CN1373772A - Gene encoding human huntingtin interacting polypeptide which comprises WW domain and its producing method and application - Google Patents

Abstract

The invention discloses a kind of human huntingtin interacting polypeptide which comprises the WW domain (WW-HIP), the polynucleotide encoding said polypeptide and a process for producing the polypeptide by recombinant methods. It also disclosed the method of applying the polypeptide for the treatment of various kinds of diseases, such as HD, immune disease, presbyophrenia, Duchenne muscular dystrophy, Liddle syndrome, DRPLA. The antibody, protagonist and antagonist of the polypeptide and their therapeutic usage for the above programmed cell death concerning diseases are also discloses. In addition, the invention also discloses the method to identify the mutation in the nucleic acid sequence of WW-HIP and the diagnostic method to examine the expression changes of the WW-HIP.

Description

Gene encoding human huntingtin interacting polypeptide which comprises WW domain and its producing method and application

The Huntington protein for encoding a kind of new people's domain containing WW is combined

The gene of polypeptide and its application and preparation method technical field

The invention belongs to biological technical field, specifically, the present invention relates to a kind of Huntington protein Binding peptide for encoding new people's domain containing WW（WW-HIP polynucleotide sequence) and the polypeptide coded by it.The polypeptide and polynucleotides are further related in diagnosis, prevention and treatment and the application in the abnormal relevant disease of the protein expression.Background technology

WW functional domains there are about 35 An bases acid Can Ji Group into due to containing two unusual conservative trp residues and a proline residue, therefore being named as WW functional domains（WW domain AVWP domains）（ Biochem.Biophys.Res.Commun. , 205:1021-1205 ) ( Trends Biochem.Sci.l9:531-533(1994) ).

The Side of WW functional domains two is mostly rich in histidine or the sour residue of half Guang, such as dystrophin（Dystrophin), the structure implies that it has the function of bind metal ion (Nature Genetics 3,283-291) and WW functional domains have β-pleated sheet structure in the encirclement of four conservative aromatic amino acid residues in itself.Hydrophobic core and a large amount of charged residues all show that this functional domain is the functional domain of albumen and protein-interacting.Although WW functional domains are present in the different albumen of function, signal transmission and the adjustment effect of cell are involved in.

The domain phase separation of WW functional domains mostly with Pro-rich, transmission signal (Proc.Natl.Acad.Sci.USA 92:7819-7823(1995).Such as FBP-11, PPLP primitives, the PY primitives of WW functional domains respectively with respective part in YAP65 are combined (J.Biol.Chem., 272,17070-17077 (1997), (EMBO J., 16,2376-2383

(1997)).A small number of WW functional domains dependence phosphorylation pathways, albumen dried meat ammonia barefoot isomerase Pinl, WW functional domains in ubiquitin binding enzyme Nedd4 are combined with phosphoprotein.It has been confirmed that, with phosphoserine or phosphothreonine with reference to being necessary to Pinl plays its physiological effect in vitro with its substrate（Science, 1999 Feb 26,283 (5406) 1325-1328).

Huntington protein（Huntingtin) repeated containing the region of polyglutamic barefoot amine more than one, enrichment proline region, a 3-4 HEAT.It is albumen necessary to embryonic development, nerve formation（Cell, 81,811-823,1995), (Nature Genet., 17,404-410).The extension of many polyglutamic barefoot amine of its mutant causes the nervous system disease Huntington's disease (HD) (Cell, 72,971-983,1993).Yeast two-hybrid assay is elicited, and Huntington protein is only enriched with proline fragment and all kinds of Huntington protein action proteins by its N-terminal（HYPs, HIPs) combine, and its its longer binding ability of neighbouring poly glumine fragment is stronger（Human Molecular Genetics, 1998, Vol.7, No.9 1463-1474)

The HYP (HIP) being currently known has HYPA (the mouse FBP11 homologous proteins for participating in albumen splicing function）（EMBO.J., 15,1045-1054,1996), HYPF, HYPG (HIP2) of protein metabolism are participated in, HYPI, HYPJ, HIP1 (yeast cells skelemin Sla2p homologous proteins of certain films function are participated in）, CBS, and function not specific HYPB, HYPC, HYPD etc..In these albumen, HYPA, HYPB, HYPC belong to WW functional domains family.HYPA is present in cytoplasm and nucleus.Northern traces elicite three kinds of protein mRNAs in adult and embryo and brain compared with horn of plenty, this albuminoid are implied from playing different physiological functions in vivo after Huntington protein effect, so as to adjust the growth and development of nervous system（Human Molecular Genetics, 1998, Vol.7, No.9 1463-1474 λ

Because the Huntington protein associated proteins of the domain containing WW as described above play an important role in the growth of regulation nervous system, development and other body critical functions, and believe that these are related to substantial amounts of albumen during adjusting, thus have always a demand for identifying the Huntington protein associated proteins of the domain containing WW of more these processes of participation in this area, particularly Identify the amino acid sequence of this albumen.The separation of the Huntington protein associated proteins encoding gene of the new domain containing WW is also providing the foundation that the determination albumen is acted under health and morbid state.This albumen may constitute the basis of exploitation medical diagnosis on disease and/or curative, therefore it is very important to separate its coding DNA.Summary of the invention

On the one hand, the invention provides a kind of Huntington protein Binding peptide of functional domain containing WW of separation, it includes SEQ ID No. 2 amino acid sequence, or its conservative variation's polypeptide or its active fragment or derivative.

In a preferred embodiment, polypeptide of the invention is the polypeptide with the amino acid sequences of SEQ ID No. 2.

On the other hand, the invention provides a kind of polynucleotides of separation, it is characterised in that it includes a kind of nucleotide sequence being selected from the group：

(a) polynucleotides of polypeptide as described above are encoded；

(b) polynucleotides complementary with polynucleotides (a)；With

(c) with（Or the polynucleotides of (b) have the polynucleotides of at least 70% phase same sex a).In a preferred embodiment, polynucleotide encoding of the invention has SEQ

The polypeptide of amino acid sequence shown in ID No.2.

In another further preferred embodiment, polynucleotides of the invention are the one kind being selected from the group：With the sequences of 575-1849 in SEQ ID No. 1 and with the sequence of 1-2507 in SEQ ID No. 1.

The host cell for converting, transfecting or transduceing the invention further relates to a kind of recombinant vector containing above-mentioned polynucleotides and with the polynucleotides or carrier.

Another further aspect, the present invention relates to a kind of preparation method of the polypeptide of the Huntington protein Binding peptide activity with people's WW functional domains, it is characterised in that this method includes：

(a) in the condition for the Huntington protein Binding peptide for being adapted to expression functional domain containing WW Under, cultivate above-mentioned host cell；With

(b) polypeptide of the Huntington protein Binding peptide activity with the functional domain containing WW is isolated from culture.

The invention further relates to the antibody that a kind of Huntington protein Binding peptide with the functional domain of the invention containing WW is specifically bound.

The invention further relates to a kind of Huntington protein Binding peptide activity for screening simulation or the regulation functional domain containing WW or the method for the compound of expression, it is characterised in that utilize the polypeptide or polynucleotides of the present invention；And simulation, promotion, antagonism or active or expression the compound for suppressing polypeptide of the present invention obtained with this method.

Moreover, it relates to a kind of detection disease related to polypeptide of the present invention or the method for disease susceptibility, it is characterised in that including：

(a) exception of the expression of polypeptides amount is detected；

(b) detect that the activity of the polypeptide is abnormal；Or

(c) detection and the variation in the expression of polypeptides amount or the abnormal related nucleic acid of activity.

Finally, the present invention provides a kind of polypeptide of the present invention containing effective dose or the simulation, promotion, antagonism or the activity of suppression polypeptide of the present invention or the compound of expression and the pharmaceutical composition of pharmaceutically acceptable carrier.

The other side of the present invention, due to the disclosure of this paper technology, is obvious to those skilled in the art.Detailed description of the invention

On the one hand, the invention provides a kind of substantially pure WW-HIP polypeptides, it is substantially made up of the amino acid sequence shown in SEQ ID No.2.A WW-HIP feature is with a WW domain being made up of about 35 amino acid. As used in the present invention, " substantially pure " is WW-HIP substantially free of natural relative other albumen, lipid, carbohydrate or other materials.Those skilled in the art can purify WW-HIP with the purified technology of protein of standard.Substantially pure polypeptide can produce single master tape on non-reduced polypropylene barefoot amine gel.WW-HIP purity can use amino acid sequence analysis.The polypeptide of the present invention can be recombinant polypeptide, natural polypeptides, synthesis polypeptide, You Xuan Chong Group polypeptides.

Present invention additionally comprises the fragment of the polypeptide, derivative and analog.As used in the present invention, term " fragment ", " derivative " and " analog " refer to be kept substantially polypeptide identical biological function of the present invention or active polypeptide.Polypeptide of the present invention includes the bioactive fragment of SEQ ID No.2 polypeptides, derivative or the like, and they can be such compared with SEQ ID No.2 polypeptides：（I) wherein one or more amino acid residues are guarded or nonconserved amino acid residues（Preferably conservative amino acid residues）The amino acid for replacing and replacing can not be and be encoded by genetic codon；Or（II) wherein one or more amino acid residues include substituent；Or（III) wherein mature polypeptide is merged with another compound；Or（IV) wherein plus Amino Acid is integrated into mature polypeptide, and it is used for Purified mature polypeptide.By this paper elaboration, such fragment, derivative and the like are considered as within the knowledge of those skilled in the art.In another embodiment, the invention provides the nucleic acid of separation（Polynucleotides）, substantially by encode with SEQ ID NO.2 amino acid sequences polypeptide Duo He Gan Suan Group into.The polynucleotide sequence of the present invention includes SEQ ID NO.l nucleotide sequence. The polynucleotides of the present invention are found from the cDNA libraries of Human fetal brain.The polynucleotide sequence total length that it is included is 2507 bases, and its open reading frame encodes 424 amino acid.According to amino acid sequence homologous it was found that, the Huntington protein action protein of this polypeptide and people（Huntingtin interating protein, HIP) there is 42% homology, and the polypeptide has HIP gene functions area, it can be inferred that the novel polypeptide has the similar 26S Proteasome Structure and Functions of HIP.As used in the present invention, " separation " refers to that material separates that (if crude, primal environment is natural surroundings from its primal environment）.As the polynucleotide and polypeptide under the native state in active somatic cell are not isolated and purified, but same polynucleotide or polypeptide such as from native state with separated in other materials existed, then isolate and purify.The polynucleotides of the present invention can be DNA forms or RNA forms.DNA forms include cDNA, genomic DNA or artificial synthesized DNA.DNA can be single-stranded or double-strand.DNA can be coding strand or noncoding strand.The coding region sequence of encoding mature polypeptide can variant identical or degeneracy with the coding region sequence shown in SEQ ID NO.l.As used in the present invention, " variant of degeneracy " refer in the present invention coding with SEQ ID N02 protein or peptide but with the differentiated nucleotide sequence of coding region sequence shown in SEQ ID NOl.The polynucleotides of coding SEQ ID NO.2 mature polypeptide include：The only coded sequence of mature polypeptide；The coded sequence of mature polypeptide and various additional coding sequences；The coded sequence of mature polypeptide（With optional additional coding sequence）And non-coding sequence.Term " polynucleotides of coded polypeptide " refers to many nucleosides for including encoding this polypeptide Acid and the polynucleotides including additional code and/or non-coding sequence.The invention further relates to the variant of foregoing description polynucleotides, it is encoded has the polypeptide of identical amino acid sequence or the segment of polypeptide, analogs and derivatives with the present invention.The variant of this polynucleotides can be the variant that the allelic variant or non-natural naturally occurred occurs.These nucleotide variants include substitution variants, Deletion variants and insert variation.As known in the art, allelic variant is the alternative forms of a polynucleotides, it be probably one or more nucleotides substitution, missing or insert, but not from substantially change its coding polypeptide function.The invention further relates to the polynucleotides with sequence hybridization described above（Have at least 50% between two sequences, preferably with the 70% phase same sex）.The present invention is more particularly directed under strict conditions with the interfertile polynucleotides of polynucleotides of the present invention.In the present invention, " stringent condition " refers to：（1) hybridization and elution under compared with low ionic strength and higher temperature, such as 0.2xSSC, 0.1%SDS, 60C;Or add during (2) hybridization and use denaturant, such as 50% (v/v) formamide, 0.1% calf serum/0.1%Ficoll, 42C etc.；Or the phase same sex of (3) only between two sequences at least more than 95%, just hybridizes when more preferably more than 97%.Also, the polypeptide of interfertile polynucleotide encoding has identical biological function and activity with the mature polypeptide shown in accompanying drawing 2.

The invention further relates to the nucleic acid fragment with sequence hybridization described above.As used in the present invention, the length of " nucleic acid fragment " at least contains more than 15 nucleotides, preferably at least 20-30 nucleotides, preferably more preferably at least 50-60 nucleotides, at least 100 nucleotides.Nucleic acid fragment can be used for the amplification technique (such as PCR) of nucleic acid to determine and/or separate coding WW-HIP polynucleotide.The present invention also relates to the carrier of the polynucleotides comprising the present invention and the load with the present invention The host cell that body is produced through genetic engineering, and the method through recombinant technique generation polypeptide of the present invention.The DNA sequence dna of the present invention can be obtained with several method.For example, separating DNA with hybridization technique well known in the art.These technologies include but is not limited to：1) with probe and genome or cDNA Library hybridizations to detect homologous nucleotide sequences, and 2) antibody screening of expression library to detect the DNA fragmentation of the common clone with architectural feature.

Coding WW-HIP specific DNA fragment sequence is produced and can also obtained with following method：1) double chain DNA sequence is separated from Ji because of Group DNA；2) chemical synthesising DNA sequence is with the double-stranded DNA of polypeptide needed for obtaining.

When known to the whole amino acid sequence of the polypeptide product of needs, it can be obtained by the direct chemical synthesis of DNA sequences.If the whole sequence of required amino acid is not known, the method for selection is the separation of cDNA sequence.Separation cDNA interested standard method is from the high donorcells separation mRNA for expressing the gene and carries out reverse transcription, forms plasmid or bacteriophage cDNA library.The mRNA existing a variety of ripe technologies of method are extracted, kit is also commercially obtained（Qiagene) and construction cDNA library is also usual way (Sambrook, et al, Molecular Cloning, a Laboratory Manual, Cold Spring Harbor Laboratory. New York, 1989).It also can obtain the cDNA library of commercial offers, the different cDNA libraries of such as Clontech companies.When polymerization hemiculeer leucisculus reaction technology is used in combination, it can also be cloned even if few expression product.The gene of the present invention can be screened from these cDNA libraries with conventional method.These methods include（But it is not limited to）：(1) DNA-DNA or DNA-R A hybridize；(2) function of marker gene occurs or lost；（3) level of WW-HIP transcript is determined；（4) applied immunology technology or measure biological activity detect gene expression Protein product.The above method can be alone, also can a variety of method use in conjunction.

（1) in kind method, hybridization probe used is any a part of homologous, at least 15 nucleotides of its length, preferably 20-30 nucleotides with the polynucleotides of the present invention, it is more than more preferably 50-60 nucleotides, preferably 100 nucleotides.Probe used herein is typically the DNA sequence dna of the chemical synthesis on the basis of the gene DNA sequence information of the present invention.The gene of the present invention is in itself or fragment is it is of course possible to being used as probe.The mark of DNA probe can use radio isotope, fluorescein or enzyme（Such as alkaline phosphatase).

（4) in kind method, the protein product of detection WW-HIP gene expressions can use immunological technique such as Western blots, radioimmunoprecipitation, enzyme linked immunosorbent assay（ELISA) etc..Using round pcr DNA amplification/RNA method（ Saiki, et al. Science 1985;230:It can 1350-1354) be preferentially used the gene for obtaining the present invention.Particularly it is difficult to when the cDNA of total length is obtained from library, preferably use RACE methods (RACE:CDNA ends rapid amplification）, primer used be able to can be properly selected according to the sequence information of invention disclosed herein in terms of above-mentioned PCR, and available conventional method synthesis.The DNA/RNA fragments of amplification such as can be separated and purified by gel electrophoresis with conventional method.The gene of the invention obtained as described above, or the measure of the nucleotide sequence of various DNA fragments etc. can use conventional method such as dideoxy chain termination（Sanger et al. PNAS, 1977, 74：This kind of nucleotide sequencings of 5463-54671 can also use business sequencing kit etc..In order to obtain the cDNA sequences of total length, sequencing need to be repeated.Sometimes for the cDNA sequence for determining multiple clones, the cDNA sequences of total length can be just spliced into. According to common recombinant DNA technology, the WW-HIP polypeptides of restructuring are can be used to express or produced using the polynucleotide sequence of the present invention（Science, 1984; 224： 1431 ).In general there are following steps：

(1) the coding WW-HIP of present invention polynucleotides（Or variant）Or the recombinant expression carrier containing polynucleotides converts suitable host cell；

(2) host cell that is cultivated in suitable culture medium；

(3) separated from culture medium or cell, protein purification.In the present invention, WW-HIP polynucleotide sequences can be plugged into recombinant expression carrier.Term " recombinant expression carrier " refers to bacterial plasmid well known in the art, bacteriophage, yeast plasmid, plant cell virus, mammalian cell virus such as adenovirus, retrovirus or other carriers.Applicable carrier includes but is not limited in the present invention：The expression vector based on T7 expressed in bacterium（ Rosenberg, et al. Gene, 1987, 56:125 )；The pMSXND expression vectors expressed in mammalian cell（ Lee and Nathans, J Bio Chem. 263:3521,1988) carrier from baculoviral and in insect cell expressed.In a word, as long as can be replicated in host and stably, any plasmid and carrier can be used.One key character of expression vector is to usually contain replication orgin, promoter, marker gene and translation control element.Method well-known to those having ordinary skill in the art can be used for the expression vector for building WW-HIP DNA sequences encodings and suitable transcription/translation control signal.These methods include the (Sambroook such as extracorporeal recombinant DNA technology, DNA synthetic technologys, In vivo recombination technology, et al. Molecular Cloning, a laboratory Manual, cold Spring Harbor laboraty. New York, 1989).Described DNA sequence dna can be effectively connected in the appropriate promoter in expression vector, to instruct mRNA to close Into.There is representational example in these promoters：Lac the or trp promoters of Escherichia coli；Bacteriophage lambda PL promoters；Eukaryotic promoter includes the promoter of the CMV expression of early stage, HSV thymidine kinases, early and late SV40, the LTRs of retroviruse and some other known controllable gene in protokaryon or eukaryotic or its virus immediately.Expression vector also includes the ribosome bind site and transcription terminator of translation initiation.In addition, expression carrying agent preferably includes one or more selected markers, to provide the phenotypic character for the host cell for being used to select conversion, dihyrofolate reductase, neomycin resistance and the green fluorescent protein of such as eukaryotic culture（), GFP or Escherichia coli tetracycline or amicillin resistance.Carrier comprising above-described appropriate DNA sequence dna and appropriate promoter or control sequence can be used for changing appropriate host cell, allow it to marking protein.Host cell can be prokaryotic, such as bacterial cell；Or low eukaryotic, such as yeast cells；Or higher eucaryotic cells, such as mammalian cell.Representational example has：Escherichia coli, streptomyces；The bacterial cell of salmonella typhimurium；The fungal cell of such as yeast；Plant cell；Drosophila S2 or Sf9 insect cell；Zooblast of CHO, COS or Bowes melanoma cells etc..When the polynucleotides of the present invention are expressed in higher eucaryotic cells, if will be strengthened transcription when inserting an enhancer sequence in the carrier.Enhancer is DNA cis-acting factors, generally about there is 10 to 300 base-pairs, acts on promoter to strengthen the transcription of gene.Can illustrated example be included in the Side of the replication origin late period one SV40 enhancers of 100 to 270 base-pairs, the polyoma in the Side of replication origin late period one Enhancer and adenovirus cancers etc..Persons skilled in the art are aware that how to select appropriate carrier, promoter, enhancer and host cell.It can be carried out with recombinant DNA conversion host cell with routine techniques well known to those skilled in the art.When host is prokaryotes such as Escherichia coli, the DNA competent cell harvesting after exponential phase of growth, use can be absorbed< 1₂Method processing, step used gathers generally well-known in the art.Alternative is to use MgCl₂.If desired, conversion can also be carried out with the method for electroporation.When host is eucaryote, following DNA transfection methods can be used：Calcium phosphate precipitation, conventional mechanical methods such as microinjection, electroporation, liposome packaging etc..The transformant of acquisition can use conventional method culture, express the polypeptide of the coded by said gene of the present invention.According to host cell used, culture medium used may be selected from various conventional mediums in culture.Cultivated under conditions of suitable for host cell growth.After host cell growth is to appropriate cell density, suitable method is used（Such as temperature transition or chemical induction）The promoter of selection is induced, cell is further cultured for a period of time.Required recombinant polypeptide is coated in intracellular, extracellular or expresses or be secreted on cell membrane and be extracellular in the above methods.If desired, can be separated using its physics, chemistry and other characteristics by various separation methods and purification of Recombinant albumen.These methods are mostly well-known to those skilled in the art.More specifically, it can mention, conventional renaturation process, be handled with protein precipitant（Salting-out method）, centrifugation, the broken bacterium of infiltration, super processing, ultracentrifugation, sieve chromatography（Gel filtration）, adsorption chromatography, ion-exchange chromatography, high performance liquid chroma- tography（HPLC) and other various liquid chromatography technologies and these The combination of method.The WW-HIP albumen or polypeptide of restructuring are of use in many ways.These purposes include（But it is not limited to）Directly as the disease caused by drug therapy WW-HIP hypofunctions or forfeiture, as pseudohypertrophy is malnutritive, Huntington's disease (HD), senile dementia, disease of immune system, Liddle syndromes, DRPLA (dentatorubral and pallidoluysian atrophy) etc., and for screening the antibody for the Huntington protein Binding peptide function of promoting or resist the functional domain containing WW, polypeptide or other parts.For example, antibody can be used for the function of activating or suppress WW-HIP.It can be used for finding the peptide molecule that can suppress or irritate WW-HIP functions for having therapeutic value with the restructuring WW-HIP screening peptide libraries of expression.

Invention also provides screening medicine to identify raising（Activator）Or check（Antagonist）The method of WW-HIP medicament.For example, mammalian cell or expression W- HIP film preparation can in the presence of medicine with the WW-HIP of mark-play culture.Then the ability that medicine improved or checked this interaction is determined.

WW-HIP antagonist includes antibody, compound, acceptor the missing thing and analog etc. filtered out.People WW-HIP antagonist can be combined with people WW-HIP and eliminate its function, or suppress people WW-HIP generation, or combined with the avtive spot of polypeptide and prevent polypeptide from playing biological function.The WW-HIP of employment antagonist can treat HD, disease of immune system etc..

When screening compounds are as antagonist, such a new people WW-HIP can be added in bioanalysis measure, determine whether compound is antagonist by determining the interaction between such a new people WW-HIP influences and its acceptor.The Shang Shu Sieve of Yi select the same method of compound, can filter out the acceptor missing thing and analog of antagonist action.

The polypeptide of the present invention can be used as peptide and know analysis well, for example, polypeptide available physical, the specific cutting of chemical or enzyme progress, and carry out one-dimensional two-dimentional or three-dimensional gel electrophoresis point Analysis.

A variety of methods can be used for production for the antibody of WW-HIP antigenic determinants.These antibody include（But it is not limited to）The fragment that polyclonal antibody, monoclonal antibody, chimeric antibody, single-chain antibody, Fab fragments and Fab expression libraries are produced.

Anti- WW-HIP antibody can be used in immunohistochemistry technology, the WW-HIR in detection biopsy specimen

The monoclonal antibody combined with WW-HIP can also use labelled with radioisotope, be injected in vivo its traceable position and distribution.This radiolabeled antibody can be used for the positioning of tumour cell as a kind of noninvasive diagnosis method and determine whether transfer.

Antibody in the present invention can be used for treating or preventing the disease related to WW-HIP.WW-HIP generation or activity can be stimulated or block by giving the antibody of suitable dosage.

Antibody can also be used for immunotoxin of the design for internal a certain privileged sites.Monoclonal antibody such as WW-HIP high-affinities can be with bacterium or phytotoxin（Such as diphtheria toxin, ricin, abrine etc.）Covalent bond.A kind of usual way is, with thiol crosslinkers such as SPDP, to attack the amino of antibody, by the exchange of disulfide bond, toxin is incorporated on antibody, and this hybrid antibody can be used for killing the positive cells of WW-HIP.

The production of polyclonal antibody can use WW-HIP or polypeptide immune animal, such as rabbit, mouse, rat etc..A variety of adjuvants can be used for enhancing immune response, including but not limited to Freund's adjuvant etc..

WW-HIP monoclonal antibodies can be produced with hybridoma technology（Kohler and Milstein. Nature, 1975,256:495-497 ).The chimeric antibody that the variable region in human constant region and inhuman source is combined can be produced with existing technology（ Morrison et al ,PNAS,1985,81:6851 ).And the technology of existing production single-chain antibody（U.S. Pat No.4946778) it can also be used for producing anti-W-HIP single-chain antibody.

The peptide molecule Ke Tong Guo Return choosings that can be combined with stomach-HIP by it is various may the amino acid that combine be incorporated into random peptide library that solid formation constitutes and obtain.During screening, it is necessary to WW-HIP molecules are marked.The polypeptide of the present invention can be applied in combination with suitable medicinal carrying agent.This composition includes the polypeptide of therapeutically effective amount, and medicinal acceptable carrier or excipients.Such carrier includes but is not limited to salt solution, Slow and rushes salt solution, glucose, water, glycol, ethanol and combinations thereof.These preparations should be suitable for method of application.The present invention also provides the Pharmaceutical composition composition equipped with one or more present invention in medicine box or kit containing one or more containers, container.Together with these containers, can there is a prompting for the instruction form that the government authorities by manufacture, use or sale medicine or biological products provide, the prompting reflect production, using or the government authorities of sale permit it to be applied on human body.In addition, the polypeptide of the present invention can be used in combination with other therapeutic compounds.Pharmaceutical composition can be administered in a convenient way, such as pass through local, intravenous, intraperitoneal, this intramuscular, subcutaneous, intranasal or intracutaneous approach.WW-HIP is to effectively treat and/or prevent the amount of specific indication to be administered.Many factors, the judgement of such as administering mode, the natural conditions of desire curer and diagnostician will be depended on by delivering medicine to the WW-HIP for the person of knowing amount and dosage range.The invention further relates to the diagnostic testing process of quantitative and detection and localization WW-HIP levels.These experiments are known in the art, and are determined and radiommunoassay including FLISH.The WW-HIP levels detected in experiment may be used as explaining importance of the WW-HIP in various diseases and for diagnosing the disease that WW-HIP can work.

WW-HIP polynucleotides can be used for the diagnosis of WW-HIP relevant diseases and control Treat.In terms of diagnosis, WW-HIP polynucleotides WW-HIP available for detection WW-HIP expression or under morbid state unconventionality expression.DNA sequences such as WW-HIP can be used for the hybridization of biopsy specimen to judge WW-HIP abnormal expression.Hybridization technique includes Southern blotting, Northern blotting, in situ hybridization etc..These technical methods are all disclosed mature technologies, and related kit is all commercially obtained.Part or all of the polynucleotides of the present invention can be fixed on microarray as probe（) or DNA chip Microarray（DNA Chip) on be used for analyze the Differential expression analysis of gene and gene diagnosis in tissue.RNA- polymeric alcohol chain reactions are carried out with the special primer of the Huntington protein Binding peptide of the functional domain containing WW（RT-PCR) amplification in vitro also can detect WW-HIP transcription product.The mutation of detection WW-HIP genes can also be used for diagnosing the related diseases of WW-HIP.The form of WW-HIP mutation includes point mutation, transposition, missing, restructuring and other any exceptions compared with normal wild type WW-HIP DNA sequence dna etc..It can be mutated with existing technology such as Southern traces, DNA sequence analysis, PCR and in situ hybridization detection.In addition, mutation is possible to influence the expression of albumen, therefore it can judge gene whether there is mutation indirectly with Northern traces, western blot.The sequence pair Chromosome Identification of the present invention is also valuable.The sequence-specific using the particular location of single human chromosome as target, and can be with this chromosomal hybridation.Moreover, needing to identify specific site on chromosome at present.Now, it is few to be based on actual sequence data（Repeat polymorphism）Chromosome marking reagent can be used for mark fine strain of millet colour solid position.According to the present invention, D N A are acted on chromosome, are with being relevant to the important first step that the base solid phase of disease is associated by these sequences.

Briefly, by preparing PCR primer by cDNA（It is preferred that 15-25bp), can be by sequence pair chromosome mapping.Promptly selected not with cDNA computer analysis Across the primer of an extron of genomic DNA, thus complicated amplification program.Then, these primers are used for PCR and screen the body cell hybrid cell containing single human chromosome.Only these hybrid cells contained corresponding to the people's gene of primer can produce the fragment of amplification.

The PCR mappings of body cell hybrid cell are the quick methods that specific DNA is assigned to specific seven colour solid.Using the present invention with identical Oligonucleolide primers, can similarly be used for making other effect strategies of its seven colour solid of date for obtaining sub- positioning from the Group experimental subjects or similar fashion lots of genes group clone collection body of the fragment of specific chromosome includes in situ hybridization, the chromosome prescreening with the airflow classification of mark and is selected by the pre- Sieve for hybridizing to the specific cDNA library of the colour solid of construct seven.

To the FISH of the cDNA clone of the metaphase chromosome of diffusion（FISH) can be for accurate chromosome mapping in one step.The summary of this technology, referring to Verma etc., Human Chromosomes:a Manual of Basic Techniques,Pergamon Press, New York(1988).

Once accurate chromosome position is arrived in sequence mapping, the physical location of this sequence on chromosome just can be associated with gene diagram data.These data are found in, for example, V.Mckusick, MendeIian Inheritance in Man I (by with Johns Hopkins University Welch Medical Library are online to obtain）And then by association analysis, determine gene and map already to the relation of the disease of some chromosomal regions.

Then, it is necessary to determine the difference of the c D N A or genome sequence between ill and non-diseased individuals.If observing mutation in some or all of diseased individuals, and do not observed in any normal individual, then mutation is probably the reason of disease.

With current physical mapping and the resolution capability of gene mapping technology, it is accurately positioned to the cDNA of the chromosomal region relevant with disease, can is one kind between 50 to 500 potential Disease-causing genes（It is assumed that 1 megabasse mapping resolution capability and every 20kb-individual gene）. Compare ill and non-diseased individuals, be usually directed to the change for first looking for structure in chromosome, such as stretch visible or with the detectable missings of PCR based on cDNA sequence or transposition from chromosome.Finally, it is that confirmation is mutated required for presence and mutation and many types of difference that several individual genes, which completely sort,.

WW-HIP polynucleotides can also be used for a variety of therapeutic purposes.Gene therapy technology can be used for cell propagation, development or metabolic disorder of the treatment caused by the expression of the WW- HIP polypeptides without expression or exception/inactive of WW-HIP polypeptides.The gene therapy vector of restructuring（Such as viral vector）The WW-HIP polypeptides of expression variance are may be designed to, for suppressing endogenic WW-HIP polypeptide actives.For example, a kind of WW-HIP polypeptides of variation can be the WW-HIP polypeptides for shortening, having lacked signal transduction functional domain, though Binding Capacity that can be with downstream, lacks signaling activity.Therefore the gene therapy vector of restructuring can be used for the disease for the treatment of WW-HIP expression of polypeptides or active caused by abnormal.From expression vector such as the retrovirus of virus, adenovirus, adeno-associated virus (AAV), herpes simplex virus, parvovirus etc. is intracellular available for WW-HIP polypeptide genes are transferred to.The method for building the recombinant viral vector for carrying WW-HIP polypeptide genes is found in existing document（ Sambrook^et al. ).In addition restructuring WW-HIP polypeptide genes can be packaged into liposome be transferred to it is intracellular.Suppress WW-HIP polypeptides mRNA oligonucleotide（Including antisense RNA and DNA) and core hemiculeer leucisculus it is also within the scope of the invention.Core alcohol is a kind of specific RNA of energy specific cleavage enzyme sample RNA molecule, and its mechanism of action is ribozyme molecule with carrying out endonuclease effect after complementary target RNA-specific hybridization.The RNA and DNA and ribozyme of antisense can be obtained with existing any synthesis RNA or DNA technology, and the technology of the bony sour barefoot amine chemical synthesis synthetic oligonucleotide of such as solid phase has been widely used.Antisense RNA molecule can transcribe acquisition in vitro or in vivo by encoding the DNA sequence dna of the RNA.This Plant the downstream that DNA sequence dna has been integrated into the RNA polymerase promoter of carrier.In order to increase the stability of nucleic acid molecules, it can be modified with a variety of methods, such as increase the connection application phosphorothioates key or peptide bond rather than bony acid diesters key between two Side sequence length, ribonucleotide.

Polynucleotide, which imports tissue or intracellular method, to be included：Polynucleotide is directly injected into in-vivo tissue；Or pass through carrier in vitro（Such as virus, bacteriophage or plasmid）First polynucleotide is imported in cell, then transplanted cells into internal etc..Brief description of the drawings

Fig. 1:Represent WW-HIP peptide sequences（Lower row) with the Huntington protein action protein of people（HIP) sequence of (upper row) compares, and comparison length is 178 amino acid.

Score=971 (341.8 bits), expect=1.8e-98, P=1.8e-98

The phase same sex=178/178 (100%), similitude=178/178 (100%), framework

= +2.Fig. 2:The photo of yeast two-hybrid assay result：

Culture medium upper semi-circle is（PLexA-HDl-425Q62) with（PB42AD-WW- HIP) be transferred to jointly during the yeast EGY48 bacterium of LEU2 and LacZ genes are smeared after in selective flat board（SD-UraHisTrpLeu the result on),

Cultivating hypobasal half Round is（PLexA-HDl-425Q62) with（PB42AD in selective flat board after) being transferred to jointly in the yeast EGY48 bacterial strains of LEU2 and LacZ genes（SD-UraHisTrpLeu the result on).The present invention will be further illustrated in the following examples, but is not to limit the present invention with this.

Embodiment 1:WW-HIP polypeptides cDNA clone Human fetal spleen total serum IgE is extracted with guanidinium isothiocyanate/phenol/chloroform one-step method.Poly (A) mRNA is separated from total serum IgE with Quik mRNA Isolation Kit (Qiegene).2ug poly (A) mRNA are through reverse transcription formation cDNA. Smart cDNA Cloning Kits（Purchased from Clontech) cDNA fragments orientation is inserted into pUC118 multiple cloning sites, conversion DH5ct bacteriums form cDNA library.3028 clones are obtained altogether.The sequence of the 5' and 3' ends of all clones is determined with dideoxy.The cDNA sequence of measure is compared with existing public DNA sequence data storehouse, it is new DNA as a result to find the DNA sequence dna for having a clone 0273H12.By synthesizing a series of primer pairs, this clones the contained two-way measure of DNA sequences progress.Computer analysis shows, full-length cDNA is a new DNA sequence dna（SEQ ID NOl), there is 1275bp ORF from 575bp to 1849bp, encode a new protein（ SEQ ID NO 2 ).This protein is named as the Huntington protein Binding peptide of the functional domain containing WW by we（ WW-HIP ).Example 2:WW-HIP polypeptides are cloned with RT-PCR method

It is template with fetus brain cell total serum IgE, reverse transcription reaction synthesis cDNA is carried out by primer of oligo-dT, after Qiagen kits, enters performing PCR with following primer and expand：

Primer 1:5 ,-TACCTACATCTGAACCAGAAGC-3, positioned at SEQ ID No.l section start l-23bp;

Primer 2:5'-GTTCTTTAATTGATTTTATTTT-3' is located at SEQ ID No.l 2485-2507bp.

The condition of amplified reaction：Contain 50mmol/L KCl, 10mmol/L Tris-Cl, (pH8.5), the μ π ι ο Ι of 1.5mmol/L MgCl 2,200/L dNTP, 25pmol primers, 2.5U Taq archaeal dna polymerases in 50 μ 1 reaction volume.Following 25 cycles of conditioned response are pressed on PE9600 type DNA thermal cyclers： 94C 30sec; 55C, 30sec; 72C 2min.Template blank is set in RT-PCR as negative control.After amplified production QIAGEN kits, it is connected to TA Cloning Kits on pCR carriers（), Invitrogen and DNA sequence dna is determined.As a result the DNA sequence dna of PCR primer is identical with SEQ ID No.l l-2507bp.Example 3:Recombinate the vivoexpression of WW-HIP polypeptides, separation and purifying

Fen Do devise pair of primers at the initiation codon of WW-HIP polypeptide genes and at terminator codon,

Primer 3: 5'TCTAGAAGGCTCAGAAACAACA-3'

Primer 4: 5'-AAGCTTCCAACAGTCACTCTAA-3'

Its 5, end be respectively provided with Xbal and Hindlll restriction enzyme sites.Enter performing PCR amplification by template of the plasmid 0273hl2 of the target gene containing total length and obtain WW-HIP polypeptide genes code area.Amplified fragments are inserted into expression vector pGEM-3Z (being purchased from Pharmacia Biotech) by digestion, and convert BL21 (DE3) pLysE, on the LB flat boards containing ampicillin and IPTG, 5 recombinant conversions for screening white carries out DNA sequence analyses, as a result identical with the gained coding sequence of example one.

Choose a ring recombinant bacterium to smear, be inoculated in 20ml LB culture mediums（Containing ampicillin

100ug/ml), 37 " shaken cultivation takes seed liquor to be transferred by 2% inoculum concentration in 4 liters of LB culture mediums, 37t shaken cultivations to thalline Α overnight as seed liquor₆When 00=0.7（Exponential phase）Add IPTG to final concentration 0.4mmoI/L, it is further cultured for culture 12 hours, thalline is collected by centrifugation, washed with lxPBS with Slow fliud flushings A (16mM Na2HP04,4mM NaH2P04, pH 6.5) by 10ml/ grams of thalline dissolving, ultrasonication in ice bath, collected after centrifugation supernatant, supernatant crosses glutathione-Sepharose 4B affinity columns, after Slow fliud flushings A elution, eluted with the eluent of the reduced glutathione containing 5mM, the WW-HIP polypeptides that can be purified. Embodiment 4：The generation of anti-WW-HIP polypeptide antibodies

Use Peptide synthesizer（PE-ABI the polypeptide of WW-HIP polypeptid specificities) is synthesized： NH₂-Gly-Tyr-Asn-Ala-Pro-His-His-Pro-Phe-Ala-Gly-Tyr-Pro- Pro-Gly-COOH.The polypeptide is coupled to form compound with hemocyanin and bovine serum albumin(BSA) respectively, method referring to： Avrameas. Immunochemistry, 1969; 6:43.Complete Freund's adjuvant immunizing rabbit is added with the above-mentioned hemocyanin polypeptide complexes of 4mg, adds incomplete Freund's adjuvant booster immunization once with hemocyanin polypeptide complex again after 15 days.The titre that ELISA determines antibody in rabbit anteserum is done using through the 15 coated titer plates of g/ml bovine serum albumin polypeptide compounds.Total I gG are separated from the rabbit anteserum of antibody positive with albumin A-Sepharose.Polypeptide is incorporated on the Sepharose 4B posts of cyanogen bromide-activated, anti-peptide antibody is separated from total G with affinity chromatography.Immuno-precipitation proves that the antibody of purifying can be combined specifically with WW-HIP polypeptides.Embodiment 5:The Homology search of cDNA clone

The sequence of polynucleotides of polypeptide that is there is provided with the present invention and its protein sequence of coding are to Genbank, the databases such as swissport carry out Homology search, and the program for retrieval is Blast (Basic local alignment search tool) (1993 Proc Nat Acasd Sci 90:5873-5877), Blast can be found out in many genes of the new WW-HIP homologous peptides of people, wherein with we have found that the maximum gene of genetic homology, its albumen encoded is AF049103 in Genbank access number.These genes retrieved and protein sequence can be recalled from Genbank.The sequence recalled can use the Pileup (multisequencing in G C G software kits）With Gap (two sequences）Program, which is done even to match somebody with somebody, to be compared.The function prediction of new albumen can be analyzed with Motif programs.The result of Homology search is illustrated in fig. 1 shown below, the new WW-HIP polypeptides of the people that the result display present invention is provided and the HIP phases same sex 178/178 (100%), similitude 178/178 (100%).This albumen is secretory protein. Embodiment 6:Northern traces

Total R A are extracted with one-step method【Anal. Biochem 1987,162,156-159】.The method includes acid guanidine thiocyanate benzene and expects-chloroform.Use 4M guanidinium isothiocyanate -25mM sodium citrates, 0.2M sodium acetates（PH4.0) to organize into be homogenized, add the stupid phenol of 1 times of volume and the chloroform-isoamyl alcohol of 1/5 volume（49:1), centrifuged after mixing.Aqueous layer is suctioned out, isopropanol is added（0.8 volume）And mixture centrifugation is obtained into RNA precipitations.

Obtained RNA precipitate is washed with 70% ethanol, it is dry and soluble in water.

20mg RNA are used, in (the N- beautiful jade generations of 3- containing 20mM）Propane sulfonic acid（PH7.0) electrophoresis is carried out on 1.2% Ago-Gel of -5mM sodium acetates-ImM EDTA-2.2M formaldehyde.It is then transferred on nitrocellulose filter.The probe marked with 32P-（About 1'106cpm/ml) in 42, " hybridized overnight, the solution includes 50% formamide -25mM KH2P04 (pH7.4) -5 ' SSC-5 ' Denhardt's solution and 200mg/ml salmon sperm DNAs in a solution.The DNA probe of 32P- marks is prepared by random priming with a-32P dATP.DNA probe used is the WW-HIP peptide coding region sequences that PCR is expanded.After hybridization, by filter membrane in 1'SSC-0.1%SDS in washing 30min.Then, analyzed and quantified with Phosphor Imager.

As a result show, WW-HIP polypeptide genes are main to express in brain tissue, testis tissue.Example 7, yeast two-hybrid assay

Utilize multiple cloning sites, plasmid pLexA merges (pLexA-HDl-425Q62) plasmid pB42AD and WW-HIP Gene Fusions (B42AD-WW-HIP) with H D gene N-terminals, it is transferred to jointly in the yeast EGY48 bacterial strains of LEU2 and LacZ genes, then with negative control * simultaneously in selective flat board（SD- UraHisTrpLeu) on express（Fig. 2), LEU2 and LacZ gene expression feelings are observed Condition, can determine whether Huntington protein and WW-HIP whether there is interaction relationship.

* negative control is the EGY48 bacterial strains that pLexA-HDl-425Q62 and pB42AD are transferred to

* plasmid used in two hybridization experiments and bacterium are smeared purchased from Clontech

As a result show that Huntington protein and WW-HIP can interact.

Claims (15)

1. Claim

   1. a kind of Huntington protein Binding peptide of functional domain containing WW of separation, it includes SEQ ID No. 2 amino acid sequence, or its conservative variation's polypeptide or its active fragment or derivative.

   2. polypeptide as claimed in claim 1, it is characterised in that the polypeptide is the polypeptide with the amino acid sequences of SEQ ID No. 2.

   3. a kind of polynucleotides of separation, it is characterised in that it includes a kind of nucleotide sequence being selected from the group：

   (a) polynucleotides of polypeptide as claimed in claim 1 or 2 are encoded；

   (b) polynucleotides complementary with polynucleotides (a)；With

   (c) with（Or the polynucleotides of (b) have the polynucleotides of at least 70% phase same sex a).

   4. polynucleotides as claimed in claim 3, it is characterised in that the polynucleotide encoding has the polypeptide of amino acid sequence shown in SEQ ID No.2.

   5. polynucleotides as claimed in claim 3, it is characterised in that one kind that the sequence of the polynucleotides is selected from the group：

   (a) there is the sequence of 575-1849 in SEQ ID No. 1；With

   (b) there is the sequence of 1-2507 in SEQ ID No. 1.

   6. a kind of recombinant vector, it is characterised in that it contains the polynucleotides described in claim 3,4 or 5.

   7. a kind of genetically engineered host cell, it is characterised in that it is the host cell for being converted or being transduceed with the carrier described in claim 6 or the host cell for being converted or being transduceed with the polynucleotides described in claim 3,4 or 5.

   8. a kind of preparation method of the polypeptide of the Huntington protein Binding peptide activity with people's WW functional domains, it is characterised in that this method includes：

   (a) in the condition for the Huntington protein Binding peptide for being adapted to expression functional domain containing WW

   - 25-replace page (yarn of detailed rules and regulations the 26th) Under, the host cell described in culture claim 7；With

   (b) polypeptide of the Huntington protein Binding peptide activity with the functional domain containing WW is isolated from culture.

   9. a kind of antibody of the Huntington protein Binding peptide specific binding of functional domain containing WW with claim 1.

   10. a kind of method of Huntington protein Binding peptide activity for screening simulation or the regulation functional domain containing WW or the compound of expression, it is characterised in that utilize the polypeptide or the polynucleotides of claim 3 of claim 1.

   11. the simulation that the method according to claim 10 is obtained, promotion, antagonism suppress the activity of polypeptide described in claim 1 or the compound of expression.

   12. compound as claimed in claim 11, it is characterised in that it is SEQ ID NO:The antisense sequences of polynucleotide sequence or its fragment shown in 1.

   13. the application of compound described in claim 11, it is characterised in that adjust the activity of the Huntington protein Binding peptide of the functional domain containing WW in vivo, external using the compound.

   14. a kind of method detected with polypeptide relevant disease or disease susceptibility described in claim 1, it is characterised in that including：

   (d) exception of the expression of polypeptides amount is detected；

   (e) detect that the activity of the polypeptide is abnormal；Or

   (f) detection and the variation in the expression of polypeptides amount or the abnormal related nucleic acid of activity.

   15. the pharmaceutical composition of compound and pharmaceutically acceptable carrier described in polypeptide described in the claim 1 containing effective dose or claim 11.

   - 26-replace page (the 26th article of detailed rules and regulations)