CN1302877A - Polypeptide-human nuclein II precursor protein 25 and polynucleotide for coding it - Google Patents

Abstract

A new polypeptide-human nuclein II precursor protein 25, the polynucleotide for encoding it, the process for preparing said polypeptide by DNA recombination, the application of said polypeptide to treating diseases (cancer, HIV infection, etc), the antagonist against the said polypeptide and its medical action, and the usage of said polynucleotide are disclosed.

Description

The polynucleotide of a kind of new polypeptide--HCP and this peptide species of coding

The invention belongs to biological technical field, specifically, the invention describes a kind of new polypeptide--HCP, and the polynucleotide sequence of this polypeptide of encoding.The invention still further relates to the preparation method and the application of these polynucleotide and polypeptide.

The main energy derive of cellular activity is in ATP.Plastosome is the place that sugar, fat and amino acid finally release energy.Nutritive substances such as sugar and fat produce pyruvic acid and lipid acid through zymohydrolysis in tenuigenin, these matter selective ground enter the mitochondrial matrix from tenuigenin, be degraded to the unit that contains 2 carbon atoms through a series of variations, combine the formation acetyl-CoA with coenzyme A and enter tricarboxylic acid cycle, the electron transfer system of the hydrogen that tricarboxylic acid cycle is taken off on the plastosome inner membrance is respiratory chain, transmits oxygen supply at last.In this process, the electronics that energy level is higher drops to the energy lower level through electron transport.Wherein the energy that is discharged, generates the ATP that contains high-energy phosphate bond and is stored in the body by the phosphorylation of ADP with the high-energy phosphate bond form.As seen from the above, respiratory chain is the important system that energy is provided to organism, and vital movement is had significant meaning.(press of chief editor such as cytobiology Wang Ren Beijing Normal University)

Respiratory chain mainly is made up of following several quasi-molecules: the desaturase that pyridine nucleotide connects, desaturase (flavoprotein), iron-sulphur protein, ubiquinone, cytopigment that flavine is relevant.

The component that experiment showed, respiratory chain all is embedded in (except that ubiquinone and cytochrome c) in the mitochondrial inner membrance with the form of polymolecular mixture.

Complex is a nadh dehydrogenase, and its effect is that 2 electron transport of catalyzing N ADH are to ubiquinone;

Complex is a succinodehydrogenase, and the catalysis electronics is passed to ubiquinone from succsinic acid by FAD and iron-sulphur protein;

The mixture III is cytochrome b-c, and its effect is that the catalysis electronics is passed to cytochrome c from ubiquinone, and the film of striding that proton takes place simultaneously shifts;

The mixture IV is a cytochrome c oxidase, is used for the electron transport oxygen supply that the catalysis cytochrome c is accepted.(cytobiology Zhai Zhonghe edits Higher Education Publishing House)

At this wherein, the mixture III has about 10-11 subunit.The nucleoprotein II is the subunit that the wherein second largest nuclear is encoded.(Shimomura,Y?and?Ozawa,T?1982?Biochem?Int?5,1-6)；(MethodsEnzymol?126,224-237)

Be cloned into the precursor of nucleoprotein II from the liver cDNA library of mouse, it is a polypeptide that 438 amino-acid residues are arranged, and its molecular weight is approximately 47KD.According to existing result of study, we have also known mouse, people and the structural identical and difference of zymic nucleoprotein II.Concrete research process please refer to relevant document with the result.(Biochemistry?International?1990?Vol?20?No?4?pp731-737)；(J?BiolChem?1989?264,13483-13488)

The nucleoprotein II does not have the redoxomorphism center, therefore it does not participate in electron transfer process directly, and the effect of nucleoprotein II in respiratory chain also is in the research process, more molecular biological studies show that, if lack the nucleoprotein II, can influence the combination of a lot of subunits of mixture III.(J?Biol?Chem?1988?263,14323-14333)；(Eur?J?Biochem?1987?163,97-103)

Though the research to nucleoprotein II 26S Proteasome Structure and Function is all unclear fully as yet, but we can know basically, the nucleoprotein II plays an important role in mixture III (cytochrome reductase), thereby directly affect the normal function of respiratory chain, indirect adjustments and controls oxidative phosphorylation process, mitochondrial function, to the energy of biology provide, vital movement has important role.

According to amino acid homology result relatively, polypeptide of the present invention is accredited as a kind of new HCP (HCPIIP25) by deduction, the nucleoprotein II precursor that its homologous protein is behaved, and albumen number is J04973.

An object of the present invention is to provide isolating new polypeptide--the HCP 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 HCP that encodes.

Another object of the present invention provides the genetically engineered host cell of the polynucleotide that contain the HCP that encodes.

Another object of the present invention provides the method for producing the HCP.

Another object of the present invention provides at polypeptide of the present invention--HCP's antibody.

Another object of the present invention has provided at polypeptide of the present invention--HCP's simulated compound, antagonist, agonist, inhibitor.

Another object of the present invention provides the method for the diagnoses and treatment disease relevant unusually with the HCP.

In a first aspect of the present invention, novel isolated HCP 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 91% homogeny with a kind of nucleotides sequence that is selected from down group: (a) the above-mentioned HCP's of coding polynucleotide; (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 2572-3249 position among the SEQ ID NO:1; (b) has the sequence of 1-3425 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 HCP " is meant that the HCP 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 HCP of standard.Basically pure polypeptide can produce single master tape on non-reduced polyacrylamide gel.The purity of HCP's polypeptide can be used amino acid sequence analysis.

The invention provides a kind of new polypeptide--HCP, it is made up of the aminoacid sequence shown in the SEQ IDNO: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 HCP's fragment, derivative and analogue.As used herein, term " fragment ", " derivative " are meant with " analogue " and keep identical biological function of HCP 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 3425 bases, its open reading frame (2572-3249) 225 amino acid of having encoded.Find relatively that according to amino acid sequence homologous this polypeptide and people's nucleoprotein II precursor have 91% homology, deducibility goes out this HCP and has the similar 26S Proteasome Structure and Function of people's nucleoprotein II precursor.

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 coding HCP's polynucleotide.

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

Coding HCP's of the present invention special polynucleotide sequence 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 mensuration HCP's 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 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 HCP's 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 HCP's encoding sequence through the genetically engineered generation, and the method that produces polypeptide of the present invention through recombinant technology.

Among the present invention, coding HCP's polynucleotide sequence 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 HCP that encodes and the expression vector of suitable transcribing/translational control element.These methods comprise (Sambroook, et al.Molecular Cloning, aLaboratory Manual, cold Spring Harbor Laboratory.New York, 1989) such as extracorporeal recombinant DNA technology, DNA synthetic technology, the interior recombinant technologys of body.Described dna sequence dna can effectively be connected on the suitable promotor in the expression vector, and is synthetic to instruct mRNA.The representative example of these promotors has: colibacillary lac or trp promotor; The 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, coding HCP's polynucleotide 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 HCP (Science, 1984 of reorganization; 224:1431).In general following steps are arranged:

(1). with coding of the present invention people HCP's polynucleotide (or varient), 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 formed people's of polypeptide of the present invention (people's nucleoprotein II precursor protein) nucleoprotein II, it is the important component of cytochrome reductase in the respiratory chain, it is one of subunit by the cytochrome reductase of nucleus coding, more molecular biological studies show that, if lack the nucleoprotein II, can influence the combination of a lot of subunits of mixture III, can infer, though the nucleoprotein II does not have the redoxomorphism center, do not participate in electron transfer process directly, but it plays an important role for the stability and the normal function that keep mixture III (cytochrome reductase).

Research to polypeptide function of the present invention and structure is still waiting further deeply, function according to above-mentioned polypeptide of the present invention, polypeptide of the present invention can be used for diagnosis and treats a lot of diseases, as malignant tumour, endocrine system disease, nervous system disorders, immunological disease, people's acquired immune deficiency syndrome (AIDS) or the like.

Polypeptide of the present invention can be regulated and control micromolecular expression such as some hormones, thereby play endocrine system treatment of diseases function, comprise: pituitary tumor (prolactinoma), gigantosoma and acromegaly, simmonds-sheehan syndrome, the pituitary dwarfism, diabetes insipidus, syndrome of inappropriate ADH secretion, hyperthyroidism, first subtracts, simple goiter, thyroiditis, hydrocortisone increases to be levied, the primary aldosterone increases to be levied, pheochromocytoma, the primary chronic adrenal cortex hypofunction is levied, and primary hyperparathyroidism is levied, parathyroid gland technical ability goes down and levies, ectopic hormone secretion syndrome or the like.

Various tumours comprise: the tumour that comprises epithelium (as substrate epithelium, squama shape epithelium, myxocyte or the like), (as fibrous tissue, fatty tissue, cartilaginous tissue, smooth muscle tissue, blood vessel and lymphatic endothelial tissue or the like), hemopoietic tissue (as B cell, T cell, histocyte or the like), central nervous tissue, peripheral nerve tissue, endocrine tissue, gonadal tissue, particular tissues (as dental tissue or the like) source, for example, cancer of the stomach, liver cancer, large bowel cancer, mammary cancer, lung cancer, prostate cancer, cervical cancer, carcinoma of the pancreas, esophagus cancer or the like.

Polypeptide of the present invention is a kind of immunomodulator, has immunological enhancement or immunosuppressive action.Polypeptide of the present invention can be used for the treatment of some diseases, and these diseases comprise immunoreactive anergy, or abnormal immune reaction, or the host defend invalid.Polypeptide of the present invention and its antibody also have effect to damage, defective or the imbalance class disease of immuning tissue, particularly for disease of hematopoietic system (as pernicious anemia), tetter (as psoriasis), autoimmune disease (as rheumatoid arthritis), radiation disease and immune lymphocytic generation and adjusting have extremely close relation.

The present invention also provides SCREENED COMPOUND to identify the method that improves (agonist) or check (antagonist) HCP's medicament.Agonist improves HCP's 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 HCP with mark cultivates with mammalian cell or the film preparation of expressing the HCP.Measure the medicine raising then or check this interactional ability.

HCP's antagonist comprises antibody, compound, acceptor disappearance thing and the analogue etc. that filter out.HCP's antagonist can combine and eliminate its function with the HCP, or suppresses the generation of this polypeptide, or combines with the avtive spot of this polypeptide and to make this polypeptide can not bring into play biological function.

In screening during as the compound of antagonist, the HCP can be added in the bioanalysis mensuration, interactional influence between HCP and its acceptor be determined whether compound is antagonist by measuring compound.With the same quadrat method of above-mentioned SCREENED COMPOUND, can filter out the acceptor disappearance thing and the analogue of antagonist action.Can be incorporated into the rondom polypeptide storehouse that solid formation forms by the various amino acid that may make up by screening with HCP's bonded peptide molecule obtains.During screening, generally tackle HCP's 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 HCP 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 HCP's 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.Preparation HCP's the technology of monoclonal antibody include but not limited to hybridoma technology (Kohler and Milstein.Nature, 1975,256:495-497), three knurl technology, people B-quadroma technology, EBV-hybridoma technology etc.With the variable region bonded chimeric antibody in human constant region and inhuman source can with existing technology production (Morrison et al, PNAS, 1985,81:6851).And the technology of existing manufacture order chain antibody (U.S.Pat No.4946778) also can be used for producing anti-HCP's single-chain antibody.

Anti-HCP's antibody can be used in the immunohistochemistry technology, detects the HCP in the biopsy specimen.

With the also available labelled with radioisotope of HCP's 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 HCP's 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 HCP's positive cells.

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

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

Coding HCP's polynucleotide 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 HCP's nothing expression or the unusual/non-activity expression.The gene therapy vector (as virus vector) of reorganization can be designed for the HCP who expresses variation, to suppress endogenic HCP's activity.For example, a kind of HCP of variation can be the HCP who 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 HCP's 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 coding HCP's polynucleotide are transferred in the cell.The method of recombinant viral vector that structure carries coding HCP's polynucleotide is found in existing document (Sambrook, et al.).Reorganization coding HCP's polynucleotide 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 HCP 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.

Coding HCP's polynucleotide can be used for the diagnosis with HCP's relative disease.The expression that coding HCP's polynucleotide can be used for detecting HCP HCP's whether or under morbid state unconventionality expression.As the HCP's that encodes dna sequence dna can be used for biopsy specimen is hybridized to judge HCP's expression situation.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 the HCP with the special primer of HCP.

The sudden change that detects HCP's gene also can be used for the disease of diagnosing the HCP to be correlated with.The form of HCP sudden change comprises that the point mutation compared with normal wild type HCP 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.The HCP 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 HCP 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 amino acid sequence homology comparison diagram of HCP of the present invention and people's nucleoprotein II precursor.The top sequence is the HCP, and the below sequence is people's nucleoprotein II precursor.Same amino acid represents with monocase amino acid that between two sequences similar amino acid is represented with "+".

Fig. 2 is isolating HCP's polyacrylamide gel electrophoresis figure (SDS-PAGE).25kDa 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:Cold Sprlng HarborLaboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.Embodiment 1: HCP's clone

Extract the total RNA of people's tire brain with guanidinium isothiocyanate/phenol/chloroform single stage method.From total RNA, separate poly (A) mRNA with Quik mRNA Isolation Kit (Qiegene company product).2ug poly (A) mRNA forms cDNA through reverse transcription.CDNA fragment orientation is inserted on the multiple clone site of pBSK (+) carrier (Clontech company product) with Smart cDNA clone's test kit (available from Clontech), transforms DH5 α, bacterium forms the cDNA library.Measure all clones' 5 ' and 3 ' terminal sequence with Dyeterminate cycle reaction sequencing kit (Perkin-Elmer company product) and ABI 377 automatic sequencers (Perkin-Elmer company).CDNA sequence and the existing public dna sequence data storehouse (Genebank) measured are compared, found that the cDNA sequence of one of them clone 0077C07 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 0077C07 clone is 3425bp (shown in Seq ID NO:1), from 2572bp to 3249bp the open reading frame (ORF) of a 678bp, the new protein (shown in Seq ID NO:2) of encoding arranged.We are with this clone's called after pBS-0077C07, encoded protein matter called after HCP.

Embodiment 2:cDNA clone's homology retrieval

With HCP's of the present invention sequence and encoded protein sequence thereof, with Blast program (Basiclocal Alignment search tool) [Altschul, SF et al.J.Mol.Biol.1990; 215:403-10], carry out the homology retrieval at databases such as Genbank, Swissport.The gene the highest with HCP's homology of the present invention is a kind of known people's nucleoprotein II precursor, and its encoded protein number is J04973 in the access of Genbank.Protein homology the results are shown in Fig. 1, both height homologies, and its homogeny is 90%.Embodiment 3: with RT-PCR method clones coding HCP's gene

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

Primer2: 5’-TTAAACAGAATGTTTTATTGAC-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 1-3425bp shown in the SEQ ID NO:1 are identical.Embodiment 4:Northern blotting is analyzed HCP's 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)-SmM 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 HCP's coding region sequence (2572bp to 3249bp) 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: reorganization HCP's vivoexpression, 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’-CCCGCTAGCATGACAGCCTGTTCGATACGTTAG-3’NheⅠ -3’(Seq?IDNo:5)

Primer4:5’-CCCCTCGAGTTACAACTCATCAACAAAAGGTGT-3’XhoⅠ -3’(Seq?IDNo:6)

5 ' end of these two sections primers contains Nhe I and Xho I restriction enzyme site respectively, be respectively the encoding sequence of target gene 5 ' end and 3 ' end thereafter, Nhe I and Xho 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-0077C07 plasmid that contains the total length goal gene is template, carries out the PCR reaction.The PCR reaction conditions is: contain pBS-0077C07 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 2 min, totally 25 circulations.Respectively amplified production and plasmid pET-28 (+) are carried out double digestion with Nhe I and Xho 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-0077C07) 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-0077C07) 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 HCP of purifying.Through the SDS-PAGE electrophoresis, obtain a single band (Fig. 2) at the 25kDa 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-HCP's production of antibodies

Synthesize the specific polypeptide of following HCP with Peptide synthesizer (PE company product):

NH ₂-Met-Thr-Ala-Phe-Ser-Leu-Thr-Ala-Glu-Arg-Ser-Ser-Ser-Leu-Cys-COOH(SEQ?ID?NO:7)。Form compoundly with hemocyanin and bovine serum albumin coupling this polypeptide respectively, method is referring to Avrameas, et al.Immunochemi stry, 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 the HCP specifically.

Sequence table (1) general information:

(ⅱ) denomination of invention: HCP and encoding sequence thereof

(ⅲ) sequence number: the information of 7 (2) SEQ ID NO:1:

(ⅰ) sequence signature:

(A) length: 3425bp

(B) type: nucleic acid

(C) chain: two strands

(D) topological framework: linearity

(ⅱ) molecule type: cDNA

( ⅹⅰ ) ：SEQ ID NO:1: 1 GCTATTTGGCAATTTATAAATCCCCACTGTCCAGATTGAAAGTGCTATCAACACTCCTAA 61 TTCATTAGTAGTTCGAGGGTTTCTCTAAAAGAGACAGACTTGGGACAAAGTTGGAAATCA121 ACCTGCATTTTCATACAGCCGCTTAAGACATTTAAGTTTGACTCCATCTACTTAGAACAA181 TTCAAAATGTGAATCTTGCAGCTCCAGTGGTCGTTATGATCATTTAGTATTGCCCACAGG241 GGAAGCAGAATTACAACCCAGGATGGAACAACCTTTTGGTCATTTCTGTTCCCATTAGTA301 TTGTTAGTTTTGTTATGCTGAGCACACTACAAAGTTGCATCTCTTGTATAAGTACAACAC361 AATTTATTAAATATTGTATACATAGAACCAAGAATCGATAGGATGCAGGGCTGCTTGATT421 GTACCAGAAACAAAACATGATGAGAATACATTGAAACACTGGATTTACTTAAGAAAAACC481 TAAGTTTTTCAAATCTAATTTATTTAAGAAAACGTATTTTCTTAATAAGTGTAACACAGT541 TCTTACAAGGTATGGTAATAAGCAGAAAGTAGAATTTTGAATATTAATGTCAAAGTATGT601 TTTCTGCCATTTTGATGGAGTTGATACTTTTTCTTGTAAGTTCTTCCTGTAGTGAAAGAG661 AGAAAGTGAGGTGTCATTGCCGAAAGGACACACTAATAACCTCTTGGTAGTTTATTGCAG721 GTAAAATCATTTTTTAATTAAGCTGACAACGTGGTAAAACTGCAGCATTAAATTGTAATT781 ATGTTTAAATGGAAGTCTTTCTGTAGAGTGTTAAAGGATGATAAACGTTTGAGGTTCAAA841 TAACTCGACTGTCTTATCAAACACCATGTCTATAAGTAATAAGTTTCTGGGAGAATTGTG901 AATTGGATTTTGTTTTATTTCAAGTGGATGCTTTTGTGCCCATATTTAAGCTGCCGTTGC 961 TTACAAGACCTGTACTGCCCTTCCCCACCTGCCTTTTCCATCATACCCTATGCTCACAGA1021 CAAATCTCAGCAAGAGGCAGGAGTTGCTTCACTAATCAAAACCTTACATTAAAATAGTTT1081 TACCTAGATCTAAGTTCTAGTCCCAAATCAAAAAGCAATCTGTGGAGTTTTCAGTATAAT1141 TTCGTACTGACAGCTTTCCAAATAGTAACGTAAACGGTTCACGTACTTGGTGAACTGAAG1201 AAATAGAATGGAGGCCATTAGCACTGTAAGTTGTTAGTTTGCCTGTTTACCAAAAATATG1261 TTGAGTTTCGGGGCACATACCTTAAAATTGCCCTGTTTCACTGTGTAAGAGGAACTGGCT1321 TGACTACTTCACCCTTGGTGAAAACAAACAGGCCCTAAAAGAGGCAGCAACAGCTGAAGT1381 GGATATCACAGAAATAAACAGACAGCAGAAGCTCTTCAGACACCCCCGAAACCTGCCAAA1441 ACTCACAGGTAATACAAGTGAAACGATAGCATAAGAAGAAAATAAACATGAGAATCTTCA1501 AACTACAAGCATTGAACGGTATCCTCCATCCAAGAAGAGAACCCACTTCATACAAGGAAA1561 AGGCAAAAGGAAAAGGTCAAGACTGTGACCTTTATAACACTGTGACCCCAGCTGTGGGCC1621 AGGTGGAGTTGTCTGTGGTGACACCAAGTACAGGCCTGACACCCTGGTTTGACAGAAGGC1681 TCTTCAAGATCCCTGGTCTTCACACTGTAGCAGCCAAAAGGTAGGTTTACAATATACACT1741 GTCCCCAACTTGCCATGGATCAATTATAATTTTTCAACTTGACTATGGAGGAAAACCATC1801 GCAATTTCAGCTTCCAGTCATGGGTCACATAACGATGGGGATACATTCTGAGAAATGTGG1861 TGTTAGGTGGTTTCATCACTGTGTGAACCTCAGTGTTCAGACAAATCTAGATGGTATAGC1921 CTACTACACACATGGGCTCTATGGTATAGTCTATTGCTCTTAGGCTACAAACCTGTATAG1981 CATGTCTATAGTACTGAATACTGTAGGCAGTTATAACACAAAGGTAAGTATTTGTGCATC2041 TAAACATAGGAAAGGTCAGTAAAAATACAGCACAAAAGATAAAAAAATGGTATGCCATTT2101 TTATGGCATAGCTCCTGCTTTCGAGTGAGTCAGTGAGTGAGTGGTGAGTAAATGGAAAGG2161 GCTGGACATTACTGTGCACTGCTGTAGATACTAAACACTGTAAACATAGGCTACGCTAAA2221 TTTATTTTTTTAAAATTGCACTACAACATTATGTTGTCAGTAAGTAATAGGAATCTTTCA2281 GCTCCCTAATAATCTTAACGGGACCACCATCATATATGTGGTCCATCATTGACCAAAAAC2341 ATCATTATGTAGTACATGACTATCCTTGAATTTCGATCTTTTCCTAGGCTAGTGATATGC2401 AGTACATGAGATAATCAATGCTTCCTTACAAAATGGGCTTATGTTAGAAGACTTTTTGCC2461 CAGCTGCAGGCTATTGTAAGTGTTCTGAGCACATATGAGATAACCTGGGCCAAGCTATGA2521 TGTTCGATACGTTAGGTGTATTAAATGCACTTTTGACTGCCATCTCAGTGGATGACAGCC2581 TTCTCACTGACAGCAGAGAGATCTTCCTCACTGTGCCAGTGGGCAGGAGAAAGAGCATGC2641 TGCGACTGGCCAGTGACATGCAGAGGATCCAGATTGCACAACCGGATCCAGAGGCCTTGG2701 GAAGCATTAGGGAGCTCTCCAGCTGTCTCACTCAAATCTGTAGCAGCATATGGACCCACA2761 AATGGAGGCCAAAGTGGGCCCTCAATTGGACACTTTGGCAACCCTGAAGAGACTTCTTCC2821 CTTCAGACTTACTGTTTTAGTATGTTCCAGAGAAGGGAGTGGCATTCTTCTTGGGGAATT2881 CAGGAAGAGGGAGACACGCTGTTACTCTATCCATTAAATAAAGCTTTGATGTTCACAGCC2941 CCCCGCCACCACTCAGAAAAAAAAATAATTGCCTTGCTCTCTAAAACTGACTTCTGCCTT3001 TTATTGGACAGGAACAAGCTGAAAGCTGGATACCTAATGTCAGTGGAGTCTTCTGAGTGT3061 TTCCTGGAAGAAGTCGGGTCCCAGGCTCTAGTTGCTGGTTCTTACATGCCACCATCCACA3121 GTCCTTCAGCAGATTGATTCAGTGGCTAATGCTGATATCATAAATGCGGCAAAGAAGTTT3181 GTTTCTGGCCAGAAGTCAATGGCAGCAAGTGGAAATTTGGGACATACACCTTTTGTTGAT3241 GAGTTGTAATACTGATGCACACATTACAGGAGAGAGCTGAACGTTCTCTCAGCCCAGAGC3301 AGCAAACACATGAAAGTCAGAAGTCTCTAATATATCATTTGTCTTTTTTCCAGTGAGGTA3361 AAATAAGGCATAAATGCAGGTAATTATTCCCAGCTGACCTAAAGTCAATAAAACATTCTG3421 TTTAA ( 3 ) SEQ ID NO:2：

(ⅰ) sequence signature:

(A) length: 952 amino acid

(B) type: amino acid

(D) topological framework: linearity

(ⅱ) molecule type: polypeptide

( ⅹⅰ ) ：SEQ ID NO:2: 1 Met Thr Ala Phe Ser Leu Thr Ala Glu Arg Ser Ser Ser Leu Cys 16 Gln Trp Ala Gly Glu Arg Ala Cys Cys Asp Trp Pro Val Thr Cys 31 Arg Gly Ser Arg Leu His Asn Arg Ile Gln Arg Pro Trp Glu Ala 46 Leu Gly Ser Ser Pro Ala Val Ser Leu Lys Ser Val Ala Ala Tyr 61 Gly Pro Thr Asn Gly Gly Gln Ser Gly Pro Ser Ile Gly His Phe 76 Gly Asn Pro Glu Glu Thr Ser Ser Leu Gln Thr Tyr Cys Phe Ser 91 Met Phe Gln Arg Arg Glu Trp His Ser Ser Trp Gly Ile Gln Glu106 Glu Gly Asp Thr Leu Leu Leu Tyr Pro Leu Asn Lys Ala Leu Met121 Phe Thr Ala Pro Arg His His Ser Glu Lys Lys Ile Ile Ala Leu136 Leu Ser Lys Thr Asp Phe Cys Leu Leu Leu Asp Arg Asn Lys Leu151 Lys Ala Gly Tyr Leu Met Ser Val Glu Ser Ser Glu Cys Phe Leu166 Glu Glu Val Gly Ser Gln Ala Leu Val Ala Gly Ser Tyr Met Pro181 Pro Ser Thr Val Leu Gln Gln Ile Asp Ser Val Ala Asn Ala Asp196 Ile Ile Asn Ala Ala Lys Lys Phe Val Ser Gly Gln Lys Ser Met211 Ala Ala Ser Gly Asn Leu Gly His Thr Pro Phe Val Asp Glu Leu ( 4 ) SEQ ID NO:3

(ⅰ) sequence signature

(A) length: 22 bases

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity

(ⅱ) molecule type: oligonucleotide

(ⅹ ⅰ) sequence description: the information of SEQ ID NO:3:5 '-TGACTACTTCACCCTTGGTGAA-3 ' 22 (5) SEQ ID NO:4

(ⅰ) sequence signature

(A) length: 22 bases

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity

(ⅱ) molecule type: oligonucleotide

(ⅹ ⅰ) sequence description: the information of SEQ ID NO:4:5 '-TTAAACAGAATGTTTTATTGAC-3 ' 22 (6) SEQ ID NO:5

(ⅰ) sequence signature

(A) length: 33 bases

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity

(ⅱ) molecule type: oligonucleotide

(ⅹ ⅰ) sequence description: the information of SEQ ID NO:5:5 '-CCCGCTAGCATGACAGCCTGTTCGATACGTTAG-3 ' Nhe I 33 (7) SEQ ID NO:6

(ⅰ) sequence signature

(A) length: 33 bases

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity

(ⅱ) molecule type: oligonucleotide

(ⅹ ⅰ) sequence description: the information of SEQ ID NO:6:5 '-CCCCTCGAGTTACAACTCATCAACAAAAGGTGT-3 ' Xho I 33 (8) 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-Thr-Ala-Phe-Ser-Leu-Thr-Ala-Glu-Arg-Ser-Ser-Ser-Leu-Cys-COOH

Claims (18)

1, a kind of isolated polypeptide-HCP is characterized in that it includes: SEQ ID NO:2

The polypeptide of shown aminoacid sequence or the active fragments of its polypeptide, analogue or derivative.

2, polypeptide as claimed in claim 1 is characterized in that the aminoacid sequence of described polypeptide, analogue or derivative

Has 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 and has the amino acid preface shown in the SEQ ID NO:2

The polypeptide of row.

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 polypeptide of aminoacid sequence shown in the SEQ ID NO:2 or its fragment, analogue, derivative

Polynucleotide;

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

(c) with (a) or the polynucleotide of at least 91% homogeny (b) are arranged.

5, polynucleotide as claimed in claim 4 is characterized in that described polynucleotide comprise coding and have SEQ ID

The polynucleotide of aminoacid sequence shown in the NO:2.

6, polynucleotide as claimed in claim 4 is characterized in that the sequence of described polynucleotide includes SEQ ID NO:1

The sequence of 1-3425 position among the sequence of middle 2572-3249 position or the SEQ ID NO:1.

7, a kind of recombinant vectors that contains exogenous polynucleotide is characterized in that it is by the arbitrary power among the claim 4-6

The recombinant vectors that profit requires described polynucleotide and plasmid, virus or vehicle expression vector establishment to form.

8, a kind of genetically engineered host cell that contains exogenous polynucleotide, it is characterized in that it 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 HCP is characterized in that described method

Comprise:

(a) expressing under HCP's condition, cultivate the described through engineering approaches host of claim 8

Cell;

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

10, a kind of can with polypeptide bonded antibody, it is characterized in that described antibody be can and the HCP

Specificity bonded antibody.

11, the compound of an analoglike or adjusting polypeptide active or expression is characterized in that they are simulation, promotion, short of money

Resist or suppress HCP's active compound.

12, compound as claimed in claim 11 is characterized in that it is the polynucleotide preface shown in the SEQ ID NO:1

Row or its segmental antisense sequences.

13, the described application of compound of a kind of claim 11 is characterized in that described compound is used for mediator's nucleoprotein

The II precursor protein 25 in vivo, the method for external activity.

14, a kind of detect with claim 1-3 in relevant disease or the disease susceptibility of the described polypeptide of arbitrary claim

Method, it is characterized in that it comprises the described polypeptide expression amount that detects, perhaps detect the activity of described polypeptide, or

The person detects and causes described expression of polypeptides amount or active unusual nucleotide diversity in the polynucleotide.

15,, it is characterized in that it is applied to screening as the application of polypeptide as described in the arbitrary claim among the claim 1-3

HCP's stand-in, agonist, antagonist or inhibitor; Perhaps be used for the peptide fingerprint image

Spectrum is identified.

16, as the application of the described nucleic acid molecule of arbitrary claim among the claim 4-6, it is characterized in that its conduct

Primer is used for nucleic acid amplification reaction, perhaps is used for hybridization as probe, perhaps is used to make gene chip or little

Array.

17, as the described polypeptide of arbitrary claim, polynucleotide or application of compound in claim 1-6 and 11,

It is characterized in that having with safety with described polypeptide, polynucleotide or its stand-in, agonist, antagonist or inhibitor

It is unusual as diagnosis or treatment and HCP to imitate dosage and pharmaceutically acceptable carrier composition

The pharmaceutical composition of relevant disease.

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 hemopathy, HIV with described polypeptide, polynucleotide or compound

The medicine of infection and immunological disease and all kinds of inflammation.

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