CN1485339A - Polypeptide-human zinc finger protein 70 and polynucleotide for coding this polypeptide - Google Patents

Abstract

The present invention provides a polypeptide-human zinc finger protein 70, polynucleotide for encoding the polypeptide and the process for producing the polypeptide by the DNA restructuring technique. The invention also discloses the method of use of the polypeptide for treating a plurality of diseases, e.g. malignant tumor, hematopathy, HIV affection, immunopathy and various types of inflammation. The invention also discloses the antagonist of the polypeptide and its therapeutic action. The invention also discloses the use of the polynucleotide for coding the new human zinc finger protein 70.

Description

The polynucleotide of a kind of polypeptide-human zinc finger protein 70 and this peptide species of coding

Technical field

The invention belongs to biological technical field, specifically, the invention describes a kind of polypeptide-human zinc finger protein 70, 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.

Background technology

The transcriptional control of eukaryotic gene is crucial for the normal expression and the performance biological function of gene, finishes this process by transcriptional regulator usually.Transcriptional regulator participates in determining gene at which kind of tissue and which kind of etap transcriptional start in vivo, encode the gene of this proteinoid as undergoing mutation, not only this gene self can not normal expression, and can not transcribe normally and express by many genes of its adjusting.Transcription factor to the regulation and control of genetic expression mainly by transcription factor combine with specific dna sequence dna, interaction between transcription factor and transcription factor and conventional interaction of transcribing mechanism finish.Difference according to structural motif, known DNA is conjugated protein can mainly to be divided into two classes: the albumen and zinc finger protein [Kamal Chowdhury, Heidi Rohdewohld et al., the Nucleic AcidsResearch that contain helix-turn-helix motif, 1988,16:9995-10011].

Zinc finger protein is that a kind of DNA is conjugated protein, finds in the proteic aminoacid sequence of 5SrRNA gene transcription factor TF III A of Africa xenopus rna plymerase iii mediation.After this find that zinc finger protein all has expression in different biological various tissues, these tissues comprise hematopoietic cell, brain, neural system, face tissue, the various tissues relevant with the secretion absorption and the tissue relevant with immortal cell line with tumour etc.Zinc finger protein gene has surpassed 1% (Bellefroid et al., 1989 on human genome according to estimates; Pellegrino andBerg, 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.and Sauer closely, R.T, 1992).This type of zinc finger protein often has 1 to 37 zinc fingers (Rhodes, D.and Klug, 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.et al., 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.

Zinc fingers all has discovery in many and " protein-nucleic acid " and " protein-protein " interaction proteins associated.Zinc finger protein can combine with double-stranded DNA (dsDNA) or single stranded RNA, is carrying out many vital role, and its most important function relates to gene transcription regulation and control, and the effect (EMBO J.12:1363-1374,1993) of signal transmission factor may be arranged simultaneously.Existing studies show that, the Zinc finger domain of C2H2 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 gene and tumour form, as WT1 in Wilm ' s tumour and congenital reproduction urethra are grown (Haber, 1990).

The suppressor gene of Hairy-wing gene (su (Hw)) coding zinc finger protein---SUHW, it can be incorporated into the gYPsy gene and stops the stimulation Transcription of genetic enhancer.Discover that SUHW just has effect when only being inserted between enhanser and the promotor.Further studies show that SUHW and and not interaction of enhanser bonded albumen.(Mol?Cell?Biol?1994?Sep；14(9)：5645-52)

The distortion of researching DNA and enhanser checked concern the time, find that by the mobility of checking gel electrophoresis SUHW can change the structure of DNA.In fact, SUHW can cause the change of dna structure, and this change is not the bending of direct dna structure, but relevant with losing of existing warp architecture.Generally speaking, SUHW can increase the snappiness of DNA, and the bending of DNA can not be checked enhanser fully, is that the snappiness of enhanced DNA can help the interaction between the interactional albumen of the enhanser of SUHW and support wide apart and promotor so SUHW stops the reason of the stimulation Transcription of genetic enhancer.(Mol?Cell?Biol?1994Sep；14(9)：5645-52)

SUHW albumen can be regulated and control the sudden change owing to the gypsy retrotransposon, and by suppressing the function of transcriptional enhancer, transcriptional enhancer is positioned the place away from su (Hw) binding site.(Genetics?1996Feb；142(2)：425-36)

By fruit bat discovered that the sudden change of the suppressor gene of Hairy-wing gene---su (Hw) can cause the Infertility of female fruit bat.The mutation inhibiting of the suppressor gene of Hairy-wing is inserted in the gyPsy retrotransposon.Gypsy can with the SUHW protein binding, to discovering of SUHW structure, SUHW contains 12 zinc fingerses, they can be divided into 4 classes: (6-10) of core; Effect is arranged but non-core (1,2,3,11); Unessential (5,12); (4) that suppress.Amino-acid residue in the 737-880 position of carboxyl terminal has been formed the functional domain that female reproduction is played an important role.Total conclusion is that SUHW can hinder the ovum generation.(Mol?CellBiol.1996?Jul；16(7)：3381-92)

The gypsy retrotransposon can cause tissue-specific phenotype sudden change, and the sudden change of su (Hw) can make the phenotype results of mutation recover, and in addition, su (Hw) gene also can be regulated transcribing of gypsy.Gypsy's RNA that total length is 7.0kb has encoded, specifically expressing in sialisterium precursor and embryo's fatty tissue.And these express the participation that all be unable to do without su (Hw) gene, prove that su (Hw) albumen is the forward stimulating factor that gypsy transcribes.The sudden change of zinc fingers can influence itself and the combining of DNA among the su (Hw), and induces transcribing of lacZ reporter gene.Aminoterminal disappearance can cause the lacZ of embryo's fatty tissue and adult's ovary tissue to be beyond expression among the su (Hw); The reduction that the sudden change in leucic zipper territory then can cause the lacZ of the raising of embryo's fatty tissue lacZ expression, the ovary tissue of being grown up to express.(Genetics?1995?Jan；139(1)：215-28)

According to amino acid homology result relatively, polypeptide of the present invention is accredited as a kind of new human zinc finger protein 70 or people Hairy-wing gene inhibition protein 70 (HZNF70/HSUHW70) by deduction.Its homologous protein is people's existing zinc finger protein dJ 322P7.1, and albumen number is AL023799.

Because human zinc finger protein 70 albumen plays an important role in regulating body critical functions such as cell fission and fetal development as mentioned above, and believe and relate to a large amount of albumen in these regulate processes, thereby need to identify the human zinc finger protein 70 albumen of more these processes of participation in this area always, particularly identify this proteic aminoacid sequence.The separation of new human zinc finger protein 70 protein coding gene also provides the foundation for determining the effect of this albumen under healthy and morbid state.This albumen may constitute the basis of exploitation medical diagnosis on disease and/or curative, and it is very important therefore separating its coding DNA.

Summary of the invention

An object of the present invention is to provide isolating new polypeptide-human zinc finger protein 70 with and fragment, analogue and derivative.

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

Another object of the present invention provides the recombinant vectors of the polynucleotide that contain the human zinc finger protein 70 of encoding.

Another object of the present invention provides the genetically engineered host cell of the polynucleotide that contain the human zinc finger protein 70 of encoding.

Another object of the present invention provides the method for producing human zinc finger protein 70.

Another object of the present invention provides the antibody at polypeptide-human zinc finger protein 70 of the present invention.

Another object of the present invention has provided at the simulated compound of polypeptide-human zinc finger protein 70 of the present invention, antagonist, agonist, inhibitor.

Another object of the present invention provides the method for the diagnoses and treatment disease relevant unusually with human zinc finger protein 70.

The present invention relates to a kind of isolated polypeptide, this polypeptide is the people source, and it comprises: have＜polypeptide or its examples of conservative variations, bioactive fragment or the derivative of 210〉2 aminoacid sequences.Preferably, this polypeptide is to have＜polypeptide of 210〉2 aminoacid sequences.

The invention still further relates to a kind of isolating polynucleotide, it comprises a kind of nucleotide sequence or its variant that is selected from down group:

(a) coding has＜polynucleotide of the polypeptide of 210〉2 aminoacid sequences;

(b) with polynucleotide (a) complementary polynucleotide;

(c) with (a) or polynucleotide sequence (b) have the polynucleotide of at least 74% homogeny.

More preferably, the sequence of these polynucleotide is be selected from down group a kind of: (a) have＜210〉1 in the sequence of 2-1924 position; (b) have＜210〉1 in the sequence of 1-3191 position.

The present invention relates to a kind of carrier that contains polynucleotide of the present invention, particularly expression vector in addition; The host cell that this carrier of a kind of usefulness is genetically engineered comprises the host cell of conversion, transduction or transfection; A kind of method for preparing polypeptide of the present invention of cultivating described host cell and reclaiming expression product that comprises.

The invention still further relates to a kind of can with polypeptid specificity bonded antibody of the present invention.

The invention still further relates to a kind of simulation, activation, antagonism of screening or suppress the method for the compound of human zinc finger protein 70 protein-active, it comprises and utilizes polypeptide of the present invention.The invention still further relates to the compound that obtains with this method.

The invention still further relates to a kind of vitro detection and express the relevant disease or the method for disease susceptibility with the human zinc finger protein 70 abnormal protein, comprise the sudden change in polypeptide described in the detection of biological sample or its coded polynucleotide sequence, perhaps the amount or the biological activity of polypeptide of the present invention in the detection of biological sample.

The present invention also relates to a kind of pharmaceutical composition, it contains polypeptide of the present invention or its stand-in, activator, antagonist or inhibitor and pharmaceutically acceptable carrier.

The invention still further relates to polypeptide of the present invention and/or polynucleotide and be used for the treatment of cancer, developmental character disease or immunological disease or other purposes owing to the medicine of human zinc finger protein 70 disease that abnormal expression causes in preparation.

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

The following term of using in this specification sheets and claims has following implication unless stated otherwise:

" nucleotide sequence " is meant oligonucleotide, Nucleotide or polynucleotide and fragment or part, also can refer to genome or synthetic DNA or RNA, and they can be strand or two strands, represent sense strand or antisense strand.Similarly, term " aminoacid sequence " is meant oligopeptides, peptide, polypeptide or protein sequence and fragment or part.When " aminoacid sequence " among the present invention related to a kind of aminoacid sequence of naturally occurring protein molecule, this " polypeptide " or " protein " did not mean that aminoacid sequence are restricted to the complete natural amino acid relevant with described protein molecule.

Protein or polynucleotide " variant " are meant a kind of polynucleotide sequence that has the aminoacid sequence of one or more amino acid or Nucleotide change or encode it.Described change can comprise disappearance, insertion or the replacement of amino acid in aminoacid sequence or the nucleotide sequence or Nucleotide.Variant can have " conservative property " and change, and wherein the amino acid of Ti Huaning has structure or the chemical property similar with original acid, as replacing Isoleucine with leucine.Variant also can have non-conservation and change, as replacing glycine with tryptophane.

" disappearance " is meant the disappearance of in aminoacid sequence or nucleotide sequence one or more amino acid or Nucleotide.

" insertion " or " interpolation " is meant that the change in aminoacid sequence or nucleotide sequence causes comparing the increase of one or more amino acid or Nucleotide with naturally occurring molecule." replacement " is meant by different amino acid or Nucleotide and replaces one or more amino acid or Nucleotide.

" biological activity " is meant the protein of structure, regulation and control or biochemical function with natural molecule.Similarly, term " immunologic competence " be meant natural, reorganization or synthetic protein and fragment thereof in suitable animal or cell, induce specific immune response and with specific antibody bonded ability.

" agonist " is meant when combining with human zinc finger protein 70, thereby a kind of this protein that causes changes the molecule of regulating this protein active.Agonist can comprise protein, nucleic acid, carbohydrate or any other can be in conjunction with the molecule of human zinc finger protein 70.

" antagonist " or " inhibition " is meant when combining with human zinc finger protein 70, a kind of sealing or the biologic activity of mediator's zinc finger protein 70 or the molecule of immunologic competence.Antagonist and inhibition can comprise protein, nucleic acid, carbohydrate or any other can be in conjunction with the molecule of human zinc finger protein 70.

" adjusting " is meant that the function of human zinc finger protein 70 changes, and comprises the change of any other biological property, function or the immune property of the change of the rising of protein active or reduction, binding characteristic and human zinc finger protein 70.

" pure basically " is meant and is substantially free of natural relative other albumen, lipid, carbohydrate or other material.Those skilled in the art can use the purified technology of protein purifying human zinc finger protein 70 of standard.Basically pure human zinc finger protein 70 can produce single master tape on the irreducibility polyacrylamide gel.The purity available amino end acid sequence of human zinc finger protein 70 polypeptide is analyzed.

" complementary " or " complementation " is meant under salt concn that allows and temperature condition by the natural combination of the polynucleotide of base pairing.For example, sequence " C-T-G-A " can combine with complementary sequence " G-A-C-T ".Complementation between two single chain molecules can be part or whole.Complementary degree between the nucleic acid chains has a significant effect for efficient of hybridizing between the nucleic acid chains and intensity.

" homology " is meant the complementary degree, can be portion homologous or complete homology." portion homologous " is meant a kind of part complementary sequence, and it can partly suppress the hybridization of complete complementary sequence and target nucleic acid at least.The inhibition of this hybridization can detect by hybridizing (Southern trace or Northern trace etc.) under the condition that reduces in the severity degree.Basically homologous sequence or hybridization probe can compete and suppress complete homologous sequence and target sequence the condition that reduces of severity degree under combine.This does not mean the conditions permit non-specific binding that the severity degree reduces because the conditional request two sequences that the severity degree reduces mutual be combined into specificity or selectivity interacts.

" homogeny percentage " be meant two or more amino acid or nucleotide sequence relatively in the same or analogous percentage of sequence.The available electron method is measured the homogeny percentage, as passing through MEGALIGN program (Lasergenesoftware package, DNASTAR, Inc., Madison Wis.).The MEGALIGN program can compare two or more sequences (Higgins, D.G. and P.M.Sharp (1988) Gene 73:237-244) according to diverse ways such as Cluster method.The Cluster method is organized the series arrangement cluster by checking the distance between all pairings with each.Then with each bunch with in pairs or become set of dispense.Homogeny percentage between two aminoacid sequences such as sequence A and the sequence B calculates by following formula:

The residue number of mating between sequence A and the sequence B

Interval residue number * 100 in the residue number-sequence B of interval in the residue number-sequence A of sequence A

Also can be by the Cluster method or with the homogeny percentage (Hein J., (1990) Methods in emzumology 183:625-645) between known method in this area such as the Jotun Hein mensuration nucleotide sequence.

" similarity " is meant the degree that the identical or conservative property of corresponding position amino-acid residue when arranging contrast between the aminoacid sequence replaces.For example be used for amino acid that conservative property replaces, electronegative amino acid can comprise aspartic acid and L-glutamic acid; Positively charged amino acid can comprise Methionin and arginine; Having uncharged head group has similar hydrophilic amino acid can comprise leucine, Isoleucine and Xie Ansuan; Glycine and L-Ala; L-asparagine and glutamine; Serine and Threonine; Phenylalanine and tyrosine.

" antisense " is meant and specific DNA or RNA sequence complementary nucleotide sequence." antisense strand " is meant and " sense strand " complementary nucleic acid chains.

" derivative " is meant HFP or encodes its chemical modification object of nucleic acid.This chemical modification object can be with alkyl, acyl group or the amino hydrogen atom of replacing.The nucleic acid derivative codified keeps the polypeptide of the main biological characteristics of natural molecule.

" antibody " is meant complete antibody molecule and fragment thereof, as Fa, F (ab ') ₂And Fv, its energy specificity is in conjunction with the antigenic determinant of human zinc finger protein 70.

" humanized antibody " is meant that the aminoacid sequence in non-antigen binding domain territory is replaced and becomes more similar to people's antibody, but still keep original in active antibody.

" isolating " speech refers to material is shifted out among its original environment (for example, if spontaneous its natural surroundings that just refers to).Such as it is exactly not to be separated that spontaneous polynucleotide or polypeptide are present in the Live Animals, but same polynucleotide or polypeptide with some or all in natural system with it the material of coexistence separately be exactly isolating.Such polynucleotide may be the parts of a certain carrier, the part that also possible such polynucleotide or polypeptide are a certain composition.Since carrier or composition are not the compositions of its natural surroundings, they remain isolating.

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

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

The invention provides a kind of polypeptide-human zinc finger protein 70, it is basically by＜210〉aminoacid sequence shown in 2 forms.Polypeptide of the present invention can be recombinant polypeptide, natural polypeptides, synthetic polypeptide, preferred recombinant polypeptide.Polypeptide of the present invention can be the product of natural purifying, or the product of chemosynthesis, or uses recombinant technology to produce from protokaryon or eucaryon host (for example, bacterium, yeast, higher plant, insect and mammalian cell).The host used according to the recombinant production scheme, polypeptide of the present invention can be glycosylated, maybe can be nonglycosylated.Polypeptide of the present invention also can comprise or not comprise initial methionine residues.

The present invention also comprises fragment, derivative and the analogue of human zinc finger protein 70.As used herein, term " fragment ", " derivative " are meant with " analogue " and keep identical biological function of human zinc finger protein 70 of the present invention or active polypeptide basically.The fragment of polypeptide of the present invention, derivative or analogue can be: (I) 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 by coding have＜polynucleotide of the polypeptide of 210〉2 aminoacid sequences form.Polynucleotide sequence of the present invention comprises＜210〉1 nucleotide sequence.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 3191 bases, its open reading frame (2-1924) 640 amino acid of having encoded.Find relatively that according to amino acid sequence homologous this polypeptide and Novel Human Zinc Finger Protein dJ 322P7.1 have 7 3% homology, deducibility goes out this human zinc finger protein 70 and has the similar 26S Proteasome Structure and Function of Novel Human Zinc Finger Protein dJ 322P7.1.

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＜210〉1 or the varient of degeneracy.As used herein, " varient of degeneracy " are meant that in the present invention coding has＜210〉2 protein or polypeptide, but with the differentiated nucleotide sequence of coding region sequence shown in＜210〉1.

The polynucleotide of the mature polypeptide of coding＜210〉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 and＜210〉and the mature polypeptide shown in 2 has identical biological function and activity.

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

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

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

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

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

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

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

In (4) kind method, detect the protein product of human zinc finger protein 70 genetic expression and can use immunological technique such as Western blotting, radioimmunoprecipitation, enzyme-linked immunosorbent assay (ELISA) etc.

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

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

The present invention also relates to comprise the carrier of polynucleotide of the present invention, and with carrier of the present invention or directly with the host cell of human zinc finger protein 70 encoding sequence through the genetically engineered generation, and the method that produces polypeptide of the present invention through recombinant technology.

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

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

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

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

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

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

By the recombinant DNA technology of routine, utilize polynucleotide sequence of the present invention to can be used to express or produce human zinc finger protein 70 (Science, 1984 of reorganization; 224:1431).In general following steps are arranged:

(1). with the polynucleotide (or varient) of coding of the present invention people human zinc finger protein 70, or with the recombinant expression vector that contains these polynucleotide proper host cell that transforms or transduce;

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

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

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

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

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

Polypeptide of the present invention (human zinc finger protein 70) is crucial for the normal expression and the performance biological function of gene, and it can be incorporated into the gypsy gene and stops the stimulation Transcription of genetic enhancer.Polypeptide of the present invention (human zinc finger protein 70) can increase the snappiness of DNA, can cause the change of dna structure.

In addition, the physiological and biochemical procedure that is found to be the differentiation of research cell under normal and pathological conditions, growth, propagation of polypeptide of the present invention (human zinc finger protein 70) provides a kind of method, also comprises that with the disease that the disorder of cytodifferentiation growing multiplication causes cancer provides a kind of new way for diagnosis, treatment.

The gene of code book invention polypeptide then may cause Infertility as undergoing mutation, because according to result of study, SUHW can hinder ovum and generate.

Human zinc finger protein 70 of the present invention 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 monster, Williams syndromes, Alagille syndromes, cleft hand; The canceration of various tissues is as leukemia, lymphoma, non_hodgkin 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, thymus neoplasms etc.; Heredopathia; Nervous system disorders is as neuroblastoma etc.; Endocrine system disease is as endocrine neoplasia; Disease of immune system.

Specifically with regard to human zinc finger protein 70 of the present invention, its protein product can be used to prevent and treats the various diseases relevant with its expression.These diseases include but not limited to following kind: the cancer of hematopoietic cell and tissue comprises leukemia, lymphoma, lymphosarcoma, myelomatosis etc.; The cancer of brain and nervous tissue comprises neuroma, neurospongioma, meningioma, neurofibroma and astrocytoma etc.; The various cancers of secretion or absorptive tissue, body of gland and organ comprise: suprarenal gland, Tiroidina, lung, pancreas, liver, prostate gland, uterus, bladder, kidney, testis and gi tract (small intestine, colon, rectum and stomach); Also have some and cellular abnormality differentiation, propagation and the relevant disease of degraded, comprise diseases such as hyperthyroidism, hypothyroidism, gastritis, polyp of colon, gastro-duodenal ulcer.

Human zinc finger protein 70 of the present invention can be used for treating imbalance and various growth and the relevant disease of pernicious disorder that causes gene transcript expression.These diseases include but not limited to following these, as heredity nervous system disorderss such as the cerebrovascular disease that ictal disorders, epilepsy, cerebral ischemia or hypertension such as spina bifida, hydrocephalus, achondroplastic dwarf's disease, renal tubule acidosis, anaemia, hypogenitalism, congenital glaucoma or cataract, congenital sensory nerve hearing loss, cerebral palsy cause, neurofibroma etc.

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

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

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

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

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

The antibody of anti-human zinc finger protein 70 can be used in the immunohistochemistry technology, detects the human zinc finger protein 70 in the biopsy specimen.

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

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

The disease that antibody among the present invention can be used for treating or prevention is relevant with human zinc finger protein 70.The antibody that gives suitable dosage can stimulate or block the generation or the activity of human zinc finger protein 70.

The invention still further relates to the diagnostic testing process of quantitative and detection and localization human zinc finger protein 70 level.These tests are known in the art, and comprise that FISH measures and radioimmunoassay.The human zinc finger protein 70 level that is detected in the test can be with laying down a definition the importance of human zinc finger protein 70 in various diseases and be used to the disease of diagnosing human zinc finger protein 70 to work.

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

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

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

Suppress the oligonucleotide (comprising sense-rna and DNA) of human zinc finger protein 70 mRNA and ribozyme also within the scope of the invention.Ribozyme is the enzyme sample RNA molecule that a kind of energy specificity is decomposed specific RNA, and its mechanism of action is to carry out the endonuclease effect after ribozyme molecule and the hybridization of complementary target RNA-specific.The RNA of antisense and DNA and ribozyme can obtain with existing any RNA or DNA synthetic technology, as the technology widespread use of solid phase phosphoamide chemical synthesis synthetic oligonucleotide.Antisense rna molecule can be transcribed acquisition by the dna sequence dna of this RNA that encodes in external or body.This dna sequence dna has been incorporated into the downstream of rna polymerase promoter of carrier.In order to increase the stability of nucleic acid molecule, available several different methods is modified it, and as increasing the sequence length of both sides, the connection between the ribonucleoside is used phosphoric acid thioester bond or peptide bond but not phosphodiester bond.

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

The sudden change that detects the human zinc finger protein 70 gene also can be used for the disease of diagnosing human zinc finger protein 70 relevant.The form of human zinc finger protein 70 sudden change comprises that the point mutation compared with normal wild type human zinc finger protein 70 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, Mendelian Inheritancein Man (can by with the online acquisition of Johns Hopkins University Welch Medical Library).Can pass through linkage analysis then, determine gene and navigated to relation between the disease on the chromosomal region already.

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

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

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

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

Description of drawings

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 inventor's zinc finger protein 70 and Novel Human Zinc Finger Protein dJ322P7.1.The top sequence is a human zinc finger protein 70, and the below sequence is Novel Human Zinc Finger Protein dJ322P7.1.Same amino acid represents with monocase amino acid that between two sequences similar amino acid is represented with "+".

Fig. 2 is the polyacrylamide gel electrophoresis figure (SDS-PAGE) of isolating human zinc finger protein 70.70kDa is proteinic molecular weight.The arrow indication is isolated protein band.

Embodiment

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

Embodiment 1: the clone of human zinc finger protein 70

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 0860B06 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 0860B06 clone is 3191bp (as＜210〉1 shown in), from 2bp to 1924bp the open reading frame (ORF) of a 1923bp arranged, the new protein of encoding (as＜210〉2 shown in).We are with this clone's called after pBS-0860B06, encoded protein matter called after human zinc finger protein 70.

Embodiment 2:cDNA clone's homology retrieval

With the sequence and the encoded protein sequence thereof of human zinc finger protein 70 of the present invention, with Blast program (BasiclocalAlignment search tool) [Altschul, SF et al.J.Mol.Biol.1990; 215:403-10], carry out the homology retrieval at databases such as Genbank, Swissport.The gene the highest with human zinc finger protein 70 homology of the present invention is a kind of known Novel Human Zinc Finger Protein dJ322P7.1, and its encoded protein number is AL023799 in the access of Genbank.Protein homology the results are shown in Fig. 1, both height homologies, and its homogeny is 73%; Similarity is 84%.

Embodiment 3: with the gene of RT-PCR method clones coding human zinc finger protein 70

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’-CATGAAACACCATTTGGAACTTGAG-3’(<210>3)

Primer2：5’-CAGAGTATTTTAATCGCCATTTAA-3’(<210>4)

Primer1 for to be positioned at＜the forward sequence that begins of 1bp of 210〉15 ' end;

Primer2 be＜210〉1 in 3 ' end reverse sequence.

The condition of amplified reaction: in the reaction volume of 50 μ l, contain 50mmol/L KCl, 10mmol/LTris-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 β simultaneously--the blank negative contrast of positive contrast of actin and template.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 the PCR product

Dna sequence dna and＜210〉1-3191bp shown in 1 is identical.

Embodiment 4:Northern blotting analyst zinc finger protein 70 expression of gene:

Extract total RNA[Anal.Biochem 1987,162,156-159 with single stage method].This method comprises acid guanidine thiocyanate phenol-chloroform extracting.Promptly use 4M guanidinium isothiocyanate-25mM Trisodium Citrate, 0.2M sodium acetate (pH4.0) carries out homogenate to tissue, adds the phenol of 1 times of volume and the chloroform-primary isoamyl alcohol (49: 1) of 1/5 volume, and is centrifugal after mixing.The sucking-off aqueous phase layer adds Virahol (0.8 volume) and with the centrifugal RNA precipitation that obtains of mixture.With RNA precipitation 70% washing with alcohol that obtains, dry and soluble in water.With 20 μ g RNA, on 1.2% sepharose that contains 20mM 3-(N-morpholino) propanesulfonic acid (pH7.0)-5mM sodium acetate-1mM EDTA-2.2M formaldehyde, carry out electrophoresis.Be transferred on the nitrocellulose filter then.With α- ³²P dATP prepares by random priming ³²The dna probe of P-mark.Used dna probe is the human zinc finger protein 70 coding region sequence (2bp to 1924bp) 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 PhosphorImager.

Embodiment 5: the vivoexpression of recombinant human zinc finger protein 70, separation and purifying

According to＜210〉1 and coding region sequence shown in Figure 1, design a pair of specificity amplification primer, sequence is as follows:

Primer3：5’-CATGCTAGCATGAAACACCATTTGGAACTTGAG-3’(<210>5)

Primer4：5’-CATGGATCCTCAACTTCTTTCTTTTTCATCTTC-3’(<210>6)

5 ' end of these two sections primers contains NheI and BamHI restriction enzyme site respectively, be respectively the encoding sequence of target gene 5 ' end and 3 ' end thereafter, NheI and BamHI 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-0860B06 plasmid that contains the total length goal gene is template, carries out the PCR reaction.The PCR reaction conditions is: contain pBS-0860B06 plasmid 10pg, primer Primer-3 and Primer-4 among the cumulative volume 50 μ l and be respectively 10pmol, Advantage polymerase Mix (Clontech company product) 1 μ l.Loop parameter: 94 ℃ of 20s, 60 ℃ of 30s, 68 ℃ of 2min, totally 25 circulations.Respectively amplified production and plasmid pET-28 (+) are carried out double digestion with NheI and BamHI, 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-0860B06) 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-0860B06) is cultured to logarithmic phase at 37 ℃, adds IPTG to final concentration 1mmol/L, continues to cultivate 5 hours.Centrifugal collection thalline, through the broken bacterium of ultrasonic wave, centrifugal collection supernatant with carrying out chromatography with 6 Histidines (6His-Tag) bonded affinity column His.Bind Quick Cartridge (Novagen company product), has obtained the target protein human zinc finger protein 70 of purifying.Through the SDS-PAGE electrophoresis, obtain a single band (Fig. 2) at the 70kDa place.This band is transferred on the pvdf membrane carries out the n terminal amino acid sequential analysis with the Edams hydrolysis method, 15 amino acid of N-end are identical with 15 amino-acid residues of N-end shown in＜210〉2 as a result.

Embodiment 6 anti-human zinc finger protein 70 production of antibodies

Synthesize the specific polypeptide of following human zinc finger protein 70 with Peptide synthesizer (PE company product):

NH ₂-Met-Lys-His-His-Leu-Glu-Leu-Glu-Lys-Gln-Ser-Ser-Glu-Ser-Trp-COOH(SEQ?ID?NO：7)。

Form compoundly with hemocyanin and bovine serum albumin coupling this polypeptide respectively, method is referring to Avrameas, etal.Immunochemistry, 1969; 6:43.Add the complete Freund's adjuvant immunizing rabbit with the above-mentioned hemocyanin polypeptide complex of 4mg, add the incomplete Freund's adjuvant booster immunization once with the hemocyanin polypeptide complex again after 15 days.Employing is done the titre that ELISA measures antibody in the rabbit anteserum through the titer plate of 15 μ g/ml bovine serum albumin polypeptide complex bag quilts.From the rabbit anteserum of antibody positive, separate total IgG with albumin A-Sepharose.Polypeptide is incorporated on the Sepharose4B post of cyanogen bromide-activated, from total IgG, separates anti-peptide antibody with affinity chromatography.Immuno-precipitation proof antibody purified can combine with human zinc finger protein 70 specifically.

Sequence table＜110〉Bode Gene Development Co., Ltd., Shanghai＜120〉peptide species--the polynucleotides＜130〉0860b06＜160〉6＜170〉PatentIn version 3.1＜210 of human zinc finger protein 70 and this peptide species of coding〉1＜211〉2100＜212〉DNA＜213〉Homo sapiens＜220〉＜221〉CDS＜222〉(2) .. (1924)＜223〉＜400〉1c atg aaa cac cat ttg gaa ctt gag aag cag agc agt gag agc tgg gaa, 49 Met Lys His His Leu Glu Leu Glu Lys Gln Ser Ser Glu Ser Trp Glu, 15 10 15aac cat acc acc tgc cag cac tgc tac cgt cag ttt ccc aca cca ttt 97Asn His Thr Thr Cys Gln His Cys Tyr Arg Gln Phe Pro Thr Pro Phe

20 25 30cag?ttg?cag?tgt?cac?atc?gaa?agt?aca?cat?acg?ccc?cat?gaa?ttt?tct 145Gln?Leu?Gln?Cys?His?Ile?Glu?Ser?Thr?His?Thr?Pro?His?Glu?Phe?Ser

35 40 45act?att?tgt?aaa?atc?tgt?gaa?ttg?tca?ttt?gaa?aca?gaa?cat?gtt?ctt 193Thr?Ile?Cys?Lys?Ile?Cys?Glu?Leu?Ser?Phe?Glu?Thr?Glu?His?Val?Leu

50 55 60tta?caa?cat?atg?aag?gac?aat?cat?aaa?cct?ggt?gaa?atg?cca?tat?gtc 241Leu?Gln?His?Met?Lys?Asp?Asn?His?Lys?Pro?Gly?Glu?Met?Pro?Tyr?Val65 70 75 80tgc?cag?gtt?tgt?aat?tat?aga?tca?tca?tca?ttt?tct?gat?gta?gaa?act 289Cys?Gln?Val?Cys?Asn?Tyr?Arg?Ser?Ser?Ser?Phe?Ser?Asp?Val?Glu?Thr

85 90 95cat?ttt?aga?aca?tcc?cat?gaa?aac?act?aag?aac?ttg?cta?tgc?ccg?ttt 337His?Phe?Arg?Thr?Ser?His?Glu?Asn?Thr?Lys?Asn?Leu?Leu?Cys?Pro?Phe

100 105 110tgc?ctc?aaa?gtt?att?aaa?att?gca?aca?cca?tat?atg?cat?cat?tat?atg 385Cys?Leu?Lys?Val?Ile?Lys?Ile?Ala?Thr?Pro?Tyr?Met?His?His?Tyr?Met

115 1205 125aag?cat?cag?aaa?aaa?gga?ata?cat?cgt?tgt?aca?aaa?tgc?agg?ctg?cag 433Lys?His?Gln?Lys?Lys?Gly?Ile?His?Arg?Cys?Thr?Lys?Cys?Arg?Leu?Gln

130 135 140ttt?ttg?aca?tgc?aag?gag?aaa?atg?gat?cat?aag?act?caa?cac?cat?cga 481Phe?Leu?Thr?Cys?Lys?Glu?Lys?Met?Asp?His?Lys?Thr?Gln?His?His?Arg145 150 155 160aca?ttt?ata?aaa?cct?aaa?caa?cta?gaa?gga?ttg?cct?cct?gga?aca?aaa 529Thr?Phe?Ile?Lys?Pro?Lys?Gln?Leu?Glu?Gly?Leu?Pro?Pro?Gly?Thr?Lys

165 170 175gtt?act?att?cga?gct?tca?gtt?gga?cct?ctg?caa?tca?gga?gct?tca?cct 577Val?Thr?Ile?Arg?Ala?Ser?Val?Gly?Pro?Leu?Gln?Ser?Gly?Ala?Ser?Pro

180 185 190aca?cct?tcc?att?agt?gca?agt?gct?tct?acc?ctt?cag?ctc?tca?cct?cca 625Thr?Pro?Ser?Ile?Ser?Ala?Ser?Ala?Ser?Thr?Leu?Gln?Leu?Ser?Pro?Pro

195 200 205agg?act?aaa?aat?ata?act?gct?aaa?aat?cct?gca?aaa?tct?aat?aca?agt 673Arg?Thr?Lys?Asn?Ile?Thr?Ala?Lys?Asn?Pro?Ala?Lys?Ser?Asn?Thr?Ser

210 215 220aaa?cct?aat?aca?atc?aaa?tcc?aac?gca?agt?aaa?cct?aat?aca?agt?aag 721Lys?Pro?Asn?Thr?Ile?Lys?Ser?Asn?Ala?Ser?Lys?Pro?Asn?Thr?Ser?Lys225 230 235 240cct?aat?gga?agt?aaa?tct?aaa?tac?aaa?cca?aaa?atc?tct?aat?atg?caa 769Pro?Asn?Gly?Ser?Lys?Ser?Lys?Tyr?Lys?Pro?Lys?Ile?Ser?Asn?Met?Gln

245 250 255aag?aaa?caa?agc?aca?ttg?gct?agt?agc?aat?aaa?aaa?agt?aaa?gtc?aat 817Lys?Lys?Gln?Ser?Thr?Leu?Ala?Ser?Ser?Asn?Lys?Lys?Ser?Lys?Val?Asn

260 265 270aca?gca?ttg?agg?aat?tta?agg?tat?cgt?cgg?ggc?att?cac?aaa?tgc?att 865Thr?Ala?Leu?Arg?Asn?Leu?Arg?Tyr?Arg?Arg?Gly?Ile?His?Lys?Cys?Ile

275 280 285gag?tgt?tgt?tcc?gaa?ata?aaa?gat?ttt?gca?aac?cac?ttt?cct?acg?tac 913Glu?Cys?Cys?Ser?Glu?Ile?Lys?Asp?Phe?Ala?Asn?His?Phe?Pro?Thr?Tyr

290 295 300gtc?cac?tgt?agt?ttt?tgc?aga?tac?aac?act?agc?tgt?agc?aaa?gcc?tat 961Val?His?Cys?Ser?Phe?Cys?Arg?Tyr?Asn?Thr?Ser?Cys?Ser?Lys?Ala?Tyr305 310 315 320gta?aat?cat?atg?atg?agc?ttt?cat?agt?aac?cgt?cca?agc?aaa?agg?ttt 1009Val?Asn?His?Met?Met?Ser?Phe?His?Ser?Asn?Arg?Pro?Ser?Lys?Arg?Phe

325 330 335tgt?att?ttt?aag?aag?cat?tca?gaa?aat?ctc?cgg?ggc?att?act?cta?gtg 1057Cys?Ile?Phe?Lys?Lys?His?Ser?Glu?Asn?Leu?Arg?Gly?Ile?Thr?Leu?Val

340 345 350tgc?ctt?aat?tgt?gat?ttc?cta?agt?gat?gtt?tct?ggc?tta?gat?aat?atg 1105Cys?Leu?Asn?Cys?Asp?Phe?Leu?Ser?Asp?Val?Ser?Gly?Leu?Asp?Asn?Met

355 360 365gct?aca?cac?tta?agt?caa?cat?aaa?act?cat?act?tgc?caa?gtt?gta?atg 1153Ala?Thr?His?Leu?Ser?Gln?His?Lys?Thr?His?Thr?Cys?Gln?Val?Val?Met

370 375 380cag?aaa?gtt?tct?gtt?tgt?atc?cca?act?tct?gag?cac?ctt?tct?gaa?tta 1201Gln?Lys?Val?Ser?Val?Cys?Ile?Pro?Thr?Ser?Glu?His?Leu?Ser?Glu?Leu385 390 395 400aaa?aaa?gaa?gct?ccc?gca?aag?gaa?caa?gaa?cct?gtg?tct?aag?gaa?att 1249Lys?Lys?Glu?Ala?Pro?Ala?Lys?Glu?Gln?Glu?Pro?Val?Ser?Lys?Glu?Ile

405 410 415gca?aga?cct?aac?atg?gct?gaa?aga?gaa?aca?gaa?aca?tca?aat?tct?gaa 1297Ala?Arg?Pro?Asn?Met?Ala?Glu?Arg?Glu?Thr?Glu?Thr?Ser?Asn?Ser?Glu

420 425 430agt?aaa?caa?gat?aaa?gct?gct?tct?tca?aaa?gaa?aaa?aat?gga?tgt?aat 1345Ser?Lys?Gln?Asp?Lys?Ala?Ala?Ser?Ser?Lys?Glu?Lys?Asn?Gly?Cys?Asn

435 440 445gca?aat?tca?ttt?gaa?ggc?tca?tca?aca?aca?aaa?agt?gaa?gaa?agc?ata 1393Ala?Asn?Ser?Phe?Glu?Gly?Ser?Ser?Thr?Thr?Lys?Ser?Glu?Glu?Ser?Ile

450 455 460aca?gtt?tca?gat?aag?gaa?aat?gaa?acc?tgt?ctt?gca?gac?cag?gaa?act 1441Thr?Val?Ser?Asp?Lys?Glu?Asn?Glu?Thr?Cys?Leu?Ala?Asp?Gln?Glu?Thr465 470 475 480ggc?tca?aaa?aac?atc?gtc?agt?tgt?gat?tca?aat?att?ggt?gca?gat?aaa 1489Gly?Ser?Lys?Asn?Ile?Val?Ser?Cys?Asp?Ser?Asn?Ile?Gly?Ala?Asp?Lys

485 490 495gtg?gaa?aag?aaa?aaa?caa?ata?caa?cac?gtt?tgt?cag?gaa?atg?gag?ttg 1537Val?Glu?Lys?Lys?Lys?Gln?Ile?Gln?His?Val?Cys?Gln?Glu?Met?Glu?Leu

500 505 510aag?atg?tgc?caa?agt?tca?gaa?aac?ata?atc?tta?tct?gat?cag?att?aaa 1585Lys?Met?Cys?Gln?Ser?Ser?Glu?Asn?Ile?Ile?Leu?Ser?Asp?Gln?Ile?Lys

515 520 525gat?cac?aac?tcc?agt?gaa?gcc?aga?ttt?tct?tca?aag?aat?att?aag?gat 1633Asp?His?Asn?Ser?Ser?Glu?Ala?Arg?Phe?Ser?Ser?Lys?Asn?Ile?Lys?Asp

530 535 540ttg?cga?tta?gca?tca?gat?aat?gta?agc?att?gat?cag?ttt?ttg?aga?aaa 1681Leu?Arg?Leu?Ala?Ser?Asp?Asn?Val?Ser?Ile?Asp?Gln?Phe?Leu?Arg?Lys545 550 555 560aga?cat?gaa?cct?gaa?tct?gtt?agt?tct?gat?gtt?agc?gag?caa?ggc?agt 1729Arg?His?Glu?Pro?Glu?Ser?Val?Ser?Ser?Asp?Val?Ser?Glu?Gln?Gly?Ser

565 570 575att?cat?ttg?gaa?cct?ctg?act?cca?tcc?gag?gta?ctt?gag?tat?gaa?gcc 1777Ile?His?Leu?Glu?Pro?Leu?Thr?Pro?Ser?Glu?Val?Leu?Glu?Tyr?Glu?Ala

580 585 590aca?gag?att?ctt?cag?aaa?ggt?agt?ggt?gat?cct?tca?gcc?aag?act?gat 1825Thr?Glu?Ile?Leu?Gln?Lys?Gly?Ser?Gly?Asp?Pro?Ser?Ala?Lys?Thr?Asp

595 600 605gaa?gta?gtg?tct?gat?caa?aca?gat?gac?att?cct?gga?gga?aat?aac?cct 1873Glu?Val?Val?Ser?Asp?Gln?Thr?Asp?Asp?Ile?Pro?Gly?Gly?Asn?Asn?Pro

610 615 620agc?aca?aca?gag?gca?aca?gta?gac?ctg?gaa?gat?gaa?aaa?gaa?aga?agt 1921Ser?Thr?Thr?Glu?Ala?Thr?Val?Asp?Leu?Glu?Asp?Glu?Lys?Glu?Arg?Ser625 630 635 640tga?aattagtcat?tttaagtttc?agtgtaccaa?cgataagggc?atttggaaca 1974gtgctatcag?gtgagctcag?tggtgctgtt?gtaggttcag?aaatggaaat?atgtaaggga 2034ggtcacacat?acgctttacc?tgtatgttca?acctatgtta?tcaaacaaat?caattcacca 2094ataata2100<210>2<211>640<212>PRT<213>Homo?sapiens<400>2Met?Lys?His?His?Leu?Glu?Leu?Glu?Lys?Gln?Ser?Ser?Glu?Ser?Trp?Glu1 5 10 15Asn?His?Thr?Thr?Cys?Gln?His?Cys?Tyr?Arg?Gln?Phe?Pro?Thr?Pro?Phe

20 25 30Gln?Leu?Gln?Cys?His?Ile?Glu?Ser?Thr?His?Thr?Pro?His?Glu?Phe?Ser

35 40 45Thr?Ile?Cys?Lys?Ile?Cys?Glu?Leu?Ser?Phe?Glu?Thr?Glu?His?Val?Leu

50 55 60Leu?Gln?His?Met?Lys?Asp?Asn?His?Lys?Pro?Gly?Glu?Met?Pro?Tyr?Val65 70 75 80Cys?Gln?Val?Cys?Asn?Tyr?Arg?Ser?Ser?Ser?Phe?Ser?Asp?Val?Glu?Thr

85 90 95His?Phe?Arg?Thr?Ser?His?Glu?Asn?Thr?Lys?Asn?Leu?Leu?Cys?Pro?Phe

100 105 110Cys?Leu?Lys?Val?Ile?Lys?Ile?Ala?Thr?Pro?Tyr?Met?His?His?Tyr?Met

115 120 125Lys?His?Gln?Lys?Lys?Gly?Ile?His?Arg?Cys?Thr?Lys?Cys?Arg?Leu?Gln

130 135 140Phe?Leu?Thr?Cys?Lys?Glu?Lys?Met?Asp?His?Lys?Thr?Gln?His?His?Arg145 150 155 160Thr?Phe?Ile?Lys?Pro?Lys?Gln?Leu?Glu?Gly?Leu?Pro?Pro?Gly?Thr?Lys

165 170 175Val?Thr?Ile?Arg?Ala?Ser?Val?Gly?Pro?Leu?Gln?Ser?Gly?Ala?Ser?Pro

180 185 190Thr?Pro?Ser?Ile?Ser?Ala?Ser?Ala?Ser?Thr?Leu?Gln?Leu?Ser?Pro?Pro

195 200 205Arg?Thr?Lys?Asn?Ile?Thr?Ala?Lys?Asn?Pro?Ala?Lys?Ser?Asn?Thr?Ser

210 215 220Lys?Pro?Asn?Thr?Ile?Lys?Ser?Asn?Ala?Ser?Lys?Pro?Asn?Thr?Ser?Lys225 230 235 240Pro?Asn?Gly?Ser?Lys?Ser?Lys?Tyr?Lys?Pro?Lys?Ile?Ser?Asn?Met?Gln

245 250 255Lys?Lys?Gln?Ser?Thr?Leu?Ala?Ser?Ser?Asn?Lys?Lys?Ser?Lys?Val?Asn

260 265 270Thr?Ala?Leu?Arg?Asn?Leu?Arg?Tyr?Arg?Arg?Gly?Ile?His?Lys?Cys?Ile

275 280 285Glu?Cys?Cys?Ser?Glu?Ile?Lys?Asp?Phe?Ala?Asn?His?Phe?Pro?Thr?Tyr

290 295 300Val?His?Cys?Ser?Phe?Cys?Arg?Tyr?Asn?Thr?Ser?Cys?Ser?Lys?Ala?Tyr305 310 315 320Val?Asn?His?Met?Met?Ser?Phe?His?Ser?Asn?Arg?Pro?Ser?Lys?Arg?Phe

325 330 335Cys?Ile?Phe?Lys?Lys?His?Ser?Glu?Asn?Leu?Arg?Gly?Ile?Thr?Leu?Val

340 345 350Cys?Leu?Asn?Cys?Asp?Phe?Leu?Ser?Asp?Val?Ser?Gly?Leu?Asp?Asn?Met

355 360 365Ala?Thr?His?Leu?Ser?Gln?His?Lys?Thr?His?Thr?Cys?Gln?Val?Val?Met

370 375 380Gln?Lys?Val?Ser?Val?Cys?Ile?Pro?Thr?Ser?Glu?His?Leu?Ser?Glu?Leu385 390 395 400Lys?Lys?Glu?Ala?Pro?Ala?Lys?Glu?Gln?Glu?Pro?Val?Ser?Lys?Glu?Ile

405 410 415Ala?Arg?Pro?Asn?Met?Ala?Glu?Arg?Glu?Thr?Glu?Thr?Ser?Asn?Ser?Glu

420 425 430Ser?Lys?Gln?Asp?Lys?Ala?Ala?Ser?Ser?Lys?Glu?Lys?Asn?Gly?Cys?Asn

435 440 445Ala?Asn?Ser?Phe?Glu?Gly?Ser?Ser?Thr?Thr?Lys?Ser?Glu?Glu?Ser?Ile

450 455 460Thr?Val?Ser?Asp?Lys?Glu?Asn?Glu?Thr?Cys?Leu?Ala?Asp?Gln?Glu?Thr465 470 475 480Gly?Ser?Lys?Asn?Ile?Val?Ser?Cys?Asp?Ser?Asn?Ile?Gly?Ala?Asp?Lys

485 490 495Val?Glu?Lys?Lys?Lys?Gln?Ile?Gln?His?Val?Cys?Gln?Glu?Met?Glu?Leu

500 505 510Lys?Met?Cys?Gln?Ser?Ser?Glu?Asn?Ile?Ile?Leu?Ser?Asp?Gln?Ile?Lys

515 520 525Asp?His?Asn?Ser?Ser?Glu?Ala?Arg?Phe?Ser?Ser?Lys?Asn?Ile?Lys?Asp

530 535 540Leu?Arg?Leu?Ala?Ser?Asp?Asn?Val?Ser?Ile?Asp?Gln?Phe?Leu?Arg?Lys545 550 555 560Arg?His?Glu?Pro?Glu?Ser?Val?Ser?Ser?Asp?Val?Ser?Glu?Gln?Gly?Ser

565 570 575Ile?His?Leu?Glu?Pro?Leu?Thr?Pro?Ser?Glu?Val?Leu?Glu?Tyr?Glu?Ala

580 585 590Thr?Glu?Ile?Leu?Gln?Lys?Gly?Ser?Gly?Asp?Pro?Ser?Ala?Lys?Thr?Asp

595 600 605Glu?Val?Val?Ser?Asp?Gln?Thr?Asp?Asp?Ile?Pro?Gly?Gly?Asn?Asn?Pro

610 615 620Ser?Thr?Thr?Glu?Ala?Thr?Val?Asp?Leu?Glu?Asp?Glu?Lys?Glu?Arg?Ser625 630 635 640<210>3<211>25<212>DNA<213>Homo?sapiens<400>3catgaaacac catttggaac ttgag25<210>4<211>24<212>DNA<213>Homo?sapiens<400>4cagagtattt taatcgccat ttaa24<210>5<211>33<212>DNA<213>Homo?sapiens<400>5catgctagca tgaaacacca tttggaactt gag33<210>6<211>33<212>DNA<213>Homo?sapiens<400>6catggatcct caacttcttt ctttttcatc ttc33

Claims (18)

1, a kind of isolated polypeptide-human zinc finger protein 70 is characterized in that it includes: the polypeptide of the aminoacid sequence shown in＜210〉2 or the active fragments of its polypeptide, analogue or derivative.

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＜210〉2.

3, polypeptide as claimed in claim 2 is characterized in that it comprises and has＜polypeptide of the aminoacid sequence shown in 210〉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 have＜210〉2 shown in the polynucleotide of the polypeptide of aminoacid sequence or its fragment, analogue, derivative;

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

(c) with (a) or the polynucleotide of at least 74% homogeny (b) are arranged.

5, polynucleotide as claimed in claim 4, it is characterized in that described polynucleotide comprise coding and have＜210〉2 shown in the polynucleotide of aminoacid sequence.

6, polynucleotide as claimed in claim 4, it is characterized in that the sequence of described polynucleotide includes＜210〉1 in the 2-1924 position sequence or＜210〉1 in the sequence of 1-3191 position.

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

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

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

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

9, a kind of preparation method with the active polypeptide of human zinc finger protein 70 is characterized in that described method comprises:

(a) under expressing human zinc finger protein 70 condition, cultivate the described through engineering approaches host cell of claim 8;

(b) from culture, isolate and have the active polypeptide of human zinc finger protein 70.

10, a kind of can with polypeptide bonded antibody, it is characterized in that described antibody be can with human zinc finger protein 70 specificity bonded antibody.

11, the compound of an analoglike or adjusting polypeptide active or expression is characterized in that they are simulation, promotion, antagonism or the active compound that suppresses human zinc finger protein 70.

12, compound as claimed in claim 11 is characterized in that it is＜polynucleotide sequence or its segmental antisense sequences shown in 210〉1.

13, the described application of compound of a kind of claim 11, it is characterized in that described compound be used for mediator's zinc finger protein 70 in vivo, the method for external activity.

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

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

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

17,, it is characterized in that forming pharmaceutical composition with safe and effective dosage and pharmaceutically acceptable carrier as diagnosis or the treatment disease relevant unusually with human zinc finger protein 70 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.