify the polypeptide expressed by the above-mentioned method from the culture of the above-mentioned transformant, an ordinary enzyme isolation and purification method may be used.

For example, when the polypeptide of the present invention is 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. From the supernatant obtained by centrifuging the 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.

 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.

 When 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. .

 That is, 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.

Alternatively, 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. For example, the method of Roura [Proc. Natl. Acad. Sci. USA, 86 5 8227 (1989), Genes & Dev., 4, 1288 (1990) ], JP-A 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. Further, the 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.

 Furthermore, the 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.

 In addition, Advanced ChemTech, Parkin Elma, Pharmacia, Protein 'Technology' Instrument Company, Synthene Cell-Vega, and Novet. ——Chemical synthesis can also be performed using a peptide synthesizer such as PerSeptive or Shimadzu Corporation.

 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. ,

[3] Preparation of an antibody that recognizes the polypeptide of the present invention

 (1) Preparation of polyclonal antibody

 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.

 -Egret, goat, rat, mouse, hamster, etc. can be used as the animal to be administered.

The dose of the antigen is preferably 50 to 100 zg per animal. In the case of using 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)

] Confirm with.

 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. (2) Preparation of monoclonal antibodies, such as chromatography using a chromatography column, anion exchange column, protein A or G-column, gel filtration column, etc.

(2-1) Preparation of antibody-producing cells

 In the above (1), 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.

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.

 (2-2) Preparation of myeloma cells

 As myeloma cells, cell lines obtained from mice or rats are used. For example, 8-azaguanine resistant mouse (derived from BALB / c) myeloma cell line

P3-X63Ag8-Ul (P3-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)] ヽ 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. 5 mm o 1 / L ), 2- mercaptoethanol ^{(5 X 10- 5 mo 1 /} L ), Genyumycin (10 µg / mL) and fetal calf serum (FCS) (10%, CSL) (hereinafter referred to as “normal medium”), followed by 8-azaguanine (15 µg / mL). )), But culture the cells in a normal medium 3 to 4 days before cell fusion, and use at least 2 x 10 ⁷ cells for fusion.

(2-3) Production of hybridoma

 The antibody-producing cells obtained in (2-1) and the myeloma cells obtained in (2-2) were combined with MEM medium or PBS (1.83 g of disodium phosphate, 0.21 g of monopotassium phosphate, and 7.65 g of salt). After washing well with 1 L of distilled water, pH 7.2), mix the cells so that the number of antibody-producing cells: myeloma cells = 5-; L 0: 1 and centrifuge at 1,200 rpm for 5 minutes. Discard the supernatant.

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.

 After the addition, add MEM medium to adjust the total volume to 5 OmL.

After centrifuging the preparation at 900 rpm for 5 minutes, discard the supernatant. The precipitated fraction of cells obtained, gently Nihoku, after, viewed suction from a graduated pipette, gently HAT medium [normal medium hypoxanthine in balloon (10- ⁴ niol / L), thymidine (1.5 ^{X 10 ~ 5 m 01 / L} ) and aminopterin (suspended in ^{1 0 "7 mo 1 / L} ) was added medium] in 10 OML 4x.

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.

 After the cultivation, a part of the culture supernatant was removed and subjected to the enzyme immunoassay described in Antibodies-A Laboratory Manual, Cold Spring Harbor Laboratory Press, Chapter 14 (1988), etc. Select hybridomas that react specifically with the polypeptide.

 The following method can be given as a specific example of the enzyme immunoassay.

 At the time of immunization, 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.

 Using the hybridoma, cloning was repeated twice by the limiting dilution method [First time, use HT medium (medium obtained by removing aminopterin from HAT medium), second time, use normal medium]. (2-4) Preparation of Monoclonal Monoclonal Antibody with High Antibody Titer

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.

 [4] Measurement of PDE activity of polypeptide of the present invention

According to the method described in [2], 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). '— Separation, quantification of nucleoside 5, monomonophosphate (eg, [ ³ H] 5, AMP or [ ³ H] 5' GMP) formed by hydrolysis of phosphoester bond, or After treatment, the product is Measured by separation and quantification [Biol. Chem., 257, 1973 (1982), Methods in Enzymology, 159, 457 (1988)] In addition, two intrinsic PDEs (pde1, pde2) are missing 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)].

[5] Search / identification of agonist or angelic gonist of the polypeptide of the present invention And therapeutic use

 Cells that can be used for the activity measurement of [4] above, or tissues and cells that have been confirmed to express the polypeptide or mRNA of the present invention by the method of [7] described below are used. A test sample is added, and PDE activity is measured by the method described in [4] above.

 Comparison of PDE activity of the polypeptide of the present invention with or without the addition of the test sample, or bioassay using endogenous PDE-deficient yeast expressing the polypeptide of the present invention, Substances that enhance the activity (agonist) and substances that inhibit the activity (angianist) can be screened. 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). Urine, body fluids, tissue extracts, cell culture supernatants, cell extracts, and other non-peptidic compounds, as well as fermented fertilizers, urine, bovine, ovine, ovine, poma, canine, cat, monkey, human, etc. Examples include products, extracts of plants and other organisms.

 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.

 It is desirable to use the most effective method for the treatment, and it is preferable to use the method by oral administration or parenteral administration such as oral, respiratory, rectal, subcutaneous, intramuscular, and intravenous administration. Can be used. 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.

 As the propellant, 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.

 Specific examples of 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.

 In these parenteral preparations, the components exemplified as additives for oral preparations can be added.

-The dose or frequency of administration varies depending on the desired therapeutic effect, administration method, treatment period, age, body weight, etc., but is usually 10 zg / kg to 8 mg / kg per adult per day. [6] Search and identification of compounds that regulate the expression of the polypeptide of the present invention (hereinafter, abbreviated as expression regulating compounds)

 (1) Search and identification of expression regulating compounds using the antibody of the present invention

 After the cells expressing the polypeptide of the present invention are brought into contact with a test sample, 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.

 As the cell, any cell, cell line, or tissue expressing the polypeptide of the present invention can be used.

 For example, 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 (¾ cells).

 As the 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.

 Wash the adherent cells with PBS buffer, add 3 mL of PBS buffer containing 0.05% trypsin and 0.02% EDTA (ethylenediamine tetraacetic acid), remove excess solution, and remove the solution. The cells are detached from the flask by incubating for 5 minutes.

Cultured cells can be used as they are for suspension cells. The cells are washed with a PBS buffer and suspended in a fixative. Examples of the fixative include a PBS buffer containing 3.7% formaldehyde. After incubating at room temperature for 30 minutes, wash with PBS buffer and suspend in the membrane permeable reaction solution. 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.

 As 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 µg / mL using an immune cell staining buffer or an immune cell staining buffer containing 10% animal serum.

 Dispense the diluted antibody to 20-500 L / well and leave under ice-cooling for 30 minutes.

 When an unlabeled antibody is used, a buffer for immunocell staining is added to the plate, and the cells are washed. Anti-mouse immunoglobulin antibody or anti-rat immunoglobulin antibody labeled with a fluorescent dye such as FITC (fluorescein isothiocyanate) or phycoerythrin at a concentration of about 0.1 to 50 μg / mL Dispense about 50-500 L / well of cell staining buffer, and leave it on ice for 30 minutes while blocking light.

 In the case of using the biotin-labeled monoclonal antibody, 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 μL / well, and the plate is iced. Leave in the light for 30 minutes under cooling.

In both cases, after standing, the plate is added with a buffer solution for staining of immune cells, the cells are thoroughly washed, and analyzed using a fluorescence microscope, Celso overnight, or the like. 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.

 (2) Search and Identification Using the Transcript Quantification System for the Polypeptide Gene of the Present Invention After Contacting Cells Expressing the Polypeptide of the Present Invention or mRNA Encoding the Polypeptide with a Test Sample By quantifying the mRNA content, expression-regulating compounds can be searched and identified.

 As 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.

 Specifically, 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.

 (-3) Search and identification using repo overnight gene

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). In addition, a fragment obtained by cutting this region using an appropriate restriction enzyme to an appropriate length can be used as a transcription control region. As 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. Alternatively, a cell line in which expression of the polypeptide mRNA has been confirmed can be used.

 The 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. .

In addition, gene selection for positive selection (such as G418 resistance gene) and 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 can also be prepared (Nature ₃ 336, 348 (1988), Analytical Biochemistry, 1, 77 (1993), Gene Targeting, The Practical Approach Series, IRL Press (1993)]. 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.

 As a detection and quantification method, in the case of CAT, for example, the method described in Molecular Cloning 2nd Edition, Chapter 16, page 60, and in the case of ^ -gal, for example, molecular cloning In the case of luc, for example, the method described in Experimental Medicine Separate Volume Bio Manual Series 4 Gene Transfer and Expression-Analysis, 89 (1994) can be used in the case of luc. In the case of P, for example, the method described in Proc. Natl. Acad. Sci. USA, 94, 4653 (1997) can be mentioned.

 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.

 [7] Utilization of DNA, polypeptide, antibody, agonist, angelic gonist and expression regulating compound of the present invention

 (1) 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). By performing the daidization, the mRNA of the polypeptide gene of the present invention in the tissue or cell can be detected or quantified. By comparing the mRNA expression levels in various tissues, the tissue expression distribution of the polypeptide of the present invention can be known.

 (2) 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). By performing RT-PCR (reverse transcription PCR; PCE Pr-otocols (1990)) on RNA, 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.

 By quantifying the mRNA in various disease model animals, the importance of the gene product in the disease can be clarified. In addition, a drug can be evaluated by comparing the expression amount of the mRNA with or without the drug.

 (3) Using the oligonucleotide of the present invention as a probe, in situ hybridization is performed on tissue sections of non-human mammals such as humans and mice [Methods in Enzymology, 254, 419 (1995)], it is possible to know a finer distribution of expression, such as identification of cells expressing the polypeptide of the present invention in a tissue.

 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.

 (4) In the present invention: 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.

 (5) 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) can be detected. By detecting the polymorphism, 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

'Diagnosis of diseases such as disease, dementia or malignancy can be performed. (6) By using the 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)], 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.

 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].

 (7) The polypeptide of the present invention can be obtained by the method described in [2] using the DNA of the present invention.

 Uses of the 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].

(8) 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.

(9) 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.

 Using the antibody against the polypeptide of the present invention, 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.

Specifically, 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 ^ί Ι 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. 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.

 Further, by using 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.

(10) By administering an antibody that inhibits the function (PDE activity) of the polypeptide of the present invention, diabetes, ischemic heart disease, hypertension, nephritis, tengitis, ulcer, allergy, It is expected to treat or prevent diseases such as asthma, rheumatism, osteoporosis, pain, anxiety, schizophrenia, manic depression, Parkinson's disease, dementia, infectious disease or malignancy.

 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].

 (11) 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.

 The present invention is specifically illustrated by the following examples, but the scope of the present invention is not limited by the examples. Example

Example 1: Cloning of a human cDNA fragment encoding a protein having PDE activity (1) Preparation of a cDNA library derived from HepG2 cells

Cytoplasmic RNA extraction and PolyA (+) RNA purification described in the literature (J. Sambrook, EF Fritsch & T. Maniatis, Molecular Cloning Second edition, Cold Spring Harbor Laboratory Press 1989) from human hepatocyte cell line HepG2. The corresponding mRNA was extracted and purified. A cDNA library was prepared from each polyA (+) RNA by an oligocap method [L Maruyama and Sr Sugano, Gene, 138, 171 (1994)]. Using Oligo-cap linker (SEQ ID NO: 3) and 01 igo dT primer (SEQ ID NO: 4), literature [Suzuki Sugano, Protein Nucleic Acid Enzyme, 1, 603 (1996), Y.Suzuki et al. ₅ Gene, 200 ₃ 149 (1997)] BAP (Bacterial Alkaline Phosphatase) treatment, TAP

(Tobacco Acid Pyrophosphatase) treatment, RM ligation, synthesis of first-strand cDNA, and removal of A. Next, the DNA was converted into double-stranded cDNA by a PClU polymerase chain reaction using PCR primers (SEQ ID NOS: 5 and 6) at the 5 'end and 3' end, and then cut with the restriction enzyme il. The cDNA was inserted into a vector PE18SFL3 (GenBank AB009864, Expression vector, 3392 bp) cut with ΠΙ to prepare a cDNA library. The cDNA was incorporated in a direction allowing expression.

 (2) Random sequence

 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). As the primer, the synthetic DNA shown in SEQ ID NOs: 7 and 8 was used.

 (3) Cloning of N-terminal region and analysis using homology search software

 The obtained base sequence was analyzed using blast search homology search software based on the gene sequence information of human PDE10A (GenBank: AB020593), and a sequence showing homology was found. As a result of determining the entire nucleotide sequence of the clone, 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

,

Penic acid was coded

 Based on the sequence information, 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.

-That is, 2 L of Human Pancreas Marathon-Ready cDNA, 0.2 mol / L of each of the primers of SEQ ID NO: 9 and API (attached to kit), dNTP (dATP, dGTP, dCTPs dTTP) mixture of 200-11101 each component, Taq Gold Polymerase (PerkinElmer) 2.5 units PCR was carried out under the following conditions using a reaction solution (50 / L) containing lx Taq Gold buffer solution.

That is, using 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. Subsequently, 2 zL of a 100-fold diluted solution of the PCR reaction solution obtained, primers of SEQ ID NO: 10 and AP2 (attached to the kit) 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.

 50 ng of the DNA fragment collected above and 50 ng of pT7Blue T-Vector (Novagen) were subjected to a binding reaction using DNA Ligation Kit Ver. 2 (Takara Shuzo) according to the manual. Escherichia coli JM109 strain was transformed with the recombinant plasmid DNA obtained by the reaction, and plasmid pT-1 was obtained by a conventional method.

 By determining the nucleotide sequence of the DNA fragment contained in plasmid ρΤ-1 by a conventional method, it was found that the inserted DNA fragment could be ligated to the Cla I site of hepl0314 at the Cla I site in the inserted fragment. The linked base sequence is shown in SEQ ID NO: 2. The novel polypeptide encoded in SEQ ID NO: 1 was encoded in the sequence.

Further, when a Sndth & Waterman search of the amino acid sequence was performed on the known protein sequence database, homology with PDE5A and PDE10A families was detected strongly. Therefore, human PDE5A amino acid sequence (GenBank: CAA06170) and human PDE10A amino acid sequence (GenBank: BM78034) were selected from each family, and an alignment was prepared. Figure 1 shows the results of the alignment with the human PDE5A sequence, and Figure 2 shows the results of the alignment with the human PDE10A sequence. An HDXXHXXXXN sequence (SEQ ID NO: 14), which is an amino acid sequence common to PDEs, was also recognized (underlined parts in FIGS. 1 and 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

 (hepl0314) 10ng, 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.

 In other words, using a TaKaRa PCR Thermal Cycler 480, heat at 95 ° C for 3 minutes, perform 25 cycles of 1 cycle at 94 ° C for 1 minute and 1 minute at 60 ° C, and further heat at 72 ° C for 8 minutes did.

 From the obtained PCR reaction solution, 5 L was taken, and after confirming that the MA fragment of about 0.2 kb was amplified by agarose gel electrophoresis, using a QIAEX II Gel Extraction Kit (manufactured by QIAGEN), The DNA fragment was purified according to the manual.

 — 2 μg of pBluescript II KS (-) (manufactured by STRATAGENE) in 50 t ol / L Tris-hydrochloric acid (pH 7.5), 10 fractions ol / L magnesium chloride, lmmol / L dithiothreitol (hereinafter referred to as DTT This was dissolved in 50 L of a buffer solution containing 100 mmol / L sodium chloride, and 10 units of ^ RV (manufactured by Takara Shuzo Co., Ltd.) were added, followed by digestion at 37 ° C for 3 hours. After phenol extraction and ethanol precipitation, the DM fragment was dissolved in 50 μl of 50 μl / L Tris-hydrochloric acid (pH 9.0), 1 μl / L magnesium chloride buffer, and 0.5 units of alcohol was added. Force phosphatase

(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 binding reaction was performed according to the manual using 50 ng of the DNA fragment collected above and the V-BAP-treated fragment derived from pBluescriptll KS (-) using DNA Blunting iat (Takara Shuzo). Using the recombinant plasmid DNA obtained by the reaction, E. coli JM109 strain was transformed. After transformation, plasmid p200 was obtained by a conventional method. '

 A buffer solution consisting of 50 μl / L Tris-HCl (pH 7.5), 10 orchid ol / L magnesium chloride, 1 orchid ol / L DTT, and 100 hol / L sodium chloride 50 μl of plasmid p200溶解 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 µg of the prepared plasmid p200-EB at 10 µl / 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 ヽ 1 ol / L ATP, lmmol / L CTP. 1 ol / L GTPs 0.65 舰 ol / L UTP, 0.35 ol / L digoxigenin-; -ϋΤΡ-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.

 After the reaction, a digoxigenin-labeled cRNA probe was recovered from the obtained reaction solution by ethanol precipitation.

Using this probe, poly (A) ⁺ RNA file of human heart, brain, placenta, lung, liver, skeletal muscle, kidney, and knee (Rile Yuichi of Human Multiple Tissue Northern Blots (Clontech)) And human heart, brain, liver, lentin, placenta, and lung poly (A) ⁺ RNA filter Human Normal Tissue mRNA blot I (Normalized) filter (Toyobo Co., Ltd.)] 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. for 2 hours.

 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.

 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.

The film is immersed in a luminescent substrate CDP-Star (Boehring-Germanheim) solution diluted 100 times with DIGIII buffer at room temperature for 15 minutes to emit a signal, and a CCD camera (Fuji Photo Film Co., Ltd.) ). Fig. 4 shows the results. Approximately 2. ^{s was} found in the kidney

Example 3 Measurement of PDE activity using E. coli expressing GST fusion protein

A. Construction of plasmid for expression of GST fusion protein

 'To confirm that the MA fragment obtained in Example 1 encodes an amino acid sequence having PDE activity, E. coli expressing a glutathione S transferase (hereinafter abbreviated as GST) fusion protein was used. Produced.

 Based on the information on the nucleotide sequence determined in Example 1, a 5'-end DNA primer shown in SEQ ID NO: 12 and a 3, DNA-end DNA primer shown in SEQ ID NO: 13 were designed and synthesized.

,

 The resulting two types of primers, each at 0.2 mol / L, the plasmid of Example 1

(hepl0314) 10 ngs Each component ZOOAimol / L dNTP (dATP, dGTP, dCTP, dTTP) mixture, PLATINUM Pfx DNA polymerase (manufactured by Lifetech) 2.5 units and lx PLATINUM-Pfx DNA polymerase buffer containing 50 L of reaction solution PCR was carried out under the following conditions.

 That is, using a Thermal Cycler PTC-200 (manufactured by MJ Risaichi Co., Ltd.), heat at 95 ° C for 3 minutes, and then perform 25 cycles of 94 ° C for 1 minute and 60 ° C for 1 minute. The mixture was further heated at 72 ° C for 8 minutes.

The obtained PCR reaction solution was fractionated, and it was confirmed by agarose electrophoresis that a DNA fragment of about l.Okb was amplified. After phenol extraction and ethanol precipitation, 20 t ol / L tris-hydrochloric acid (pH 7.5), 10 t ol / L magnesium chloride, 1 hi ol / L DTT, 100 t ol / L chloride After dissolving in 50 μL 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. After phenol extraction and ethanol precipitation, 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 μL 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). '

 On the other hand, 2 〃g of plasmid pVL1393 (Pharmingen) 20 ol / L tris monohydrochloride (pH 7.5), 10 ol / L magnesium chloride, 1 fiber ol / L DTT ヽ 100 employment ol / L Buffer solution consisting of potassium chloride Dissolve, add 10 units of ^ iHi, perform digestion reaction at 37 ° C for 3 hours d After phenol extraction and ethanol precipitation, transfer the DNA fragment to 10 t ol / L Tris monosalt Dissolve in a buffer consisting of acid (pH 7.5), 10 ol / L magnesium chloride, 1 ol / L DTT, 50 t ol / L sodium chloride, and 0.01% ゥ serum albumin. In addition, 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).

 50 ng of the PCR-amplified 1-Xba I fragment (1.0 kb) recovered above and pVL1393-derived

 One fragment (9.6 kb) was subjected to a binding reaction 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 plasmid pPDE-1393 was obtained by a conventional method. 2 μg 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. O Triton X-100, and 10 units of Kk and I were added to each, followed by digestion at 37 ° C for 3 hours. The reaction solution was subjected to agarose electrophoresis, and the -MI fragment (l. Okb) was purified using a QIAEX II Gel Extraction Kit (manufactured by QIAGEN).

 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).

On the other hand, 2 μg of plasmid pGEX-5X-1 (Pharmacia) was added to 33 μl / 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. After phenol extraction and ethanol precipitation, the DNA fragment was used in 50 ol / L tris monohydrochloride (pH 7.5), 10 ol / L magnesium chloride, 1 ol / L DTT ヽ 100 ol / L sodium chloride Was dissolved in 50 μL 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).

 50 ng of ^ I-I fragment (1.0 kb) derived from pPDE-1393, 50 ng of EcoRV-Gla I fragment (0.3 kb) derived from pT-1 and I-Not I fragment derived from pGEX-5X-1

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.

 Expression of B. GST fusion protein in Escherichia coli and measurement of PDE activity

 To 50 mL of LB medium containing 200 ig / mL of ampicillin, add 1/100 of the E. coli preculture solution (30 ° C; culture once in LB medium containing 200 / g / fflL of ampicillin), and add at 25 ° C. The cells were cultured with shaking. When the 0D value reached 0.5, isopropyl-/?-D-thiogalactobilanoside (2 ol / L) was added, and the expression was induced at 25 ° C for 3 hours. 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 μg / 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.

 C. Measurement of PDE activity

PDE activity was determined by the method of Kincaid, RL and Manganiello, VC Ofethod. Enzymol., 159, 457-470 (1988)). [¾] -cAMP or [¾] -cGMP as a substrate, 300 L reaction solution [50 ol / L of Ν, · Ν-bis (2-hydroxyethyl) -2_aminoethanesulfonate (ρΗ7. 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. After incubating the reaction solution at 30 ° C. for 15 minutes, HC1 was added to stop the reaction. The reaction product was converted to adenosine with 5'-nucleotidase (Sigma) and separated from unreacted substances on a DEAE-Sephadex A-25 column (Pharmacia). The eluate was transferred to a scintillation vial, 6 mL of Ultima Gold (manufactured by Packard) was added, and the radioactivity was measured using a liquid scintillation counter (Beckman LS6500). Non-catalytic water dissociation was observed when no E. coli soluble fraction was added.

[¾] -cAMP and [¾] -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

 (1) Cloning from human fetal kidney-derived cDNA library

 Buffer containing 30 μg of plasmid pGST-PDE, 20 t ol / L Tris-HCl (pH 8.5), 10 w / ol / L magnesium chloride, 100 μl / L potassium chloride, 1 w / ol / L DTT 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).

23-1). The operation of the hybrid is all performed by ECL direct nucleic acid labeling.

-Performed according to the manual of the detection system (Amersham). 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.

 5 g of pBluescriptll KS (-) (manufactured by STRATAGE) 50 t ol / L Tris-HCl (pH7.5), 10 ol / L magnesium chloride, 100 ol / L sodium chloride, 1 ol / L DTT Was dissolved in 30 L of a buffer solution containing, and 15 units of! ^ RI (manufactured by Takara Shuzo Co., Ltd.) was added, followed by a 2 hour digestion reaction at 37 ° C. ^ -Digested DNA fragments were recovered from the reaction solution by ethanol precipitation. '

 The DNA fragment was dissolved in 30 zL of a buffer containing 50 t / ol / L Tris-monohydrochloride (ρΗ90) 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.

Approximately 2.0 kb of the ^ M DNA fragment of about l. And pBluescriptll KS (-)! ^ RI—BAP-treated fragment 50 ng / 66 ol / L Tris monohydrochloride (pH 7.5), 6.6 ol / L magnesium chloride, 10 mmol / L DTT, O.lmmol / L adenosine triphosphate buffer 20 Then, 175 units of T4 DNA ligase (Takara Shuzo) was added, and the binding reaction was performed at 16 ° C for 16 hours.

 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.

 As a result of determining the base sequence of the cDNA fragment contained in the plasmid, 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.

 When the amino acid sequence of the polypeptide was compared with a known PDE sequence, high homology with PDE5A was observed. FIG. 8 shows an alignment between the amino acid sequence and the human PDE5A amino acid sequence (GenBank: CM06170).

 From the above results, it is considered that the sequence encodes a novel PDE.

(2) Expression analysis using RT-PCR

 Based on the information on the nucleotide sequence determined in (1) of Example 4 described above, a 5, DNA primer shown in SEQ ID NO: 17 and a 3, DNA primer shown in SEQ ID NO: 18 were designed. , Synthesized.

 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).

In other words, after heating at 95 ° C for 10 minutes using a thermal cycler PTC-200 (manufactured by MJ Risaichi Co., Ltd.), a process consisting of 94 ° C for 1 minute and 60 ° C for 1 minute is defined as one cycle. The cycle was performed, and heating was further performed at 72 ° C for 8 minutes. From the obtained PCR reaction solution, lOL was fractionated, and amplification of the expected DM fragment of about 400 bp was confirmed by agarose electrophoresis. Expression in testis, prostate, mammary gland, and rat. The results of the electrophoresis are shown in FIG. Industrial applicability

 By using 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.

Claims

1. A polypeptide consisting of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 15.  2. Encoding the polypeptide of claim 1: DNA.  3. DNA having the nucleotide sequence of SEQ ID NO: 2 or SEQ ID NO: 16.  4. A recombinant cell containing the DNA of claim 2 or 3.  5. A transformant having the recombinant vector according to claim 4.  6. The transformant according to claim 5, wherein the transformant is a transformant selected from the group consisting of a microorganism, an animal cell, a plant cell, and an insect cell.  7. The transformant according to claim 6, wherein the microorganism is a microorganism belonging to the genus Escherichia.  8. The transformant according to claim 7, which has an accession number FERM BP-6976.  9. The transformant according to any one of claims 5 to 8 is cultured in a medium, the polypeptide according to claim 1 is produced and accumulated in the culture, and the polypeptide is extracted from the culture. 2. The method for producing a polypeptide according to claim 1, wherein the method comprises sampling a tide.  10. A sense oligonucleotide having the same sequence as a continuous 5-β0 base in the base sequence of the DNA according to claim 2 or 3, an antisense oligonucleotide having a sequence complementary to the sense oligonucleotide, And oligonucleotides selected from the group consisting of oligonucleotide derivatives of these sense or antisense oligonucleotides. 11. oligonucleotide derivatives, O oligo nucleotide derivative phosphate Jiesu ether bond is converted into Hosuhorochioe Ichito bond in an oligonucleotide, O Rigo phosphodiester bond New 3 in the nucleotide '-Ρ ⁵ 5 Hosuhoamide one DOO Oligonucleotide derivative converted into a bond, oligonucleotide derivative in which ribose and phosphodiester bond in the oligonucleotide are converted into peptide nucleic acid bond Conductor, 'oligonucleotide derivative in which peracyl in oligonucleotide is substituted with C-5 propynylperacyl, oligonucleotide derivative in which peracyl in oligonucleotide is substituted with C-5 thiazo-peruracil, cytosine in oligonucleotide is Oligonucleotide derivatives substituted with C-5 propynylcytosine, cytosine in the oligonucleotide is phenoxazine {decorated cytosine ( Oligonucleotide derivative substituted with phenoxazine-modified cytosine), ribose in DNA is 2'-〇-oligonucleotide derivative substituted with 1-provyl ribose, and ribose in oligonucleotide is 2,1-methoxyethoxyribose 2. The oligonucleotide according to claim 1, wherein the oligonucleotide is an oligonucleotide derivative selected from the group consisting of oligonucleotide derivatives substituted with a nucleotide. ·  12. A method for detecting mRNA encoding the polypeptide according to claim 1, characterized by using the oligonucleotide according to claim 10 or 11.  13. A method for suppressing the expression of a polypeptide according to claim 1, wherein the method according to claim 1 is characterized by using the oligonucleotide according to L0 or 11.  14. An antibody that recognizes the polypeptide of claim 1.  15. The method for immunologically detecting a polypeptide according to claim 1, wherein the antibody according to claim 14 is used.  16. The method for immunohistochemical staining of the polypeptide according to claim 1, wherein the antibody according to claim 14 is used. '  17. An immunohistochemical, comprising the antibody according to claim 14.  18. A method for screening for a compound that alters the phosphodiesterase activity of the polypeptide according to claim 1, which comprises contacting the polypeptide according to claim 1 with a test sample.  19. A compound obtained by the method according to claim 18. 20. 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 claim 1 with a test sample. 21. Detection of mRNA encoding the polypeptide of claim 1 using the method of claim 12 to detect fluctuations in the expression of the gene encoding the polypeptide The screening method according to claim 20, characterized in that:  22. By detecting the polypeptide of claim 1 using the method of claim 15, a change in the expression of a gene encoding the polypeptide is detected. A screening method according to claim 20.  23. Compound c obtained by the method according to any one of claims 20 to 22 24. A promoter-DNA that controls transcription of a gene encoding the polypeptide according to claim 1.  25. A test sample and a transformant having a plasmid containing the promoter DNA of claim 24 and a repo overnight gene linked downstream of the promoter overnight DNA are used. A method for screening a compound that changes the efficiency of transcription by said promoter, comprising contacting and measuring the translation product content of said repo overnight gene.  26. The repo overnight genes are the chloramphenic acid acetyltransferase gene, the? -Galactosidase gene, the luciferase gene, the beta-glucanidase gene, the aequorin gene and the green fluorescein gene. 26. The screening method according to claim 25, wherein the screening method is a gene selected from the group consisting of oral tin genes.  27. A compound obtained by the method according to claim 25 or 26.  28. A medicament comprising the polypeptide according to claim 1.  29. A medicament comprising the oligonucleotide according to claim 10 or 11. 30. A medicament comprising the antibody according to claim 14. '  31. A medicament comprising the compound according to claim 19, 23 or 27. 3 2. Diabetes, ischemic heart disease, hypertension, nephritis, nephritis, ulcer, allergy, asthma, rheumatism, osteoporosis, pain, anxiety, schizophrenia, manic depression, Parkinson's disease, dementia 30. The medicament according to any one of claims 28 to 31, which is a medicament for prevention or treatment of baldness, infectious disease or malignant tumor.  3 3. 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 The medicament according to any one of claims 28 to 31, which is a medicament for diagnosis of: