CN1302889A - Polypeptide-human protein 70 containing plasmolemma regulation function and polynucleotide for coding it - Google Patents

Abstract

A new polypeptide-plasmalemma regulation function-contained protein 70, the polynucleotide for encoding it, the process for preparing said polypeptide by DNA recombination, the application of said polypeptide to treating several diseases (cancer, HIV infection, etc.), the antagonist against the said polypeptide and its medical action, and the usage of said polynucleotide for encoding this polypeptide are disclosed.

Description

A kind of new polypeptide-human contains the protein 70 of plasma membrane adjusting function and the polynucleotide of this peptide species of coding

The invention belongs to biological technical field, specifically, the invention describes a kind of new polypeptide-human and contain the protein 70 of plasma membrane adjusting function, and the polynucleotide sequence of this polypeptide of encoding.The invention still further relates to the preparation method and the application of these polynucleotide and polypeptide.

The albumen that MUPP1 is made up of 13 PDZ domain is with the relevant PDZ domain height of the pdz protein homology of Human genome hINADL (human INAD like) and nematode gene C52A11.4 coding.These three kinds of albumen all are made up of a plurality of PDZ domain, and do not have tangible catalytic subunit.

PDZ domain has 90 amino-acid residues approximately, and often single pdz protein comprises a plurality of PDZ domain, and its title comes from three albumen: PSD95/DlgA/Z0-1 that find to comprise this functional domain at first.Most pdz proteins are attached on the plasma membrane, usually be limited in specific subcellular area, as the cell surface of cynapse, cell binding or top, cardinal extremity, side, infer that thus PDZ domain plays an important role in the function controlling in plasma membrane zone.The research of crystallography has disclosed the molecular structure of PDZ domain, and PDZ domain comprises 2 antiparallel laminated structures that 6 beta sheets constitute, and utilizes 2 alpha-helixs to connect.The proteic C end of great majority and PDZ domain effect has the characteristic sequence of T/SXV.New experimental evidence shows that there is more mode in PDZ domain with combining of specific protein, the S/TXV order inner as INAD identification TRP (transientreceptor potential); Directly effect takes place between PDZ domain, forms the homotype or the special-shaped polymers of pdz protein.By control with contain the proteic effect of PDZ domain regulation and control and comprise the temporary gathering of PDZdomain mixture, the activity of membrane-spanning protein, and propagate and wear film or along the signal transduction incident of plasma membrane.

The prevailing function of PDZ domain is with bonded transmembrane protein guiding with it, accumulates in specific ubcellular functional area.In the polar epithelium, pdz protein is positioned the plasma membrane zone that specific top, side and cell are connected, corresponding and its bonded membrane-spanning protein and the cytoplasmic protein the same area that also is positioned.Another basic function of pdz protein is in specific ubcellular territory membrane-spanning protein to be assembled and grappling, and the pdz protein of this function in epithelial cell and neuronal cell all is confirmed.Receptor protein and the tunnel protein aggregation of wearing film there is the important function meaning.The gathering of acceptor is considered to the prerequisite of receptor activation, and assembling simultaneously provides that collaborative activatory may not between isoacceptor and ionic channel.

PDZ domain and many plasmosin effects are by the additional membrane complex of the reaction of PDZ mediation.Wherein, PDZ domain is supplemented to specific ubcellular territory as membrane-spanning protein and endochylema fastening means with plasmosin.Many and plasmosin PDZ domain effect has participated in the approach of signal conduction, and these albumen comprise protein kinase, the proteic modulin of G, phospho-esterase c or the like.This shows the medium of PDZ domain as contact channel albumen, transmembrane receptor and downstream signal element.With INAD is example, and a series of heredity and biochemical test show that in most light transduction processes, the protein mediated signaling molecule of G all is incorporated into the PDZ domain of INAD, and INAD is made into the mixture that a signal transmits with these protein groups.Carry out the universal functionality that signal transduction is a cell by the proteic tissue of many PDZ domain, this molecular structure has the meaning of important function.At first, utilize the connection of physics to say that the signaling molecule in donor and downstream combines the efficient that has improved the signal transmission.The second, guaranteed the fidelity that signal transmits.The signaling molecule in many downstreams also participates in other reaction path, and signaling molecule is connected to acceptor in the mode of physical connection, makes it to separate from other reaction paths, has guaranteed specificity and fidelity that signal transmits.

Show cytoskeletal protein effects such as pdz protein and microtubule, Actin muscle by experimental evidence.Like this, by directly or indirectly acting on the pdz protein that is combined on the cytoskeleton, other albumen enrich in the reaction support, and formation one is the network system efficiently.

According to amino acid homology result relatively, polypeptide of the present invention is accredited as many PDZ of people domain albumen (Multi-PDZ domain protein), a kind of protein 70 that contains the plasma membrane adjusting function of called after by deduction.The homologous protein of polypeptide of the present invention is MUPP1, and albumen number is aj001319.Many PDZ of people domain protein 70

An object of the present invention is to provide protein 70 that isolating new polypeptide-human contains the plasma membrane adjusting function with and fragment, analogue and derivative.

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

Another object of the present invention provides and contains the recombinant vectors of polynucleotide that the coding people is contained the protein 70 of plasma membrane adjusting function.

Another object of the present invention provides and contains the genetically engineered host cell of polynucleotide that the coding people is contained the protein 70 of plasma membrane adjusting function.

Another object of the present invention provides the method that the people is contained the protein 70 of plasma membrane adjusting function of producing.

Another object of the present invention provides the antibody that contains the protein 70 of plasma membrane adjusting function at polypeptide-human of the present invention.

Another object of the present invention has provided simulated compound, antagonist, agonist, the inhibitor that contains the protein 70 of plasma membrane adjusting function at polypeptide-human of the present invention.

Another object of the present invention provides the method that diagnoses and treatment and people are contained the unusual relevant disease of the protein 70 of plasma membrane adjusting function.

In a first aspect of the present invention, provide novel isolated people to contain the protein 70 of plasma membrane adjusting function, this polypeptide is the people source, and it comprises: have the polypeptide of SEQ ID NO:2 aminoacid sequence or its conservative property variation polypeptide or its active fragments or its reactive derivative, analogue.Preferably, this polypeptide is the polypeptide with SEQ IDNO:2 aminoacid sequence.

In a second aspect of the present invention, the polynucleotide of isolating these polypeptide of coding are provided, these polynucleotide comprise a nucleotide sequence, and this nucleotide sequence is shown at least 70% homogeny with a kind of nucleotides sequence that is selected from down group: (a) the above-mentioned people of coding is contained the polynucleotide of the protein 70 of plasma membrane adjusting function; (b) with polynucleotide (a) complementary polynucleotide.Preferably, this polynucleotide encoding has the polypeptide of aminoacid sequence shown in the SEQ ID NO:2.More preferably, the sequence of these polynucleotide is be selected from down group a kind of: the sequence that (a) has 236-2134 position among the SEQ ID NO:1; (b) has the sequence of 1-3737 position among the SEQ ID NO:1.

In a third aspect of the present invention, the carrier that contains above-mentioned polynucleotide is provided, and has been transformed or host cell of transduceing or the host cell that is directly transformed or transduce by above-mentioned polynucleotide by this carrier.

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

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

As used herein, " isolating people is contained the protein 70 of plasma membrane adjusting function " is meant that the people contains the protein 70 of plasma membrane adjusting function and be substantially free of natural relative other albumen, lipid, carbohydrate or other material.Those skilled in the art can be contained the protein 70 of plasma membrane adjusting function with the purified technology of protein purifying people of standard.Basically pure polypeptide can produce single master tape on non-reduced polyacrylamide gel.The purity that the people is contained the protein 70 polypeptide of plasma membrane adjusting function can be used amino acid sequence analysis.

The invention provides the protein 70 that a kind of new polypeptide-human contains the plasma membrane adjusting function, it is made up of the aminoacid sequence shown in the SEQ ID NO:2 basically.Polypeptide of the present invention can be recombinant polypeptide, natural polypeptides, synthetic polypeptide, preferred recombinant polypeptide.Polypeptide of the present invention can be the product of natural purifying, or the product of chemosynthesis, or uses recombinant technology to produce from protokaryon or eucaryon host (for example, bacterium, yeast, higher plant, insect and mammalian cell).The host used according to the recombinant production scheme, polypeptide of the present invention can be glycosylated, maybe can be nonglycosylated.Polypeptide of the present invention also can comprise or not comprise initial methionine residues.

The present invention comprises that also the people contains the fragment of the protein 70 of plasma membrane adjusting function, derivative and analogue.As used herein, term " fragment ", " derivative " are meant identical biological function or the active polypeptide of protein 70 that keeps people of the present invention to contain the plasma membrane adjusting function basically with " analogue ".The fragment of polypeptide of the present invention, derivative or analogue can be: (I) is a kind of like this, wherein one or more amino-acid residues are replaced by conservative or non-conservative amino acid residues (preferably conservative amino acid residues), and the amino acid that replaces can be also can not encoded by genetic codon; Perhaps (II) is a kind of like this, and certain group on wherein one or more amino-acid residues is replaced by other group and comprises substituting group; Perhaps (III) is a kind of like this, and wherein mature polypeptide and another kind of compound (such as the compound that prolongs the polypeptide transformation period, for example polyoxyethylene glycol) merge; Perhaps (IV) is a kind of like this, wherein additional aminoacid sequence is integrated into mature polypeptide and the peptide sequence that forms (as leader sequence or secretion sequence or be used for the sequence or the proteinogen sequence of this polypeptide of purifying) by the elaboration of this paper, such fragment, derivative and analogue are considered within those skilled in the art's ken.

The invention provides isolating nucleic acid (polynucleotide), substantially the polynucleotide that have a polypeptide of SEQ ID NO:2 aminoacid sequence by coding are formed.Polynucleotide sequence of the present invention comprises the nucleotide sequence of SEQ ID NO:1.Polynucleotide of the present invention are to find from the cDNA library of people's fetal brain tissue.The polynucleotide sequence total length that it comprises is 3737 bases, its open reading frame (236-2134) 632 amino acid of having encoded.Find relatively that according to amino acid sequence homologous this polypeptide and homologous protein MUPP1 have 33% homology, deducibility goes out the protein 70 that this people contains the plasma membrane adjusting function and has the similar 26S Proteasome Structure and Function of homologous protein MUPP1.

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

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

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

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

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

The invention still further relates to nucleic acid fragment with sequence hybridization described above.As used herein, " nucleic acid fragment " length contain 10 Nucleotide at least, better be 20-30 Nucleotide at least, be more preferably 50-60 Nucleotide at least, preferably more than at least 100 Nucleotide.The amplification technique (as PCR) that nucleic acid fragment also can be used for nucleic acid is to determine and/or to separate the polynucleotide that the coding people is contained the protein 70 of plasma membrane adjusting function.

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

The special polynucleotide sequence that coding people of the present invention is contained the protein 70 of plasma membrane adjusting function can obtain with several different methods.For example, separate polynucleotide with hybridization technique well known in the art.These technology including, but not limited to: 1) with probe and genome or the hybridization of cDNA library to detect homologous polynucleotide sequence and 2) antibody screening of expression library to be to detect the polynucleotide passage of the clone with common structure feature.

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

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

Available ordinary method is screened gene of the present invention from these cDNA libraries.These methods include, but is not limited to: (1) DNA-DNA or DNA-RNA hybridization; (2) appearance of marker gene function or forfeiture; (3) measure the level of transcript that the people is contained the protein 70 of plasma membrane adjusting function; (4), detect the protein product of genetic expression by immunological technique or mensuration biologic activity.Aforesaid method can singly be used, but also several different methods combined utilization.

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

In (4) kind method, detect the people and contain the protein product of the protein 70 genetic expression of plasma membrane adjusting function and can use immunological technique such as Western blotting, radioimmunoprecipitation, enzyme-linked immunosorbent assay (ELISA) etc.

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

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

The present invention also relates to comprise the carrier of polynucleotide of the present invention, and with carrier of the present invention or directly personnel selection contain the host cell that the protein 70 encoding sequence of plasma membrane adjusting function produces through genetically engineered, and the method that produces polypeptide of the present invention through recombinant technology.

Among the present invention, the polynucleotide sequence that the coding people is contained the protein 70 of plasma membrane adjusting function can be inserted in the carrier, contains the recombinant vectors of polynucleotide of the present invention with formation.Term " carrier " refers to that bacterial plasmid well known in the art, phage, yeast plasmid, vegetable cell virus, mammalian cell virus are as adenovirus, retrovirus or other carrier.The carrier of Shi Yonging includes but not limited in the present invention: and the expression vector based on the T7 promotor of in bacterium, expressing (Rosenberg, et al.Gene, 1987,56:125); The pMSXND expression vector of in mammalian cell, expressing (Lee and Nathans, J Bio Chem.263:3521,1988 and at the carrier that derives from baculovirus of expressed in insect cells.In a word, as long as can duplicate in host and stablize, any plasmid and carrier may be used to make up recombinant expression vector.A key character of expression vector is to contain replication origin, promotor, marker gene and translational control element usually.

Method well-known to those having ordinary skill in the art can be used to make up and contain the coding people and contain the dna sequence dna of protein 70 of plasma membrane adjusting function and the expression vector of suitable transcribing/translational control element.These methods comprise (Sambroook, et al.Molecular Cloning, a Laboratory Manual, cold Spring Harbor Laboratory.New York, 1989) such as extracorporeal recombinant DNA technology, DNA synthetic technology, the interior recombinant technologys of body.Described dna sequence dna can effectively be connected on the suitable promotor in the expression vector, and is synthetic to instruct mRNA.The representative example of these promotors has: colibacillary lac or trp promotor; The PL promotor of lambda particles phage; Eukaryotic promoter comprises LTRs and some other known may command gene expression promoter in prokaryotic cell prokaryocyte or eukaryotic cell or its virus of CMV immediate early promoter, HSV thymidine kinase promoter, early stage and late period SV40 promotor, retrovirus.Expression vector also comprises ribosome bind site that translation initiation is used and transcription terminator etc.Inserting enhancer sequence in carrier will make its transcribing in higher eucaryotic cells be enhanced.Enhanser is the cis acting factor that DNA expresses, and nearly 10 to 300 base pairs act on promotor transcribing with enhancing gene usually.Can for example be included in the SV40 enhanser of 100 to 270 base pairs of replication origin side in late period one, at the polyoma enhanser of replication origin side in late period one and adenovirus enhanser etc.

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

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

Among the present invention, the coding people is contained the polynucleotide of protein 70 of plasma membrane adjusting function or the recombinant vectors that contains these polynucleotide can transform or transduce into host cell, contains the genetically engineered host cell of these polynucleotide or recombinant vectors with formation.Term " host cell " refers to prokaryotic cell prokaryocyte, as bacterial cell; Or eukaryotic cell such as low, as yeast cell; Or higher eucaryotic cells, as mammalian cell.Representative example has: intestinal bacteria, streptomyces; Bacterial cell such as Salmonella typhimurium; Fungal cell such as yeast; Vegetable cell; Insect cell such as fruit bat S2 or Sf9; Zooblast such as CHO, COS or Bowes melanoma cells etc.

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

By the recombinant DNA technology of routine, utilize polynucleotide sequence of the present invention to can be used to express or the people that produces reorganization is contained protein 70 (Science, 1984 of plasma membrane adjusting function; 224:1431).In general following steps are arranged:

(1). everybody contains the polynucleotide (or varient) of the protein 70 of plasma membrane adjusting function with coding of the present invention, or transforms or the transduction proper host cell with the recombinant expression vector that contains these polynucleotide;

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

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

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

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

Application Areas:

According to the function of homologous protein, infer that polypeptide of the present invention has participated in the tissue regulation and control of cytoplasmic membrane regional function, has mediated intercellular signal transmission.Signal transmission between the neuronal cell cynapse of pdz protein mediation is relevant with the learning and memory function.May have certain effect for the obstacle that improves correlation function.

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

The present invention also provides SCREENED COMPOUND to identify the method that improves (agonist) or check the medicament of the protein 70 that (antagonist) people contains the plasma membrane adjusting function.Agonist improves protein 70 that the people contains plasma membrane adjusting function biological function such as stimulate cellular proliferation, and antagonist prevention disorder such as the various cancer relevant with cell hyperproliferation with treatment.For example, can be in the presence of medicine, mammalian cell or expressing human are contained the protein 70 that the film preparation of the protein 70 of plasma membrane adjusting function contains the plasma membrane adjusting function with the people of mark cultivate.Measure the medicine raising then or check this interactional ability.

The antagonist that the people is contained the protein 70 of plasma membrane adjusting function comprises antibody, compound, acceptor disappearance thing and the analogue etc. that filter out.The antagonist that the people is contained the protein 70 of plasma membrane adjusting function can combine and eliminate its function with the protein 70 that the people is contained the plasma membrane adjusting function, or suppresses the generation of this polypeptide, or combines with the avtive spot of this polypeptide and to make this polypeptide can not bring into play biological function.

In screening during as the compound of antagonist, the protein 70 that the people can be contained the plasma membrane adjusting function adds during bioanalysis measures, and the people is contained between the protein 70 of plasma membrane adjusting function and its acceptor interactional influence determines whether compound is antagonist by measuring compound.With the same quadrat method of above-mentioned SCREENED COMPOUND, can filter out the acceptor disappearance thing and the analogue of antagonist action.Can be incorporated into the rondom polypeptide storehouse that solid formation forms by the various amino acid that may make up by screening with the protein 70 bonded peptide molecule that the people is contained the plasma membrane adjusting function obtains.During screening, generally tackle the people and contain the protein 70 molecule of plasma membrane adjusting function and carry out mark.

The invention provides and use polypeptide, and fragment, derivative, analogue or their cell are as the method for antigen with production antibody.These antibody can be polyclonal antibody or monoclonal antibody.The present invention also provides the antibody that contains the protein 70 antigenic determinant of plasma membrane adjusting function at the people.These antibody include, but is not limited to: the fragment that polyclonal antibody, monoclonal antibody, chimeric antibody, single-chain antibody, Fab fragment and Fab expression library produce.

Can the choose method of the protein 70 direct injection immune animal (as rabbit, mouse, rat etc.) that contains the plasma membrane adjusting function of the production of polyclonal antibody obtains, and multiple adjuvant can be used for the enhancing immunity reaction, includes but not limited to freund's adjuvant etc.The technology of monoclonal antibody that the preparation people is contained the protein 70 of plasma membrane adjusting function includes but not limited to hybridoma technology (Kohler and Milstein. Nature, 1975,256:495-497), three knurl technology, people B-quadroma technology, EBV-hybridoma technology etc.With the variable region bonded chimeric antibody in human constant region and inhuman source can with existing technology production (Morrison et al, PNAS, 1985,81:6851).And the technology of existing manufacture order chain antibody (U.S. Pat No.4946778) also can be used for producing the single-chain antibody that anti-people is contained the protein 70 of plasma membrane adjusting function.

The antibody that anti-people is contained the protein 70 of plasma membrane adjusting function can be used in the immunohistochemistry technology, and the people in the detection biopsy specimen is contained the protein 70 of plasma membrane adjusting function.

Contain the also available labelled with radioisotope of protein 70 bonded monoclonal antibody of plasma membrane adjusting function with the people, inject in the body and can follow the tracks of its position and distribution.This radiolabeled antibody can be used as a kind of atraumatic diagnostic method and is used for the location of tumour cell and has judged whether transfer.

Antibody also can be used for designing the immunotoxin at a certain privileged sites in the body.Contain as the people plasma membrane adjusting function the protein 70 high-affinity monoclonal antibody can with bacterium or plant poison (as diphtheria toxin, ricin, abrine etc.) covalent attachment.A kind of usual method is with sulfydryl linking agent such as SPDP, attacks the amino of antibody, by the exchange of disulfide linkage, toxin is incorporated on the antibody, and this hybrid antibody can be used for killing the protein 70 positive cells that the people is contained the plasma membrane adjusting function.

Antibody among the present invention can be used for treating or prevention and people are contained the relevant disease of protein 70 of plasma membrane adjusting function.The antibody that gives suitable dosage can stimulate or block generation or the activity that the people is contained the protein 70 of plasma membrane adjusting function.

The invention still further relates to quantitatively and the detection and localization people is contained the diagnostic testing process of the protein 70 level of plasma membrane adjusting function.These tests are known in the art, and comprise that FISH measures and radioimmunoassay.The people who is detected in the test is contained the protein 70 level of plasma membrane adjusting function, can be with the disease that the people that lays down a definition is contained the importance of protein 70 in various diseases of plasma membrane adjusting function and the protein 70 that is used to diagnose the people to contain the plasma membrane adjusting function works.

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

The polynucleotide that the coding people is contained the protein 70 of plasma membrane adjusting function also can be used for multiple therapeutic purpose.Gene therapy technology can be used for treating because the people contains that the nothing of the protein 70 of plasma membrane adjusting function is expressed or cell proliferation, growth or the metabolic disturbance of unusual/non-activity due to expressing.The gene therapy vector (as virus vector) of reorganization can be designed for the protein 70 that the people who expresses variation is contained the plasma membrane adjusting function, contains the protein 70 activity of plasma membrane adjusting function to suppress endogenic people.For example, the protein 70 that a kind of people of variation is contained the plasma membrane adjusting function can be that the people who shortens, lacked signal conduction function territory is contained the protein 70 of plasma membrane adjusting function, though can combine with the substrate in downstream, lacks signaling activity.Therefore the gene therapy vector of reorganization can be used for treating the people and contains the protein 70 expression of plasma membrane adjusting function or the disease of active caused by abnormal.Deriving from the polynucleotide that the expression vector of virus such as retrovirus, adenovirus, adeno-associated virus (AAV), hsv, parvovirus etc. can be used for the coding people is contained the protein 70 of plasma membrane adjusting function is transferred in the cell.The method of recombinant viral vector that structure carries the polynucleotide of the protein 70 that the coding people contains the plasma membrane adjusting function is found in existing document (Sambrook, et al.).The reorganization coding people polynucleotide that contain the protein 70 of plasma membrane adjusting function can be packaged in the liposome and be transferred in the cell in addition.

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

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

The polynucleotide that the coding people is contained the protein 70 of plasma membrane adjusting function can be used for containing with the people diagnosis of relative disease of the protein 70 of plasma membrane adjusting function.The polynucleotide that the coding people is contained the protein 70 of plasma membrane adjusting function can be used for detecting the people and contain the unconventionality expression that expression people whether or under morbid state of the protein 70 of plasma membrane adjusting function is contained the protein 70 of plasma membrane adjusting function.As the people that the encodes dna sequence dna that contains the protein 70 of plasma membrane adjusting function can be used for biopsy specimen is hybridized to judge that the people contains the expression situation of the protein 70 of plasma membrane adjusting function.Hybridization technique comprises the Southern blotting, Northern blotting, in situ hybridization etc.These technological methods all are disclosed mature technologies, and relevant test kit all can obtain from commercial channels.Part or all of polynucleotide of the present invention can be used as probe stationary on microarray (Microarray) or DNA chip (being called " gene chip " again), is used for analyzing the differential expression analysis and the gene diagnosis of tissue gene.The special primer of protein 70 that personnel selection contains the plasma membrane adjusting function carries out RNA-polymerase chain reaction (RT-PCR) amplification in vitro and also can detect the transcription product that the people is contained the protein 70 of plasma membrane adjusting function.

Detecting the people contains the sudden change of the protein 70 gene of plasma membrane adjusting function and also can be used for the relevant disease of protein 70 of diagnosing the people to contain the plasma membrane adjusting function.The form that the people is contained the protein 70 sudden change of plasma membrane adjusting function comprises that to contain point mutation that the protein 70 dna sequence dna of plasma membrane adjusting function compares, transposition, disappearance, reorganization and other any unusual etc. with the normal wild type people.Available existing technology such as Southern blotting, dna sequence analysis, PCR and in situ hybridization detect sudden change.In addition, sudden change might influence proteic expression, therefore can judge indirectly that with Northern blotting, Western blotting gene has or not sudden change.

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

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

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

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

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

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

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

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

Pharmaceutical composition can be with mode administration easily, as by in part, intravenously, intraperitoneal, intramuscular, subcutaneous, the nose or the route of administration of intracutaneous.The protein 70 that the people is contained the plasma membrane adjusting function comes administration with the amount that treats and/or prevents concrete indication effectively.Amount and dosage range that the people who is applied to the patient is contained the protein 70 of plasma membrane adjusting function will depend on many factors, as administering mode, person's to be treated healthiness condition and diagnostician's judgement.

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

Fig. 1 is that the inventor is contained the protein 70 of plasma membrane adjusting function and the amino acid sequence homology comparison diagram of homologous protein MUPP1.The top sequence is the protein 70 that the people is contained the plasma membrane adjusting function, and the below sequence is homologous protein MUPP1.Same amino acid represents with monocase amino acid that between two sequences similar amino acid is represented with "+".

Fig. 2 contains the polyacrylamide gel electrophoresis figure (SDS-PAGE) of the protein 70 of plasma membrane adjusting function for isolating people.70kDa is proteinic molecular weight.The arrow indication is isolated protein band.

Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually according to people such as normal condition such as Sambrook, molecular cloning: laboratory manual (New York:ColdSpring Harbor Laboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.Embodiment 1: the people is contained the clone of the protein 70 of plasma membrane adjusting function

Extract the total RNA of people's tire brain with guanidinium isothiocyanate/phenol/chloroform single stage method.From total RNA, separate poly (A) mRNA with Quik mRNA Isolation Kit (Qiegene company product).2ug poly (A) mRNA forms cDNA through reverse transcription.CDNA fragment orientation is inserted on the multiple clone site of pBSK (+) carrier (Clontech company product) with Smart cDNA clone's test kit (available from Clontech), transforms DH5 α, bacterium forms the cDNA library.Measure all clones' 5 ' and 3 ' terminal sequence with Dyeterminate cycle reaction sequencing kit (Perkin-Elmer company product) and ABI 377 automatic sequencers (Perkin-Elmer company).CDNA sequence and the existing public dna sequence data storehouse (Genebank) measured are compared, found that one of them clone's 0409 cDNA sequence is new DNA.By synthetic a series of primers the contained insertion cDNA fragment of this clone is carried out two-way mensuration.The result shows, the contained full-length cDNA of 0409 clone is 3737bp (shown in Seq ID NO:1), from 236bp to 2134bp the open reading frame (ORF) of a 632bp, the new protein (shown in Seq IDNO:2) of encoding arranged.We are with this clone's called after pBS-0409, and the name of encoded protein matter is behaved and contained the protein 70 of plasma membrane adjusting function.

Embodiment 2:cDNA clone's homology retrieval

The sequence and the encoded protein sequence thereof that people of the present invention are contained the protein 70 of plasma membrane adjusting function are with Blast program (Basiclocal Alignment search tool) [Altschul, SF et al.J.Mol.Biol.1990; 215:403-10], carry out the homology retrieval at databases such as Genbank, Swissport.Containing the highest gene of the protein 70 homology of plasma membrane adjusting function with people of the present invention is a kind of known MUPP1, and its encoded protein number is aj001319 in the access of Genbank.Protein homology the results are shown in Fig. 1, both height homologies, and its homogeny is 33%; Similarity is 54%.Embodiment 3: the gene that contains the protein 70 of plasma membrane adjusting function with RT-PCR method clones coding people

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

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

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

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

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

The condition of amplified reaction: in the reaction volume of 50 μ l, contain 50mmol/L KCl, 10mmol/L Tris-Cl, (pH8.5), 1.5mmol/L MgCl ₂, 200 μ mol/L dNTP, 10pmol primer, the Taq archaeal dna polymerase of 1U (Clontech company product).Go up by 25 cycles of following conditioned response at PE9600 type DNA thermal cycler (Perkin-Elmer company): 94 ℃ of 30sec; 55 ℃ of 30sec; 72 ℃ of 2min.When RT-PCR, establish the blank negative contrast of positive contrast of β-actin and template simultaneously.Amplified production is connected to (Invitrogen company product) on the pCR carrier with the test kit purifying of QIAGEN company with TA clone test kit.The dna sequence analysis result shows that the dna sequence dna of PCR product and the 1-3737bp shown in the SEQ ID NO:1 are identical.Embodiment 4:Northern blotting analyst is contained the protein 70 expression of gene of plasma membrane adjusting function:

Extract total RNA[Anal.Biochem 1987,162,156-159 with single stage method].This method comprises acid guanidine thiocyanate phenol-chloroform extracting.Promptly use 4M guanidinium isothiocyanate-25mM Trisodium Citrate, 0.2M sodium acetate (pH4.0) carries out homogenate to tissue, adds the phenol of 1 times of volume and the chloroform-primary isoamyl alcohol (49: 1) of 1/5 volume, and is centrifugal after mixing.The sucking-off aqueous phase layer adds Virahol (0.8 volume) and with the centrifugal RNA precipitation that obtains of mixture.With RNA precipitation 70% washing with alcohol that obtains, dry and soluble in water.With 20 μ g RNA, on 1.2% sepharose that contains 20mM 3-(N-morpholino) propanesulfonic acid (pH7.0)-5mM sodium acetate-1mM EDTA-2.2M formaldehyde, carry out electrophoresis.Be transferred on the nitrocellulose filter then.With α- ³²P dATP prepares by random priming ³²The dna probe of P-mark.Used dna probe is the protein 70 coding region sequence (236bp to 2134bp) that the people of pcr amplification shown in Figure 1 is contained the plasma membrane adjusting function.Probe (about 2 * 10 with the 32P-mark ⁶Cpm/ml) spend the night in 42 ℃ of hybridization in a solution with the nitrocellulose filter that has shifted RNA, this solution comprises 50% methane amide-25mM KH ₂PO ₄(pH7.4)-5 * SSC-5 * Denhardt ' s solution and 200 μ g/ml salmon sperm DNAs.After the hybridization, filter membrane is washed 30min in 55 ℃ in 1 * SSC-0.1%SDS.Then, analyze with quantitative with Phosphor Imager.Embodiment 5: recombinant human contains vivoexpression, separation and the purifying of the protein 70 of plasma membrane adjusting function

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

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

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

5 ' end of these two sections primers contains Nde I and BamH I restriction enzyme site respectively, be respectively the encoding sequence of target gene 5 ' end and 3 ' end thereafter, Nde I and BamH I restriction enzyme site are corresponding to expression vector plasmid pET-28b (+) (Novagen company product, Cat.No.69865.3) the selectivity restriction enzyme site on.With the pBS-0409 plasmid that contains the total length goal gene is template, carries out the PCR reaction.The PCR reaction conditions is: contain pBS-0409 plasmid 10pg, primer Primer-3 and Primer-4 among the cumulative volume 50 μ l and divide other to be 10pmol, Advantage polymera se Mix (Clontech company product) 1 μ l.Loop parameter: 94 ℃ of 20s, 60 ℃ of 30s, 68 ℃ of 2min, totally 25 circulations.Respectively amplified production and plasmid pET-28 (+) are carried out double digestion with Nde I and BamH I, reclaim big fragment respectively, and connect with the T4 ligase enzyme.Connect product and transform, after the dull and stereotyped overnight incubation of the LB that contains kantlex (final concentration 30 μ g/ml), use the colony polymerase chain reaction (PCR) method screening positive clone, and check order with the big enterobacterial DH5 of Calcium Chloride Method α.Select the correct positive colony of sequence (pET-0409) with Calcium Chloride Method with recombinant plasmid transformed e. coli bl21 (DE3) plySs (Novagen company product).In the LB liquid nutrient medium that contains kantlex (final concentration 30 μ g/ml), host bacterium BL21 (pET-0409) is cultured to logarithmic phase at 37 ℃, adds IPTG to final concentration 1mmol/L, continues to cultivate 5 hours.Centrifugal collection thalline, through the broken bacterium of ultrasonic wave, centrifugal collection supernatant, with carrying out chromatography with 6 Histidines (6His-Tag) bonded affinity column His.Bind Quick Cart ridge (Novagen company product), the target protein people who has obtained purifying is contained the protein 70 of plasma membrane adjusting function.Through the SDS-PAGE electrophoresis, obtain a single band (Fig. 2) at the 70kDa place.This band is transferred on the pvdf membrane carries out the n terminal amino acid sequential analysis with the Edams hydrolysis method, 15 amino acid of N-end hold 15 amino-acid residues identical with the N-shown in the SEQ ID NO:2 as a result.Embodiment 6 anti-people are contained the protein 70 production of antibodies of plasma membrane adjusting function

The specific polypeptide of protein 70 that contains the plasma membrane adjusting function with the synthetic following people of Peptide synthesizer (PE company product):

NH ₂-Met-Lys-Ala-Leu-Leu-Leu-Leu-Val-Leu-Pro-Trp-Leu-Ser-Pro-Ala-COOH(SEQ?ID?NO:7)。Form compoundly with hemocyanin and bovine serum albumin coupling this polypeptide respectively, method is referring to Avrameas, et al. Immunochemistry, 1969; 6:43.Add the complete Freund's adjuvant immunizing rabbit with the above-mentioned hemocyanin polypeptide complex of 4mg, add the incomplete Freund's adjuvant booster immunization once with the hemocyanin polypeptide complex again after 15 days.Employing is done the titre that ELISA measures antibody in the rabbit anteserum through the titer plate of 15 μ g/ml bovine serum albumin polypeptide complex bag quilts.From the rabbit anteserum of antibody positive, separate total IgG with albumin A-Sepharose.Polypeptide is incorporated on the Sepharose4B post of cyanogen bromide-activated, from total IgG, separates anti-peptide antibody with affinity chromatography.The protein 70 that immuno-precipitation proof antibody purified can be specifically with the people is contained the plasma membrane adjusting function combines.

Sequence table (1) general information: (ⅱ) denomination of invention: the people is contained the protein 70 and encoding sequence (ⅲ) the sequence number thereof of plasma membrane adjusting function: the information of 7 (2) SEQ ID NO:1: (ⅰ) sequence signature:

(A) length: 3737bp

(B) type: nucleic acid

(C) chain: two strands

( D ) ： ( ⅱ ) ：cDNA ( ⅹⅰ ) ：SEQ ID NO:1： 1 CCAACCGTGGTGGCTCCTTGCGTTCCTACATCCTCTCATCTGAGAATCAGAGAGCATAAT 61 CTTCTTACGGGCCCGTGATTTATTAACGTGGCTTAATCTGAAGGTTCTCAGTCAAATTCT121 TTGTGATCTACTGATTGTGGGGGCATGGCAAGGTTTGCTTAAAGGAGCTTGGCTGGTTTG181 GGCCCTTGTAGCTGACAGAAGGTGGCCAGGGAGAAGGCAGCACACTGCTCGGAGAATGAA241 GGCGCTTCTGTTGCTGGTCTTGCCTTGGCTCAGTCCTGCTAACTACATTGACAATGTGGG301 CAACCTGCACTTCCTGTATTCAGAACTCTGTAAAGGTGCCTTCCACTACGGGCTGACCAA361 AGATAGGAAGAGGCGCTCACAAGATGGCTGTCCAGACGGCTGTGCGAGCCTCACAGCCAC421 GGCTCCCTCCCCAGAGGTTTCTGCAGCTGCCACCATCTCCTTAATGACAGACGAGCCTGG481 CCTAGACAACCCTGCCTACGTGTCCTCGGCAGAGGACGGGCAGCCAGCAATCAGCCCAGT541 GGACTCTGGCCGGAGCAACCGAACTAGGGCACGGCCCTTTGAGAGATCCACTATTAGAAG601 CAGATCATTTAAAAAAATAAATCGAGCTTTGAGTGTTCTTCGAAGGACAAAGAGCGGGAG661 TGCAGTTGCCAACCATGCCGACCAGGGCAGGGAAAATTCTGAAAACACCACTGCCCCTGA721 AGTCTTTCCAAGGTTGTACCACCTGATTCCAGATGGTGAAATTACCAGCATCAAGATCAA781 TCGAGTAGATCCCAGTGAAAGCCTCTCTATTAGGCTGGTGGGAGGTAGCGAAACCCCACT841 GGTCCATATCATTATCCAACACATTTATCGTGATGGGGTGATCGCCAGAGACGGCCGGCT901 ACTGCCAGGAGACATCATTCTAAAGGTCAACGGGATGGACATCAGCAATGTCCCTCACAA 961 CTACGCTGTGCGTCTCCTGCGGCAGCCCTGCCAGGTGCTGTGGCTGACTGTGATGCGTGA1021 ACAGAAGTTCCGCAGCAGGAACAATGGACAGGCCCCGGATGCCTACAGACCCCGAGATGA1081 CAGCTTTCATGTGATTCTCAACAAAAGTAGCCCCGAGGAGCAGCTTGGAATAAAACTGGT1141 GCGCAAGGTGGATGAGCCTGGGGTTTTCATCTTCAATGTGCTGGATGGCGGTGTGGCATA1201 TCGACATGGTCAGCTTGAGGAGAATGACCGTGTGTTAGCCATCAATGGACATGATCTTCG1261 ATATGGCAGCCCAGAAAGTGCGGCTCATCTGATTCAGGCCAGTGAAAGACGTGTTCACCT1321 CGTCGTGTCCCGCCAGGTTCGGCAGCGGAGCCCTGACATCTTTCAGGAAGCCGGCTGGAA1381 CAGCAATGGCAGCTGGTCCCCAGGGCCAGGGGAGAGGAGCAACACTCCCAAGCCCCTCCA1441 TCCTACAATTACTTGTCATGAGAAGGTGGTAAATATCCAAAAAGACCCCGGTGAATCTCT1501 CGGCATGACCGTCGCAGGGGGAGCATCACATAGAGAATGGGATTTGCCTATCTATGTCAT1561 CAGTGTTGAGCCCGGAGGAGTCATAAGCAGAGATGGAAGAATAAAAACAGGTGACATTTT1621 GTTGAATGTGGATGGGGTCGAACTGACAGAGGTCAGCCGGAGTGAGGCAGTGGCATTATT1681 GAAAAGAACATCATCCTCGATAGTACTCAAAGCTTTGGAAGTCAAAGAGTATGAGCCCCA1741 GGAAGACTGCAGCAGCCCAGCAGCCCTGGACTCCAACCACAACATGGCCCCACCCAGTGA1801 CTGGTCCCCATCCTGGGTCATGTGGCTGGAATTACCACGGTGCTTGTATAACTGTAAAGA1861 TATTGTATTACGAAGAAACACAGCTGGAAGTCTGGGCTTCTGCATTGTAGGAGGTTATGA1921 AGAATACAATGGAAACAAACCTTTTTTCATCAAATCCATTGTTGAAGGAACACCAGCATA1981 CAATGATGGAAGAATTAGATGTGGTGATATTCTTCTTGCTGTCAATGGTAGAAGTACATC2041 AGGAATGATACATGCTTGCTTGGCAAGACTGCTGAAAGAACTTAAAGGAAGAATTACTCT2101 AACTATTGTTTCTTGGCCTGGCACTTTTTTATAGAATCAATGATGGGTCAGAGGAAAACA2161 GAAAAATCACAAATAGGCTAAGAAGTTGAAACACTATATTTATCTTGTCAGTTTTTATAT2221 TTAAAGAAAGAATACATTGTAAAAATGTCAGGAAAAGTATGATCATCTAATGAAAGCCAG2281 TTACACCTCAGAAAATATGATTCCAAAAAAATTAAAACTACTAGTTTTTTTTCAGTGTGG2341 AGGATTTCTCATTACTCTACAACATTGTTTATATTTTTTCTATTCAATAAAAAGCCCTAA2401 AACAACTAAAATGATTTGTATACCCCACTGAATTCAAGCTGATTTAAATTTAAAATTTGG2461 TATATGCTGAAGTCTGCCAAGGGTACATTATGGCCATTTTTAATTTACAGCTAAAATATT2521 TTTTAAAATGCATTGCTGAGAAACGTTGCTTTCATCAAACAAGAATAAATATTTTTCAGA2581 AGTTATAGTTGTCTTTTAGTATGTGATACTAATTAAGATTACTTTTGTATTATCACTATT2641 TAAAAGATCCTAGTAATTTATTCTTTCAAATACCATGTTATTTGTTACCATCACCGATGA2701 ATACCTCCTAGGCTTATCCCTAAAAATGCTCGCTCAGAGAATTAATTATAAACTTGTTTT2761 GTTTTTAGTAAGAAATGGCTAAAGCTCTTTTTTTTCCACAATCGTTAGTAACTGTATAAA2821 AACTCATGCTGCTCCACCAGTGGGCCTTGGAAAATGCATCAAGAAGGCCAAACCAGCTTG2881 ACCCTGGCTCACAGACATGGTCATGAGGCGATTTAAATTTGTGCCACAATGAAGAGTGTG2941 TGTACACATGCCTCCTAGAAACTACACTATGGGTAATTAATTTATTTCATAGAGGCCACC3001 CAGATGCCTTATAGGTTTTATTAATTTGGATATGAAAGTGTACCCCATTTGGTTTCACCA3061 GGAACCCAAATTTAGAATATTGAAAAGCCATCAAAAAGTTGTGATATCAAAAATGTATGA3121 GTCTCTTAATATACTAAGCAAGAGTGTCACAGCAGTAATGATAAAGACTAGTTTTAATCT3181 CAAGCCTTAGAGGGGCCCTTTGTTGCCTTTTGTGGTGCAGCCTCTTAAGAGAGTGGTGTT3241 TGATTAACAAAAAAACTGTGGCCCAAGTGGAACCCTTGACCTTTTCTCAGATAATCTGTG3301 TATGTACACAGCTAACACAGCTCTTTAGATTCCCTGTTAAGTGACTCATTCACATTCCTT3361 TCTTGGATATAAAGTCATTGCTGTCTTTTTATTTTTGAAATAGTACAAGACAAAGATTTT3421 TAACTTAACATGAAAAATTCACTCTTTTATTTTGGAAAAAAAGTTAACTTTTCATACTAA3481 CAAACAGAACAAGATTTAAGGTAAATTTCTTAAACATTATCCAGAAAAATAACAAGATTT3541 ATAGTATCTACTTCTGGTACTAATATACACAAAAGGCCAAAACCATGCCTATTCTGCAGG3601 TGTAGCTTCGGTGCTCTCCTGTTCAGGGGCAGGCTCACTGCCCGCTTCTTTTCCTTCTTT3661 GCTTCTTTTAGATTTTTTGTGTTTGTGTCTCCTGTGACTATCTCCTTCTTCACTTTCATG3721 GCGACGTCTACTATTAC ( 3 ) SEQ ID NO:2： ( ⅰ ) ：

(A) length: 632 amino acid

(B) type: amino acid

( D ) ： ( ⅱ ) ： ( ⅹⅰ ) ：SEQ ID NO:2： 1 Met Lys Ala Leu Leu Leu Leu Val Leu Pro Trp Leu Ser Pro Ala 16 Asn Tyr Ile Asp Asn Val Gly Asn Leu His Phe Leu Tyr Ser Glu 31 Leu Cys Lys Gly Ala Phe His Tyr Gly Leu Thr Lys Asp Arg Lys 46 Arg Arg Ser Gln Asp Gly Cys Pro Asp Gly Cys Ala Ser Leu Thr 61 Ala Thr Ala Pro Ser Pro Glu Val Ser Ala Ala Ala Thr Ile Ser 76 Leu Met Thr Asp Glu Pro Gly Leu Asp Asn Pro Ala Tyr Val Ser 91 Ser Ala Glu Asp Gly Gln Pro Ala Ile Ser Pro Val Asp Ser Gly106 Arg Ser Asn Arg Thr Arg Ala Arg Pro Phe Glu Arg Ser Thr Ile121 Arg Ser Arg Ser Phe Lys Lys Ile Asn Arg Ala Leu Ser Val Leu136 Arg Arg Thr Lys Ser Gly Ser Ala Val Ala Asn His Ala Asp Gln151 Gly Arg Glu Asn Ser Glu Asn Thr Thr Ala Pro Glu Val Phe Pro166 Arg Leu Tyr His Leu Ile Pro Asp Gly Glu Ile Thr Ser Ile Lys181 Ile Asn Arg Val Asp Pro Ser Glu Ser Leu Ser Ile Arg Leu Val196 Gly Gly Ser Glu Thr Pro Leu Val His Ile Ile Ile Gln His Ile211 Tyr Arg Asp Gly Val Ile Ala Arg Asp Gly Arg Leu Leu Pro Gly226 Asp Ile Ile Leu Lys Val Asn Gly Met Asp Ile Ser Asn Val Pro241 His Asn Tyr Ala Val Arg Leu Leu Arg Gln Pro Cys Gln Val Leu256 Trp Leu Thr Val Met Arg Glu Gln Lys Phe Arg Ser Arg Asn Asn271 Gly Gln Ala Pro Asp Ala Tyr Arg Pro Arg Asp Asp Ser Phe His286 Val Ile Leu Asn Lys Ser Ser Pro Glu Glu Gln Leu Gly Ile Lys301 Leu Val Arg Lys Val Asp Glu Pro Gly Val Phe Ile Phe Asn Val316 Leu Asp Gly Gly Val Ala Tyr Arg His Gly Gln Leu Glu Glu Asn331 Asp Arg Val Leu Ala Ile Asn Cly His Asp Leu Arg Tyr Gly Ser346 Pro Glu Ser Ala Ala His Leu Ile Gln Ala Ser Glu Arg Arg Val361 His Leu Val Val Ser Arg Gln Val Arg Gln Arg Ser Pro Asp Ile376 Phe Gln Glu Ala Gly Trp Asn Ser Asn Gly Ser Trp Ser Pro Gly391 Pro Gly Glu Arg Ser Asn Thr Pro Lys Pro Leu His Pro Thr Ile406 Thr Cys His Glu Lys Val Val Asn Ile Gln Lys Asp Pro Gly Glu421 Ser Leu Gly Met Thr Val Ala Gly Gly Ala Ser His Arg Glu Trp436 Asp Leu Pro Ile Tyr Val Ile Ser Val Glu Pro Gly GIy Val Ile451 Ser Arg Asp Gly Arg Ile Lys Thr Gly Asp Ile Leu Leu Asn Val466 Asp Gly Val Glu Leu Thr Glu Val Ser Arg Ser Glu Ala Val Ala481 Leu Leu Lys Arg Thr Ser Ser Ser Ile Val Leu Lys Ala Leu Glu496 Val Lys Glu Tyr Glu Pro Gln Glu Asp Cys Ser Ser Pro Ala Ala511 Leu Asp Ser Asn His Asn Met Ala Pro Pro Ser Asp Trp Ser Pro526 Ser Trp Val Met Trp Leu Glu Leu Pro Arg Cys Leu Tyr hsn Cys541 Lys Asp Ile Val Leu Arg Arg Asn Thr Ala Gly Ser Leu Gly Phe556 Cys Ile Val Gly Gly Tyr Glu Glu Tyr Asn Gly Asn Lys Pro Phe571 Phe Ile Lys Ser Ile Val Glu Gly Thr Pro Ala Tyr Asn Asp Gly586 Arg Ile Arg Cys Gly Asp Ile Leu Leu Ala Val Asn Gly Arg Ser601 Thr Ser Gly Met Ile His Ala Cys Leu Ala Arg Leu Leu Lys Glu616 Leu Lys Gly Arg Ile Thr Leu Thr Ile Val Ser Trp Pro Gly Thr631 Phe Leu ( 4 ) SEQ ID NO:3 ( ⅰ )

(A) length: 25 bases

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linear (ⅱ) molecule type: oligonucleotide (ⅹ ⅰ) sequence description: information (ⅰ) sequence signature of SEQ ID NO:3:CCAACCGTGGTGGCTCCTTGCGTTC 25 (5) SEQ ID NO:4

(A) length: 25 bases

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linear (ⅱ) molecule type: oligonucleotide (ⅹ ⅰ) sequence description: information (ⅰ) sequence signature of SEQ ID NO:4:GTAATAGTAGACGTCGCCATGAAAG 25 (6) SEQ ID NO:5

(A) length: 34 bases

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linear (ⅱ) molecule type: oligonucleotide (ⅹ ⅰ) sequence description: information (ⅰ) sequence signature of SEQ ID NO:5:CCCCATATGATGAAGGCGCTTCTGTTGCTGGTCT 34 (7) SEQ ID NO:6

(A) length: 32 bases

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linear (ⅱ) molecule type: oligonucleotide (ⅹ ⅰ) sequence description: the information of SEQ ID NO:6:CCCGGATCCCTATAAAAAAGTGCCAGGCCAAG 32 (8) SEQ ID NO:7: (ⅰ) sequence signature:

(A) length: 15 amino acid

(B) type: amino acid

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

Claims (18)

1, a kind of isolated polypeptide-people's the new protein 70 that contains the plasma membrane adjusting function (many PDZ of people domain protein 70) is characterized in that it includes: the polypeptide of the aminoacid sequence shown in the SEQ ID NO:2 or active fragments, analogue or the derivative of its polypeptide.

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

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

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

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

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

(c) with (a) or the polynucleotide of at least 70% homogeny (b) are arranged.

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

6, polynucleotide as claimed in claim 4, the sequence that it is characterized in that described polynucleotide include the sequence of 1-3737 position among the sequence of 236-2134 position among the SEQ IDNO:1 or the SEQ ID NO:1.

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

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

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

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

9, a kind of people of having is contained the preparation method of the active polypeptide of protein 70 of plasma membrane adjusting function, it is characterized in that described method comprises:

(a) under the new protein 70 condition that contains the plasma membrane adjusting function of expressing human, cultivate the described through engineering approaches host cell of claim 8;

(b) from culture, isolate and have the active polypeptide of protein 70 that the people is contained the plasma membrane adjusting function.

10, a kind of can with polypeptide bonded antibody, it is characterized in that described antibody is the protein 70 specificity bonded antibody that can contain the plasma membrane adjusting function with the people.

11, the compound of an analoglike or adjusting polypeptide active or expression is characterized in that they are simulation, promotion, antagonism or suppress the active compound that the people is contained the protein 70 of plasma membrane adjusting function.

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

13, the described application of compound of a kind of claim 11, it is characterized in that described compound be used for protein 70 that the mediator contains the plasma membrane adjusting function in vivo, the method for external activity.

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

15, as the application of polypeptide as described in the arbitrary claim among the claim 1-3, it is characterized in that it is applied to screen the people and contains the stand-in of the protein 70 of plasma membrane adjusting function, agonist, antagonist or inhibitor; Perhaps be used for the peptide finger print identification.

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

17,, it is characterized in that forming the pharmaceutical composition that contains the relevant unusually disease of the protein 70 of plasma membrane adjusting function as diagnosis or treatment and people with safe and effective dosage and pharmaceutically acceptable carrier with described polypeptide, polynucleotide or its stand-in, agonist, antagonist or inhibitor as the described polypeptide of arbitrary claim, polynucleotide or application of compound in claim 1-6 and 11.

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