CN1303864A - Novel polypeptide-zinc finger protein 60 and polynucleotide for coding said polypeptide - Google Patents

Abstract

The present invention discloses a novel polypeptide-zinc-finger protein 60, polynucleotide coding said polypeptide and its method for producing this polypeptide by using DNA recombination technology. Said invention also discloses the method for curing several diseases, such as malignant tumor, blood disease, HIV infection, immunological diseases and various inflammations by using said polypeptide. Said invention also discloses antagonist resisting said polypeptide and its therapeutic effect. Said invention also discloses the application of the polynucleotide for coding this zinc-finger protein 60.

Description

The polynucleotide of a kind of new polypeptide---zinc-finger protein 60 and this peptide species of coding

The invention belongs to biological technical field, specifically, the invention describes a kind of new polypeptide---zinc-finger protein 60, 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.

Zinc finger protein is that a kind of DNA is conjugated protein, finds in the proteic aminoacid sequence of 5S rRNA gene transcription factor TF III A of Africa xenopus RNA polymerase III mediation.After this find that zinc finger protein all has and express that these tissues comprise that neural system such as brain, hematopoietic cell, face tissue, various and secretion absorb the tissues that relevant tissues reach and tumour is correlated with immortal cell line etc. in the mankind in different biological various tissues as fruit bat cell fission gene Kruppel.Zinc finger protein gene has surpassed 1% (Bellefroid et al., 1989 on human genome according to estimates; Pellegrino and Berg, 1991).The zinc fingers of zinc finger protein mainly contains following several: C2H2 configuration, C2C2 configuration, C2HC configuration, C2HC4C configuration, C3H configuration, C3HC4 configuration (Dai KS et al., 1998).The zinc finger protein gene that wherein contains the C2H2 configuration constitutes gang's gene (Berker et al., 1995) of human genome maximum.The C2H2 configuration of zinc finger protein is as follows:

Typical zinc fingers approximately is made of 28 amino-acid residues on length, is Cys-Cys ... His-His arranges, and zinc refers to that unit pack contains one section conservative relatively sequence: Cys-X _2-4-Cys-X ₃-Phe-X ₅-Leu-X ₂-His-X _2-4-His (Bray, p.et al., 1993).Two conservative Cys participate in forming an antiparallel βZhe Die in the zinc fingers, two conservative His participate in forming the α spiral, and these two structures combine with the Zn atom of core and form spheric region (Pabo, a C.A.andSauer closely, R.T, 1992).This type of zinc finger protein often has 1 to 37 zinc fingers (Rhodes, D.andKlug, 1993).Except conservative zinc fingers, the C2H2 configuration zinc finger protein that connects two adjacent Kruppel usually but always do not comprise conservative specific amino acid TGEKP (Bray, p.etal., 1993).In zinc finger protein, the effect of Zn is protein molecule to be formed refer to that the skeleton of type conformation combines with DNA.

Studies show that zinc fingers all has discovery in many and " protein-nucleic acid " and " protein-protein " interaction proteins associated.The zinc finger protein of C2H2 configuration by in conjunction with DNA and or RNA on special sequence come regulatory gene to transcribe (Engelke, D.R et al., 1980; Kadonaga, J.T etal., 1987; Stanojevic, D.et al., 1989), the zinc finger protein of number of C 2H2 configuration comes regulatory gene to transcribe and express (Lee, J-S.eta1., 1993 by the interaction with some other zinc finger protein; Seto, E.et al., 1993; Maheswaran.S.et al., 1993), other has the zinc finger protein of number of C 2H2 configuration to play adjusting growth, rna regulation metabolism, also may influence chromatinic packing (E1-Baradi, T and Pieler.T., 1991).Existing studies show that, C ₂H ₂The Zinc finger domain of type plays a crucial role in developmental regulation.For example, in Drosopila Kruppel (Rosenberg, 1986) and hunchback (Tautz, 1987) zinc finger protein, this structural domain forms relevant with embryo's pattern; In mouse Krox-20 zinc finger protein, this structural domain forms with the back brain connection pattern and cell proliferation control relevant (Chavrier, 1988; Wilkinson, 1989); GL13 is in autosomal dominant heteroplasia Greig syndromes in (vortkamp, 1991); It is relevant that some zinc finger protein and tumour form, and WT1 is in Wilm ' s tumour and congenital reproduction urethra are grown (Haber, 1990).

People's of the present invention gene protein and Novel Human Zinc Finger Protein C2H2-25 have 99% homology (homologous protein U38904), through separating and accurately mapping, it is the C2H2 configuration that this polypeptide has Zinc finger domain, belong to Novel Human Zinc Finger Protein Kruppel gene family, so called after human zinc-finger protein 60, and infer that with this its structural domain is similar to Kruppel family gene structural domain, have similar biological function.

In Novel Human Zinc Finger Protein Kruppel gene family, contain human zinc finger protein 13 1-140,142,143,148,151,154,155 (Tommerup and Vissing, Genomics 27:259-264,1995).Human zinc finger protein 13 8,139,143 is relevant with Williams syndromes, ulcuscuris, Bei Wei two syndromes respectively; Human zinc finger protein 13 2,134,135,137,154,155 all can cause solid tumor such as thyroid adenoma, human zinc finger protein 15 1 is closely related with neuroblastoma, colorectal carcinoma, mammary cancer or other tumour; Human zinc finger protein 13 9,148,151 can cause blood malignant disease such as leukemia, non_hodgkin lymphoma (Genomics 27:259-264,1995).

Novel Human Zinc Finger Protein C2H2-25 also belongs to Novel Human Zinc Finger Protein Kruppel gene family (Becker et al.Mammalian Genome 8:287-289,1997), be a kind of C2H2 configuration zinc finger protein of human developmental regulation, be positioned 19 karyomit(e)s by 2 people-dull and stereotyped pcr analysis of rodent sarcoma cell hybridization.People such as Becker obtain 118 kinds of C2H2 configuration zinc finger proteins from the separation of people hippocampus tissue cDNA library and wherein comprise Novel Human Zinc Finger Protein C2H2-25, expression level and the importance (Kevin G.Becker et al., 1995) thereof of these genes in neural system has been pointed out in a large amount of different expression of C2H2 configuration zinc finger protein in human brain.

Therefore infer, human zinc-finger protein 60 is with following but to be not limited to following disease relevant: solid tumor such as thyroid adenoma, hysteromyoma, nervous system disorders such as extrapyramidal system dysfunction, parkinson's syndrome, ataxia, neurocytoma, glioma, blood malignant disease such as leukemia, non_hodgkin lymphoma, the Williams syndromes, ulcuscuris, Bei Wei two syndromes, other tumour such as neuroblastoma, colorectal carcinoma, mammary cancer or the like.

The polynucleotide of encoding zinc finger protein, and coded zinc finger protein be found to be the differentiation of research cell under normal and pathological conditions, the physiological and biochemical procedure of propagation provides a kind of method, also comprises that with the disease that the disorder of cytodifferentiation propagation causes cancer provides a kind of new way for diagnosis, treatment.

An object of the present invention is to provide isolating new polypeptide---zinc-finger protein 60 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 encoding zinc finger protein 60.

Another object of the present invention provides the genetically engineered host cell of the polynucleotide that contain encoding zinc finger protein 60.

Another object of the present invention provides the method for producing zinc-finger protein 60.

Another object of the present invention provides at polypeptide of the present invention---the antibody of zinc-finger protein 60.

Another object of the present invention has provided at polypeptide of the present invention---the simulated compound of zinc-finger protein 60, antagonist, agonist, inhibitor.

Another object of the present invention provides the method for the diagnoses and treatment disease relevant unusually with zinc-finger protein 60.

In a first aspect of the present invention, novel isolated zinc-finger protein 60 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 99% homogeny with a kind of nucleotides sequence that is selected from down group: (a) polynucleotide of the above-mentioned zinc-finger protein 60 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 241-1875 position among the SEQ ID NO:1; (b) has the sequence of 1-2458 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 zinc-finger protein 60 " is meant that zinc-finger protein 60 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 zinc-finger protein 60 of standard.Basically pure polypeptide can produce single master tape on non-reduced polyacrylamide gel.The purity of zinc-finger protein 60 polypeptide can be used amino acid sequence analysis.

The invention provides a kind of new polypeptide---zinc-finger protein 60, 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 zinc-finger protein 60.As used herein, term " fragment ", " derivative " are meant with " analogue " and keep identical biological function of zinc-finger protein 60 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 2458 bases, and its open reading frame (241---1875) 544 amino acid of having encoded.Find relatively that according to amino acid sequence homologous this polypeptide and people's zinc finger protein C2H2-25 has 99% homology, deducibility goes out the similar 26S Proteasome Structure and Function of zinc finger protein C2H2-25 that this zinc-finger protein 60 has the people.

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 encoding zinc finger protein 60.

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

The special polynucleotide sequence of encoding zinc finger protein 60 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 zinc-finger protein 60; (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 zinc-finger protein 60 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 zinc-finger protein 60 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 encoding zinc finger protein 60 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 encoding zinc finger protein 60 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 encoding zinc finger protein 60 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 zinc-finger protein 60 (Science, 1984 of reorganization; 224:1431).In general following steps are arranged:

(1). with the polynucleotide (or varient) of coding human zinc-finger protein 60 of the present invention, or transform or the transduction proper host cell with the recombinant expression vector that contains these polynucleotide;

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

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

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

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

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.

Discover, most of members in the zinc finger protein are the transcriptional regulator of genetic expression in the organism, relevant with gene transcription regulation and control, inventor's zinc-finger protein 60 will cause various growth disorder diseases, various tumour, some nervous system disorders, heredopathia, hemopathy, endocrine system disease and disease of immune system etc. in gene transcription regulation, genetic expression, rna regulation metabolism and the abnormal expression that may influence the function aspects of chromatinic packing.

Inventor's zinc-finger protein 60 can be used for treating or prevent because the disease that this protein abnormal expression caused.These diseases include but not limited to: grow disorder disease, split pedopathy and shellfish syndrome Weis two etc. as extrapyramidal system dysfunction, monster, Williams syndromes, Alagille syndromes, cleft hand; The canceration of various tissues is as leukemia, lymphoma, malignant histocytosis, melanoma, sarcoma, myelomatosis, teratoma etc., adrenal carcinoma, bladder cancer, osteocarcinoma, bone marrow cancer, the cancer of the brain, mammary cancer, uterus carcinoma, carcinoma of gallbladder, liver cancer, lung cancer, thyroid tumor, hysteromyoma, thymus neoplasms etc.; Some heredopathia; Nervous system disorders is as parkinson's syndrome, ataxia, neurocytoma, glioma etc.; Endocrine system disease is as endocrine neoplasia; Disease of immune system.

Inventor's zinc-finger protein 60 forms it in human brain development and function probably and important effect, and its gene transcription regulation, genetic expression, rna regulation metabolism and the function that may influence chromatinic packing are grown aspects such as the asynchronism that causes and tumour and will be applied at neural physiological function such as normal extrapyramidal system Coordinating Activity etc. and pathology function such as unusual extrapyramidal system.

The present invention also provides SCREENED COMPOUND to identify the method that improves (agonist) or check the medicament of (antagonist) zinc-finger protein 60.Agonist improves zinc-finger protein 60 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 expression zinc-finger protein 60 be cultivated with the zinc-finger protein 60 of mark.Measure the medicine raising then or check this interactional ability.

The antagonist of zinc-finger protein 60 comprises antibody, compound, acceptor disappearance thing and the analogue etc. that filter out.The antagonist of zinc-finger protein 60 can combine and eliminate its function with zinc-finger protein 60, 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, zinc-finger protein 60 can be added during bioanalysis measures, by measuring compound interactional influence between zinc-finger protein 60 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 zinc-finger protein 60 bonded peptide molecule obtains.During screening, generally tackle the zinc-finger protein 60 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 zinc-finger protein 60 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 zinc-finger protein 60 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 zinc-finger protein 60 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-zinc-finger protein 60.

The antibody of anti-zinc-finger protein 60 can be used in the immunohistochemistry technology, detects the zinc-finger protein 60 in the biopsy specimen.

With the also available labelled with radioisotope of zinc-finger protein 60 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 zinc-finger protein 60 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 zinc-finger protein 60 positive cells.

The disease that antibody among the present invention can be used for treating or prevention is relevant with zinc-finger protein 60.The antibody that gives suitable dosage can stimulate or block the generation or the activity of zinc-finger protein 60.

The invention still further relates to the diagnostic testing process of quantitative and detection and localization zinc-finger protein 60 level.These tests are known in the art, and comprise that FISH measures and radioimmunoassay.The zinc-finger protein 60 level that is detected in the test can be with laying down a definition the importance of zinc-finger protein 60 in various diseases and be used to the disease of diagnosing zinc-finger protein 60 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 encoding zinc finger protein 60 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 zinc-finger protein 60 or the unusual/non-activity expression.The gene therapy vector (as virus vector) of reorganization can be designed for the zinc-finger protein 60 of expressing variation, to suppress endogenic zinc-finger protein 60 activity.For example, a kind of zinc-finger protein 60 of variation can be the zinc-finger protein 60 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 zinc-finger protein 60 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 encoding zinc finger protein 60 are transferred in the cell.The method of recombinant viral vector that structure carries the polynucleotide of encoding zinc finger protein 60 is found in existing document (Sambrook, et al.).The polynucleotide of encoding zinc finger protein 60 of recombinating in addition can be packaged in the liposome and be transferred in the cell.

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 zinc-finger protein 60 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 encoding zinc finger protein 60 can be used for the diagnosis with the relative disease of zinc-finger protein 60.The unconventionality expression of the expression that the polynucleotide of encoding zinc finger protein 60 can be used for detecting zinc-finger protein 60 zinc-finger protein 60 whether or under morbid state.Dna sequence dna as encoding zinc finger protein 60 can be used for biopsy specimen is hybridized to judge the expression situation of zinc-finger protein 60.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 zinc-finger protein 60 with the special primer of zinc-finger protein 60.

The sudden change that detects the zinc-finger protein 60 gene also can be used for the disease of diagnosing zinc-finger protein 60 relevant.The form of zinc-finger protein 60 sudden change comprises that the point mutation compared with normal wild type zinc-finger protein 60 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.Zinc-finger protein 60 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 zinc-finger protein 60 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 zinc-finger protein 60 of the present invention and people's zinc finger protein C2H2-25.The top sequence is a zinc-finger protein 60, and the below sequence is people's zinc finger protein C2H2-25.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 zinc-finger protein 60.60kDa 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 zinc-finger protein 60

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 0085d03 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 0085d03 clone is 2458bp (shown in Seq ID NO:1), from 241bp to 1875bp the open reading frame (ORF) of a 1635bp, the new protein (shown in Seq ID NO:2) of encoding arranged.We are with this clone's called after pBS-0085d03, encoded protein matter called after zinc-finger protein 60.

Embodiment 2:cDNA clone's homology retrieval

With the sequence and the encoded protein sequence thereof of zinc-finger protein 60 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 zinc-finger protein 60 homology of the present invention is a kind of known people's zinc finger protein C2H2-25, and its encoded protein number is U38904 in the access of Genbank.Protein homology the results are shown in Fig. 1, both height homologies, and its homogeny is 99%; Similarity is 99%.Embodiment 3: with the gene of RT-PCR method clones coding zinc-finger protein 60

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

Primer2:5’-TTAAATAATAATGCTTTTATTATTG-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 1-2458bp shown in the SEQ ID NO:1 are identical.Embodiment 4:Northern blotting is analyzed the zinc-finger protein 60 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 zinc-finger protein 60 coding region sequence (241bp to 1875bp) 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: vivoexpression, separation and the purifying of reorganization zinc-finger protein 60

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

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

5 ' end of these two sections primers contains Nde I and BamH I restriction enzyme site respectively, be respectively the encoding sequence of target gene 5 ' end and 3 ' end thereafter, Nde I and BamH I restriction enzyme site are corresponding to expression vector plasmid pET-28b (+) (Novagen company product, Cat.No.69865.3) the selectivity restriction enzyme site on.With the pBS-0085d03 plasmid that contains the total length goal gene is template, carries out the PCR reaction.The PCR reaction conditions is: contain pBS-0085d03 plasmid 10pg, primer Primer-3 and Primer-4 among the cumulative volume 50 μ l and be respectively 10pmol, Advantage polymera se Mix (Clontech company product) 1 μ l.Loop parameter: 94 ℃ of 20s, 60 ℃ of 30s, 68 ℃ of 2min, totally 25 circulations.Respectively amplified production and plasmid pET-28 (+) are carried out double digestion with Nde I and BamH I, reclaim big fragment respectively, and connect with the T4 ligase enzyme.Connect product and transform, after the dull and stereotyped overnight incubation of the LB that contains kantlex (final concentration 30 μ g/ml), use the colony polymerase chain reaction (PCR) method screening positive clone, and check order with the big enterobacterial DH5 of Calcium Chloride Method α.Select the correct positive colony of sequence (pET-0085d03) 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-0085d03) 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 zinc-finger protein 60 of purifying.Through the SDS-PAGE electrophoresis, obtain a single band (Fig. 2) at the 60kDa 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-zinc-finger protein 60 production of antibodies

Synthesize the specific polypeptide of following zinc-finger protein 60 with Peptide synthesizer (PE company product):

NH ₂-Met-Ala-Thr-Ala-Leu-Arg-Tyr-Pro-Ala-Ser-Gly-Ser-Val-Thr-Phe-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 zinc-finger protein 60 specifically.

Sequence table

(1) general information:

(ⅱ) denomination of invention: zinc-finger protein 60 and encoding sequence thereof

(ⅲ) sequence number: 7

(2) information of SEQ ID NO:1:

(ⅰ) sequence signature:

(A) length: 2458bp

(B) type: nucleic acid

(C) chain: two strands

(D) topological framework: linearity

(ⅱ) molecule type: cDNA

( ⅹⅰ ) ：SEQ ID NO:1： 1 GCAGGGCGGGACTTCTGGCGTCTTCGCGGCGGTCATTTTGGCTGCCCTCCCGGAGGTCCG 61 TTCTGTCTGTCAGCCGCTTTGGTACGCTGCATCGGGATCGAAGTGACGGACCGTGAAGGC121 GCGAGAGTCAGGTCTGAGGGTCGGGGGCAGAGCCGCCGCGAGGCCGGCCTGGGGATAGCG181 GTGCTCGGGTTCCCCCCGGGTCGCCCGCAGCTCCCGGTCCAGCTCCGCCCACAGACTCGG241 ATGGCGACCGCACTGAGGTACCCGGCTTCGGGAAGTGTGACCTTTGAAGATGTGGCCGTG301 TACTTCTCCTGGGAGGAATGGGATCTCCTTGATGAGGCTCAGAAACACCTGTACTTCGAT361 GTGATGCTGGAGAACTTTGCACTTACGTCCTCCCTGGGTTGTTGGTGTGGAGTGGAACAT421 GAGGAAACACCTTCTGAACAGAGAATTTCTGGAGAAAGAGTGCCACAGTTCAGGACTTCC481 AAAGAAGGTTCATCTTCCCAGAATGCCGACTCCTGTGAAATATGTTGCCTGGTCTTGAGA541 GATATTTTGCACTTGGCTGAACACCAAGGAACAAACTGCGGGCAGAAACTACACACATGT601 GGAAAACAATTCTACATCAGTGCAAATCTTCAACAGCACCAGAGGCAGCACATTACAGAG661 GCACCTTTCAGAAGTTATGTGGACACAGCCTCGTTTACACAGAGTTGCATAGTCCATGTG721 TCGGAGAAACCCTTTACCTGCAGGGAGATCAGGAAAGACTTCCTGGCCAACATGAGGTTT781 CTCCATCAAGACGCCACTCAAACAGGGGAGAAGCCAAATAACAGTAACAAGTGTGCGGTG841 GCCTTTTACAGTGGAAAAAGTCATCACAACTGGGGAAAATGCAGTAAAGCCTTTAGCCAC901 ATAGACACACTTGTTCAGGACCAGAGAATCCTCACTAGAGAAGGACTTTTTGAGTGCAGT 961 AAATGTGGGAAAGCATGTACGCGAAGATGTAACCTCATTCAGCACCAGAAAGTCCACAGT1021 GAAGAAAGGCCTTATGAATGCAATGAATGTGGAAAATTCTTTACCTACTACTCCAGTTTC1081 ATTATACATCAGAGAGTTCATACTGGAGAAAGGCCTTATGTGTGCCCTGAATGTGGGAAA1141 TCCTTTAGTCAGATATACAGCCTCAATAGCCATAGGAAAGTTCACACTGGAGAAAGGCCT1201 TATGAATGTGGGGAATGTGGGAAATCTTTTAGCCAAAGGTCCAACCTCATGCAGCATCGC1261 AGAGTTCACACTGGAGAAAGGCCTTATGAATGCAGCGAATGTGGGAAATCTTTTAGCCAA1321 AACTTCAGCCTGATTTACCACCAGAGAGTTCACACTGGAGAAAGACCTCATGAGTGCAAT1381 GAATGTGGAAAATCCTTTAGCCGAAGCTCCAGCCTCATTCACCACCGGAGACTTCACACT1441 GGAGAAAGACCCTATGAGTGCAGTAAATGTGGGAAGTCATTTAAGCAAAGCTCCAGCTTC1501 AGTTCACATCGGAAAGTCCACACAGGGGAAAGGCCTTATGTGTGTGGGGAATGTGGGAAA1561 TCCTTTAGCCATAGCTCCAACCTTAAGAACCACCAGAGAGTTCACACTGGAGAAAGACCT1621 GTTGAGTGCAGTGAATGTAGCAAATCCTTTAGCTGTAAATCTAACCTCATTAAACACCTG1681 AGAGTTCACACTGGAGAAAGGCCTTATGAGTGCAGTGAATGTGGGAAATCCTTTAGCCAA1741 AGTTCTAGCCTCATTCAACACCGCAGAGTTCACACGGGAAAAAGGCCTTATCAGTGCAGT1801 CAATGTGGGAAATCCTTTGGCTGCAAATCTGTCCTCATTCAACACCAGAGAGTTCACATT1861 GGAGAAAAGCCTTAGCTGTACTGAGAATATGCAATTTCCTTTCAGTGTAATTATACTGAA1921 GTAGTACACCTGTGAGAGAGACAAGTACCTGATTTGGAAGCCCCAACATCTAAGGATATA1981 CGGTGGGCGGATTCCCCTTAAGTTCCAGGTATGTGTTACACTTTCTAACATGCCATTTAG2041 AAAGTGTTAGACTTTCTCACCTGCCATTTATGGCTCTTGCCATTTATGTCACTGACAGTT2101 TCTGAGGCAGAAGCCGTATCATGTCTACCACCTGTGAGGTCCACACAGTGTGTATCATTT2161 ACCTCCTGAACCTGCTCAAGGAAGCAGACCTCTGCTTCTCCCCATTTGCTAGAAGAAATC2221 ATGAATAGTCTGAGTCTTCCTCTCTGACAAGTTAGGGCATGGACTTGACCCAGCTTTGTG2281 CCAGAGAACCCAATATGAGTGTTGTTGGCAGCTTGCCAAGAAGGACTGTCTTTTTCAAGA2341 CATACTGGTTTCATGTGACACCTCCATGGATTTTTTTCCAGCCTCTAAGTCACCAACTTG2401 GGAACTGCTTGTCTCACATTGCTTTGTTTTTTACAATAATAAAAGCATTATTATTTAA

(3) information of SEQ ID NO:2:

(ⅰ) sequence signature:

(A) length: 544 amino acid

(B) type: amino acid

(D) topological framework: linearity

(ⅱ) molecule type: polypeptide

( ⅹⅰ ) ：SEQ ID NO:2： 1 Met Ala Thr Ala Leu Arg Tyr Pro Ala Ser Gly Ser Val Thr Phe 16 Glu Asp Val Ala Val Tyr Phe Ser Trp Glu Glu Trp Asp Leu Leu 31 Asp Glu Ala Gln Lys His Leu Tyr Phe Asp Val Met Leu Glu Asn 46 Phe Ala Leu Thr Ser Ser Leu Gly Cys Trp Cys Gly Val Glu His 61 Glu Glu Thr Pro Ser Glu Gln Arg Ile Ser Gly Glu Arg Val Pro 76 Gln Phe Arg Thr Ser Lys Glu Gly Ser Ser Ser Gln Asn Ala Asp 91 Ser Cys Glu Ile Cys Cys Leu Val Leu Arg Asp Ile Leu His Leu106 Ala Glu His Gln Gly Thr Asn Cys Gly Gln Lys Leu His Thr Cys121 Gly Lys Gln Phe Tyr Ile Ser Ala Asn Leu Gln Gln His Gln Arg136 Gln His Ile Thr Glu Ala Pro Phe Arg Ser Tyr Val Asp Thr Ala151 Ser Phe Thr Gln Ser Cys Ile Val His Val Ser Glu Lys Pro Phe166 Thr Cys Arg Glu Ile Arg Lys Asp Phe Leu Ala Asn Met Arg Phe181 Leu His Gln Asp Ala Thr Gln Thr Gly Glu Lys Pro Asn Asn Ser196 Asn Lys Cys Ala Val Ala Phe Tyr Ser Gly Lys Ser His His Asn211 Trp Gly Lys Cys Ser Lys Ala Phe Ser His lle Asp Thr Leu Val226 Gln Asp Gln Arg Ile Leu Thr Arg Glu Gly Leu Phe Glu Cys Ser241 Lys Cys Gly Lys Ala Cys Thr Arg Arg Cys Asn Leu Ile Gln His256 Gln Lys Val His Ser Glu Glu Arg Pro Tyr Glu Cys Asn Glu Cys271 Gly Lys Phe Phe Thr Tyr Tyr Ser Ser Phe Ile Ile His Gln Arg286 Val His Thr Gly Glu Arg Pro Tyr Val Cys Pro Glu Cys Gly Lys301 Ser Phe Ser Gln Ile Tyr Ser Leu Asn Ser His Arg Lys Val His316 Thr Gly Glu Arg Pro Tyr Glu Cys Gly Glu Cys Gly Lys Ser Phe331 Ser Gln Arg Ser Asn Leu Met Gln His Arg Arg Val His Thr Gly346 Glu Arg Pro Tyr Glu Cys Ser Glu Cys Gly Lys Ser Phe Ser Gln361 Asn Phe Ser Leu Ile Tyr His Gln Arg Val His Thr Gly Glu Arg376 Pro His Glu Cys Asn Glu Cys Gly Lys Ser Phe Ser Arg Ser Ser391 Ser Leu Ile His His Arg Arg Leu His Thr Gly Glu Arg Pro Tyr406 Glu Cys Ser Lys Cys Gly Lys Ser Phe Lys Gln Ser Ser Ser Phe421 Ser Ser His Arg Lys Val His Thr Gly Glu Arg Pro Tyr Val Cys436 Gly Glu Cys Gly Lys Ser Phe Ser His Ser Ser Asn Leu Lys Asn451 His Gln Arg Val His Thr Gly Glu Arg Pro Val Glu Cys Ser Glu466 Cys Ser Lys Ser Phe Ser Cys Lys Ser Asn Leu Ile Lys His Leu481 Arg Val His Thr Gly Glu Arg Pro Tyr Glu Cys Ser Glu Cys Gly496 Lys Ser Phe Ser Gln Ser Ser Ser Leu Ile Gln His Arg Arg Val511 His Thr Gly Lys Arg Pro Tyr Gln Cys Ser Gln Cys Gly Lys Ser526 Phe Gly Cys Lys Ser Val Leu Ile Gln His Gln Arg Val His Ile541 Gly Glu Lys Pro

(4) information of SEQ ID NO:3

(ⅰ) sequence signature

(A) length: 25 bases

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity

(ⅱ) molecule type: oligonucleotide

(ⅹ ⅰ) sequence description: SEQ ID NO:3:GCAGGGCGGGACTTCTGGCGTCTTC 25

(5) information of SEQ ID NO:4

(ⅰ) sequence signature

(A) length: 25 bases

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity

(ⅱ) molecule type: oligonucleotide

(ⅹ ⅰ) sequence description: SEQ ID NO:4:TTAAATAATAATGCTTTTATTATTG 25

(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:CCCCATATGATGGCGACCGCACTGAGGTACCCGG 34

(7) information of 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: SEQ ID NO:6:CCCGGATCCCTAAGGCTTTTCTCCAATGTGAAC 33

(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:Met-Ala-Thr-Ala-Leu-Arg-Tyr-Pro-Ala-Ser-Gly-Ser-Val-Thr-Phe 15

Claims (18)

1, a kind of isolated polypeptide-zinc-finger protein 60 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 99% 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 1-2458 position among the sequence of 241-1875 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 zinc-finger protein 60 is characterized in that described method comprises:

(a) expressing under the zinc-finger protein 60 condition, cultivate the described through engineering approaches host cell of claim 8;

(b) from culture, isolate and have the active polypeptide of zinc-finger protein 60.

10, a kind of can with polypeptide bonded antibody, it is characterized in that described antibody be can with zinc-finger protein 60 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 zinc-finger protein 60.

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 to regulate zinc-finger protein 60 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 zinc-finger protein 60, 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 zinc-finger protein 60 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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