CN1297049A - Polypeptide-human cyclophosphate guanine suppressed phosphodiesterase 17 and polynucleotide for coding said polypeptide - Google Patents

Abstract

A new polypeptide-human cyclophosphate guanine suppressed phosphodiesterase 17, the polynucleotide for coding it, the process for preparing said polypeptide by DNA recombination, the application of said polypeptide in treating more diseases (cancer, HIV infection, etc), the antagonist to resist said polypeptide and its therapeutic action, and the application of said polynucleotide are disclosed.

Description

The polynucleotide of the phosphodiesterase 17 that a kind of new polypeptide--people's cyclic phosphoric acid guanine suppresses and this peptide species of coding

The invention belongs to biological technical field, specifically, the invention describes a kind of new polypeptide--the phosphodiesterase 17 that people's cyclic phosphoric acid guanine suppresses, and the polynucleotide sequence of this polypeptide of encoding.The invention still further relates to the preparation method and the application of these polynucleotide and polypeptide.

Nucleolus glycosides PDE (PDEs) relates to the super enzyme family of many regulatory pathway, the characteristic of PDE3 family is with cAMP height affinity, is suppressed by cGMP, to special supressor cilostamide and milrinone, sensitivity, by phosphorylation and activation response Regular Insulin and surrogate to improve the cAMP level.PDE3 family is called the cGI PDE of cGMP-inhibited again.(Miki?et?al.,1996；Lobbert?etal.,1996)。

The C end of cGI PDE contains conservative structural domain, and it is proved to be the catalytic site region.Insertion that representational PDE3B and PDE3A are made up of one 44 amino acid in this conservative catalyst structure domain among the PDE3.This insertion relates to the susceptibility of catalytic site to cGMP.(Meacci?E?etal.,1992；He?R?et?al.,1996)。

CGIP1 is PDE3B, form by 16 exons, have the polymorphism of G/A in the genetic decoding zone, in 2 introns (intron 5 and introne 1 2), dinucleotides multiple polymorphism is arranged, 1112 amino acid of PDE3B genetic decoding, it and mouse PDE3B have 81% homology.The northern probe show it in many fat stores tissues than high expression level, low expression (Miki etal., 1996) in liver, spleen, lung, kidney.CGIP2 is PDE3A, and its cAMP hydrolytic activity can be suppressed (Meacci etal., 1992) by cGMP, and PDE3A is high expression level in thrombocyte, myocardial cell, and the disappearance of the N end of PDE3A can improve the cAMP hydrolytic activity.Research thinks that the N end difference of cGI PDE relates to the specificity (Tang etal., 1997) of substrate identification.

People such as Tang find PDE3A high expression level (Tang et al., 1997) in thrombocyte, myocardial cell.

People such as Miki and Lobbert thinks that the chromosomal region that the PDE3B gene exists relates to obesity and noninsulin dependent diabetes (Miki et al., 1996; Lobbert et al., 1996).

Discover that optionally cGI PDE supressor can be used for treating bronchial asthma and chronic obstructive pulmonary disease (Schmidt D, et al., 1999)

The PDE family gene that people's of the present invention polypeptide gene and people's cyclic phosphoric acid guanine suppresses has 98% homology on protein level, (homologous protein AF151819, homologous protein CGI-121PROTEIN, this protein 157aa, 17.3KD), its structural domain is similar in appearance to the characteristic structural domain of the PDE family that the cyclic phosphoric acid guanine suppresses--and-C end contains conservative structural domain, and it is the catalytic site region; Insertion of in this conservative catalyst structure domain, often forming by one 44 amino acid.Based on above each point, so think that new gene of the present invention is the gene of the PDE family of a coding people cyclic phosphoric acid guanine inhibition, names the PDE 17 that suppresses into people's cyclic phosphoric acid guanine.And infer that with this it has the similar biological function of PDE family that the cyclic phosphoric acid guanine suppresses.

The polynucleotide of the PDE 17 that coding people cyclic phosphoric acid guanine suppresses, and the PDE 17 that suppresses of coded people's cyclic phosphoric acid guanine be found to be the differentiation of research cell under normal and pathological conditions, the physiological and biochemical procedure of propagation provides a kind of method, also comprises that with the disease that the disorder of cytodifferentiation propagation causes cancer provides a kind of new way for diagnosis, treatment.

Because the phosphodiesterase 17 albumen of people's cyclic phosphoric acid guanine inhibition plays an important role in regulating body critical functions such as cell fission and fetal development as mentioned above, and believe and relate to a large amount of albumen in these regulate processes, thereby the phosphodiesterase 17 albumen that suppresses of the people's cyclic phosphoric acid guanine that always needs to identify more these processes of participation in this area, particularly identify this proteic aminoacid sequence.The separation of the phosphodiesterase 17 protein coding gene that new person's cyclic phosphoric acid guanine suppresses also provides the foundation for determining the effect of this albumen under healthy and morbid state.This albumen may constitute the basis of exploitation medical diagnosis on disease and/or curative, and it is very important therefore separating its coding DNA.

An object of the present invention is to provide isolating new polypeptide--phosphodiesterase 17 that people's cyclic phosphoric acid guanine suppresses with and fragment, analogue and derivative.

Another object of the present invention provides the polynucleotide of this polypeptide of coding.

Another object of the present invention provides the recombinant vectors of the polynucleotide of the phosphodiesterase 17 that contains the inhibition of coding people cyclic phosphoric acid guanine.

Another object of the present invention provides the genetically engineered host cell of the polynucleotide of the phosphodiesterase 17 that contains the inhibition of coding people cyclic phosphoric acid guanine.

Another object of the present invention provides the method for the phosphodiesterase 17 of producing the inhibition of people's cyclic phosphoric acid guanine.

Another object of the present invention provides at polypeptide of the present invention--the antibody of the phosphodiesterase 17 that people's cyclic phosphoric acid guanine suppresses.

Another object of the present invention has provided at polypeptide of the present invention--simulated compound, antagonist, agonist, the inhibitor of the phosphodiesterase 17 that people's cyclic phosphoric acid guanine suppresses.

Another object of the present invention provides the method for the diagnoses and treatment disease relevant unusually with the phosphodiesterase 17 of people's cyclic phosphoric acid guanine inhibition.

The present invention relates to a kind of isolated polypeptide, this polypeptide is the people source, and it comprises: polypeptide or its examples of conservative variations, bioactive fragment or derivative with SEQ ID No.2 aminoacid sequence.Preferably, this polypeptide is the polypeptide with SEQ ID NO:2 aminoacid sequence.

The invention still further relates to a kind of isolating polynucleotide, it comprises a kind of nucleotide sequence or its variant that is selected from down group:

(a) coding has the polynucleotide of the polypeptide of SEQ ID No.2 aminoacid sequence;

(b) with polynucleotide (a) complementary polynucleotide;

(c) with (a) or polynucleotide sequence (b) have the polynucleotide of at least 99% homogeny.

More preferably, the sequence of these polynucleotide is be selected from down group a kind of: the sequence that (a) has 63-536 position among the SEQ ID NO:1; (b) has the sequence of 1-799 position among the SEQ ID NO:1.

The present invention relates to a kind of carrier that contains polynucleotide of the present invention, particularly expression vector in addition; The host cell that this carrier of a kind of usefulness is genetically engineered comprises the host cell of conversion, transduction or transfection; A kind of method for preparing polypeptide of the present invention of cultivating described host cell and reclaiming expression product that comprises.

The invention still further relates to a kind of can with polypeptid specificity bonded antibody of the present invention.

The invention still further relates to a kind of simulation, activation, antagonism of screening or suppress the method for the compound of the phosphodiesterase 17 protein-active that people's cyclic phosphoric acid guanine suppresses, it comprises and utilizes polypeptide of the present invention.The invention still further relates to the compound that obtains with this method.

The invention still further relates to a kind of vitro detection and express the relevant disease or the method for disease susceptibility with the phosphodiesterase 17 abnormal protein that people's cyclic phosphoric acid guanine suppresses, comprise the sudden change in polypeptide described in the detection of biological sample or its coded polynucleotide sequence, perhaps the amount or the biological activity of polypeptide of the present invention in the detection of biological sample.

The present invention also relates to a kind of pharmaceutical composition, it contains polypeptide of the present invention or its stand-in, activator, antagonist or inhibitor and pharmaceutically acceptable carrier.

The invention still further relates to polypeptide of the present invention and/or polynucleotide and be used for the treatment of cancer, developmental character disease or immunological disease or other purposes owing to the medicine of the phosphodiesterase 17 disease that abnormal expression causes of people's cyclic phosphoric acid guanine inhibition in preparation.

Others of the present invention are because disclosing of the technology of this paper is conspicuous to those skilled in the art.

The following term of using in this specification sheets and claims has following implication unless stated otherwise:

" nucleotide sequence " is meant oligonucleotide, Nucleotide or polynucleotide and fragment or part, also can refer to genome or synthetic DNA or RNA, and they can be strand or two strands, represent sense strand or antisense strand.Similarly, term " aminoacid sequence " is meant oligopeptides, peptide, polypeptide or protein sequence and fragment or part.When " aminoacid sequence " among the present invention related to a kind of aminoacid sequence of naturally occurring protein molecule, this " polypeptide " or " protein " did not mean that aminoacid sequence are restricted to the complete natural amino acid relevant with described protein molecule.

Protein or polynucleotide " variant " are meant a kind of polynucleotide sequence that has the aminoacid sequence of one or more amino acid or Nucleotide change or encode it.Described change can comprise disappearance, insertion or the replacement of amino acid in aminoacid sequence or the nucleotide sequence or Nucleotide.Variant can have " conservative property " and change, and wherein the amino acid of Ti Huaning has structure or the chemical property similar with original acid, as replacing Isoleucine with leucine.Variant also can have non-conservation and change, as replacing glycine with tryptophane.

" disappearance " is meant the disappearance of in aminoacid sequence or nucleotide sequence one or more amino acid or Nucleotide.

" insertion " or " interpolation " is meant that the change in aminoacid sequence or nucleotide sequence causes comparing the increase of one or more amino acid or Nucleotide with naturally occurring molecule." replacement " is meant by different amino acid or Nucleotide and replaces one or more amino acid or Nucleotide.

" biological activity " is meant the protein of structure, regulation and control or biochemical function with natural molecule.Similarly, term " immunologic competence " be meant natural, reorganization or synthetic protein and fragment thereof in suitable animal or cell, induce specific immune response and with specific antibody bonded ability.

" agonist " is meant when the phosphodiesterase 17 with the inhibition of people's cyclic phosphoric acid guanine combines, thereby a kind of this protein that causes changes the molecule of regulating this protein active.Agonist can comprise the molecule of protein, nucleic acid, carbohydrate or any phosphodiesterase 17 that other can suppress in conjunction with people's cyclic phosphoric acid guanine.

" antagonist " or " inhibition " is meant when the phosphodiesterase 17 with the inhibition of people's cyclic phosphoric acid guanine combines, a kind of sealing or the biologic activity of the phosphodiesterase 17 that mediator's cyclic phosphoric acid guanine suppresses or the molecule of immunologic competence.Antagonist and inhibition can comprise the molecule of protein, nucleic acid, carbohydrate or any phosphodiesterase 17 that other can suppress in conjunction with people's cyclic phosphoric acid guanine.

" adjusting " is meant that the function of the phosphodiesterase 17 that people's cyclic phosphoric acid guanine suppresses changes, and comprises the change of any other biological property, function or the immune property of the rising of protein active or reduction, the change of binding characteristic and the phosphodiesterase 17 that people's cyclic phosphoric acid guanine suppresses.

" pure basically " is meant and is substantially free of natural relative other albumen, lipid, carbohydrate or other material.The phosphodiesterase 17 that those skilled in the art can suppress with the purified technology of protein purifying people cyclic phosphoric acid guanine of standard.Basically the phosphodiesterase 17 that pure people's cyclic phosphoric acid guanine suppresses can produce single master tape on the irreducibility polyacrylamide gel.The purity available amino end acid sequence of the phosphodiesterase 17 polypeptide that people's cyclic phosphoric acid guanine suppresses is analyzed.

" complementary " or " complementation " is meant under salt concn that allows and temperature condition by the natural combination of the polynucleotide of base pairing.For example, sequence " C-T-G-A " can combine with complementary sequence " G-A-C-T ".Complementation between two single chain molecules can be part or whole.Complementary degree between the nucleic acid chains has a significant effect for efficient of hybridizing between the nucleic acid chains and intensity.

" homology " is meant the complementary degree, can be portion homologous or complete homology." portion homologous " is meant a kind of part complementary sequence, and it can partly suppress the hybridization of complete complementary sequence and target nucleic acid at least.The inhibition of this hybridization can detect by hybridizing (Southern trace or Northern trace etc.) under the condition that reduces in the severity degree.Basically homologous sequence or hybridization probe can compete and suppress complete homologous sequence and target sequence the condition that reduces of severity degree under combine.This does not mean the conditions permit non-specific binding that the severity degree reduces because the conditional request two sequences that the severity degree reduces mutual be combined into specificity or selectivity interacts.

" homogeny percentage " be meant two or more amino acid or nucleotide sequence relatively in the same or analogous percentage of sequence.The available electron method is measured the homogeny percentage, as passing through MEGALIGN program (Lasergenesoftware package, DNASTAR, Inc., Madison Wis.).The MEGALIGN program can compare two or more sequences (Higgins, D.G. and P.M.Sharp (1988) Gene 73:237-244) according to diverse ways such as Cluster method.The Cluster method is organized the series arrangement cluster by checking the distance between all pairings with each.Then with each bunch with in pairs or become set of dispense.Homogeny percentage between two aminoacid sequences such as sequence A and the sequence B calculates by following formula: residue number--------------------------------------------------------------100 of mating between sequence A and the sequence B

Interval residue number in the residue number-sequence B of interval in the residue number-sequence A of sequence A

Also can be by the Cluster method or with the homogeny percentage (Hein J., (1990) Methods in emzumology 183:625-645) between known method in this area such as the Jotun Hein mensuration nucleotide sequence.

" similarity " is meant the degree that the identical or conservative property of corresponding position amino-acid residue when arranging contrast between the aminoacid sequence replaces.For example be used for amino acid that conservative property replaces, electronegative amino acid can comprise aspartic acid and L-glutamic acid; Positively charged amino acid can comprise Methionin and arginine; Having uncharged head group has similar hydrophilic amino acid can comprise leucine, Isoleucine and Xie Ansuan; Glycine and L-Ala; L-asparagine and glutamine; Serine and Threonine; Phenylalanine and tyrosine.

" antisense " is meant and specific DNA or RNA sequence complementary nucleotide sequence." antisense strand " is meant and " sense strand " complementary nucleic acid chains.

" derivative " is meant HFP or encodes its chemical modification object of nucleic acid.This chemical modification object can be with alkyl, acyl group or the amino hydrogen atom of replacing.The nucleic acid derivative codified keeps the polypeptide of the main biological characteristics of natural molecule.

" antibody " is meant complete antibody molecule and fragment thereof, as Fa, F (ab ') ₂And Fv, its energy specificity is in conjunction with the antigenic determinant of the phosphodiesterase 17 of people's cyclic phosphoric acid guanine inhibition.

" humanized antibody " is meant that the aminoacid sequence in non-antigen binding domain territory is replaced and becomes more similar to people's antibody, but still keep original in active antibody.

" isolating " speech refers to material is shifted out among its original environment (for example, if spontaneous its natural surroundings that just refers to).Such as it is exactly not to be separated that spontaneous polynucleotide or polypeptide are present in the Live Animals, but same polynucleotide or polypeptide with some or all in natural system with it the material of coexistence separately be exactly isolating.Such polynucleotide may be the parts of a certain carrier, the part that also possible such polynucleotide or polypeptide are a certain composition.Since carrier or composition are not the compositions of its natural surroundings, they remain isolating.

As used herein, " isolating " are meant that material separates (if natural substance, primal environment promptly is a natural surroundings) from its primal environment.Do not have separation and purification as polynucleotide under the native state in the active somatic cell and polypeptide, but same polynucleotide or polypeptide as from native state with in other materials that exist separately, then for separation and purification.

As used herein, " phosphodiesterase 17 that isolating people's cyclic phosphoric acid guanine suppresses " is meant that the phosphodiesterase 17 that people's cyclic phosphoric acid guanine suppresses is substantially free of natural relative other albumen, lipid, carbohydrate or other material.The phosphodiesterase 17 that those skilled in the art can suppress with the purified technology of protein purifying people cyclic phosphoric acid guanine of standard.Basically pure polypeptide can produce single master tape on non-reduced polyacrylamide gel.The purity of the phosphodiesterase 17 polypeptide that people's cyclic phosphoric acid guanine suppresses can be used amino acid sequence analysis.

The invention provides a kind of new polypeptide--the phosphodiesterase 17 that people's cyclic phosphoric acid guanine suppresses, it is made up of the aminoacid sequence shown in the SEQ ID NO:2 basically.Polypeptide of the present invention can be recombinant polypeptide, natural polypeptides, synthetic polypeptide, preferred recombinant polypeptide.Polypeptide of the present invention can be the product of natural purifying, or the product of chemosynthesis, or uses recombinant technology to produce from protokaryon or eucaryon host (for example, bacterium, yeast, higher plant, insect and mammalian cell).The host used according to the recombinant production scheme, polypeptide of the present invention can be glycosylated, maybe can be nonglycosylated.Polypeptide of the present invention also can comprise or not comprise initial methionine residues.

The present invention also comprises fragment, derivative and the analogue of the phosphodiesterase 17 that people's cyclic phosphoric acid guanine suppresses.As used herein, term " fragment ", " derivative " are meant identical biological function or the active polypeptide of phosphodiesterase 17 that keeps people's cyclic phosphoric acid guanine of the present invention to suppress basically with " analogue ".The fragment of polypeptide of the present invention, derivative or analogue can be: (I) is a kind of like this, wherein one or more amino-acid residues are replaced by conservative or non-conservative amino acid residues (preferably conservative amino acid residues), and the amino acid that replaces can be also can not encoded by genetic codon; Perhaps (II) is a kind of like this, and certain group on wherein one or more amino-acid residues is replaced by other group and comprises substituting group; Perhaps (III) is a kind of like this, and wherein mature polypeptide and another kind of compound (such as the compound that prolongs the polypeptide transformation period, for example polyoxyethylene glycol) merge; Perhaps (IV) is a kind of like this, wherein additional aminoacid sequence is integrated into mature polypeptide and the peptide sequence that forms (as leader sequence or secretion sequence or be used for the sequence or the proteinogen sequence of this polypeptide of purifying) by the elaboration of this paper, such fragment, derivative and analogue are considered within those skilled in the art's ken.

The invention provides isolating nucleic acid (polynucleotide), substantially the polynucleotide that have a polypeptide of SEQ ID NO:2 aminoacid sequence by coding are formed.Polynucleotide sequence of the present invention comprises the nucleotide sequence of SEQ ID NO:1.Polynucleotide of the present invention are to find from the cDNA library of people's fetal brain tissue.The polynucleotide sequence total length that it comprises is 799 bases, its open reading frame (63-536) 157 amino acid of having encoded.Find relatively that according to amino acid sequence homologous this polypeptide and CGI-121 albumen have 98% homology, deducibility goes out the 26S Proteasome Structure and Function that phosphodiesterase 17 that this people's cyclic phosphoric acid guanine suppresses has the CGI-121 protein similar.

Polynucleotide of the present invention can be dna form or rna form.Dna form comprises the DNA of cDNA, genomic dna or synthetic.DNA can be strand or double-stranded.DNA can be coding strand or noncoding strand.The coding region sequence of encoding mature polypeptide can be identical with the coding region sequence shown in the SEQ ID NO:1 or the varient of degeneracy.As used herein, " varient of degeneracy " are meant that in the present invention coding has protein or the polypeptide of SEQ ID NO:2, but with the differentiated nucleotide sequence of coding region sequence shown in the SEQ ID NO:1.

The polynucleotide of the mature polypeptide of coding SEQ ID NO:2 comprise: the encoding sequence that has only mature polypeptide; The encoding sequence of mature polypeptide and various additional code sequence; Encoding sequence of mature polypeptide (with optional additional code sequence) and non-coding sequence.

Term " polynucleotide of coded polypeptide " is meant polynucleotide that comprise this polypeptide of encoding and the polynucleotide that comprise additional code and/or non-coding sequence.

The invention still further relates to the varient of foregoing description polynucleotide, its coding has the polypeptide of identical aminoacid sequence or segment, analogue and the derivative of polypeptide with the present invention.The varient of these polynucleotide can be the allelic variant of natural generation or the varient that non-natural takes place.These nucleotide diversity bodies comprise and replace varient, deletion mutation body and insert varient.As known in the art, allelic variant is the replacement form of polynucleotide, and it may be replacement, disappearance or the insertion of one or more Nucleotide, but can be from not changing the function of its encoded polypeptides in fact.

The invention still further relates to and the polynucleotide (have at least 50% between two sequences, preferably have 70% homogeny) of sequence hybridization described above.The present invention be more particularly directed under stringent condition and the interfertile polynucleotide of polynucleotide of the present invention.In the present invention, " stringent condition " is meant: (1) than hybridization under low ionic strength and the comparatively high temps and wash-out, as 0.2 * SSC, and 0.1%SDS, 60 ℃; Or (2) hybridization the time adds and to use denaturing agent, as 50% (v/v) methane amide, 0.1% calf serum/0.1%Ficoll, 42 ℃ etc.; Or (3) only at the homogeny between the two sequences at least more than 95%, be more preferably 97% and just hybridize when above.And the polypeptide of interfertile polynucleotide encoding has identical biological function and activity with the mature polypeptide shown in the SPQ ID NO:2.

The invention still further relates to nucleic acid fragment with sequence hybridization described above.As used herein, " nucleic acid fragment " length contain 10 Nucleotide at least, better be 20-30 Nucleotide at least, be more preferably 50-60 Nucleotide at least, preferably more than at least 100 Nucleotide.Nucleic acid fragment also can be used for the amplification technique (as PCR) of nucleic acid to determine and/or to separate the polynucleotide of the phosphodiesterase 17 that coding people cyclic phosphoric acid guanine suppresses.

Polypeptide among the present invention and polynucleotide preferably provide with isolating form, more preferably are purified to homogeneous.

The special polynucleotide sequence of the phosphodiesterase 17 that coding people cyclic phosphoric acid guanine of the present invention suppresses can obtain with several different methods.For example, separate polynucleotide with hybridization technique well known in the art.These technology including, but not limited to: 1) with probe and genome or the hybridization of cDNA library to detect homologous polynucleotide sequence and 2) antibody screening of expression library to be to detect the polynucleotide passage of the clone with common structure feature.

Sequence dna fragment of the present invention also can obtain with following method: 1) separate double chain DNA sequence from genomic dna; 2) the chemical synthesising DNA sequence is to obtain the double-stranded DNA of described polypeptide.

In the above-mentioned method of mentioning, isolation of genomic DNA is least commonly used.The direct chemical of dna sequence dna is synthetic to be the method for often selecting for use.The more frequent method of selecting for use is the separation of cDNA sequence.The standard method that separates interested cDNA is from the donorcells separating mRNA of this gene of high expression level and carries out reverse transcription, forms plasmid or phage cDNA library.Extract the existing multiple proven technique of method of mRNA, test kit also can obtain (Qiagene) from commercial channels.And the construction cDNA library also is usual method (Sambrook, et al., MolecularCloning, A Laboratory Manual, Cold Spring Harbor Laboratory.New York, 1989).Also can obtain the cDNA library of commercial offers, as the different cDNA library of Clontech company.When being used in combination the polymeric enzyme reaction technology, even few expression product also can be cloned.

Available ordinary method is screened gene of the present invention from these cDNA libraries.These methods include, but is not limited to: (1) DNA-DNA or DNA-RNA hybridization; (2) appearance of marker gene function or forfeiture; (3) level of the transcript of the phosphodiesterase 17 of mensuration people cyclic phosphoric acid guanine inhibition; (4), detect the protein product of genetic expression by immunological technique or mensuration biologic activity.Aforesaid method can singly be used, but also several different methods combined utilization.

In (1) kind method, hybridizing used probe is and any a part of homology of polynucleotide of the present invention that at least 10 Nucleotide of its length better are at least 30 Nucleotide, are more preferably at least 50 Nucleotide, preferably at least 100 Nucleotide.In addition, within 2000 Nucleotide, preferable is within 1000 Nucleotide to the length of probe usually.Probe used herein is the dna sequence dna of chemosynthesis on the basis of gene order information of the present invention normally.Gene of the present invention itself or fragment are certainly as probe.The mark of dna probe can be used radio isotope, fluorescein or enzyme (as alkaline phosphatase) etc.

In (4) kind method, detect the protein product of the phosphodiesterase 17 genetic expression of people's cyclic phosphoric acid guanine inhibition and can use immunological technique such as Western blotting, radioimmunoprecipitation, enzyme-linked immunosorbent assay (ELISA) etc.

Use method (Saiki, the et al.Science1985 of round pcr DNA amplification/RNA; 230:1350-1354) be optimized for acquisition gene of the present invention.When particularly being difficult to from the library, obtain the cDNA of total length, can preferably use RACE method (the terminal rapid amplifying method of RACE-cDNA), the primer that is used for PCR can suitably be selected according to polynucleotide sequence information of the present invention disclosed herein, and available ordinary method is synthetic.Available ordinary method is as the DNA/RNA fragment by gel electrophoresis separation and purifying amplification.

The gene of the present invention that obtains as mentioned above, perhaps the polynucleotide sequence of various dna fragmentations etc. can with ordinary method such as dideoxy chain termination (Sanger et al.PNAS, 1977,74:5463-5467) measure.This class polynucleotide sequence is measured also available commercial sequencing kit etc.In order to obtain the cDNA sequence of total length, order-checking need be carried out repeatedly.Sometimes need to measure a plurality of clones' cDNA sequence, just can be spliced into the cDNA sequence of total length.

The present invention also relates to comprise the carrier of polynucleotide of the present invention, and the host cell that produces through genetically engineered of the phosphodiesterase 17 encoding sequence that suppresses with the carrier of the present invention or the cyclic phosphoric acid guanine of directly choosing, and the method that produces polypeptide of the present invention through recombinant technology.

Among the present invention, the polynucleotide sequence of the phosphodiesterase 17 that coding people cyclic phosphoric acid guanine suppresses can be inserted in the carrier, contains the recombinant vectors of polynucleotide of the present invention with formation.Term " carrier " refers to that bacterial plasmid well known in the art, phage, yeast plasmid, vegetable cell virus, mammalian cell virus are as adenovirus, retrovirus or other carrier.The carrier of Shi Yonging includes but not limited in the present invention: and the expression vector based on the T7 promotor of in bacterium, expressing (Rosenberg, et al.Gene, 1987,56:125); The pMSXND expression vector of in mammalian cell, expressing (Lee and Nathans, J Bio Chem.263:3521,1988) and at the carrier that derives from baculovirus of expressed in insect cells.In a word, as long as can duplicate in host and stablize, any plasmid and carrier may be used to make up recombinant expression vector.A key character of expression vector is to contain replication origin, promotor, marker gene and translational control element usually.

Method well-known to those having ordinary skill in the art can be used to make up the dna sequence dna of the phosphodiesterase 17 that contains the inhibition of coding people cyclic phosphoric acid guanine and the expression vector of suitable transcribing/translational control element.These methods comprise (Sambroook, et al.MolecularCloning, a Laboratory Manual, cold Spring Harbor Laboratory.New York, 1989) such as extracorporeal recombinant DNA technology, DNA synthetic technology, the interior recombinant technologys of body.Described dna sequence dna can effectively be connected on the suitable promotor in the expression vector, and is synthetic to instruct mRNA.The representative example of these promotors has: colibacillary lac or trp promotor; The P of lambda particles phage _LPromotor; Eukaryotic promoter comprises LTRs and some other known may command gene expression promoter in prokaryotic cell prokaryocyte or eukaryotic cell or its virus of CMV immediate early promoter, HSV thymidine kinase promoter, early stage and late period SV40 promotor, retrovirus.Expression vector also comprises ribosome bind site that translation initiation is used and transcription terminator etc.Inserting enhancer sequence in carrier will make its transcribing in higher eucaryotic cells be enhanced.Enhanser is the cis acting factor that DNA expresses, and nearly 10 to 300 base pairs act on promotor transcribing with enhancing gene usually.Can for example be included in the SV40 enhanser of 100 to 270 base pairs of replication origin side in late period one, at the polyoma enhanser of replication origin side in late period one and adenovirus enhanser etc.

In addition, expression vector preferably comprises one or more selected markers, to be provided for selecting the phenotypic character of transformed host cells, cultivate Tetrahydrofolate dehydrogenase, neomycin resistance and the green fluorescent protein (GFP) of usefulness as eukaryotic cell, or be used for colibacillary tsiklomitsin or amicillin resistance etc.

Persons skilled in the art all know how to select appropriate carriers/transcriptional regulatory element (as promotor, enhanser etc.) and selected marker.

Among the present invention, the polynucleotide of the phosphodiesterase 17 that coding people cyclic phosphoric acid guanine suppresses or the recombinant vectors that contains these polynucleotide can transform or transduce into host cell, contain the genetically engineered host cell of these polynucleotide or recombinant vectors with formation.Term " host cell " refers to prokaryotic cell prokaryocyte, as bacterial cell; Or eukaryotic cell such as low, as yeast cell; Or higher eucaryotic cells, as mammalian cell.Representative example has: intestinal bacteria, streptomyces; Bacterial cell such as Salmonella typhimurium; Fungal cell such as yeast; Vegetable cell; Insect cell such as fruit bat S2 or Sf9; Zooblast such as CHO, COS or Bowes melanoma cells etc.

Can carry out with routine techniques well known to those skilled in the art with dna sequence dna of the present invention or the recombinant vectors transformed host cell that contains described dna sequence dna.When the host was prokaryotic organism such as intestinal bacteria, the competent cell that can absorb DNA can be used CaCl in exponential growth after date results ₂Method is handled, and used step is well-known in this area.Alternative is to use MgCl ₂If desired, transforming also the method for available electroporation carries out.When the host is an eukaryote, can select following DNA transfection method for use: coprecipitation of calcium phosphate method, perhaps conventional mechanical method such as microinjection, electroporation, liposome packing etc.

By the recombinant DNA technology of routine, utilize polynucleotide sequence of the present invention to can be used to express or produce phosphodiesterase 17 (Science, 1984 of people's cyclic phosphoric acid guanine inhibition of reorganization; 224:1431).In general following steps are arranged:

(1). the polynucleotide (or varient) of the phosphodiesterase 17 that suppresses with everybody cyclic phosphoric acid guanine of coding of the present invention, or with the recombinant expression vector that contains these polynucleotide proper host cell that transforms or transduce;

(2). in suitable medium, cultivate host cell;

(3). separation, protein purification from substratum or cell.

In step (2), according to used host cell, used substratum can be selected from various conventional substratum in the cultivation.Under the condition that is suitable for the host cell growth, cultivate.After host cell grows into suitable cell density, induce the promotor of selection with suitable method (as temperature transition or chemical induction), cell is cultivated for some time again.

In step (3), recombinant polypeptide can wrap and be expressed or be secreted into the extracellular in cell or on cytolemma.If desired, can utilize its physics, the separating by various separation methods with other characteristic and the albumen of purification of Recombinant of chemistry.These methods are well-known to those skilled in the art.These methods include, but are not limited to: conventional renaturation is handled, protein precipitant is handled (salt analysis method), centrifugal, the broken bacterium of infiltration, the combination of ultrasonication, super centrifugal, sieve chromatography (gel-filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC) and other various liquid chromatography (LC) technology and these methods.

The antagonist of polypeptide of the present invention and this polypeptide, agonist and inhibitor can be directly used in disease treatment, for example, can treat malignant tumour, adrenal gland defect, tetter, all kinds of inflammation, HIV infection and immunological disease etc.

Miki discovery PDE3B gene than high expression level, hangs down in liver, spleen, lung, kidney and expresses in many fat stores tissues.People such as Miki and Lobbert thinks that the chromosomal region that the PDE3B gene exists relates to obesity and noninsulin dependent diabetes (Miki et al., 1996; Lobbert et al., 1996).This shows that the abnormal expression of the PDE 17 that people's cyclic phosphoric acid guanine of the present invention suppresses will produce various diseases especially lipodystrophy disease, noninsulin dependent diabetes, these diseases include but not limited to:

Fatty deposits disease: fatty liver, steatosis myocardosis, steatosis ephrosis

Cardiovascular disorder: coronary atherosclerotic heart disease such as invisible heart trouble, stenocardia, myocardial infarction, overworked dead property coronary heart disease, hypertension

Steroid derivatives metabolic disturbance disease: the sexual development obstacle of (1) bile acid biosynthesis obstacle disease such as cholehepatocirrhosis (2) growth and development stage: sexual prematurity, sexual development postpones, sex differentiation disorder, other (3) internal secretion of genitalia developmental defect and metabolism syndrome: hyperinterrenopathy such as Cushing syndromes, aldosteronism, hypoadrenocorticism is sick as acute hypoadrenocorticism disease, chronic adrenocortical hypofunction disease

Tumour: lipoma, Lipoblastoma, liposarcoma

Non insulin dependent diabetes: non-endomorphy type non insulin dependent diabetes, endomorphy type non insulin dependent diabetes

People such as Tang find PDE3A high expression level (Tang et al., 1997) in thrombocyte, myocardial cell.Discover that optionally cGI PDE supressor can be used for treating bronchial asthma and chronic obstructive pulmonary disease (Schmidt D, et al., 1999).

This shows; the abnormal expression of the PDE 17 that people's cyclic phosphoric acid guanine of the present invention suppresses also will produce the thrombocyte obstacle disease; respiratory system disease; these diseases include but not limited to: thrombocyte dependency hemorrhagic diseases such as spy's property sent out thrombocytopenia purpura; respiratory system disease such as bronchial asthma; chronic obstructive pulmonary disease; chronic bronchitis; the obstructive bronchitis; idiopathic pulmonary fibrosis; sarcoidosis; primary arm lung cancer; hydrothorax; bronchiectasis is levied; the adult breathes embarrassed syndromes

The abnormal expression of the PDE 17 that people's cyclic phosphoric acid guanine of the present invention suppresses also will produce some and grow disorder disease, tumour, heredopathia, nervous system disorders, hemopathy and disease of immune system etc.

The present invention also provides SCREENED COMPOUND to identify the method that improves (agonist) or check the medicament of the phosphodiesterase 17 that (antagonist) people cyclic phosphoric acid guanine suppresses.Agonist improves phosphodiesterase 17 that people's cyclic phosphoric acid guanine suppresses biological function such as stimulate cellular proliferation, and antagonist prevention disorder such as the various cancer relevant with cell hyperproliferation with treatment.For example, can be in the presence of medicine, the film preparation of the phosphodiesterase 17 that mammalian cell or expressing human cyclic phosphoric acid guanine are suppressed is cultivated with the phosphodiesterase 17 that people's cyclic phosphoric acid guanine of mark suppresses.Measure the medicine raising then or check this interactional ability.

The antagonist of the phosphodiesterase 17 that people's cyclic phosphoric acid guanine suppresses comprises antibody, compound, acceptor disappearance thing and the analogue etc. that filter out.The antagonist of the phosphodiesterase 17 that people's cyclic phosphoric acid guanine suppresses can combine and eliminate its function with the phosphodiesterase 17 that people's cyclic phosphoric acid guanine suppresses, or suppress the generation of this polypeptide, or combine with the avtive spot of this polypeptide and to make this polypeptide can not bring into play biological function.

In screening during as the compound of antagonist, the phosphodiesterase 17 that people's cyclic phosphoric acid guanine can be suppressed adds during bioanalysis measures, and determines by measuring between phosphodiesterase 17 that compound suppresses people's cyclic phosphoric acid guanine and its acceptor interactional influence whether compound is antagonist.With the same quadrat method of above-mentioned SCREENED COMPOUND, can filter out the acceptor disappearance thing and the analogue of antagonist action.The phosphodiesterase 17 bonded peptide molecule that can suppress with people's cyclic phosphoric acid guanine can be incorporated into the rondom polypeptide storehouse that solid formation forms by the various amino acid that may make up by screening and obtain.During screening, the phosphodiesterase 17 molecule of generally tackling the inhibition of people's cyclic phosphoric acid guanine carries out mark.

The invention provides and use polypeptide, and fragment, derivative, analogue or their cell are as the method for antigen with production antibody.These antibody can be polyclonal antibody or monoclonal antibody.The present invention also provides the antibody of the phosphodiesterase 17 antigenic determinant that suppresses at people's cyclic phosphoric acid guanine.These antibody include, but is not limited to: the fragment that polyclonal antibody, monoclonal antibody, chimeric antibody, single-chain antibody, Fab fragment and Fab expression library produce.

Can the choose method of the phosphodiesterase 17 direct injection immune animal (as rabbit, mouse, rat etc.) that the cyclic phosphoric acid guanine suppresses of the production of polyclonal antibody obtains, and multiple adjuvant can be used for the enhancing immunity reaction, includes but not limited to freund's adjuvant etc.The technology of the monoclonal antibody of the phosphodiesterase 17 that preparation people cyclic phosphoric acid guanine suppresses includes but not limited to hybridoma technology (Kohler and Milstein.Nature, 1975,256:495-497), three knurl technology, people B-quadroma technology, EBV-hybridoma technology etc.With the variable region bonded chimeric antibody in human constant region and inhuman source can with existing technology production (Morrison etal, PNAS, 1985,81:6851).And the technology of existing manufacture order chain antibody (U.S.Pat No.4946778) also can be used for producing the single-chain antibody of the phosphodiesterase 17 that anti-people's cyclic phosphoric acid guanine suppresses.

The antibody of the phosphodiesterase 17 that anti-people's cyclic phosphoric acid guanine suppresses can be used in the immunohistochemistry technology, detects the phosphodiesterase 17 that the people's cyclic phosphoric acid guanine in the biopsy specimen suppresses.

The also available labelled with radioisotope of phosphodiesterase 17 bonded monoclonal antibody with people's cyclic phosphoric acid guanine suppresses injects in the body and can follow the tracks of its position and distribution.This radiolabeled antibody can be used as a kind of atraumatic diagnostic method and is used for the location of tumour cell and has judged whether transfer.

Antibody also can be used for designing the immunotoxin at a certain privileged sites in the body.The monoclonal antibody of the phosphodiesterase 17 high-affinity that suppresses as people's cyclic phosphoric acid guanine can with bacterium or plant poison (as diphtheria toxin, ricin, abrine etc.) covalent attachment.A kind of usual method is with sulfydryl linking agent such as SPDP, attacks the amino of antibody, by the exchange of disulfide linkage, toxin is incorporated on the antibody, and this hybrid antibody can be used for killing the phosphodiesterase 17 positive cells that people's cyclic phosphoric acid guanine suppresses.

The relevant disease of phosphodiesterase 17 that antibody among the present invention can be used for treating or prevention and people's cyclic phosphoric acid guanine suppress.The antibody that gives suitable dosage can stimulate or block the generation or the activity of the phosphodiesterase 17 of people's cyclic phosphoric acid guanine inhibition.

The invention still further relates to diagnostic testing process quantitative and the phosphodiesterase 17 level that detection and localization people cyclic phosphoric acid guanine suppresses.These tests are known in the art, and comprise that FISH measures and radioimmunoassay.The phosphodiesterase 17 level that people's cyclic phosphoric acid guanine of being detected in the test suppresses, the disease that the importance of phosphodiesterase 17 in various diseases that can suppress with people's cyclic phosphoric acid guanine that lays down a definition and the phosphodiesterase 17 that is used to diagnose people's cyclic phosphoric acid guanine to suppress work.

Polypeptide of the present invention also can be used as the peptide spectrum analysis, for example, the polypeptide available physical, chemistry or enzyme carry out the specificity cutting, and carries out the two-dimentional or three-dimensional gel electrophoresis analysis of one dimension, be more preferably and carry out mass spectroscopy.

The polynucleotide of the phosphodiesterase 17 that coding people cyclic phosphoric acid guanine suppresses also can be used for multiple therapeutic purpose.Gene therapy technology can be used for treating because cell proliferation, growth or the metabolic disturbance due to the nothing expression of the phosphodiesterase 17 that people's cyclic phosphoric acid guanine suppresses or the unusual/non-activity expression.The gene therapy vector (as virus vector) of reorganization can be designed for the phosphodiesterase 17 of people's cyclic phosphoric acid guanine inhibition of expressing variation, to suppress the phosphodiesterase 17 activity that endogenic people's cyclic phosphoric acid guanine suppresses.For example, the phosphodiesterase 17 that a kind of people's cyclic phosphoric acid guanine of variation suppresses can be the phosphodiesterase 17 that people's cyclic phosphoric acid guanine shortening, that lacked signal conduction function territory suppresses, though can combine with the substrate in downstream, lacks signaling activity.Therefore the gene therapy vector of reorganization can be used for treating the phosphodiesterase 17 expression of people's cyclic phosphoric acid guanine inhibition or the disease of active caused by abnormal.Deriving from the polynucleotide that the expression vector of virus such as retrovirus, adenovirus, adeno-associated virus (AAV), hsv, parvovirus etc. can be used for phosphodiesterase 17 that coding people cyclic phosphoric acid guanine is suppressed is transferred in the cell.The method of recombinant viral vector that structure carries the polynucleotide of the phosphodiesterase 17 that coding people cyclic phosphoric acid guanine suppresses be found in existing document (Sambrook, etal.).The polynucleotide of the phosphodiesterase 17 of reorganization coding people cyclic phosphoric acid guanine inhibition can be packaged in the liposome and be transferred in the cell in addition.

Polynucleotide import tissue or intracellular method comprises: directly be injected into polynucleotide in the in-vivo tissue; Or external by carrier (as virus, phage or plasmid etc.) earlier with the polynucleotide transfered cell in, again cell is transplanted in the body etc.

Suppress the oligonucleotide (comprising sense-rna and DNA) of the phosphodiesterase 17 mRNA that people's cyclic phosphoric acid guanine suppresses and ribozyme also within the scope of the invention.Ribozyme is the enzyme sample RNA molecule that a kind of energy specificity is decomposed specific RNA, and its mechanism of action is to carry out the endonuclease effect after ribozyme molecule and the hybridization of complementary target RNA-specific.The RNA of antisense and DNA and ribozyme can obtain with existing any RNA or DNA synthetic technology, as the technology widespread use of solid phase phosphoamide chemical synthesis synthetic oligonucleotide.Antisense rna molecule can be transcribed acquisition by the dna sequence dna of this RNA that encodes in external or body.This dna sequence dna has been incorporated into the downstream of rna polymerase promoter of carrier.In order to increase the stability of nucleic acid molecule, available several different methods is modified it, and as increasing the sequence length of both sides, the connection between the ribonucleoside is used phosphoric acid thioester bond or peptide bond but not phosphodiester bond.

The polynucleotide of the phosphodiesterase 17 that coding people cyclic phosphoric acid guanine suppresses can be used for the diagnosis of the relative disease of the phosphodiesterase 17 that suppresses with people's cyclic phosphoric acid guanine.Whether or the unconventionality expression of the phosphodiesterase 17 that people's cyclic phosphoric acid guanine suppresses under morbid state the expression that the polynucleotide of the phosphodiesterase 17 that coding people cyclic phosphoric acid guanine suppresses can be used for detecting the phosphodiesterase 17 that people's cyclic phosphoric acid guanine suppresses.As the dna sequence dna of the phosphodiesterase 17 of people's cyclic phosphoric acid guanine inhibition of encoding can be used for biopsy specimen is hybridized to judge the expression situation of the phosphodiesterase 17 that people's cyclic phosphoric acid guanine suppresses.Hybridization technique comprises the Southern blotting, Northern blotting, in situ hybridization etc.These technological methods all are disclosed mature technologies, and relevant test kit all can obtain from commercial channels.Part or all of polynucleotide of the present invention can be used as probe stationary on microarray (Microarray) or DNA chip (being called " gene chip " again), is used for analyzing the differential expression analysis and the gene diagnosis of tissue gene.The special primer of phosphodiesterase 17 that personnel selection cyclic phosphoric acid guanine suppresses carries out the transcription product that RNA-polymerase chain reaction (RT-PCR) amplification in vitro also can detect the phosphodiesterase 17 of people's cyclic phosphoric acid guanine inhibition.

The sudden change that detects the phosphodiesterase 17 gene of people's cyclic phosphoric acid guanine inhibition also can be used for diagnosing the relevant disease of phosphodiesterase 17 of people's cyclic phosphoric acid guanine inhibition.The form of the phosphodiesterase 17 sudden change that people's cyclic phosphoric acid guanine suppresses comprises that point mutation that the phosphodiesterase 17 dna sequence dna that suppresses with normal wild type people cyclic phosphoric acid guanine compares, transposition, disappearance, reorganization and other are any unusual etc.Available existing technology such as Southern blotting, dna sequence analysis, PCR and in situ hybridization detect sudden change.In addition, sudden change might influence proteic expression, therefore can judge indirectly that with Northern blotting, Western blotting gene has or not sudden change.

Sequence of the present invention identifies it also is valuable to karyomit(e).This sequence can be specifically at certain bar human chromosome particular location and and can with its hybridization.At present, need to identify the concrete site of each gene on the karyomit(e).Now, have only chromosomal marker thing seldom to can be used for the marker chromosomes position based on actual sequence data (repetition polymorphism).According to the present invention, for these sequences are associated with disease related gene, its important the first step is positioned these dna sequence dnas on the karyomit(e) exactly.

In brief, prepare PCR primer (preferred 15-35bp), sequence can be positioned on the karyomit(e) according to cDNA.Then, these primers are used for the somatocyte hybrid cell that the PCR screening contains each bar human chromosome.Have only those hybrid cells that contain corresponding to the people's gene of primer can produce the fragment of amplification.

The PCR localization method of somatocyte hybrid cell is that DNA is navigated to concrete chromosomal quick method.Use Oligonucleolide primers of the present invention,, can utilize one group to realize inferior location from specific chromosomal fragment or a large amount of genomic clone by similar approach.Other the similar strategy that can be used for chromosomal localization comprises in situ hybridization, uses the karyomit(e) prescreen and the hybridization preliminary election of the airflow classification of mark, thereby makes up the special cDNA storehouse of karyomit(e).

The cDNA clone is carried out fluorescence in situ hybridization (FISH) with Metaphase Chromosome, can in a step, accurately carry out chromosomal localization.The summary of this technology is referring to Verma etc., Human Chromosomes:a Manualof Basic Techniques, Pergamon Press, New York (1988).

In case sequence is positioned to chromosome position accurately, the physical location of this sequence on karyomit(e) just can be associated with the gene map data.These data for example are found in, V.Mckusick, MendelianInheritance in Man (can by with the online acquisition of Johns Hopkins University Welch Medical Library).Can pass through linkage analysis then, determine gene and navigated to relation between the disease on the chromosomal region already.

Then, need to measure ill and not cDNA between diseased individuals or genome sequence difference.If observe certain sudden change in some or all of diseased individuals, and this sudden change is not observed in any normal individual, then this sudden change may be the cause of disease of disease.More ill and diseased individuals not is usually directed at first seek the variation of structure in the karyomit(e), as from the horizontal visible of karyomit(e) or use based on detectable disappearance of the PCR of cDNA sequence or transposition.Resolving power according to present physical mapping and assignment of genes gene mapping technology, being accurately positioned to the cDNA of the chromosomal region relevant with disease, can be a kind of (the supposing that 1 megabasse mapping resolving power and every 20kb are corresponding to a gene) between 50 to 500 potential Disease-causing genes.

Polypeptide of the present invention, polynucleotide and stand-in thereof, agonist, antagonist and inhibitor and suitable pharmaceutical carrier combination back can be used.These carriers can be water, glucose, ethanol, salt, damping fluid, glycerine and their combination.Composition comprises the polypeptide or the antagonist of safe and effective amount and carrier and the vehicle that does not influence effect of drugs.These compositions can be used as medicine and are used for disease treatment.

The present invention also provides medicine box or the test kit that contains one or more containers, and one or more medicinal compositions compositions of the present invention are housed in the container.With these containers, can have by the given indicative prompting of government authorities of making, using or selling medicine or biological products, the government authorities that this prompting reflects production, uses or sells permits it to use on human body.In addition, polypeptide of the present invention can be used in combination with other treatment compound.

Pharmaceutical composition can be with mode administration easily, as by in part, intravenously, intraperitoneal, intramuscular, subcutaneous, the nose or the route of administration of intracutaneous.The phosphodiesterase 17 that people's cyclic phosphoric acid guanine suppresses comes administration with the amount that treats and/or prevents concrete indication effectively.The amount and the dosage range that are applied to the phosphodiesterase 17 that people's cyclic phosphoric acid guanine of patient suppresses will depend on many factors, as administering mode, person's to be treated healthiness condition and diagnostician's judgement.

Following accompanying drawing is used to illustrate specific embodiments of the present invention, and is not used in qualification by the scope of the invention that claims defined.

Fig. 1 is phosphodiesterase 17 and the proteic amino acid sequence homology comparison diagram of CGI-121 that inventor's cyclic phosphoric acid guanine suppresses.The top sequence is the phosphodiesterase 17 that people's cyclic phosphoric acid guanine suppresses, and the below sequence is a CGI-121 albumen.Same amino acid represents with monocase amino acid that between two sequences similar amino acid is represented with "+".

Fig. 2 is the polyacrylamide gel electrophoresis figure (SDS-PAGE) of the phosphodiesterase 17 of isolating people's cyclic phosphoric acid guanine inhibition.17kDa is proteinic molecular weight.The arrow indication is isolated protein band.

{ embodiment }

Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually according to people such as normal condition such as Sambrook, molecular cloning: laboratory manual (New York:ColdSpring Harbor Laboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.

Embodiment 1: the clone of the phosphodiesterase 17 that people's cyclic phosphoric acid guanine suppresses

Extract the total RNA of people's tire brain with guanidinium isothiocyanate/phenol/chloroform single stage method.From total RNA, separate poly (A) mRNA with Quik mRNA Isolation Kit (Qiegene company product).2ug poly (A) mRNA forms cDNA through reverse transcription.CDNA fragment orientation is inserted on the multiple clone site of pBSK (+) carrier (Clontech company product) with Smart cDNA clone's test kit (available from Clontech), transforms DH5 α, bacterium forms the cDNA library.Measure all clones' 5 ' and 3 ' terminal sequence with Dyeterminate cycle reaction sequencing kit (Perkin-Elmer company product) and ABI 377 automatic sequencers (Perkin-Elmer company).CDNA sequence and the existing public dna sequence data storehouse (Genebank) measured are compared, found that the cDNA sequence of one of them clone 1164G04 is new DNA.By synthetic a series of primers the contained insertion cDNA fragment of this clone is carried out two-way mensuration.The result shows, the contained full-length cDNA of 1164G04 clone is 799bp (shown in Seq ID NO:1), from 63bp to 536bp the open reading frame (ORF) of a 474bp, the new protein (shown in Seq IDNO:2) of encoding arranged.We are with this clone's called after pBS-1164G04, the phosphodiesterase 17 that the name of encoded protein matter suppresses for people's cyclic phosphoric acid guanine.

Embodiment 2:cDNA clone's homology retrieval

The sequence and the encoded protein sequence thereof of the phosphodiesterase 17 that people's cyclic phosphoric acid guanine of the present invention is suppressed are with Blast program (Basiclocal Alignment search tool) [Altschul, SF et al.J.Mol.Biol.1990; 215:403-10], carry out the homology retrieval at databases such as Genbank, Swissport.The gene that the phosphodiesterase 17 homology that suppresses with people's cyclic phosphoric acid guanine of the present invention is the highest is a kind of known CGI-121 albumen, and its encoded protein number is AF151879 in the access of Genbank.Protein homology the results are shown in Fig. 1, both height homologies, and its homogeny is 98%; Similarity is 100%.

Embodiment 3: the gene of using the phosphodiesterase 17 of RT-PCR method clones coding people cyclic phosphoric acid guanine inhibition

Total RNA is a template with fetus brain cell, is that primer carries out the synthetic cDNA of reverse transcription reaction with oligo-dT, with behind the test kit purifying of Qiagene, carries out pcr amplification with following primer:

Primer1:5’-GGGAGCGGGAGCTCTTCCGAGAC-3’(SEQ?ID?NO:3)

Primer2:5’-TTTTAAAAGCATACTAAATTTATT-3’(SEQ?ID?NO:4)

Primer1 is the forward sequence that begins of 1bp that is positioned at the 5 ' end of SEQ ID NO:1;

Primer2 be SEQ ID NO:1 in 3 ' end reverse sequence.

The condition of amplified reaction: in the reaction volume of 50 μ l, contain 50mmol/L KCl, 10mmol/LTris-Cl, (pH8.5), 1.5mmol/L MgCl ₂, 200 μ mol/L dNTP, 10pmol primer, the Taq archaeal dna polymerase of 1U (Clontech company product).Go up by 25 cycles of following conditioned response at PE9600 type DNA thermal cycler (Perkin-Elmer company): 94 ℃ of 30sec; 55 ℃ of 30sec; 72 ℃ of 2min.When RT-PCR, establish the blank negative contrast of positive contrast of β-actin and template simultaneously.Amplified production is connected to (Invitrogen company product) on the pCR carrier with the test kit purifying of QIAGEN company with TA clone test kit.The dna sequence analysis result shows that the dna sequence dna of PCR product and the 1-799bp shown in the SEQ ID NO:1 are identical.

The phosphodiesterase 17 expression of gene that embodiment 4:Northern blotting analyst cyclic phosphoric acid guanine suppresses:

Extract total RNA[Anal.Biochem 1987,162,156-159 with single stage method].This method comprises acid guanidine thiocyanate phenol-chloroform extracting.Promptly use 4M guanidinium isothiocyanate-25mM Trisodium Citrate, 0.2M sodium acetate (pH4.0) carries out homogenate to tissue, adds the phenol of 1 times of volume and the chloroform-primary isoamyl alcohol (49: 1) of 1/5 volume, and is centrifugal after mixing.The sucking-off aqueous phase layer adds Virahol (0.8 volume) and with the centrifugal RNA precipitation that obtains of mixture.With RNA precipitation 70% washing with alcohol that obtains, dry and soluble in water.With 20 μ g RNA, on 1.2% sepharose that contains 20mM 3-(N-morpholino) propanesulfonic acid (pH7.0)-5mM sodium acetate-1mM EDTA-2.2M formaldehyde, carry out electrophoresis.Be transferred on the nitrocellulose filter then.With α- ³²P dATP prepares by random priming ³²The dna probe of P-mark.Used dna probe is the phosphodiesterase 17 coding region sequence (63bp to 536bp) that people's cyclic phosphoric acid guanine of pcr amplification shown in Figure 1 suppresses.Probe (about 2 * 10 with the 32P-mark ⁶Cpm/ml) spend the night in 42 ℃ of hybridization in a solution with the nitrocellulose filter that has shifted RNA, this solution comprises 50% methane amide-25mM KH ₂PO ₄(pH7.4)-5 * SSC-5 * Denhardt ' s solution and 200 μ g/ml salmon sperm DNAs.After the hybridization, filter membrane is washed 30min in 55 ℃ in 1 * SSC-0.1%SDS.Then, analyze with quantitative with Phosphor Imager.

Embodiment 5: vivoexpression, separation and the purifying of the phosphodiesterase 17 that recombinant human cyclic phosphoric acid guanine suppresses

According to SEQ ID NO:1 and coding region sequence shown in Figure 1, design a pair of specificity amplification primer, sequence is as follows:

Primer3:5’-CCCCATATGATGCAGTTAACACATCAGCTGGACC-3’(Seq?ID?No:5)

Primer4:5’-CATGGATCCTCACACTCTTTCTAAAAATATGGAC-3’(Seq?ID?No:6)

5 ' end of these two sections primers contains Nde I and BamH I restriction enzyme site respectively, be respectively the encoding sequence of target gene 5 ' end and 3 ' end thereafter, Nde I and BamH I restriction enzyme site are corresponding to expression vector plasmid pET-28b (+) (Novagen company product, Cat.No.69865.3) the selectivity restriction enzyme site on.With the pBS-1164G04 plasmid that contains the total length goal gene is template, carries out the PCR reaction.The PCR reaction conditions is: contain pBS-1164G04 plasmid 10pg, primer Primer-3 and Primer-4 among the cumulative volume 50 μ l and be respectively 10pmol, Advantage polymerase Mix (Clontech company product) 1 μ l.Loop parameter: 94 ℃ of 20s, 60 ℃ of 30s, 68 ℃ of 2min, totally 25 circulations.Respectively amplified production and plasmid pET-28 (+) are carried out double digestion with Nde I and BamH I, reclaim big fragment respectively, and connect with the T4 ligase enzyme.Connect product and transform, after the dull and stereotyped overnight incubation of the LB that contains kantlex (final concentration 30 μ g/ml), use the colony polymerase chain reaction (PCR) method screening positive clone, and check order with the big enterobacterial DH5 of Calcium Chloride Method α.Select the correct positive colony of sequence (pET-1164G04) with Calcium Chloride Method with recombinant plasmid transformed e. coli bl21 (DE3) plySs (Novagen company product).In the LB liquid nutrient medium that contains kantlex (final concentration 30 μ g/ml), host bacterium BL21 (pET-1164G04) is cultured to logarithmic phase at 37 ℃, adds IPTG to final concentration 1mmol/L, continues to cultivate 5 hours.Centrifugal collection thalline, through the broken bacterium of ultrasonic wave, centrifugal collection supernatant, with carrying out chromatography, obtained the phosphodiesterase 17 of the target protein people cyclic phosphoric acid guanine inhibition of purifying with 6 Histidines (6His-Tag) bonded affinity column His.Bind Quick Cartridge (Novagen company product).Through the SDS-PAGE electrophoresis, obtain a single band (Fig. 2) at the 17kDa place.This band is transferred on the pvdf membrane carries out the n terminal amino acid sequential analysis with the Edams hydrolysis method, 15 amino acid of N-end hold 15 amino-acid residues identical with the N-shown in the SEQ ID NO:2 as a result.

The phosphodiesterase 17 production of antibodies that embodiment 6 anti-people's cyclic phosphoric acid guanines suppress

The specific polypeptide of phosphodiesterase 17 with the synthetic following people's cyclic phosphoric acid guanine inhibition of Peptide synthesizer (PE company product):

NH ₂-Met-Gln-Leu-Thr-His-Gln-Leu-Asp-Leu-Phe-Pro-Glu-Cys-Arg-Val-COOH(SEQ?ID?NO:7)。Form compoundly with hemocyanin and bovine serum albumin coupling this polypeptide respectively, method is referring to Avrameas, et al.Immunochemistry, 1969; 6:43.Add the complete Freund's adjuvant immunizing rabbit with the above-mentioned hemocyanin polypeptide complex of 4mg, add the incomplete Freund's adjuvant booster immunization once with the hemocyanin polypeptide complex again after 15 days.Employing is done the titre that ELISA measures antibody in the rabbit anteserum through the titer plate of 15 μ g/ml bovine serum albumin polypeptide complex bag quilts.From the rabbit anteserum of antibody positive, separate total IgG with albumin A-Sepharose.Polypeptide is incorporated on the Sepharose4B post of cyanogen bromide-activated, from total IgG, separates anti-peptide antibody with affinity chromatography.Immuno-precipitation proof antibody purified can combine with the phosphodiesterase 17 that people's cyclic phosphoric acid guanine suppresses specifically.

Sequence table (1) general information: (ⅱ) denomination of invention: phosphodiesterase 17 and encoding sequence (ⅲ) sequence number thereof that people's cyclic phosphoric acid guanine suppresses: the information of 7 (2) SEQ ID NO:1: (ⅰ) sequence signature:

(A) length: 799bp

(B) type: nucleic acid

(C) chain: two strands

( D ) ： ( ⅱ ) ：cDNA ( ⅹⅰ ) ：SEQ ID NO:1: 1 GGGAGCGGGAGCTCTTCCGAGACGCACTGGGGGCCGGATGTAGAATCCTGCTTATCTGTG 61 AAATGCAGTTAACACATCAGCTGGACCTATTTCCCGAATGCAGGGTAACCCTTCTGTTAT121 TTAAAGATGTAAAAAATGCGGGAGACTTGAGAAGAAAGGCCATGGAAGGCACCATCGATG181 GATCACTGGTAAATCCTACAGTGATTGTTGATCCATTTCAGATACTTGTGGCAGCAAACA241 AAGCAGTTCACCTCTACAAACTGGGAAAAATGAAGACAAGAACTCTATCTACTGAAATTA301 TTTTCAACCTTTCCCCAAATAACAATATTTCAGAGGCTTTGAAAAAATTTGGTATCTCAG361 CAAATGACACTTCAATTCTAATTGTTTACATTGAAGAGGGAGAAAAACAAATAAATCAAG421 AATACCTAATATCTCAAGTAGAAGGTCATCAGGTTTCTCTGAAAAATCTTCCTGAAATAA481 TGAATATTACAGAAGTCAAAAAGGTTTGCCAGTCCATATTTTTAGAAAGAGTGTGATAGA541 TGAGCAATAATGCTGATGCCGTTTGCCTATTGATCACCAGAGCTCCTTGTTTTTTGAACT601 TTTGAGAAAGTTAATCTAATTTTAAAAATTACTTAAAAATTACAGATACAAAGAACTGGG661 AGGTGTGATGGTTTATGTAAAATATAATTACAAACTCAGCTGACATAACCATTCTCCACA721 TTAAGAAAGAGAATACCTTTAAATATCAAACTTACATAGTATATTTAAAATGTAAAATAA781 ATTTAGTATGCTTTTAAAA ( 3 ) SEQ ID NO:2： ( ⅰ ) ：

(A) length: 157 amino acid

(B) type: amino acid

( D ) ： ( ⅱ ) ： ( ⅹⅰ ) ：SEQ ID NO:2: 1 Met Gln Leu Thr His Gln Leu Asp Leu Phe Pro Glu Cys Arg Val 16 Thr Leu Leu Leu Phe Lys Asp Val Lys Asn Ala Gly Asp Leu Arg 31 Arg Lys Ala Met Glu Gly Thr Ile Asp Gly Ser Leu Val Asn Pro 46 Thr Val Ile Val Asp Pro Phe Gln Ile Leu Val Ala Ala Asn Lys 61 Ala Val His Leu Tyr Lys Leu Gly Lys Met Lys Thr Arg Thr Leu 76 Ser Thr Glu Ile Ile Phe Asn Leu Ser Pro Asn Asn Asn Ile Ser 91 Glu Ala Leu Lys Lys Phe Gly Ile Ser Ala ASn Asp Thr Ser Ile106 Leu Ile Val Tyr Ile Glu Glu Gly Glu Lys Gln Ile Asn Gln Glu121 Tyr Leu Ile Ser Gln Val Glu Gly His Gln Val Ser Leu Lys Asn136 Leu Pro Glu Ile Met Asn Ile Thr Glu Val Lys Lys Val Cys Gln151 Ser Ile Phe Leu Glu Arg Val ( 4 ) SEQ ID NO:3 ( ⅰ )

(A) length: 23 bases

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linear (ⅱ) molecule type: oligonucleotide (ⅹ ⅰ) sequence description: information (ⅰ) sequence signature of SEQ ID NO:3:GGGAGCGGGAGCTCTTCCGAGAC 23 (5) SEQ ID NO:4

(A) length: 24 bases

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linear (ⅱ) molecule type: oligonucleotide (ⅹ ⅰ) sequence description: information (ⅰ) sequence signature of SEQ ID NO:4:TTTTAAAAGCATACTAAATTTATT 24 (6) SEQ ID NO:5

(A) length: 32 bases

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linear (ⅱ) molecule type: oligonucleotide (ⅹ ⅰ) sequence description: information (ⅰ) sequence signature of SEQ ID NO:5:CAGCCATGGCGGGGAAGAAGAATGTTCTGTCG 32 (7) SEQ ID NO:6

(A) length: 29 bases

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linear (ⅱ) molecule type: oligonucleotide (ⅹ ⅰ) sequence description: the information of SEQ ID NO:6:CCCGGATCCCGCTGCTTGGCCTTCTTCAC 29 (8) SEQ ID NO:7: (ⅰ) sequence signature:

(A) length: 15 amino acid

(B) type: amino acid

(D) topological framework: linear (ⅱ) molecule type: polypeptide (ⅹ ⅰ) sequence description: SEQ ID NO:7:Met-Ala-Gly-Lys-Lys-Asn-Val-Leu-Ser-Ser-Leu-Ala-Val-Tyr-Ala 15

Claims (18)

1, the phosphodiesterase 17 of a kind of isolated polypeptide-people's cyclic phosphoric acid guanine inhibition is characterized in that it includes: the polypeptide of the aminoacid sequence shown in the SEQ ID NO:2 or active fragments, analogue or the derivative of its polypeptide.

2, polypeptide as claimed in claim 1, the aminoacid sequence that it is characterized in that described polypeptide, analogue or derivative has the homogeny with the aminoacid sequence at least 95% shown in the SEQ ID NO:2.

3, polypeptide as claimed in claim 2 is characterized in that it comprises the polypeptide with the aminoacid sequence shown in the SEQ ID NO:2.

4, a kind of isolating polynucleotide, it is characterized in that described polynucleotide comprise be selected from down the group in a kind of:

(a) coding has the polynucleotide of the polypeptide of aminoacid sequence shown in the SEQ ID NO:2 or its fragment, analogue, derivative;

(b) with polynucleotide (a) complementary polynucleotide; Or

(c) with (a) or the polynucleotide of at least 99% homogeny (b) are arranged.

5, polynucleotide as claimed in claim 4 is characterized in that described polynucleotide comprise the polynucleotide that coding has aminoacid sequence shown in the SEQID NO:2.

6, polynucleotide as claimed in claim 4, the sequence that it is characterized in that described polynucleotide include the sequence of 1-799 position among the sequence of 63-536 position among the SEQ IDNO:1 or the SEQ ID NO:1.

7, a kind of recombinant vectors that contains exogenous polynucleotide is characterized in that it is the recombinant vectors that is formed by the described polynucleotide of arbitrary claim among the claim 4-6 and plasmid, virus or vehicle expression vector establishment.

8, a kind of genetically engineered host cell that contains exogenous polynucleotide is characterized in that it is to be selected from following a kind of host cell:

(a) host cell that transforms or transduce with the described recombinant vectors of claim 7; Or

(b) host cell that transforms or transduce with the described polynucleotide of the arbitrary claim among the claim 4-6.

9, a kind of preparation method with active polypeptide of phosphodiesterase 17 of people's cyclic phosphoric acid guanine inhibition is characterized in that described method comprises:

(a) under the phosphodiesterase 17 condition that expressing human cyclic phosphoric acid guanine suppresses, cultivate the described through engineering approaches host cell of claim 8;

(b) from culture, isolate the active polypeptide of phosphodiesterase 17 with the inhibition of people's cyclic phosphoric acid guanine.

10, a kind of can with polypeptide bonded antibody, it is characterized in that described antibody is the phosphodiesterase 17 specificity bonded antibody that can suppress with people's cyclic phosphoric acid guanine.

11, the compound of an analoglike or adjusting polypeptide active or expression is characterized in that they are simulation, promotion, antagonism or the active compound that suppresses the phosphodiesterase 17 of people's cyclic phosphoric acid guanine inhibition.

12, compound as claimed in claim 11 is characterized in that it is the polynucleotide sequence shown in the SEQ ID NO:1 or its segmental antisense sequences.

13, the described application of compound of a kind of claim 11, it is characterized in that described compound be used for phosphodiesterase 17 that mediator's cyclic phosphoric acid guanine suppresses in vivo, the method for external activity.

14, a kind of disease relevant or method of disease susceptibility of detecting with the described polypeptide of arbitrary claim among the claim 1-3, it is characterized in that it comprises the described polypeptide expression amount that detects, perhaps detect the activity of described polypeptide, perhaps detect and cause described expression of polypeptides amount or active unusual nucleotide diversity in the polynucleotide.

15,, it is characterized in that it is applied to screen stand-in, the agonist of the phosphodiesterase 17 that people's cyclic phosphoric acid guanine suppresses, antagonist or inhibitor as the application of polypeptide as described in the arbitrary claim among the claim 1-3; Perhaps be used for the peptide finger print identification.

16, as the application of the described nucleic acid molecule of arbitrary claim among the claim 4-6, it is characterized in that it is used for nucleic acid amplification reaction as primer, perhaps be used for hybridization, perhaps be used to make gene chip or microarray as probe.

17,, it is characterized in that forming with safe and effective dosage and pharmaceutically acceptable carrier the pharmaceutical composition of the relevant unusually disease of the phosphodiesterase 17 that suppresses as diagnosis or treatment and people's cyclic phosphoric acid guanine with described polypeptide, polynucleotide or its stand-in, agonist, antagonist or inhibitor as the described polypeptide of arbitrary claim, polynucleotide or application of compound in claim 1-6 and 11.

18, the described polypeptide of arbitrary claim, polynucleotide or the application of compound among the claim 1-6 and 11, it is characterized in that being used for the treatment of as malignant tumour with described polypeptide, polynucleotide or compound, hemopathy, the medicine of HIV infection and immunological disease and all kinds of inflammation.

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