CN1303865A - Novel polypeptide-human vasicentric protein 69 and polynucleotide for coding said polypeptide - Google Patents

Abstract

The present invention discloses a novel polypeptide-human vasicentric protein 69, polynucleotide coding said polypeptide and its method for producing this polypeptide by means of DNA recombination technology. Said inventino also discloses method for curing several diseases, such as malignant tumor, blood disease, HIV infection, immunological disease and various inflammations by using said polypeptide. Said invention also discloses an antagonist resisting said polypeptide and its therapeutic effect. Said invention also discloses the application of said polynucleotide for coding this novel human vosicentric protein 69.

Description

A kind of new polypeptide-human vasicentric protein 69 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 vasicentric protein 69, 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.

Mutual communication contact between the cell plays a part in cellulous organism and is important.Because iuntercellular exists the meticulous division of labor, some cell colonys depend on other cell colonys, and require other cell colonys that it is produced responsing reaction.The cell-cell communication network of this exquisiteness can control cell growth, division, death, be differentiated to form tissue and various other vital process.

In many kinds is in the sperm and ovum growth course of biology, and developmental sperm or ovum obtain nutritive substance by endless tube in its cell on every side.The formation of endless tube is relevant with the Actin muscle fiber.Now known the assembling and the function that have some genes to participate in endless tube, wherein a class is the gene family that contains the Kelch structural domain.The Kelch protein family all contains two common structures, at the N latter end BTB/POZ zone is arranged, and at the C latter end kelch tumor-necrosis factor glycoproteins [Soltysik-Espanola M et al, Mol biol Cell 1999 Jul] is arranged.

The kelch protein family that has been found that now is an actin binding protein, or relevant with Actin muscle.The Kelch protein family is at play an important role in the physiological process relevant with cytoskeleton in growth course [Soltysik-espanola M, Mol biol Cell1999 such as endless tube formation, nerve fiber location, cytogamy, cellular form formation; Philips J, J Cell biol 1998; ].For example, in fruit bat, kelch albumen is positioned in the medial surface of endless tube, and it makes the endless tube side keep the state of tight bundle.The Kelch protein mutation can cause the nurse cell that escorts around the ovum to carry this process of kytoplasm to be subjected to hindering [Xue F, Cooley L, Cell 1993Mar 12] by endless tube to ovum.The proteic disappearance of Kel-1 can cause the early stage cessation of growth cessation of larva [Ohmachi M, genes Cells1999].Owing in the sperm of the many biologies that comprise the people, ovum forming process, all found endless tube structure and albumen, therefore can think that kelch albumen is of universal significance to the effect of the avette one-tenth of essence with kelch structure.The kelch albumen Mayven that and for example in brain, finds in the dynamic organization of brain cell actin cytoskeleton, to work [Soltysik-Espanola M, Mol Biol Cell 1999].

People's of the present invention gene and fruit bat kelch albumen have 74% homology on protein level.Itself and kelch protein family the characteristic structural domain---kelch structural domain is closely similar, and contain the amino-acid residue of the several high conservatives in the kelch structural domain.Based on above each point, so think that new gene of the present invention is a coding people and the similar gene of kelch protein family kelch structural domain, called after human vasicentric protein 69.And infer that with this it is similar to the kelch structural domain, have similar biological function.

An object of the present invention is to provide isolating new polypeptide-human vasicentric protein 69 with and fragment, analogue and derivative.

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

Another object of the present invention provides the recombinant vectors of the polynucleotide that contain the human vasicentric protein 69 of encoding.

Another object of the present invention provides the genetically engineered host cell of the polynucleotide that contain the human vasicentric protein 69 of encoding.

Another object of the present invention provides the method for producing human vasicentric protein 69.

Another object of the present invention provides the antibody at polypeptide-human vasicentric protein 69 of the present invention.

Another object of the present invention has provided at the simulated compound of polypeptide-human vasicentric protein 69 of the present invention, antagonist, agonist, inhibitor.

Another object of the present invention provides the method for the diagnoses and treatment disease relevant unusually with human vasicentric protein 69.

In a first aspect of the present invention, novel isolated human vasicentric protein 69 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 isolating these polypeptide of coding are provided, these polynucleotide comprise a nucleotide sequence, and this nucleotide sequence is shown at least 75% homogeny with a kind of nucleotides sequence that is selected from down group: (a) polynucleotide of the above-mentioned human vasicentric protein 69 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 462-2342 position among the SEQ ID NO:1; (b) has the sequence of 1-3488 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 human vasicentric protein 69 " is meant that human vasicentric protein 69 is substantially free of natural relative other albumen, lipid, carbohydrate or other material.Those skilled in the art can use the purified technology of protein purifying human vasicentric protein 69 of standard.Basically pure polypeptide can produce single master tape on non-reduced polyacrylamide gel.The purity of human vasicentric protein 69 polypeptide can be used amino acid sequence analysis.

The invention provides a kind of new polypeptide-human vasicentric protein 69, it is made up of the aminoacid sequence shown in the SEQ ID NO:2 basically.Polypeptide of the present invention can be recombinant polypeptide, natural polypeptides, synthetic polypeptide, preferred recombinant polypeptide.Polypeptide of the present invention can be the product of natural purifying, or the product of chemosynthesis, or uses recombinant technology to produce from protokaryon or eucaryon host (for example, bacterium, yeast, higher plant, insect and mammalian cell).The host used according to the recombinant production scheme, polypeptide of the present invention can be glycosylated, maybe can be nonglycosylated.Polypeptide of the present invention also can comprise or not comprise initial methionine residues.

The present invention also comprises fragment, derivative and the analogue of human vasicentric protein 69.As used herein, term " fragment ", " derivative " are meant with " analogue " and keep identical biological function of human vasicentric protein 69 of the present invention or active polypeptide basically.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 3488 bases, and its open reading frame (462---2342) 626 amino acid of having encoded.Find relatively that according to amino acid sequence homologous this polypeptide and fruit bat Kelch albumen have 74% homology, deducibility goes out the 26S Proteasome Structure and Function that this human vasicentric protein 69 has fruit bat Kelch protein similar.

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

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

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

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

The invention still further relates to and the polynucleotide (have at least 50% between two sequences, preferably have 70% homogeny) of sequence hybridization described above.The present invention be more particularly directed under stringent condition and the interfertile polynucleotide of polynucleotide of the present invention.In the present invention, " stringent condition " is meant: (1) than hybridization under low ionic strength and the comparatively high temps and wash-out, as 0.2 * SSC, and 0.1%SDS, 60 ℃; Or (2) hybridization the time adds and to use denaturing agent, as 50% (v/v) methane amide, 0.1% calf serum/0.1%Ficoll, 42 ℃ etc.; Or (3) only at the homogeny between the two sequences at least more than 95%, be more preferably 97% and just hybridize when above.And the polypeptide of interfertile polynucleotide encoding has identical biological function and activity with the mature polypeptide shown in the 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.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 human vasicentric protein 69.

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

The special polynucleotide sequence of coding human vasicentric protein 69 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 the polymeric enzyme reaction technology, even few expression product also can be cloned.

Available ordinary method is screened gene of the present invention from these cDNA libraries.These methods include, but is not limited to: (1) DNA-DNA or DNA-RNA hybridization; (2) appearance of marker gene function or forfeiture; (3) level of the transcript of mensuration human vasicentric protein 69; (4), detect the protein product of genetic expression by immunological technique or mensuration biologic activity.Aforesaid method can singly be used, but also several different methods combined utilization.

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

In (4) kind method, detect the protein product of human vasicentric protein 69 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, 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 with the host cell of human vasicentric protein 69 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 human vasicentric protein 69 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 human vasicentric protein 69 of encoding and the expression vector of suitable transcribing/translational control element.These methods comprise (Sambroook, et al.Molecular Cloning, a LaboratoryManual, cold Spring Harbor Laboratory.New York, 1989) such as extracorporeal recombinant DNA technology, DNA synthetic technology, the interior recombinant technologys of body.Described dna sequence dna can effectively be connected on the suitable promotor in the expression vector, and is synthetic to instruct mRNA.The representative example of these promotors has: colibacillary lac or trp promotor; The P of lambda particles phage _LPromotor; Eukaryotic promoter comprises LTRs and some other known may command gene expression promoter in prokaryotic cell prokaryocyte or eukaryotic cell or its virus of CMV immediate early promoter, HSV thymidine kinase promoter, early stage and late period SV40 promotor, retrovirus.Expression vector also comprises ribosome bind site that translation initiation is used and transcription terminator etc.Inserting enhancer sequence in carrier will make its transcribing in higher eucaryotic cells be enhanced.Enhanser is the cis acting factor that DNA expresses, and nearly 10 to 300 base pairs act on promotor transcribing with enhancing gene usually.Can for example be included in the SV40 enhanser of 100 to 270 base pairs of replication origin side in late period one, at the polyoma enhanser of replication origin side in late period one and adenovirus enhanser etc.

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

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

Among the present invention, the polynucleotide of coding human vasicentric protein 69 or the recombinant vectors that contains these polynucleotide can transform or transduce into host cell, contain the genetically engineered host cell of these polynucleotide or recombinant vectors with formation.Term " host cell " refers to prokaryotic cell prokaryocyte, as bacterial cell; Or eukaryotic cell such as low, as yeast cell; Or higher eucaryotic cells, as mammalian cell.Representative example has: intestinal bacteria, streptomyces; Bacterial cell such as Salmonella typhimurium; Fungal cell such as yeast; Vegetable cell; Insect cell such as fruit bat S2 or Sf9; Zooblast such as CHO, COS or Bowes melanoma cells etc.

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

By the recombinant DNA technology of routine, utilize polynucleotide sequence of the present invention to can be used to express or produce human vasicentric protein 69 (Science, 1984 of reorganization; 224:1431).In general following steps are arranged:

(1). with the polynucleotide (or varient) of coding of the present invention people human vasicentric protein 69, or with the recombinant expression vector that contains these polynucleotide proper host cell that transforms or transduce;

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

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

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

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

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

Kelch albumen sperm, ovum form and growth course in endless tube formation, nerve fiber location, cytogamy, cellular form keep etc. in the process and play an important role.The Kelch protein mutation can cause diseases such as ovum forms unusually, dysplasia.

Polypeptide fragment or derivatives thereof of the present invention can be used for treating and prevent and can also be used to treating and preventing dysontogenesis and associated various heredopathias owing to sperm, ovum form diseases such as infertility that not normal or smart ovum causes in conjunction with obstacle and congenital dysplasia; Various organs, tissue development are bad etc.

Polypeptide fragment or derivatives thereof of the present invention can also be used to the disease for the treatment of and preventing to cause owing to nervous system development is not normal, as neuromuscular disease, includes but not limited to: congenital dyskinesia, congenital paralysis, spinal muscular atrophy etc.; Neuropathy, diseases such as congenital mental retardation.

The present invention also provides SCREENED COMPOUND to identify the method that improves (agonist) or check the medicament of (antagonist) human vasicentric protein 69.Agonist improves human vasicentric protein 69 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 in the presence of medicine, the film preparation of mammalian cell or expressing human vasicentric protein 69 be cultivated with the human vasicentric protein 69 of mark.Measure the medicine raising then or check this interactional ability.

The antagonist of human vasicentric protein 69 comprises antibody, compound, acceptor disappearance thing and the analogue etc. that filter out.The antagonist of human vasicentric protein 69 can combine and eliminate its function with human vasicentric protein 69, 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, human vasicentric protein 69 can be added during bioanalysis measures, by measuring compound interactional influence between human vasicentric protein 69 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 human vasicentric protein 69 bonded peptide molecule obtains.During screening, generally tackle the human vasicentric protein 69 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 human vasicentric protein 69 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 human vasicentric protein 69 direct injection of the production of polyclonal antibody immune animal (as rabbit, mouse, rat etc.) 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 of preparation human vasicentric protein 69 include but not limited to hybridoma technology (Kohler andMilstein.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-human vasicentric protein 69.

The antibody of anti-human vasicentric protein 69 can be used in the immunohistochemistry technology, detects the human vasicentric protein 69 in the biopsy specimen.

With the also available labelled with radioisotope of human vasicentric protein 69 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 human vasicentric protein 69 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 human vasicentric protein 69 positive cells.

The disease that antibody among the present invention can be used for treating or prevention is relevant with human vasicentric protein 69.The antibody that gives suitable dosage can stimulate or block the generation or the activity of human vasicentric protein 69.

The invention still further relates to the diagnostic testing process of quantitative and detection and localization human vasicentric protein 69 level.These tests are known in the art, and comprise that FISH measures and radioimmunoassay.The human vasicentric protein 69 level that is detected in the test can be with laying down a definition the importance of human vasicentric protein 69 in various diseases and be used to the disease of diagnosing human vasicentric protein 69 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 human vasicentric protein 69 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 human vasicentric protein 69 or the unusual/non-activity expression.The gene therapy vector (as virus vector) of reorganization can be designed for the human vasicentric protein 69 of expressing variation, to suppress endogenic human vasicentric protein 69 activity.For example, a kind of human vasicentric protein 69 of variation can be the human vasicentric protein 69 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 human vasicentric protein 69 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 human vasicentric protein 69 are transferred in the cell.The method of recombinant viral vector that structure carries the polynucleotide of coding human vasicentric protein 69 is found in existing document (Sambrook, et al.).The polynucleotide of reorganization coding human vasicentric protein 69 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 human vasicentric protein 69 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 human vasicentric protein 69 can be used for the diagnosis with the relative disease of human vasicentric protein 69.The unconventionality expression of the expression that the polynucleotide of coding human vasicentric protein 69 can be used for detecting human vasicentric protein 69 human vasicentric protein 69 whether or under morbid state.As the dna sequence dna of the human vasicentric protein 69 of encoding can be used for biopsy specimen is hybridized to judge the expression situation of human vasicentric protein 69.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 human vasicentric protein 69 with the special primer of human vasicentric protein 69.

The sudden change that detects the human vasicentric protein 69 gene also can be used for the disease of diagnosing human vasicentric protein 69 relevant.The form of human vasicentric protein 69 sudden change comprises that the point mutation compared with normal wild type human vasicentric protein 69 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).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.Human vasicentric protein 69 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 human vasicentric protein 69 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 the proteic amino acid sequence homology comparison diagram of inventor's vasicentric protein 69 and fruit bat Kelch.The top sequence is a human vasicentric protein 69, and the below sequence is a fruit bat Kelch albumen.Same amino acid represents with monocase amino acid that between two sequences similar amino acid is represented with "+".

Fig. 2 is the polyacrylamide gel electrophoresis figure (SDS-PAGE) of isolating human vasicentric protein 69.69kDa 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 clone of human vasicentric protein 69

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 sequenclng kit (Perkin-Elmer company product) and ABI 377 automatic sequencers (Perkin-Elmer company).CDNA sequence and the existing public dna sequence data storehouse (Genebank) measured are compared, found that the cDNA sequence of one of them clone 0934e08 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 0934e08 clone is 3488bp (shown in Seq ID NO:1), from 462bp to 2342bp the open reading frame (ORF) of a 1881bp, the new protein (shown in Seq ID NO:2) of encoding arranged.We are with this clone's called after pBS-0934e08, encoded protein matter called after human vasicentric protein 69.

Embodiment 2:cDNA clone's homology retrieval

With the sequence and the encoded protein sequence thereof of human vasicentric protein 69 of the present invention, with Blast program (BasiclocalAlignment search tool) [Altschul, SF et al.J.Mol.Biol.1990; 215:403-10], carry out the homology retrieval at databases such as Genbank, Swissport.The gene the highest with human vasicentric protein 69 homology of the present invention is a kind of known fruit bat Kelch albumen, and its encoded protein number is AL035424 in the access of Genbank.Protein homology the results are shown in Fig. 1, both height homologies, and its homogeny is 74%; Similarity is 86%.Embodiment 3: with the gene of RT-PCR method clones coding human vasicentric protein 69

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’-TGTTTCATCTTTATTCATTATCC-3’(SEQ?ID?NO:3)

Primer2:5’-CGCCGCTTATATCTTATAGGATT-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 1-3488bp shown in the SEQ ID NO:1 are identical.Embodiment 4:Northern blotting analyst vasicentric protein 69 expression of gene:

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 human vasicentric protein 69 coding region sequence (462bp to 2 342bp) of pcr amplification shown in Figure 1.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: the vivoexpression of recombinant human vasicentric protein 69, separation and purifying

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’-CATCCATGGCATCCACCTCTGAAGTCCCTGCATT-3’(Seq?ID?No:5)

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

5 ' end of these two sections primers contains Nco I and BamH I restriction enzyme site respectively, be respectively the encoding sequence of target gene 5 ' end and 3 ' end thereafter, Nco 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-0934e08 plasmid that contains the total length goal gene is template, carries out the PCR reaction.The PCR reaction conditions is: contain pBS-0934e08 plasmid 10pg, primer Primer-3 and Primer-4 among the cumulative volume 50 μ l and be respectively 10pmol, Advantage polymerase Mix (Clontech company product) 1 μ l.Loop parameter: 94 ℃ of 20s, 60 ℃ of 30s, 68 ℃ of 2min, totally 25 circulations.Respectively amplified production and plasmid pET-28 (+) are carried out double digestion with Nco 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-0934e08) 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-0934e08) 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 Cartridge (Novagen company product), has obtained the target protein human vasicentric protein 69 of purifying.Through the SDS-PAGE electrophoresis, obtain a single band (Fig. 2) at the 69kDa 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-human vasicentric protein 69 production of antibodies

Synthesize the specific polypeptide of following human vasicentric protein 69 with Peptide synthesizer (PE company product):

NH ₂-Met-Ala-Ser-Thr-Ser-Glu-Val-Pro-Ala-Phe-Glu-Phe-Thr-Ala-Glu-COOH(SEQ?ID?NO:7)。Form compoundly with hemocyanin and bovine serum albumin coupling this polypeptide respectively, method is referring to Avrameas, et al.Immunochemistry, 1969; 6:43.Add the complete Freund's adjuvant immunizing rabbit with the above-mentioned hemocyanin polypeptide complex of 4mg, add the incomplete Freund's adjuvant booster immunization once with the hemocyanin polypeptide complex again after 15 days.Employing is done the titre that ELISA measures antibody in the rabbit anteserum through the titer plate of 15 μ g/ml bovine serum albumin polypeptide complex bag quilts.From the rabbit anteserum of antibody positive, separate total IgG with albumin A-Sepharose.Polypeptide is incorporated on the Sepharose4B post of cyanogen bromide-activated, from total IgG, separates anti-peptide antibody with affinity chromatography.Immuno-precipitation proof antibody purified can combine with human vasicentric protein 69 specifically.

Sequence table

(1) general information:

(ⅱ) denomination of invention: human vasicentric protein 69 and encoding sequence thereof

(ⅲ) sequence number: 7

(2) information of SEQ ID NO:1:

(ⅱ) sequence signature:

(A) length: 3488bp

(B) type: nucleic acid

(C) chain: two strands

(D) topological framework: linearity

(ⅱ) molecule type: cDNA

( ⅹⅰ ) ：SEQ ID NO：1： 1 TGTTTCATCTTTATTCATTATCCTTTGTTCTTTAAAATCTGATATATTGGCATAAAAGTA 61 ATTGTACATATATATATGAATGTGATTTATTTTCCTTTACATCTTTTTGTTGTGTACAGC121 AGGGCATATACTTCTCTTGTCTTGGTTGGATGCACAAATCTGTGTGCAGTGCTTTTTGCC181 CGTTGCCTAGACGATCACTTGGTTTCTCTGAGGATGTCTGGTTCTCGTAAAGAGTTTGAT241 GTGAAACAGATTTTGAAAATCAGATGGAGGTGGTTTGGTCATCAAGCATCATCTCCTAAT301 TCTACAGTTGACAGCCAGCAGGGAGAATTTTGGAACCGAGGACAGACTGGAGCAAACGGT361 GGGAGAAAGTTTTAGATCCATGTAGCCTACAATTGCCTTTGGCTTCAATTGGTTACCGAA421 GGTCCAGCCAACTGGATTTTCAGAATTCACCTTCTTGGCCAATGGCATCCACCTCTGAAG481 TCCCTGCATTTGAGTTTACAGCAGAAGATTGTGGCGGTGCACATTGGCTGGATAGACCAG541 AAGTGGATGATGGCACTAGTGAAGAAGAAAATGAATCTGATTCCAGTTCATGCAGGACTT601 CCAATAGTAGTCAGACATTATCATCCTGTCATACTATGGAGCCATGTACATCAGATGAAT661 TTTTCCAAGCCCTTAATCATGCCGAGCAAACATTTAAAAAAATGGAAAACTATTTGAGAC721 ATAAACAGTTGTGTGATGTAATTTTAGTCGCTGGTGATCGCAGAATTCCAGCTCACAGAT781 TGGTGCTCTCCTCTGTCTCAGACTATTTTGCTGCCATGTTTACTAATGATGTCAGAGAGG841 CAAGACAAGAAGAAATAAAAATGGAAGGTGTAGAACCAAATTCGTTGTGGTCCTTGATCC901 AGTATGCTTATACAGGCCGCCTTGAATTAAAAGAAGATAATATTGAGTGCCTGTTATCTA 961 CAGCTTGCCTTCTTCAGCTTTCACAGGTTGTAGAAGCATGCTGTAAGTTTTTAATGAAAC1021 AGCTTCATCCATCCAACTGTCTTGGAATTCGTTCTTTTGCTGATGCCCAAGGTTGTACAG1081 ATTTGCATAAAGTGGCTCACAATTATACTATGGAGCATTTCATGGAAGTAATCAGAAACC1141 AGGAATTTGTATTATTACCAGCCAGCGAAATTGCAAAGCTCTTGGCTAGTGATGACATGA1201 ACATTCCTAATGAGGAGACAATATTGAATGCACTTCTTACTTGGGTCCGTCATGATTTGG1261 AACAGAGACGGAAAGATCTAAGTAAACTTTTGGCTTATATTAGGCTACCTCTTCTTGCAC1321 CACAGTTCCTGGCAGACATGGAAAATAATGTACTTTTTCGGGATGATATAGAATGTCGGA1381 AACTCATTATGGAAGCAATGAAGTACCATTTATTACCAGAGAGACGACCCATGTTACAAA1441 GTCCTCGGACAAAACCTAGGAAGTCAACTGTTGGTACATTATTTGCAGTTGGGGGAATGG1501 ATTCAACAAAAGGAGCAACAAGCATTGAAAAGTATGATCTCCGTACAAATATGTGGACTC1561 CAGTAGCAAATATGAATGGGAGGAGGCTACAGTTCGGTGTTGCAGTGCTAGATGACAAAC1621 TGTATGTGGTTGGAGGAAGAGATGGACTGAAGACTTTGAATACTGTAGAGTGCTACAACC1681 CCAAAACAAAAACTTGGAGTGTGATGCCACCTATGTCCACACATAGACATGGCCTTGGTG1741 TGGCTGTACTGGAAGGTCCCATGTATGCCGTAGGAGGACATGATGGCTGGAGCTATCTGA1801 ACACAGTGGAAAGATGGGACCCTCAGGCTCGCCAGTGGAATTTTGTTGCCACTATGTCTA1861 CCCCTAGGAGTACAGTAGGTGTGGCAGTACTAAGTGGAAAACTTTATGCAGTTGGTGGTC1921 GTGATGGAAGTTCTTGTCTCAAATCAGTAGAATGTTTTGATCCTCATACTAATAAGTGGA1981 CACTGTGTGCACAGATGTCAAAAAGGAGAGGTGGCGTAGGAGTGACGACCTGGAATGGAC2041 TGCTGTATGCTATAGGGGGGCACGATGCTCCCGCATCCAACTTGACTTCCAGACTCTCAG2101 ACTGTGTGGAAAGATATGATCCCAAAACAGACATGTGGACTGCAGTAGCATCCATGAGCA2161 TCAGCAGAGATGCAGTGGGGGTCTGTTTACTTGGTGATAAGTTATATGCTGTTGGGGGGT2221 ATGATGGACAGGCATACCTTAATACCGTGGAGGCTTATGATCCCCAGACAAATGAGTGGA2281 CCCAGGTTGCTCCACTGTGCCTAGGAAGAGCTGGAGCTTGTGTTGTGACTGTAAAATTAT2341 AATTTAGTGCCCTGTTTTCTACATGAAGACACCGTCTTCCTTTATTAATTTAGTATAATT2401 ATTCTATCAATGGATACATTTTTAGTAAATGTGCATTGTCACAATCCTGGGCACAAAGTG2461 CCTGATGTCAAAATGAAGATAGTAAAACAAGGGAGGAAGCAGTGGATGGACCAGGATTAA2521 TTCCTTTCATTTCTTAGTAAATTAAAACCTGCAGCTGGTGGATTGTGAACACACATTCCC2581 GAAGTAATAAGTGAGGACGAATGCACTGCTCTGGAACATAACCCAGTGCTAACTGGGGGT2641 TTCATTTATTCAGTCAAGCACATCTTACTCACATCCAGATTTATTTTCCTACAGTGCAAA2701 CACACCAGATGAAACTTTAAAATGTTACTTTTTGTAAGCTTATCATAAATGAGTTGCAGT2761 AATTTGTTTGCTTGTTTGTTTAACCACAACCACTATTTTAATGATATACTAAAGATAACA2821 CTATTTAGTTTTTTCAGAAACATCTGCATTATATGTGTGTTGGTTGTGGATTTTGTTTCT2881 AAAATTGGCTTAGTCCAATAAATAAAGAAAAGCATTAAGGACTTAAAGCAACAATAACCA2941 AATAAAAACTTGATAGGATCTTTGAAGTCTATTTAAATATTCATTCCATTACATCTAGAC3001 TCACCAAGAACTACATGTTATGATGTTAAGTTGAAGTTGAAACATGATGTTTTGCATTAA3061 ATTTAAGATATGCAAATTTATGTAGAGAAAATAAATGTTATATACCCTATAATCTTTCAC3121 CTAATTAGTATTTAATTATATGGATTTGTTTTATATTATAAAAGATGTTTTGATTTTGTC3181 TTTTGATATTGACAAAATTGTTTGGATATCCTTATGTTCTCAAGTCTGTATCTGCCTCCC3241 CTGCCTTATTTCTTATGTTTTGCCACAGTTAACCCATTGTGCTTCTTTGTAATCAAACAG3301 TTTGTGGGAGAATGGGCTTATTGAATGTCTAAAAAATAAGTTTAAAGTGTTTGTTACCCT3361 AAGTTTTTTGCATTTTTAAACTCTAATTACATATGTGAATGTTATTACTCTCAGTGAATT3421 GTTATTGTTTGCAAAAATGCACTGGGCAGTAACATTTTGTGATAAATCCTATAAGATATA3481 AGCGGCGN

(3) information of SEQ ID NO:2:

(ⅰ) sequence signature:

(A) length: 626 amino acid

(B) type: amino acid

(D) topological framework: linearity

(ⅱ) molecule type: polypeptide

( ⅹⅰ ) ：SEQ ID NO:2： 1 Met Ala Ser Thr Ser Glu Val Pro Ala Phe Glu Phe Thr Ala Glu 16 Asp Cys Gly Gly Ala His Trp Leu Asp Arg Pro Glu Val Asp Asp 31 Gly Thr Ser Glu Glu Glu Asn Glu Ser Asp Ser Ser Ser Cys Arg 46 Thr Ser Asn Ser Ser Gln Thr Leu Ser Ser Cys His Thr Met Glu 61 Pro Cys Thr Ser Asp Glu Phe Phe Gln Ala Leu Asn His Ala Glu 76 Gln Thr Phe Lys Lys Met Glu Asn Tyr Leu Arg His Lys Gln Leu 91 Cys Asp Va1 Ile Leu Val Ala Gly Asp Arg Arg Ile Pro Ala His106 Arg Leu Val Leu Ser Ser Val Ser Asp Tyr Phe Ala Ala Met Phe121 Thr Asn Asp Val Arg Glu Ala Arg Gln Glu Glu Ile Lys Met Glu136 Gly Val Glu Pro Asn Ser Leu Trp Ser Leu Ile Gln Tyr Ala Tyr151 Thr Gly Arg Leu Glu Leu Lys Glu Asp Asn Ile Glu Cys Leu Leu166 Ser Thr Ala Cys Leu Leu Gln Leu Ser Gln Val Val Glu Ala Cys181 Cys Lys Phe Leu Met Lys Gln Leu His Pro Ser Asn Cys Leu Gly196 Ile Arg Ser Phe Ala Asp Ala Gln Gly Cys Thr Asp Leu His Lys211 Val Ala His Asn Tyr Thr Met Glu His Phe Met Glu Val Ile Arg226 Asn Gln Glu Phe Val Leu Leu Pro Ala Ser Glu Ile Ala Lys Leu241 Leu Ala Ser Asp Asp Met Asn Ile Pro Asn Glu Glu Thr Ile Leu256 Asn Ala Leu Leu Thr Trp Val Arg His Asp Leu Glu Gln Arg Arg271 Lys Asp Leu Ser Lys Leu Leu Ala Tyr Ile Arg Leu Pro Leu Leu286 Ala Pro Gln Phe Leu Ala Asp Met Glu Asn Asn Val Leu Phe Arg301 Asp Asp Ile Glu Cys Arg Lys Leu Ile Met Glu Ala Met Lys Tyr316 His Leu Leu Pro Glu Arg Arg Pro Met Leu Gln Ser Pro Arg Thr331 Lys Pro Arg Lys Ser Thr Val Gly Thr Leu Phe Ala Val Gly Gly346 Met Asp Ser Thr Lys Gly Ala Thr Ser Ile Glu Lys Tyr Asp Leu361 Arg Thr Asn Met Trp Thr Pro Val Ala Asn Met Asn Gly Arg Arg376 Leu Gln Phe Gly Val Ala Val Leu Asp Asp Lys Leu Tyr Val Val391 Gly Gly Arg Asp Gly Leu Lys Thr Leu Asn Thr Val Glu Cys Tyr406 Asn Pro Lys Thr Lys Thr Trp Ser Val Met Pro Pro Met Ser Thr421 His Arg His Gly Leu Gly Val Ala Val Leu Glu Gly Pro Met Tyr436 Ala Val Gly Gly His Asp Gly Trp Ser Tyr Leu Asn Thr Val Glu451 Arg Trp Asp Pro Gln Ala Arg Gln Trp Asn Phe Val Ala Thr Met466 Ser Thr Pro Arg Ser Thr Val Gly Val Ala Val Leu Ser Gly Lys481 Leu Tyr Ala Val Gly Gly Arg Asp Gly Ser Ser Cys Leu Lys Ser496 Val Glu Cys Phe Asp Pro His Thr Asn Lys Trp Thr Leu Cys Ala511 Gln Met Ser Lys Arg Arg Gly Gly Val Gly Val Thr Thr Trp Asn526 Gly Leu Leu Tyr Ala Ile Gly Gly His Asp Ala Pro Ala Ser Asn541 Leu Thr Ser Arg Leu Ser Asp Cys Val Glu Arg Tyr Asp Pro Lys556 Thr Asp Met Trp Thr Ala Val Ala Ser Met Ser Ile Ser Arg Asp571 Ala Val Gly Val Cys Leu Leu Gly Asp Lys Leu Tyr Ala Val Gly586 Gly Tyr Asp Gly Gln Ala Tyr Leu Asn Thr Val Glu Ala Tyr Asp601 Pro Gln Thr Asn Glu Trp Thr Gln Val Ala Pro Leu Cys Leu Gly616 Arg Ala Gly Ala Cys Val Val Thr Val Lys Leu

(4) information of SEQ ID NO:3

(ⅰ) sequence signature

(A) length: 23 bases

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity

(ⅱ) molecule type: oligonucleotide

(ⅹ ⅰ) sequence description: SEQ ID NO:3:TGTTTCATCTTTATTCATTATCC 23

(5) information of SEQ ID NO:4

(ⅰ) sequence signature

(A) length: 23 bases

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity

(ⅱ) molecule type: oligonucleotide

(ⅹ ⅰ) sequence description: SEQ ID NO:4:CGCCGCTTATATCTTATAGGATT 23

(6) information of SEQ ID NO:5

(ⅰ) sequence signature

(A) length: 34 bases

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity

(ⅱ) molecule type: oligonucleotide

(ⅹ ⅰ) sequence description: SEQ ID NO:5:CATCCATGGCATCCACCTCTGAAGTCCCTGCATT 34

(7) information of SEQ ID NO:6

(ⅰ) sequence signature

(A) length: 34 bases

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity

(ⅱ) molecule type: oligonucleotide

(ⅹ ⅰ) sequence description: SEQ ID NO:6:CATGGATCCCCTAATTTTACAGTCACAACACAAG 34

(8) information of SEQ ID NO:7:

(ⅰ) sequence signature:

(A) length: 15 amino acid

(B) type: amino acid

(D) topological framework: linearity

(ⅱ) molecule type: polypeptide

(ⅹ ⅰ) sequence description: SEQ ID NO:7:NH ₂-Met-Ala-Ser-Thr-Ser-Glu-Val-Pro-Ala-Phe-Glu-Phe-Thr-Ala-Glu-COOH 15

Claims (18)

1, a kind of isolated polypeptide-human vasicentric protein 69 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 75% 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 1-3488 position among the sequence of 462-2342 position among the SEQ IDNO:1 or the SEQ ID NO:1.

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

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

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

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

9, a kind of preparation method with the active polypeptide of human vasicentric protein 69 is characterized in that described method comprises:

(a) under expressing human vasicentric protein 69 condition, cultivate the described through engineering approaches host cell of claim 8;

(b) from culture, isolate and have the active polypeptide of human vasicentric protein 69.

10, a kind of can with polypeptide bonded antibody, it is characterized in that described antibody be can with human vasicentric protein 69 specificity bonded antibody.

11, the compound of an analoglike or adjusting polypeptide active or expression is characterized in that they are simulation, promotion, antagonism or the active compound that suppresses human vasicentric protein 69.

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 mediator's vasicentric protein 69 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 stand-in of human vasicentric protein 69, 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 pharmaceutical composition with safe and effective dosage and pharmaceutically acceptable carrier as diagnosis or the treatment disease relevant unusually with human vasicentric protein 69 with described polypeptide, polynucleotide or its stand-in, agonist, antagonist or inhibitor as the described polypeptide of arbitrary claim, polynucleotide or application of compound in claim 1-6 and 11.

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

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