CN1373773A - Polynucleotides encoding human angiotensin-II-1 receptor proteins and method of preparation and its use - Google Patents

Abstract

The invention discloses a new kind of human angiotensin II-1 receptor proteins and polynucleotides encoding this polypeptides and a process for producing the polypeptides by recombinant methods. It also discloses the method of applying the polypeptides and polynucleotides for the prevention or treatment of various kinds of diseases, such as cardiovascular disease, coronary heart disease, essential hypertension and non-insulin dependent diabetes. The antibody and antagonist of the polypeptide and the therapeutic use of the same is also discloses. In addition, it refers to the use of polynucleotides encoding novel human angiotensin II-1 receptor proteins.

Description

Polynucleotides encoding human angiotensin-II-1 receptor proteins and method of preparation and its use

It is a kind of to encode the new polynucleotides of the receptor-related proteins of human angiotensin I 1-1 and its preparation method and application invention field

The present invention relates to a kind of polynucleotide sequence for encoding new human angiotensin Π -1 receptor-related proteins.The invention further relates to the purposes of the preparation method of the polynucleotide sequence and its protein of coding, and the protein of the polynucleotides and its coding.Background of invention

Angiotensin-ii receptor (Angiotensin II receptors, Ang II) is a kind of vasoactive peptide, can be with target tissue（Such as gland, blood vessel on Shen, Kidney）Special membrane-bound receptor interaction.The acceptor of three kinds of Angiotensin IIs such as 1 type, 2 types and 3 types has been obtained at present（It is referred to as AT1, AT2, AT3), seven hydrophobic transmembrane structure domain structures are all presented in they in structure, can be in combination with a pair of Gq albumen, and its main biological function is signal transduction.ATI and AT2 amino acid has 46% homology, and wherein ATI is made up of ATla and ATlb（Both have identical code area and different noncoding regions, amino acid identity is higher than 90%), mainly express, wide expression and can drastically decline with the birth of fetus in the whole body of fetus in the cardiovascular system, adrenal gland and kidney of adult；AT2 is then main to express in the brain, adrenal medella and ovary of adult.ATI mainly produces the two kinds of results i.e. activation of protein kinase C and Ca in the signal transduction of intracellular by activating bony lipase C²⁺Fixation realize, recently, it was found that dark tyrosine-kinase is also one of approach of ATI signal transductions.Clinical research has shown that, ATI antagonist can cause the rising of Angll levels in blood plasma, and optionally stimulate AT2, and AT2 has anti-ATI effect in itself, so AT2 has great importance [Miserey S as the function of ATI antagonists in clinical treatment, Deng Therapie 1998; 53: 205-11]. Tight element Π -1 receptor GAP-associated protein GAPs of Xue Guan（Hereinafter referred to as ATRAP) be latest find a kind of ATI acceptors GAP-associated protein GAP, the ATRAP molecular weight of mouse is 18kDa, it only interacts with the carboxy-terminal cytosolic domain of ATla acceptors, without being interacted with AT2, relax Slow kassinin kinins Β 2, beta 2 adrenoreceptor etc..ATRAP mRNA is expressed in the kidney, heart and testis of mouse, and is not expressed in the lungs in mouse, liver, spleen and brain.The interaction of ATRAP and ATla acceptors can be determined by the specific immune precipitation method of affinity chromatography, fluorescence microscopy and two kinds of albumen.Overexpressions of the ATRAP in COS-7 cells inhibits ATla acceptors to activation [the Daviet L, etc. journal of biological chemistry of phospholipase C（ J Biol Chem ) 1999； 274:17058-62] .

Because ATI is renin-angiotensin system（RA systems）Important composition composition, can mediate vascular relax the physiological effects such as contracting, water-electrolyte metabolism and vascular smooth muscle hyperplasia and function point analysis, it is RA systemic effects in the committed step of effector, it is also one of Disease-causing gene of hypertension genetic, therefore, ATRAP can be detected and for treating by the interaction with ATI to the morbidity of a variety of cardiovascular diseases, such as detecting and treating coronary heart disease, essential hypertension and Non-Insulin Dependent Diabetes Mellitus, particularly preferably for the treatment to coronary heart disease.Therefore, the research and development ATRAP polypeptides and its antibody, activator, antagonist for the purpose for the treatment of, in physiology, pharmacologically important in inhibiting（Soubrier F etc., hypertension magazine（J Hypertension), 1993,11. Suppl 5: s20-27 ) .Summary of the invention

It is an object of the present invention to provide the polypeptide of the receptor GAP-associated protein GAP of Angiotensin II -1 of new separation.

It is a further object to provide the polynucleotide sequence of encoding both Angiotensin Converting Enzyme II-1 receptor GAP-associated protein GAPs.

It is a further object to provide include encoding both Angiotensin Converting Enzyme Π -1 receptors The recombinant expression carrier of the polynucleotide sequence of GAP-associated protein GAP.

It is a further object to provide include the host cell that sanction body is expressed with encoding both Angiotensin Converting Enzyme Π -1 receptor GAP-associated protein GAP polynucleotide sequence Chong Group.

It is a further object to provide the method for the production receptor GAP-associated protein GAP of Angiotensin II -1.

It is a further object to provide the antibody and antagonist of the receptor GAP-associated protein GAP of Angiotensin II -1 for the present invention.

It is a further object to provide the method for diagnosis, the treatment disease related to the receptor GAP-associated protein GAP dysfunction of Angiotensin II -1.

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

In one embodiment of the invention, the invention provides a kind of substantially pure receptor GAP-associated protein GAP of Angiotensin II -1, it is substantially by SEQ ID NO:Amino acid sequence composition shown in 2.The GAP-associated protein GAP of angiotensins Π -1 acceptors is characterized in have a membrane spaning domain.The polynucleotides of the present invention are found from the cDNA library of Human fetal brain.The polynucleotide sequence total length that it is included is 1108 bases, and its open reading frame is SEQ ID NO:56-535 in 1, the human angiotensin II-1 receptor-related proteins of 159 amino acid are encoded.According to amino acid sequence homologous it was found that, this polypeptide has 76% homology with the ATRAP in rat testis, and the polypeptide has the conservative base of ATRAP gene families, it can be inferred that the new people ATRAP has the similar 26S Proteasome Structure and Function of ATRAP gene families.

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 polynucleotides and polypeptide under the native state in active somatic cell are not isolated and purified, but same polynucleotides or polypeptide such as from native state with dividing in other materials existed Open, then isolate and purify.

As used in the present invention, " receptor-related proteins of Angiotensin II -1 or polypeptide of separation " refer to the receptor-related proteins of Angiotensin II -1 substantially free of natural relative other albumen, lipid, carbohydrate or other materials.Those skilled in the art can purify the receptor-related proteins of Angiotensin II -1 with the purified technology of protein of standard.Substantially pure polypeptide can produce single master tape on non-reduced polypropylene barefoot amine gel.The purity of the receptor-related proteins of Angiotensin II -1 can use amino acid sequence analysis.

The polypeptide of the present invention can be recombinant polypeptide, natural polypeptides, synthesis polypeptide, preferably recombinant polypeptide.

Present invention additionally comprises the fragment of human angiotensin II -1 receptor-related proteins, derivative and analog.As used in the present invention, term " fragment ", " derivative " and

" analog " refers to be kept substantially the receptor-related proteins identical biological function of natural human Angiotensin II -1 with the present invention or the polypeptide of activity.Polypeptide fragment, the derivative or the like of the present invention can be that (i) has one or more conservative or substituted polypeptides of non-conservative amino acid residue (preferably conservative amino acid), and such substituted amino acid residue can may not be by genetic code encoding, or (ii) has the polypeptide of substituted radical, or (iii) mature polypeptide and another compound in one or more amino acid residues（Such as extend the compound of polypeptide half-life period, such as polyethylene glycol）The formed polypeptide of fusion, or（Iv) additional amino acid sequence is fused to polypeptide formed by this peptide sequence (such as targeting sequencing, secretion sequence or sequence or proprotein sequence for being easy to purify this polypeptide）.These fragments, derivative and analog belong to scope known to those skilled in the art.

In another embodiment, the invention provides the polynucleotides of separation, it substantially has SEQ ID NO by coding:The polynucleotides composition of the polypeptide of 2 amino acid sequences.In another embodiment, polynucleotide sequence of the invention includes SEQ ID NO:Nucleotide sequence shown in 1. The polynucleotides of the present invention can be DNA form or rna form.DNA form includes cDNA, genomic DNA or artificial synthesized DNA.DNA can be single-stranded or chain.DNA can be coding strand or noncoding strand.The coding region sequence of encoding mature polypeptide can be with SEQ ID Ν Ο:Coding region sequence shown in 1 is identical or its degeneracy variant." degeneracy variant " refers to because the degeneracy coding of genetic code has SEQ ID NO in the present invention:2 protein or polypeptide but with SEQ ID Ν Ο:The different polynucleotide sequence of polynucleotide sequence shown in 1.

Encode SEQ ID NO:The polynucleotides of 2 mature polypeptide include：The polynucleotide sequence of encoding mature polypeptide；The polynucleotides of encoding mature polypeptide and various appended sequences；Encoding mature polypeptide（With optional additional coding sequence）And non-translated sequence.

Term " polynucleotides of coded polypeptide " refers to include to encode the polynucleotides of this polypeptide and the polynucleotides including additional coding sequence and/or non-coding sequence.

The invention further relates to the variant of foregoing description polynucleotides, it is encoded has the polypeptide of substantially the same amino acid sequence or the fragment 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 that can hybridize under certain condition with polynucleotide sequence described above, at least have 50% between the sequence and the polynucleotide sequence of the present invention, preferably with 70%, the more preferably phase same sex with 90 %）.The present invention is more particularly directed under strict conditions can be 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, 60;Or（2) add when hybridizing and use denaturant, such as 50% (v/v) formamide, 0.1% calf serum / 0.1%Ficoll, 42 " C etc.；Or（3) the phase same sex only between two sequences just hybridizes at least more than 95% when more preferably more than 97%.Also, the polypeptide of interfertile polynucleotide encoding and SEQ ID NO:Mature polypeptide shown in 2 has identical biological function and activity.

The invention also 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 the polynucleotides of encoding both Angiotensin Converting Enzyme II-1 receptor GAP-associated protein GAPs.

The host cell produced the present invention also relates to the carrier of the polynucleotides comprising the present invention and with the carrier of the present invention through gene engineering method, and the method through recombinant technique generation polypeptide of the present invention.

Based on disclosure of the invention, DNA sequence dna of the invention can be obtained with a variety of methods.For example, separating DNA with hybridization technique well known in the art.These technologies include but is not limited to：1) hybridized to detect homologous nucleotide sequences with probe and genome or cDNA library, and 2) antibody screening of expression library to detect the DNA fragmentation of the clone with structural features.

The specific DNA fragment sequence of encoding both Angiotensin Converting Enzyme II-1 receptor GAP-associated protein GAPs is produced and can also obtained with following method：1) double chain DNA sequence is separated from genomic DNA；2) chemical synthesising DNA sequence is with the double-stranded DNA of polypeptide needed for obtaining.

In method mentioned above, isolated genes group DNA is the most commonly used., also can be by the method for the direct chemical synthesis of DNA sequence dna when known to the whole amino acid sequence of the polypeptide product of needs.If the whole sequence of required amino acid is not known, the direct chemical synthesis of DNA sequence dna is impossible, and available method 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 phagocytosis Body 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 polymeric enzyme 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 including but is not limited to：（1) DNA-DNA or DNA-RNA hybridization；(2) function of marker gene occurs or lost；（3) level of the transcript of the receptor GAP-associated protein GAP of Angiotensin II -1 is determined；（4) applied immunology technology or measure biological activity detect the protein product of gene expression.The above method can be alone, also can a variety of method use in conjunction.

（1) in kind method, hybridize more than any a part of homologous, at least 15 nucleotides of its length that probe used should be with the polynucleotides of the present invention, preferably 30 nucleotides, more preferably 50 nucleotides, preferably 100 nucleotides.In addition, the length of probe is generally within 2kb, within preferably lkb.Probe used herein is typically the DNA sequences 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.DNA probe can use radio isotope, fluorescein or hemiculeer leucisculus（Such as the bony sour enzyme of alkalescence）Deng mark.（4) in kind method, the protein product of the detection receptor of Angiotensin II -1 related protein gene expression can use immunological technique such as western blot, 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）, it is used in terms of above-mentioned PCR to draw Thing be able to can be properly selected according to the sequence information of invention disclosed herein, 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 fragmentations etc. can use conventional method such as dideoxy chain termination（The such as Sanger PNAS, 1977,74： 5463-5467 ) .This kind of nucleotide sequencing can also use business sequencing kit etc..In order to obtain the cDNA sequence of total length, sequencing need to be repeated.Sometimes for the cDNA sequence for determining multiple clones, the cDNA sequence of total length can be just spliced into.

According to common recombinant DNA technology, the receptor GAP-associated protein GAP of Angiotensin II -1 of restructuring is can be used to express or produced using the polynucleotide sequence of the present invention（Science, 1984; 224： 1431 ) .In general there are Yi Xia Bu Sudden：

(1) polynucleotides (or variant) of the receptor-related proteins of encoding human Angiotensin II -1 of the present invention are used, or are converted or suitable host cell of transduceing with the recombinant expression carrier containing the polynucleotides；

(2) host cell cultivated in suitable culture medium；

(3) protein of the present invention is separated, purified from culture medium or cell.

In the present invention, encoding both Angiotensin Converting Enzyme II-1 receptor GAP-associated protein GAP polynucleotide sequences can be plugged into recombinant expression carrier.Recombinant expression carrier used in the present invention includes but is not limited to bacterial plasmid, 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, etc. gene, 1987,56:125 ) ；The pMSXND expression vectors expressed in mammalian cell（Lee and Nathans, journal of biological chemistry 263:3521,1988) carrier from baculoviral and in insect cell expressed.In a word, as long as can be stabilized and replicate in host, any plasmid and carrier can be used.The key character of expression vector is to usually contain replication orgin, opened Mover, marker gene and translation control element.

Method well-known to those having ordinary skill in the art can be used to build the receptor GAP-associated protein GAP DNA sequences encodings of the Π -1 containing angiotensins and the expression vector of suitable transcription/translation control signal.These methods include the (Sambroook such as recombinant DNA technology in vi, DNA synthetic technologys, In vivo recombination technology, et al. Molecular Cloning, a Laboratory Manual, Cold Spring Harbor Laboratory. New York, 1989).Described DNA sequence dna can be effectively connected in the appropriate promoter in expression vector, to instruct mRNA to synthesize.There is representational example in these promoters：Lac the or trp promoters of large intestine pestle bacterium；Bacteriophage lambda P_LPromoter；Eukaryotic promoter includes CMV immediate early promoters, HSV thymidines and swashs hemiculeer leucisculus promoter, the promoter of the expression of early and late SV40 promoters, the LTRs of retroviruse and some other known controllable gene in protokaryon or eukaryotic or its virus.Expression carrying agent also includes the ribosome bind site and transcription terminator of translation initiation.

In addition, expression vector 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 large intestine pestle bacterium tetracycline or amicillin resistance.

Carrier comprising above-described appropriate DNA sequence dna and appropriate promoter or control sequence can be used for converting 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 Sf 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 have 10 to 300 base-pairs, act on promoter to strengthen the transcription of gene.Can illustrated example be included in the Side of replication origin late period one 100 to 270 base-pairs SV40 enhancers, in the Side of replication origin late period one polyoma 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, can be harvested after exponential phase of growth can absorb DNA competent cell, use CaCl₂Method processing, step used is 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.

In the above methods, purpose recombinant polypeptide can be coated in intracellular or express or be secreted on cell membrane and be extracellular.If desired, can be separated using its physics, chemistry and other characteristics by various separation methods and pureization weighs Group albumen.These methods are well known to those skilled in the art.More specifically, these methods include but is not limited to：The renaturation process of routine, salting-out method, centrifugation, osmotic shock break bacterium, ultrasonication, ultracentrifugation, sieve chromatography（Gel filtration）, adsorption chromatography, ion-exchange chromatography, high performance liquid chroma- tography（HPLC) and other various liquid chromatography technologies and these methods combination.

The receptor of Angiotensin II -1 GAP-associated protein GAP or polypeptide of restructuring have many use On the way, particularly preferably for the detection and treatment of a variety of angiocardiopathies, such as detecting and treat coronary heart disease, essential hypertension and Non-Insulin Dependent Diabetes Mellitus.These purposes include but is not limited to：Directly element II-1 receptor GAP-associated protein GAP hypofunctions or the caused disease of forfeiture are opened as drug therapy Xue Guan Tight and for screening the antibody, polypeptide or the other parts that promote or resist the receptor GAP-associated protein GAP function of Angiotensin II -1.For example, antibody can be used for the function of activating or suppress angiotensins Π -1 receptor GAP-associated protein GAPs.It can be used for finding the peptide molecule that can suppress or stimulate the receptor GAP-associated protein GAP function of Angiotensin II -1 for having therapeutic value with the recombinant vascular Angiotensin Converting Enzyme Π -1 receptors GAP-associated protein GAP screening peptide library of expression.

Improved present invention provides screening compounds, especially identification（Activator）Or check（Antagonist）The method of the compound of the receptor GAP-associated protein GAP of Angiotensin II -1, these activators or antagonist are particularly suitable for use in the detection and treatment of a variety of angiocardiopathies, such as detecting and treat coronary heart disease, essential hypertension and Non-Insulin Dependent Diabetes Mellitus.Activator improves the biological functions such as the signal transmission of the receptor of Angiotensin II -1 GAP-associated protein GAP in the cell, and antagonist prevents the disorder relevant with Intracellular signals transmission with treatment, such as various cancers.For example, mammalian cell or the expression receptor of Angiotensin II -1 GAP-associated protein GAP can be cultivated in the presence of medicine together with the angiotensins Π -1 receptor GAP-associated protein GAPs of mark.Then the ability that medicine improved or checked this interaction is determined.

The antagonist of angiotensins Π -1 receptor GAP-associated protein GAPs includes antibody, compound, acceptor the missing thing and analog etc. filtered out.The antagonist of human angiotensin II-1 receptor GAP-associated protein GAPs can be combined with human angiotensin II-1 receptor GAP-associated protein GAPs and eliminate its function, or suppress the generation of human angiotensin II-1 receptor GAP-associated protein GAPs, or combined with the avtive spot of polypeptide and prevent polypeptide from playing biological function.The antagonist of the receptor GAP-associated protein GAP of Angiotensin II -1 of employment can treat a variety of angiocardiopathies, such as coronary heart disease, essential hypertension and Non-Insulin Dependent Diabetes Mellitus. In compound of the screening as antagonist, the receptor-related proteins of Angiotensin II -1 can be added in bioanalysis measure, whether it is antagonist that compound is determined by determining the interaction between the receptor-related proteins of compounds affect Angiotensin II -1 and its acceptor.With the above-mentioned same method for selecting compound, the acceptor missing thing and analog of antagonist action can have been selected.

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

There is the antibody that a variety of methods can be used for production for the receptor GAP-associated protein GAP antigenic determinant of Angiotensin II -1.These antibody include but is not limited to the fragment that polyclonal antibody, monoclonal antibody, chimeric antibody, single-chain antibody, Fab fragments and Fab expression libraries are produced.

The antibody of antiangiotensin II-1 receptor GAP-associated protein GAPs can be used in immunohistochemistry technology, the receptor GAP-associated protein GAP of Angiotensin II -1 in detection biopsy specimen.

The monoclonal antibody combined with the receptor GAP-associated protein GAP of Angiotensin II -1 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 judge whether it shifts.

Antibody in the present invention can be used for treatment or the diagnosis disease related to angiotensins Π -1 receptor-related proteins, particularly preferably with a variety of angiocardiopathies, such as coronary heart disease, essential hypertension and Non-Insulin Dependent Diabetes Mellitus.Give generation or activity that the antibody of suitable dosage can stimulate or block the receptor GAP-associated protein GAP of Angiotensin II -1.

Antibody can also be used for immunotoxin of the design for internal a certain privileged sites.Monoclonal antibody such as the receptor GAP-associated protein GAP high-affinity of Angiotensin II -1 can be with bacterium or phytotoxin（Such as diphtheria toxin, ricin, abrine etc.）Covalently tie Close.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 cell of the receptor of Angiotensin II -1 GAP-associated protein GAP.

The production of polyclonal antibody can use angiotensins Π -1 receptors GAP-associated protein GAP 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..

The receptor GAP-associated protein GAP of Angiotensin II -1 monoclonal antibody can produce (Kohler and Milstein. natures (ature), 1975,256 with hybridoma technology: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 etc., 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 the single-chain antibodies of antiangiotensin Π -1 receptor GAP-associated protein GAPs.

The peptide molecule that can be combined with the receptor of Angiotensin II -1 GAP-associated protein GAP can by screen 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 which the receptor GAP-associated protein GAP molecule of Angiotensin II -1 is marked.

The polypeptide of the present invention can be applied in combination with suitable pharmaceutical carrier.This composition includes the polypeptide of therapeutically effective amount, and medicinal acceptable carrier or excipient.Such carrier includes but is not limited to salt solution, Slow and rushes salt solution, glucose, water, glycol, ethanol and combinations thereof.Those skilled in the art know how to make these preparations 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, the indicative prompting that can there are the government authorities by manufacture, use or sale medicine or biological products to provide, the prompting reflect production, using or sale government authorities 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, intramuscular, subcutaneous, intranasal or intracutaneous approach.The receptor of Angiotensin II -1 GAP-associated protein GAP is to effectively treat and/or prevent the amount of specific indication to be administered.The judgement of many factors, such as natural conditions of administering mode, subject and diagnostician will be depended on by delivering medicine to the amount and dosage range of the receptor GAP-associated protein GAP of Angiotensin II -1 of patient.

The invention further relates to the diagnostic testing process of the quantitative and receptor GAP-associated protein GAP level of detection and localization Angiotensin II -1.These experiments are known in the art, and are determined and radiommunoassay including FISH.Angiotensins Π -1 receptor GAP-associated protein GAP the levels detected in experiment may be used as explaining importance of the receptor of Angiotensin II -1 GAP-associated protein GAP in various diseases and for diagnosing the disease that ATRAP can work.

The polynucleotides of encoding both Angiotensin Converting Enzyme Π -1 receptor GAP-associated protein GAPs can also be used for the diagnosis and treatment of angiotensins Π -1 receptor GAP-associated protein GAP relevant diseases, particularly preferably with a variety of angiocardiopathies, such as detecting and treat coronary heart disease, essential hypertension and Non-Insulin Dependent Diabetes Mellitus.In terms of diagnosis, the unconventionality expression of the receptor GAP-associated protein GAP of Angiotensin II -1 polynucleotides receptor of Angiotensin II -1 GAP-associated protein GAP available for the expression of the detection receptor GAP-associated protein GAP of Angiotensin II -1 or under morbid state.As the receptor GAP-associated protein GAP of Angiotensin II -1 DNA sequence dna can be used for the hybridization of biopsy specimen to judge the abnormal expression of the receptor GAP-associated protein GAP of Angiotensin II -1.Hybridization technique includes Southern traces, Northern traces, 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（Microarray) or in DNA chip it is used for Differential expression analysis and gene diagnosis that Fen Xi Group knit middle gene.RNA- polymerase chain reactions are carried out with the special primer of the receptor GAP-associated protein GAP of Angiotensin II -1（RT-PCR) amplification in vitro also can detect the transcription product of the receptor GAP-associated protein GAP of Angiotensin II -1. The mutation of detection Angiotensin II-l receptor related protein genes can also be used for diagnosing the related disease of the receptor of Angiotensin II -1 GAP-associated protein GAP, particularly preferably with a variety of angiocardiopathies, such as detecting and treat coronary heart disease, essential hypertension and Non-Insulin Dependent Diabetes Mellitus.The form of the receptor of Angiotensin II -1 GAP-associated protein GAP mutation includes point mutation, transposition, missing, restructuring and other any exceptions compared with the receptor GAP-associated protein GAP DNA sequence dna of normal wild type Angiotensin II -1 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 P C R primers by c D N A（It is preferred that 15- 25bp), can be by sequence pair chromosome mapping.Ji is not spanned across because of the primer of a Group D N A extron with c D N A computer analysis promptly selection, thus complicated amplification program.Then, these primers are used for P C R 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 positioning modes of body cell hybrid cell, are the quick methods that DNA is navigated to specific chromosome.Using the Oligonucleolide primers of the present invention, by similar approach, sub- positioning is realized using one group of fragment or lots of genes group clone from specific chromosome.Chromosome prescreening and hybridization of other similar strategies including in situ hybridization, with the airflow classification of mark available for chromosome mapping is preselected, so that structure Build the special cDNA storehouses of chromosome.

CDNA clone and metaphase chromosome are subjected to FISH (FISH), chromosome mapping can be accurately carried out 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 sequence is accurately positioned the particular location of chromosome, then 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, Mendelian Inheritance in Man I (by with Johns Hopkins University Welch Medical Library are online to obtain）.Then it can determine gene by linkage analysis 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 c D N A 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 P C R based on c D N A sequences 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,.

The receptor GAP-associated protein GAP polynucleotides of Angiotensin II -1 can also be used for a variety of therapeutic purposes, particularly preferably with a variety of angiocardiopathies, such as detecting and treat coronary heart disease, essential hypertension and Non-Insulin Dependent Diabetes Mellitus.Gene therapy technology can be used for cell of the treatment caused by the expression of the receptor GAP-associated protein GAP of Angiotensin II -1 without expression or exception/inactive of the receptor GAP-associated protein GAP of Angiotensin II -1 to increase Grow, develop or metabolic disorder.The gene therapy vector of restructuring（Such as viral vector）Angiotensins Π -1 receptor the GAP-associated protein GAPs of expression variance can be designed to, for suppressing the endogenic receptor of Angiotensin II -1 GAP-associated protein GAP activity.For example, a kind of receptor of Angiotensin II -1 GAP-associated protein GAP of variation can be the receptor GAP-associated protein GAP of Angiotensin II -1 for truncating, having lacked signal transduction 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 of the treatment receptor of Angiotensin II -1 correlative protein expression or active caused by abnormal.Expression vector such as retrovirus, adenovirus, adeno-associated virus, herpes simplex virus, parvovirus etc. from virus is intracellular available for the receptor related protein gene of Angiotensin II 1 is transferred to.Build and carry angiotensins

The method of the recombinant viral vector of II-1 receptor related protein genes is found in existing document（Sambrook, etc.）.Other recombinant vascular Angiotensin Converting Enzyme II-1 receptors related protein gene can be packaged into liposome be transferred to it is intracellular.

Suppress the receptor GAP-associated protein GAP of Angiotensin II -1 mRNA oligonucleotides（Including antisense R A and DNA) and ribozyme it is also within the scope of the invention.Ribozyme is a kind of specific RNA of energy specificity cutting enzyme sample RNA molecule, and its mechanism of action is to carry out endonuclease effect after ribozyme molecule hybridizes with complementary target RNA-specific.The RNA and DNA and ribozyme of antisense can be obtained with existing any synthesis RNA or DNA technology, and the technology of such as solid phase phosphoamide chemical synthesis synthetic oligonucleotide 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 DNA sequence dna has been integrated into the downstream of 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 phosphodiester bond between two Side sequence length, ribonucleotide.

Polynucleotides are imported into tissue or intracellular method includes：Polynucleotides are directly injected into in-vivo tissue；Or pass through carrier in vitro（Such as virus, bacteriophage or plasmid）First polynucleotides are imported in cell, then transplanted cells into internal etc.. Polypeptide and polynucleotides of the present invention also have other purposes, for example, polypeptide of the present invention can be used for peptide fingerprinting to compose identification, coded sequence of the invention or its fragment can be used for manufacture genetic chip or microarray.According to the teachings of the present invention, these applications have been obvious for those skilled in the art.The present invention will be further illustrated in the following examples, but is not to limit the present invention with this.Embodiment

Embodiment 1:The clone of the polynucleotides of the receptor GAP-associated protein GAP of encoding human Angiotensin II -1

Human fetal spleen total serum IgE is extracted with guanidinium isothiocyanate/phenol/chloroform one-step method.With Quik mRNA separating kits（Qiagene g poly (A) mRNA of poly (A) mRNA 2) are separated from total serum IgE through reverse transcription formation cDNA. Smart cDNA clone kits（Purchased from Clontech) cDNA fragments orientation is inserted into the multiple cloning sites of carrier, conversion escherichia coli DH5a formation cDNA library.3028 clones are obtained altogether.With the sequence of the 5 of all clones of dideoxy measure, and 3' ends.The cDNA sequences of measure are compared with existing public DNA sequence data storehouse, as a result find there is a clone（DNA sequence dna 0894e01) is new DNA.Two-way measure is carried out by synthesizing a series of contained DNA sequence dna of primer pair 0894e01 clones.Computer analysis shows, the contained full-length cDNA of 0894e01 clones is a new DNA sequence dna（As shown in Seq ID Nol）, from the 56th to 535 ORF for having a 479bp, encode a new protein（As shown in Seq ID No 2）, the sequence is named as pBS-0894e01.This protein is named as human angiotensin Π -1 receptor GAP-associated protein GAPs.Embodiment 2:With the related egg of RT-PCR method clone human angiotensin Π -1 receptors In vain：

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：Forward primer Fl 5 ,-GGGAGCCTAGGAGCCCCC-3, positioned at SEQ ID NOl section start；Reverse primer R1:5'- GAATTCCCCAAACTTTAATG -3' are located at SEQ ID NOl 1088- 1108bp.The condition of amplified reaction：Contain 50mmol/L KCl, 10mmol/L Tris-Cl, (pH8.5), 1.5mmol/L MgCl in 50 μ 1 reaction volume₂, 20 (^mol/L dNTP, 25pmol primers, 2.5U Taq archaeal dna polymerases.By following 25 circulations of conditioned response on PE9600 type DNA thermal cyclers：94TC 30 seconds；55X, 30 seconds; 72 X：2 minutes.Set that beta-actin is positive control and template blank is negative control simultaneously in RT-PCR.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 NOl l-1108bp.Embodiment 3, the Homology search of cDNA clones

The database such as the sequence of the polynucleotides of the human angiotensin II-1 receptor-related proteins provided with the present invention and its protein sequence of coding and Genbank^Swissport carries out tetraploid rice, program for retrieval is Blast (Basic local Alignment search tool) (1993 Proc. of US National Academy of Sciences, 90:5873-5877), Blast can find out homologous many genes, wherein the gene maximum with the genetic homology of the present invention, its albumen encoded is AF102548 in Genbank access number.These genes retrieved or protein sequence can be recalled from Genbank databases.The sequence recalled can use the Pileup (multisequencing in GCG software kits）With Gap (two sequences）Program, which is done even to match somebody with somebody, to be compared.The function prediction of protein can be analyzed with Motif programs.The human angiotensin II-1 receptors GAP-associated protein GAP that the result display present invention of Homology search is provided is related to the receptor of Angiotensin II -1 of rat Albumen has 76% homology, as follows with originality comparative result.

>Gb | AAD25997.1 | AF102548_l (AF102548) ATI receptor-related proteins [Mus musculus]

Score=622 (219.0 bits) of length=161, expect=9.7e-61, P=9.7e-61

The phase same sex=123/160 (76%), similitude=134/160 (83%)

Query : 1 MELPAVNLKVILLGHWLLTTWGCIVFSGSYAWANFTILALGVWAVAQRDSIDAISMFLGG 60

MELPAVNLKVILL HWLLTTWGC+VFS SYAW NFTILALGVWAVAQRDSIDAI MFLGG Sbjct : 1 MELPAVNLKVILLVHWLLTTWGCLVFSSSYAWGNFTILALGVWAVAQRDSIDAIGMFLGG 60

Query : 61 LLATIFLDIVHISIFYPRVSLTDTGRFGVGMAILSLLLKPLSCCFVYHMYRERGGELLVH 120

L+ATIFLDI++ISIFY V+ DTGRFG GMAILSLLLKP SCC VYHM+RERGGEL + Sbjct : 61 LVATIFLDIIYISIFYSSVATGDTGRFGAGMAILSLLLKPFSCCLVYHMHRERGGELPLR 120

Query : 121 TGFLGSSQDRSAYQTIDSA-EAPADPFAVPEGRSQDA-RG 158

F G SQ+ SAYQTIDS+ +A ADPFA E + Q A RG

Sbjct :The embodiments 4 of 121 PDFFGPSQEHSAYQTIDSSSDAAADPFASLENKGQAAPRG 160:The gene expression of Northern engram analysis human angiotensin II-l receptor GAP-associated protein GAPs

Total serum IgE [analytical chemistry is extracted with one-step method（ Anal. Biochem ) 1987, 162, 156-159】.The method includes acid guanidine thiocyanate phenol-chloroform extraction.Use 4M guanidinium isothiocyanate -25mM sodium citrates, 0.2M sodium acetates（PH4.0) tissue is homogenized, the stupid phenol of 1 times of volume of addition 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 by mixture Centrifugation obtains RNA precipitate.

Obtained RNA precipitate is washed with 70% ethanol, it is dry and soluble in water.With 2 (^g RNA, in (the N- morpholinoes 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.With³²The probe of P- marks（About 2xl0⁶Cpm/ml) in 42 in a solution, " hybridized overnight, the solution includes 50% formamide -25mM KH₂P0₄(pH7.4) -5xSSC-5xDenhardt, s solution and 200 g/ml salmon sperm DNAs.With α-³²Ρ dATP are prepared by random priming³²The DNA probe of Ρ-mark.DNA probe used is the receptor GAP-associated protein GAP coding region sequences of human angiotensin II- 1 or its fragment that PCR is expanded.After hybridization, filter membrane is washed 30 minutes in lxSSC-0.1%SDS in 65.Then, analyzed and quantified with Phosphor Imager '

As a result show, human angiotensin II-1 receptors related protein gene can specificity expression in testis tissue.Embodiment 5:Vivoexpression, separation and the purifying of the human angiotensin II-1 receptor GAP-associated protein GAPs of restructuring

Devise pair of primers at the initiation codon of the receptor related protein gene of Angiotensin II -1 and at terminator codon respectively, its 5, end be respectively provided with BamHI and EcoRI restriction enzyme sites.Enter performing PCR amplification by template of plasmid pBS-0894e01, obtain the receptor related protein gene of Angiotensin II -1 code area.Amplified fragments are inserted into expression vector PGEX-2T (being purchased from Pharmacia Biotech) by digestion, and convert escherichia coli DH5a, on the LB flat boards containing ampicillin and IPTG, 5 recombinant conversions for screening white carries out DNA sequence analysis, as a result identical with the coding sequence in pBS-0894e01.The recombinant clone can express GST- ATRAP fusion proteins, and clone's mark is pATRAP.

A ring escherichia coli DH5a bacterial strain is chosen, being inoculated in 20ml LB culture mediums, (benzyl containing ammonia is blue or green Mycin 100ug/ml), 37 " C shaken cultivations take seed liquor to be transferred by 2% inoculum concentration in 4 liters of LB culture mediums, 37 overnight as seed liquor " C shaken cultivations to thalline A₆₀₀When=0.7（Exponential phase）, IPTG is added to final concentration 0.4mmol/L, 25* is further cultured for and cultivates 12 hours, thalline is collected by centrifugation, and is washed with lxPBS with Slow fliud flushings A (16mM Na₂HP0₄, 4mM NaH₂P0₄, pH 6.5) and by 10ml/ grams of thalline dissolving, ultrasonication in water-bath, collected after centrifugation supernatant, supernatant crosses glutathione-Sepharose 4B chromatographic columns, after being eluted with Slow fliud flushings A, is eluted with the eluent of the reduced glutathione containing 5mM.Eluent shows that (there are GST- ATRAP fusion protein products at place to about 17.5kDa through SDS-PAGE.Embodiment 6：The preparation of the antibody of antiangiotensin Π -1 receptor GAP-associated protein GAPs

Use Peptide synthesizer（PE-ABI following specific polypeptides of the receptor of Angiotensin II -1 GAP-associated protein GAP) are synthesized：Ala-Pro-Ala-Asp-Pro-Phe-Ala-Val-Pro- Glu (141-150）.The polypeptide is coupled to form compound with hemocyanin and bovine serum albumin(BSA) respectively, method is referring to Avrameas. immunochemistries (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 IgG is 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 IgG with affinity chromatography.Immuno-precipitation proves that the receptor GAP-associated protein GAP of Angiotensin II -1 that the antibody of purifying can be specifically with the present invention is combined.Embodiment 7:Yeast two-hybrid assay

Plasmid prepares：

Mouse ATla acceptors（：Terminal cytoplasmic domain（ ATla C-ter;Amino acid 297-359) the Gal4 binding structural domains merged into yeast shuttle vector pBD-Gal4 (Stratagene) are expanded by PCR.Same method, and ATla acceptors tail (ATla C-ter 349,339, and 329) it is subcloned into pBD-Gal4 by PCR amplifications using the antisense primer of the terminator codon containing specific site.

Utilize 11^The tails of Jie 0, using the primers of XhoI- (dT) 18 and EcoR I connectors, the receptor of Angiotensin II -1 GAP-associated protein GAP is inserted into plasmid pAD- Gal4 (Stratagene).Double cross is screened：

Reporter gene containing two Gal4 inductions（HIS3 and LacZ) yeast strains YRG-2 (Stratagene) and ATla C-ter angiotensins Π -1 receptor GAP-associated protein GAP hybrid expression plasmids transcribe jointly, two kinds of transformants are in auxotroph（Try- Leu'His) medium culture three days, His+ colonies are added in selective medium, cellulose nitrate Membrance cuiture, surveys the dark activity of P-galactoside, the results are shown in Table the interaction of the ATRAP and ATla acceptor tail deletion mutants of a table one

Gal4 binding structural domain heterozygote Gal4 active structure domain heterozygote Gal units °/.With reference to %

ATla C-ter wild types ATRAP 160 100

ATla C-ter △ 349 ATRAP 60 37.5

ATla C-ter △ 339 ATRAP 0 0

ATla C-ter Δ 329 ATRAP 0 0

Claims (1)

1. 

   1. a kind of human angiotensin II -1 receptor GAP-associated protein GAPs of separation, it, which is included, has the polypeptide of amino acid sequence or its conservative variant shown in SEQ ID No. 2 or its active fragment or its reactive derivative.

   2. polypeptide as claimed in claim 1, it is the polypeptide with amino acid sequence shown in SEQ ID No. 2.

   3. a kind of polynucleotides of separation, its nucleotides sequence for including with being selected from the group is shown to the nucleotide sequence of the 70 % phase same sexes：

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

   (b) with（A) polynucleotides of the polynucleotides complementation in.

   4. polynucleotides as claimed in claim 3, it encodes the polypeptide with amino acid sequence shown in SEQ ID No. 2.

   5. polynucleotides as claimed in claim 3, it is a kind of polynucleotide sequence being selected from the group：

   (a) there is the polynucleotide sequence of 53-535 in SEQ ID No. 1；

   (b) there is the polynucleotide sequence of 1-1108 in SEQ ID No. 1.

   6. a kind of carrier, it contains the polynucleotides described in claim 3.

   7. a kind of genetically engineered host cell, it is a kind of host cell being selected from the group：

   (a) with the carrier conversion described in claim 6 or the host cell of transduction；

   (b) with the polynucleotides conversion described in claim 3 or the host cell of transduction.

   8. the preparation method of-kind of the polypeptide with human angiotensin II -1 receptors GAP-associated protein GAP activity, this method includes：

   (a) under conditions of the expression receptor GAP-associated protein GAP of Angiotensin II -1 is adapted to, the host cell described in culture claim 7；

   (b) separation has the receptor of Angiotensin II -1 GAP-associated protein GAP activity from culture Polypeptide.

   9. a kind of antibody that can be specifically bound with human angiotensin II -1 receptors GAP-associated protein GAP described in claim 1.

   10. a kind of screen simulation, promotion, antagonism or the method for suppressing the active compound of human angiotensin Π -1 receptor GAP-associated protein GAPs described in claim 1, including the use of the polypeptide described in claim 1.

   11. the compound that method according to claim 10 is obtained, it has simulation, promotion, antagonism or the activity for suppressing human angiotensin II -1 receptor GAP-associated protein GAPs described in claim 1.

   12. compound described in a kind of application claim 11 come adjust the receptor GAP-associated protein GAP of Angiotensin II -1 albumen in vivo, the method for external activity.

   13. the method for the neurological susceptibility of-kind of the detection disease related to the angiotensins Π -1 receptor GAP-associated protein GAP unconventionality expressions described in claim 1 or disease, it is characterised in that the mutation in the nucleotide sequence of the receptor GAP-associated protein GAP of Angiotensin II -1 described in detection coding claim 1.

   14. the purposes of polypeptide as claimed in claim 1, it is used to screening the activator for promoting the receptor of Angiotensin II -1 GAP-associated protein GAP activity, or screening suppresses the antagonist of angiotensins Π -1 receptors GAP-associated protein GAP activity or be used for peptide fingerprinting to know identification well.

   15.-kind of pharmaceutical composition, it contains the polypeptide and pharmaceutically acceptable carrier described in the claim 1 of therapeutically effective amount.

   16. the polypeptide of claim 1 or the compound of claim 11 are preparing the purposes in being used to treat the pharmaceutical composition with the abnormal relevant disease of activity of the receptor GAP-associated protein GAP of Angiotensin II -1.

   17. the purposes described in claim 16, wherein the disease is coronary heart disease, essential hypertension and Non-Insulin Dependent Diabetes Mellitus.