CN1297943A

CN1297943A - Human brain expressed X chromosome linkage protein and its code sequence

Info

Publication number: CN1297943A
Application number: CN 99124179
Authority: CN
Inventors: 毛裕民; 谢毅
Original assignee: BORONG GENE DEVELOPMENT CO LTD SHANGHAI
Current assignee: BORONG GENE DEVELOPMENT CO LTD SHANGHAI
Priority date: 1999-11-30
Filing date: 1999-11-30
Publication date: 2001-06-06
Also published as: AU1688301A; WO2001040286A1

Abstract

The present invention discloses a novel polypeptide, human brain expressed X chromosome-linked protein, polynucleotides encoding this polypeptide and DNA recombination process to produce the polypeptide. The present invention also discloses the method of applying the polypeptide in treating various diseases, such as embryonic muldevelopment, acquired and hereditary diseases; tumors and cancers; deafness, X-chromosome-linked intelligence obtuseness and agyri. The present invention also discloses the antagonist resisting the polypeptide and its treatment effect. The present invention also discloses the application of the polynucleotides encoding this polypeptide.

Description

Human brain expressed X-linkage albumen and encoding sequence thereof

The invention belongs to biological technical field, specifically, the invention describes a kind of new polypeptide-human brain expressed X-linkage albumen (human brain expressed X-linked protein is called for short " hBex "), 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.

Imprinted gene (imprinted genes) is exactly gene or the karyomit(e) that exists some in vivo, and they keep some feature of gamete and carry out selective difference and express.These imprinted genes specifically expressing is in vivo all finished by a genomic imprinting process.Genomic imprinting (genomic imprinting) is that indivedual allelotrope of a certain gene are expressed specifically according to the different sources of his father's female parent.These allelotrope are in the gamete forming process or growing in early days by sign specifically, and this sign is heritable, and generation upon generation of is reversible, and whether it expresses and have stable outer genetic modification probably.The different imprinted gene of alleged occurrence 100-200 kind wherein has only the 20-30 kind to be determined in vivo.Now there are 25 kinds of imprinted genes to be proved approximately to have stable outer genetic modification, some of them to be indicated in work in the generating process of tumour [Joyce JA, Schofield PN; Mol Pathol 1998Aug; 51:185-90].Discover, genomic imprinting has a lot of important biological functions in vivo, itself and fetal development, embryo growth, various hyperplasia are grown the generation of layer disease and the generation of various human body cancers all has certain relation [ChilosiM, Lestani M et al., Adv Clin Path 1998 Jan; 2:15-24].

The CAAX motif connects two aliphatic residues after by a cysteine residues and a C-terminal X residue is formed, and wherein X can be [Taparowsky, E.etal., Cell, 1983 among C, S, M, Q or the A; 34:581-86].This motif is the target site of modifying before the protein transport, organism very important biological function is arranged.Discover that the isoprenylation of cysteine residues directly affects the biological function that albumen is brought into normal play in this motif.Have only through isoprenylation, albumen could correctly combine with cytolemma, regulates the differentiation and the propagation of cell.Usually by GGTase (prenyltransferases) it is modified, with farnesyl or yak group-transfer to terminal Cys residue, utilize the C-terminal of height change to finish combining and interaction [Hancock JF of albumen and cytolemma after the modification, Cadwallader K, et al., EMBO J1991Dev; 10; 4033-4039].Studies confirm that the known various albumen (as Ras albumen, G albumen etc.) that contain the CAAX motif all exist this transhipment preceding modification [Brown AL, Kay GF; Hum Mol Genet, 1999, Apr; 8:611-619].The C-terminal of these albumen height change has determined their location on different organoid films.If C-terminal is removed, then these albumen and membrane-bound specificity also will disappear.In addition, CAAX motif isoprenylation is modified also and may be crossed high disease relevant [Schafer, W.R. with tumour generation and blood lipidol of gallbladder; Rine, J.; Annu.Rev.Genet., 1992,26:209-237].

Show that imprinted gene is relevant with some diseases, for example fetal development, embryo growth, various hyperplasia are grown a layer disease.Therefore, be therapeutic purpose research and development people's imprinted gene and proteins encoded thereof, and relevant agonist/inhibitor is significant.

An object of the present invention is to provide X-linkage albumen (human brain expressed X-linked protein is called for short " hBex ") that isolating new polypeptide-human's brain expresses with and fragment, analogue and derivative.

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

Another object of the present invention provides the recombinant vectors that contains the human brain expressed proteic polynucleotide of X-linkage of coding.

Another object of the present invention provides the genetically engineered host cell that contains the human brain expressed proteic polynucleotide of X-linkage of coding.

Another object of the present invention provides the human brain expressed proteic method of X-linkage of producing.

Another object of the present invention provides the proteic antibody of X-linkage of expressing at polypeptide-human's brain of the present invention.

Another object of the present invention has provided the proteic simulated compound of X-linkage, antagonist, agonist, the inhibitor of expressing at polypeptide-human's brain of the present invention.

Another object of the present invention provides the method for the diagnosis disease relevant with human brain expressed X-linkage abnormal protein with treatment.

In a first aspect of the present invention, novel isolated human brain expressed X-linkage albumen is provided, 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 ID NO:2 aminoacid sequence.

In a second aspect of the present invention, the polynucleotide of these polypeptide of separated coding are provided, these polynucleotide comprise a nucleotide sequence, and this nucleotide sequence is shown at least 74% homogeny with a kind of nucleotides sequence that is selected from down group: (a) the proteic polynucleotide of the above-mentioned human brain expressed X-linkage of coding; (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 173-550 position among the SEQ ID NO:1; (b) has the sequence of 1-792 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.

In a fourth aspect of the present invention, provide preparation to have the preparation method of the active polypeptide of hBex, this method comprises: (a) under the condition that is fit to expression hBex, cultivate the above-mentioned host cell that is transformed or transduce; (b) from culture, isolate and have the active polypeptide of hBex.

In a fifth aspect of the present invention, provide and above-mentioned hBex polypeptid specificity bonded antibody.The nucleic acid molecule that can be used for detecting also is provided, and it contains a successive 10-792 Nucleotide in the above-mentioned polynucleotide.

In a sixth aspect of the present invention, the compound of simulation, promotion, antagonism hBex polypeptide active is provided, and the compound that suppresses the hBex polypeptide expression.The method of screening and/or prepare these compounds also is provided.Preferably, this compound is encoding sequence or its segmental antisense sequences of hBex polypeptide.

In a seventh aspect of the present invention, provide with above-claimed cpd regulate hBex in vivo, the method for external activity.

In a eighth aspect of the present invention, a kind of disease relevant with hBex polypeptide unconventionality expression or method of disease susceptibility of detecting is provided, this method comprises: whether have sudden change in the nucleotide sequence of detection coding said polypeptide.

In a ninth aspect of the present invention, provide the purposes of polypeptide of the present invention and encoding sequence.Polypeptide for example of the present invention can be used to screen the agonist that promotes the hBex polypeptide active, and perhaps screening suppresses the antagonist of hBex polypeptide active or is used to the peptide finger print identification.The encoding sequence of hBex of the present invention or its fragment can be used as primer and be used for pcr amplification reaction, perhaps are used for hybridization as probe, perhaps are used to make gene chip or microarray.

In a tenth aspect of the present invention, a kind of pharmaceutical composition is provided, it contains the hBex polypeptide of the present invention and the pharmaceutically acceptable carrier of safe and effective amount.These pharmaceutical compositions can be treated dysontogenesis and acquired and heredopathia; Hyperplasia is grown a layer disease; The generation of various tumours and cancer; Hearing is become deaf; The backwardness of X-linkage and agyria disease.

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

In the accompanying drawing, Fig. 1 be people and albumen X-linkage (mBex2, following row) that in brain, express that in brain, express with albumen X-linkage (hBex, last row) and mouse amino acid relatively.

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 human brain expressed X-linkage albumen " is meant that human brain expressed X-linkage albumen is substantially free of natural relative other albumen, lipid, carbohydrate or other material.Those skilled in the art can use the human brain expressed X-linkage albumen of purified technology of protein purifying of standard.Basically pure polypeptide can produce single master tape on non-reduced polyacrylamide gel.The purity of human brain expressed X-linkage protein polypeptide can be used amino acid sequence analysis.

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

The present invention also comprises the proteic fragment of human brain expressed X-linkage, derivative and analogue.As used herein, term " fragment ", " derivative " are meant with " analogue " and keep human brain expressed identical biological function or the active polypeptide of X-linkage albumen of the present invention basically.The fragment of polypeptide of the present invention, derivative or analogue can be: one or more conservative or substituted polypeptide of non-conservation amino-acid residue (preferred conservative amino acid residue) (i) are arranged, and the amino-acid residue of such replacement can be also can not encoded by genetic code, or (ii) in one or more amino-acid residues, has a polypeptide of substituted radical, or (iii) mature polypeptide and another compound (such as the compound that prolongs the polypeptide transformation period, polyoxyethylene glycol for example) merge formed polypeptide, or (iv) additional aminoacid sequence is fused to this peptide sequence and the polypeptide that forms (as leader sequence or secretion sequence or be used for the sequence or the proteinogen sequence of this polypeptide of purifying).According to the instruction of this paper, these fragments, derivative and analogue belong to the known scope of those skilled in the art.

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.

In an example of the present invention, the proteic polynucleotide of X-linkage of encoding human brain expressed are to find from the cDNA library of people's fetal brain tissue.The polynucleotide sequence total length that it comprises is 792 bases, its open reading frame (173-550 position) 125 amino acid of having encoded.Find relatively that according to amino acid sequence homologous this polypeptide has 72% identity and 85% similarity with the proteic gene of X-linkage (Bex2) of expressing with mouse in brain on protein level.And polypeptide of the present invention contains at C-terminal and comes across similar CAAX motif.

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 above-mentioned polynucleotide, its coding has the polypeptide of identical aminoacid sequence or fragment, 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 a kind of 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) when hybridization add 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, the length of " nucleic acid fragment " contain 10 Nucleotide at least, better are at least 30 Nucleotide, are more preferably at least 50 Nucleotide, preferably more than at least 100 Nucleotide.Nucleic acid fragment also can be used for the amplification technique (as PCR) of nucleic acid to determine and/or to separate the polynucleotide of coding hBex.

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

The special polynucleotide sequence of coding hBex of the present invention 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 polymerase chain reaction technique, even few expression product also can be cloned.

Available ordinary method is screened gene of the present invention from these cDNA libraries.These methods include, but is not limited to: (1) DNA-DNA or DNA-RNA hybridization; (2) appearance of marker gene function or forfeiture; (3) level of mensuration hBex transcript; (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 1000 Nucleotide, preferable is within 500 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 carry out with radio isotope, fluorescein or enzyme (as alkaline phosphatase) etc.

In (4) kind method, detect the protein product of hBex genetic expression 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 or the sequence of its various dna fragmentations, and available 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.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 with the host cell of hBex encoding sequence through the genetically engineered generation, and the method that produces polypeptide of the present invention through recombinant technology.

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

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

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

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

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

Can carry out with routine techniques well known to those skilled in the art with dna sequence dna of the present invention or the recombinant vectors transformed host cell that contains described dna sequence dna.When the host was prokaryotic organism such as intestinal bacteria, the competent cell that can absorb DNA can be used CaCl in exponential growth after date results ₂Method is handled, and used step is well-known in this area.Another kind method is to use MgCl ₂Handle.If 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.

Recombinant DNA technology (Science, 1984 by routine; 224:1431), utilize polynucleotide sequence of the present invention to can be used to express or produce the hBex of reorganization.In general following steps are arranged:

(1). with the polynucleotide (or varient) of coding of the present invention hBex, or with the recombinant expression vector that contains these polynucleotide proper host cell that transforms or transduce;

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

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

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

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

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

The present invention also provides SCREENED COMPOUND to identify the method that improves (agonist) or check the medicament of (antagonist) hBex.Agonist improves hBex biological function such as stimulate cellular proliferation, and antagonist prevention disorder such as the various cancer relevant with cell hyperproliferation with treatment.For example, can be in the presence of medicine, the hBex with mark cultivates with mammalian cell.Measure the ability that medicine improves or check hBex then.

The antagonist of hBex comprises antibody, compound, acceptor disappearance thing and the analogue etc. that filter out.The antagonist of hBex can combine and eliminate its function with the hBex polypeptide, 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, hBex can be added during bioanalysis measures, by measuring compound interactional influence between hBex and its acceptor is determined whether compound is antagonist.With the same quadrat method of above-mentioned SCREENED COMPOUND, can filter out the acceptor disappearance thing and the analogue of antagonist action.Can be incorporated into the rondom polypeptide storehouse that solid formation forms by the various amino acid that may make up by screening with hBex bonded peptide molecule obtains.During screening, generally tackle the hBex molecule 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 at the hBex antigenic determinant.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.

The method of the available hBex direct injection of the production of polyclonal antibody immune animal (as rabbit, mouse, rat etc.) obtains.Multiple adjuvant can be used for the enhancing immunity reaction, includes but not limited to freund's adjuvant etc.The technology of the monoclonal antibody of preparation hBex includes but not limited to: and 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 of anti-hBex.

The antibody of anti-hBex can be used in the immunohistochemistry technology, detects the hBex in the biopsy specimen.

With the also available labelled with radioisotope of hBex bonded monoclonal antibody, 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.As the monoclonal antibody of hBex high-affinity 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 hBex positive cells.

The disease that antibody among the present invention can be used for treating or prevention is relevant with hBex.The antibody that gives suitable dosage can stimulate or block generation or the activity of hBex.

The invention still further relates to the diagnostic testing process of quantitative and detection and localization hBex level.These tests are known in the art, and comprise that FISH measures and radioimmunoassay.The hBex level that is detected in the test can be with laying down a definition the importance of hBex in various diseases and be used to the disease of diagnosing hBex to work.

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

The polynucleotide of coding hBex also can be used for multiple therapeutic purpose.Gene therapy technology can be used for treating because cell proliferation, growth or the metabolic disturbance due to the nothing expression of hBex or the unusual/non-activity expression.The gene therapy vector (as virus vector) of reorganization can be designed for the hBex that expresses variation, to suppress endogenic hBex activity.For example, a kind of hBex of variation can be the hBex that shortens, lacked signal conduction function territory, though can combine with the substrate in downstream, lacks signaling activity.Therefore the gene therapy vector of reorganization can be used for treating the disease of hBex expression or active caused by abnormal.Deriving from viral expression vector such as retrovirus, adenovirus, adeno-associated virus (AAV), hsv, parvovirus etc. can be used for the polynucleotide of coding hBex are transferred in the cell.The method of recombinant viral vector that structure carries the polynucleotide of coding hBex be found in existing document (Sambrook, etal.).The polynucleotide of reorganization coding hBex can be packaged in the liposome and then 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 hBex 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 of coding hBex can be used for diagnosing the relative disease with hBex.The unconventionality expression of the expression that the polynucleotide of coding hBex can be used for detecting hBex hBex whether or under morbid state.As the dna sequence dna of the hBex that encodes can be used for biopsy specimen is hybridized to judge the expression situation of hBex.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.Carry out the transcription product that RNA-polymerase chain reaction (RT-PCR) amplification in vitro also can detect hBex with the special primer of hBex.

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

Sequence of the present invention identifies it also is valuable to karyomit(e).This sequence can be specifically at certain bar human chromosome particular location and and can with its hybridization.At present, need to identify the concrete site of each gene on the karyomit(e).Yet, have only chromosomal marker thing seldom to can be used for the marker chromosomes position now based on actual sequence data (repetition polymorphism).According to the present invention, for these sequences are associated with disease related gene, the wherein 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, Mendelian Inheritancein 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.HBex comes administration with the amount that treats and/or prevents concrete indication effectively.The amount and the dosage range that are applied to patient's hBex will depend on many factors, as administering mode, person's to be treated healthiness condition and diagnostician's judgement.

In addition, because hBex of the present invention has the natural acid sequence that is derived from the people, therefore, compare, estimate to have higher active and/or lower side effect (for example in the intravital immunogenicity of people lower or do not have) being applied to man-hour with the albumen of the same clan that derives from other species.

With regard to hBex albumen, polypeptide of the present invention or its fragment or derivatives thereof can be used for treating dysontogenesis and associated various acquired and heredopathia; Hyperplasia is grown a layer disease; The generation of various tumours and cancer; The method of diseases such as the backwardness of X-linkage and agyria disease.Because of the hBex gene is a kind of gene of X-linkage of expressing in cerebral tissue, its abnormal expression also may cause the disease of hearing and intellectual aspect.

Because the isoprenylation of cysteine residues has determined its location on different organoid films in the terminal CAAX motif of hBex PROTEIN C (concrete aminoacid sequence is CLMP).Therefore, the correct location and the fusion of the transportation of proteic transhipment and film bubble all have important effect between this albumen pair cell device.Thereby be very useful by genetically engineered production or the transhipment that utilizes hBex albumen to regulate intracellular protein.In addition, the isoprenylation of CAAX motif modify also may take place with tumour and the blood lipidol of gallbladder to cross high disease relevant.

HBex albumen must be by GGTase (isoprene transhipment enzyme) catalysis, prenyl is transported to the normal biological function of competence exertion on the cysteine residues of its C-terminal, so the inhibitor of GGTase can be blocked the biological activity of hBex effectively.The various tumours and the cancer that cause for being expressed by the hBex abnormal protein, the inhibitor of GGTase will be a kind of very valuable drug.

In addition, hBex protein polypeptide or its fragment or derivatives thereof are added in the clone and can break up and propagation by irritation cell, can also directly join in the active somatic cell by means such as liposome, electroporations.

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:Cold SpringHarbor Laboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.

Embodiment 1:

The clone of hBex albumen cDNA

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 Quick mRNA separating kit (Qiegene).2ug poly (A) mRNA forms cDNA. through reverse transcription and 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), transform DH5 α, bacterium forms the cDNA library.Obtain 3028 clones altogether.Measure the sequence of all clones' 5 ＇ and 3 ＇ ends with dideoxy method.CDNA sequence and the existing public dna sequence data storehouse measured are compared, and the dna sequence dna that found that a clone (286g05) is new DNA.By synthetic a series of primers the contained dna sequence dna of hBex albumen clone is carried out two-way mensuration.Computer Analysis shows, the contained full-length cDNA of hBex albumen clone is a new dna sequence dna (shown in SEQ IDNO:1), from 173bp to 550bp the ORF of a 378bp, the 125 new amino acid whose protein (shown in SEQ ID NO:2) of encoding are arranged.The name of this protein is that express in brain and albumen X-linkage (human brain expressed X-linked protein, abbreviation hBex) for the people.

Embodiment 2

CDNA clone's homology retrieval:

Sequence and encoded protein sequence thereof with the hBex protein gene arrive Genbank, databases such as Swissport carry out the homology retrieval, the program that is used to retrieve is Blast (Basic local Alignment searchtool) (1993Proc Nat Acad Sci90:5873-5877), Blast can find out many genes of the albumen homology of the X-linkage of expressing with the people in brain, be AF097439 (this be Genbank access number) with the gene of hBex dna homolog maximum of the present invention wherein.Gene that these retrieve or protein sequence can access from the Genbank database.The sequence that accesses can be made of Pileup in the GCG software package (multisequencing) and Gap (two sequences) program and connect proportioning.New proteic function prediction can be analyzed with the Motif program.The results are shown in accompanying drawing 1.

This result shows that hBex gene and mouse Bex2 gene have the highest homology, both homogenies=94/129 (72%), similarity=110/129 (85%).

Embodiment 3 embodiment 3: with the human brain expressed proteic gene of X-linkage of RT-PCR method clones coding

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:

Primer 1:5 ＇-GACTCGTGTCTCGCTACCAGCTCCC-3 ＇ (SEQ ID NO:3)

Primer 2: 5 ＇-TTTTTAAAGGTGCTTTATTAAC-3 ＇ (SEQ ID NO:4)

Primer 1 is corresponding to the 1st the forward sequence that base begins of the 5 ＇ end that is positioned at SEQ ID NO:1;

Primer 2 is corresponding to the 3 ＇ end reverse sequence that is SEQ ID NO:1.

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 beta-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 1-792bp shown in the SEQ ID NO:1 are identical.

Embodiment 4

Expression and the purifying of hBex albumen in the intestinal bacteria system

According to the corresponding gene order of this new hBex albumen, design a pair of specificity amplification primer, sequence is as follows:

Primer A1:5 ＇-AAGCTTGGGCATAAGGCAAAACTCATCATGA-3 ＇ (shown in SEQ ID NO:5)

Primer A2:5 ＇-CCATGGCAGATGGAGTCCAAAGAGAAACTAG-3 ＇ (shown in SEQ ID NO:6)

These two sections sequences contain HindIII and NcoI restriction enzyme site respectively, are respectively the encoding sequence of goal gene 3 ＇ end and 5 ＇ end thereafter, are template with the PBS plasmid that contains the total length goal gene, carry out the PCR reaction.The restriction enzyme site of HindIII and NcoI is corresponding to the selectivity restriction enzyme site on the expression vector plasmid PET-28b (+) (Novagen company provides Cat.No.69865.3).Respectively extension increasing sequence and plasmid PET-28 (+) being carried out enzyme with HindIII and NcoI cuts and is connected.Recombinant plasmid transformed is gone into host bacterium e. coli bl21 (DE3) plySs, and induce with IPTG and to express.Expression product is through carrying out ultrasonic bacteria breaking, contains the affinity column (Pharmacia company provides) of 6 Histidines on again, obtained the purpose hBex albumen of purifying.Through the SDS-PAGE electrophoresis, obtain a single band at the 13.8KD 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 5

The expression of hBex albumen in eukaryotic cell (Chinese hamster ovary celI strain)

According to the corresponding gene order of this hBex albumen, design a pair of specificity amplification primer, sequence is as follows:

Primer 1:5 '-AAGCTTGGGCATAAGGCAAAACTCATCATGA-3 ＇ (shown in SEQ ID NO:5)

Primer 2: 5 ＇-CCATGGCAGATGGAGTCCAAAGAGAAACTAG-3 ＇ (shown in SEQ ID NO:6)

These two sections sequences contain HindIII and NcoI restriction enzyme site respectively, are respectively the encoding sequence of goal gene 3 ＇ end and 5 ＇ end thereafter.

The restriction enzyme site of the restriction enzyme on the primer is corresponding to the restriction enzyme digestion sites on the expressing cho cell carrier pcDNA3, this plasmid vector coding antibiotics resistance (Amp ^rAnd Neo ^r), a phage replication starting point (fl ori), a virus replication starting point (SV40 ori), T7 promotor, a viral promotors (P-CMV), a Sp6 promotor, a SV40 promotor, a SV40 tailing signal and corresponding polyA order, a BGH tailing signal and a corresponding polyA order.

With HindIII, NcoI digestion pcDNA3 carrier, will insert fragment subsequently and be connected to the pcDNA3 carrier.Subsequently with connecting mixture Transformed E .coli DH5 α bacterial strain.Screen transformant containing on the LB culture dish of Amp, incubated overnight (O/N) contains the clone of required construction in the LB liquid nutrient medium of adding Amp (100 μ g/ml).The extracting plasmid is cut with the PstI enzyme and identify to be inserted clip size and direction, and sequence verification result shows that the proteic cDNA of hBex inserts the fragment carrier of correctly packing into.

Plasmid transfection is to adopt lipofection, carries out with Lipofectin test kit (GiBco Life).After the transfection 48 hours, through the lasting G418 pressurization screening in 2-3 week, collecting cell and cell conditioned medium are measured the expressing protein enzyme activity.Remove G418, continuous passage is cultivated; To mixing the clone cell Method of Limited Dilution, select to have the cell subclone of higher protein-active.The above-mentioned positive subclone of a large amount of according to a conventional method cultivations.After 48 hours, beginning collecting cell and supernatant are with ultrasonic degradation method smudge cells.With 50mM TrisHCl (pH7.6) solution that contains 0.05%Triton is balance liquid and elutriant, uses through the Superdex of pre-equilibration G-75 post and collects above-mentioned proteic active peak.Using 50mM TrisHCl (pH8.0) equilibrated DEAE-Sepharose post again, is that elutriant carries out gradient elution with 50mMTrisHCl (pH8.0) solution that contains 0-1M NaCl, collects above-mentioned proteic active peak.Be that dialyzate is dialysed to expressing protein solution with PBS (pH7.4) then.Last freeze-drying is preserved.

Embodiment 6

The proteic phraseology of hBex in people's cell

Carry out the Northern engram analysis, measure hBex protein expression level in people's cell. (Houston TX77033) separates total cell RNA sample for Biotecx Laboratories, Inc.6023South Loop East with BNAzolTM B system.Total RNA from each concrete about 10ug of people's separate tissue separates on 1% sepharose, and trace (Sambrook, fritsch and Maniatis, MolecularCloning, Cold Spring HarborPress, (1989) to NF.According to Stratagene Prime-It test kit with the reaction that makes marks of 50ngDNA fragment.The DNA of mark Select-G-50 column purification (5Prime-3prime, Inc.5603 Arapahoe Road, Boulder, Co 80303).Then, filter is with radiolabeled total length hBex gene, and under 65 ℃, with 1,000, the speed of 000cpm/ml is at 0.5M NaPO ₄, hybridize among pH7.4 and the 7%SDS and spend the night. at room temperature wash twice and after twice of 60 ℃ of flushing, filter be exposed under-70 ℃ with strengthening window of tube with 0.5XSSC, 0.1%SDS.The proteic messenger RNA(mRNA) of HBex is rich in cells such as brain cell, liver cell, splenocyte.

Embodiment 7

The Northern blotting is analyzed the expression of hBex protein gene:

Extract total RNA[Anal.Biochem1987,162,156-159 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.

Use 20g RNA, on 1.2% sepharose that contains 20mM3-(N-morpholino) propanesulfonic acid (pH7.0)-5mM sodium acetate-1mMEDTA-2.2M formaldehyde, carry out electrophoresis.Be transferred on the nitrocellulose filter then.With ³²(about 2 * 106cpm/ml) spend the night in 42 ℃ of hybridization in a solution probe of P-mark, and 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.With α- ³²P dATP prepares by random priming ³²The dna probe of P-mark.Used dna probe is the hBex coding region sequence of pcr amplification shown in Figure 2.After the hybridization, filter membrane is washed 30min in 65 ℃ in 1 * SSC-0.1%SDS.Then, analyze with quantitative with Phosphor Imager.

The result shows, hBex protein gene high expression level in cerebral tissue, and simultaneously, it is also expressed in tissues such as liver,spleen,kidney specifically.

Embodiment 8

The generation of anti-hBex protein antibodies

With the synthetic specific polypeptide of following hBex: the Ser-Lys-Glu-Lys-Leu-Ala-Val-Asn-Ser-Leu-Ser-Met-Glu-Asn-Ala-Asn-Gln-Glu-Asn-Glu (shown in SEQ ID NO:5) of Peptide synthesizer (PE-ABI).Form compoundly with hemocyanin and bovine serum albumin coupling this polypeptide respectively, method is referring to Avrameas.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 Sepharose 4B post of cyanogen bromide-activated, from total IgG, separates anti-peptide antibody with affinity chromatography.Immuno-precipitation proof antibody purified can combine with hBex specifically.

All quote in this application as a reference at all documents that the present invention mentions, just quoted as a reference separately as each piece document.Should be understood that in addition those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims institute restricted portion equally.

Sequence table (1) general information: (ii) denomination of invention: human brain expressed X-linkage albumen and encoding sequence thereof be the sequence number (iii): the information of 7 (2) SEQID NO:1: (i) sequence signature:

(A) length: 792bp

(B) type: nucleic acid

(C) chain: two strands

( D ) ： ( ii ) ：cDNA ( xi ) ：SEQ ID NO：1：GACTCGTGTC TCGCTACCAG CTCCCCGCTG CCCTGCGCTC GGCGGGCTGG CATCCGGCCC 60GGGGGAAAGC GGACCAGCCC TTCTGCAGGT CTGCGGGGCC AAGTGTCCCG GCGGCGCACC 120TCGTGGCGAG AATCGGGAGA AGGAGGAGAC TACAAGGATA GGCCCAGGAG TAATGGAGTC 180CAAAGAGAAA CTAGCAGTAA ACAGTCTCAG CATGGAAAAT GCCAACCAAG AAAATGAAGA 240AAAGGAGCAA GTTGCTAATA AAGGGGAGCC CTTGGCCCTC CCTTTGGATG CTGGTGAATA 300CTGTGTGCCT AGAGGAAATC GTAGGCGGTT CCGCGTTAGG CAGCCCATCC TGCAGTATAG 360ATGGGATATG ATGCATAGGC TTGGAGAACC ACAGGCAAGG ATGAGAGAAG AGAATATGGA 420AAGGATTGGG GAGGGGGTGA GACAGCTGAT GGAAAAGCTG AGGGAAAAGC AGTTGAGTCA 480TAGTCTGCGG GCAGTCAGCA CTGACCCCCC TCACCATGAC CATCATGATG AGTTTTGCCT 540TATGCCCTGA ATCCTGATGG TTTCCCTAAA GTTATTACGG AAACAGACCC CTGCTTTCGA 600ATTTACATGT TCATGATGTG CCCTTGTTGT AAACCTTTAC CTGTCACTTG TTTACGTGGG 660TCTCCTATTA CCAGCTTCTA ATTGAATATT GTGTTTTTGA ACCAGTCTGT AAGATTTTTG 720TTAGCAGAAG AATTTTACCT ATTGCATGGA AAGATGCTCA TTATAGTGAA GTTAATAAAG 780CACCTTTAAA AA 792 ( 2 ) SEQ ID NO：2： ( i ) ：

(A) length: 125 amino acid

(B) type: amino acid

( D ) ： ( ii ) ： ( xi ) ：SEQ ID NO：2：Met Glu Ser Lys Glu Lys Leu Ala Val Asn Ser Leu Ser Met Glu 15Asn Ala Asn Gln Glu Asn Glu Glu Lys Glu Gln Val Ala Asn Lys 30Gly Glu Pro Leu Ala Leu Pro Leu Asp Ala Gly Glu Tyr Cys Val 45Pro Arg Gly Asn Arg Arg Arg Phe Arg Val Arg Gln Pro Ile Leu 60Gln Tyr Arg Trp Asp Met Met His Arg Leu Gly Glu Pro Gln Ala 75Arg Met Arg Glu Glu Asn Met Glu Arg Ile Gly Glu Gly Val Arg 90Gln Leu Met Glu Lys Leu Arg Glu Lys Gln Leu Ser His Ser Leu 105Arg Ala Val Ser Thr Asp Pro Pro His His Asp His His Asp Glu 120Phe Cys Leu Met Pro 125 ( 2 ) SEQ ID NO：3 ( i )

(A) length: 25 bases

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity is molecule type (ii): oligonucleotide (xi) sequence description: information (i) sequence signature of SEQ ID NO:3:GACTCGTGTC TCGCTACCAG CTCCC 25 (2) SEQ ID NO:4

(A) length: 22 bases

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity is molecule type (ii): oligonucleotide (xi) sequence description: information (i) sequence signature of SEQ ID NO:4:TTTTTAAAGG TGCTTTATTA AC 22 (2) SEQ ID NO:5

(A) length: 31 bases

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity is molecule type (ii): oligonucleotide (xi) sequence description: information (i) sequence signature of SEQ ID NO:5:AAGCTTGGGC ATAAGGCAAA ACTCATCATG A 31 (2) SEQ ID NO:6

(A) length: 31 bases

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity is molecule type (ii): oligonucleotide (xi) sequence description: the information of SEQ ID NO:6:CCATGGCAGA TGGAGTCCAA AGAGAAACTA G 31 (2) SEQ ID NO:7: (i) sequence signature:

(A) length: 19 amino acid

(B) type: amino acid

(D) topological framework: linearity is molecule type (ii): polypeptide (xi) sequence description: SEQ ID NO:7:Ser Lys Glu Lys Leu Ala Val Asn Ser Leu Ser Met Glu Asn Ala Asn 15Gln Glu Asn Glu 19

Claims

1. isolating hBex polypeptide, it comprises: polypeptide or its conservative property variation polypeptide or its active fragments or its reactive derivative with SEQ ID No.2 aminoacid sequence.

2. polypeptide as claimed in claim 1 is characterized in that, this polypeptide is the polypeptide with SEQ ID No.2 aminoacid sequence.

3. isolating polynucleotide is characterized in that, it comprises a nucleotide sequence, and this nucleotide sequence is shown at least 72% homogeny with a kind of nucleotides sequence that is selected from down group:

(a) coding is as the polynucleotide of polypeptide as described in claim 1 and 2;

(b) with polynucleotide (a) complementary polynucleotide.

4. polynucleotide as claimed in claim 3 is characterized in that, this polynucleotide encoding has the polypeptide of aminoacid sequence shown in the SEQ ID No.2.

5. polynucleotide as claimed in claim 3 is characterized in that, the sequence of these polynucleotide is selected from down a kind of of group:

(a) has the sequence of 173-550 position among the SEQ ID No.1;

(b) has the sequence of 1-792 position among the SEQ ID No.1.

6. a carrier is characterized in that, it contains the described polynucleotide of claim 3.

7. a genetically engineered host cell is characterized in that, it is a kind of host cell that is selected from down group:

(a) host cell that transforms or transduce with the described carrier of claim 6;

(b) host cell that transforms or transduce with the described polynucleotide of claim 3.

8. the preparation method with the active polypeptide of people hBex is characterized in that, this method comprises:

(a) being fit to express under the condition of hBex, cultivate the described host cell of claim 7;

(b) from culture, isolate and have the active polypeptide of hBex.

9. energy and the described hBex polypeptid specificity of claim 1 bonded antibody.

10. nucleic acid molecule, it contains a successive 10-780 Nucleotide in the described polynucleotide of claim 3.

11. a compound is characterized in that, it is selected from down group:

(a) the active compound of the described polypeptide of simulation claim 1,

(b) the active compound of the described polypeptide of promotion claim 1,

(c) the active compound of the described polypeptide of antagonism claim 1,

(d) compound of the described polypeptide expression of inhibition claim 1.

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. an application rights require 11 described compounds regulate hBex albumen in vivo, the method for external activity.

14, a kind of method that detects the susceptibility of disease relevant with the described polypeptide unconventionality expression of claim 1 or disease is characterized in that, comprises the sudden change in the nucleotide sequence that detects coding said polypeptide.

15, the purposes of polypeptide as claimed in claim 1 is characterized in that, it is used to screen the agonist that promotes the hBex polypeptide active, and perhaps screening suppresses the antagonist of hBex polypeptide active or is used to the peptide finger print identification.

As the purposes of the described nucleic acid molecule of claim 1O, it is characterized in that 16, it is used as primer and is used for pcr amplification reaction, perhaps is used for hybridization as probe, perhaps be used to make gene chip or microarray.

17, a kind of pharmaceutical composition is characterized in that, it contains the described polypeptide of claim 1 and the pharmaceutically acceptable carrier of safe and effective amount.