CN1177862C - Human NIP2 associated protein and coding sequence and application thereof - Google Patents

Abstract

The present invention discloses new human NIP2 associated protein, polynucleotide used for coding the polypeptide, and a method of generating the polypeptide through a recombination technology. The present invention also discloses a method of using the polypeptide for treating various diseases, such as a cancer. The present invention also discloses an antagonist against the polypeptide and a therapeutic function of the antagonist. The present invention also discloses an application of the polynucleotide used for coding the new human NIP2 associated protein.

Description

Human NIP 2 associated protein and encoding sequence and uses thereof

The invention belongs to biological technical field, specifically, the present invention relates to the polynucleotide of new coding human NIP 2 associated protein, and the polypeptide of this polynucleotide encoding.The invention still further relates to the Use and preparation method of these polynucleotide and polypeptide.

The full name of NIP is NCBP (Nuclear cap binding protein) interaction protein (NCBPinteracting protein).NIP is a family.NIP1 wherein, NIP2 have proved not only influences the montage (splicing) of Pre-mRNA and the transhipment of mRNA by NCBP in conjunction with the combination that participates in mRNA cap; And NIP1, NIP2 are important member (the Katoka N et al.Nucleic Acid Res.23:3638-3641 in the apoptosis path; 1995).NIP2 participates in by TNF α inductive apoptosis.Simultaneously, NIP1 and NIP2 all interact with Bcl2: the anti-apoptotic effect of NIP2 antagonism Bcl2, and Bcl2 can suppress the short apoptosis (Vegeto E.et al.FASEB J 13:793-803,1999) of NIP2.

Cancer is one of principal disease of harm humans health.In order to treat effectively and prophylaxis of tumours, people more and more pay close attention to genetic treatment of tumor at present.Therefore, this area presses for albumen and the agonist/inhibitor thereof that development research has cancer suppressing action.Yet, before the application, the amino acid or the nucleotide sequence of new NIP2 associated protein involved in the present invention still do not disclosed.

The purpose of this invention is to provide the new NIP2 associated protein of a class (NIP-2 associated protein abbreviates " NIP2 AP " as) polypeptide with and fragment, analogue and derivative.

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

Another object of the present invention provides the method for these polypeptide of production and the purposes of this polypeptide and encoding sequence.

In a first aspect of the present invention, novel isolated NIP2 associated protein polypeptide is provided, it comprises the polypeptide of the aminoacid sequence with SEQ ID NO:2, or its conservative property variation polypeptide or its active fragments or its reactive derivative.

Preferably, this polypeptide is the polypeptide with aminoacid sequence of SEQ ID NO:2.

In a second aspect of the present invention, a kind of isolating polynucleotide are provided, it comprises a nucleotide sequence, and this nucleotide sequence is shown at least 85% homogeny with a kind of nucleotides sequence that is selected from down group: the polynucleotide of the above-mentioned NIP2 associated protein polypeptide of (a) encoding; (b) with polynucleotide (a) complementary polynucleotide.Preferably, the polypeptide of this polynucleotide encoding has SEQ ID NO:2 aminoacid sequence.More preferably, the sequence of these polynucleotide is selected from down group: coding region sequence or the full length sequence of SEQ ID NO:3.

In a third aspect of the present invention, the carrier that contains above-mentioned polynucleotide is provided, and has been transformed or host cell of transduceing or the host cell that is directly transformed or transduce by above-mentioned polynucleotide by this carrier.

In a fourth aspect of the present invention, provide preparation to have the preparation method of the active polypeptide of human NIP 2 associated protein, this method comprises: (a) under the condition that is fit to expression NIP2 associated protein, cultivate the above-mentioned host cell that is transformed or transduce; (b) from culture, isolate and have the active polypeptide of human NIP 2 associated protein.

In a fifth aspect of the present invention, provide and above-mentioned NIP2 associated protein polypeptid specificity bonded antibody.The nucleic acid molecule that can be used for detecting also is provided, and it contains a successive 10-800 Nucleotide in the above-mentioned polynucleotide.

In a sixth aspect of the present invention, a kind of pharmaceutical composition is provided, it contains the NIP2 associated protein polypeptide of the present invention and the pharmaceutically acceptable carrier of safe and effective amount.These pharmaceutical compositions can be treated illnesss such as cancer and cellular abnormality propagation.

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

In the accompanying drawings, Fig. 1 has shown the express spectra of human NIP 2 associated protein of the present invention (NIP2 AP).

Fig. 2 has shown the gel electrophoresis figure of intestinal bacteria T7 S30 external translating system expression NIP2 AP.Wherein, swimming lane 1 is a blank; Swimming lane 2 is PinPoint ^TMContrast DNA (coding CAT fusion rotein, about 39kDa); Swimming lane 3 is pT7X-NIP2 AP (about 41kDa).

The present invention adopts large-scale cDNA clone transfection cancer cell, has on the basis of cancer suppressing action in acquisition, proves new gene through order-checking, further obtains full length cDNA clone. DNA transfection evidence, human NIP 2 associated protein of the present invention has the effect that the clone forms that suppresses to cancer cell (HCC).

In one embodiment of the invention, the human NIP 2 associated protein gene is the cDNA that separates from placenta, and it and NIP2 have certain homology (homogeny 47%, similitude 73%) at amino acid sequence. It can suppress liver cancer cell growth and promote the effect of cancer cell-apoptosis. Therefore, this is a new gene with function, and has using value in oncotherapy.

As used herein, " separation " refers to that material separates (if crude, primal environment namely is natural surroundings) from its primal environment. Do not have separation and purification such as the 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, " NIP2 GAP-associated protein GAP or the polypeptide of separation " refers to that NIP2 GAP-associated protein GAP polypeptide 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 NIP2 GAP-associated protein GAP of standard. Basically pure polypeptide can produce single master tape on non-reduced polyacrylamide gel. The purity of NIP2 GAP-associated protein GAP polypeptide can be used amino acid sequence analysis.

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 chemical synthesis, or uses recombinant technique 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 analog of human NIP 2 associated protein. As used herein, term " fragment ", " derivative " refer to basically keep the identical biological function of natural human NIP 2 associated protein of the present invention or active polypeptide with " analog ". Polypeptide fragment of the present invention, derivative or analog can be that (i) has one or more conservative or substituted polypeptide of non-conservation amino acid residue (preferred conservative amino acid residue), and the amino acid residue of such replacement can be also can not encoded by genetic code, or (ii) in one or more amino acid residues, has a polypeptide of substituted radical, or (iii) mature polypeptide and another compound (such as the compound that prolongs the polypeptide half-life, polyethylene glycol for example) merges formed polypeptide, or (iv) additional amino acid sequence is fused to this peptide sequence and the polypeptide that forms (such as targeting sequencing or secretion sequence or be used for sequence or the proteinogen sequence of this polypeptide of purifying, or with the fusion of the formation of antigen I gG fragment). According to the instruction of this paper, these fragments, derivative and analog belong to the known scope of those skilled in the art.

In the present invention, term " human NIP 2 associated protein polypeptide " or " people NIP2 AP polypeptide " are used interchangeably, and all refer to have the polypeptide of the SEQ ID NO.2 sequence of human NIP 2 associated protein activity. This term also comprises having and the variant form human NIP 2 associated protein identical function, SEQ ID NO.2 sequence. These variant forms comprise (but being not limited to): several (are generally 1-50, preferably 1-30, more preferably 1-20,1-10 best) amino acid whose disappearance, insertion and/or replacement, and add one or several and (be generally in 20 in that C end and/or N are terminal, preferably being in 10, more preferably is in 5) amino acid. For example, in the art, when replacing with the close or similar amino acid of performance, usually can not change the function of protein. Again such as, add the function that or several amino acid also can not change protein usually in that C end and/or N are terminal. This term also comprises active fragment and the reactive derivative of human NIP 2 associated protein.

The variant form of this polypeptide comprises: homologous sequence, conservative variant, allelic variant, natural mutation, induced mutation body, under high or low stringency condition can with the coded albumen of the DNA of human NIP 2 associated protein DNA hybridization and the polypeptide or the albumen that utilize the antiserum of anti-human NIP2 GAP-associated protein GAP polypeptide to obtain. The present invention also provides other polypeptide, as comprises the fusion (as comprising the fusion of sequence shown in the SEQ ID NO:2) of human NIP 2 associated protein polypeptide or its fragment. Except the polypeptide of total length almost, the present invention has also comprised the soluble fragments of human NIP 2 associated protein polypeptide. Usually, this fragment have the human NIP 2 associated protein peptide sequence at least about 10 continuous amino acids, usually at least about 30 continuous amino acids, preferably at least about 50 continuous amino acids, more preferably at least about 80 continuous amino acids, best at least about 100 continuous amino acids.

Invention also provides the analog of human NIP 2 associated protein or polypeptide. The difference of these analogs and natural human NIP 2 associated protein polypeptide can be the difference on the amino acid sequence, also can be the difference that does not affect on the modified forms of sequence, perhaps haves both at the same time. These polypeptide comprise genetic variant natural or that induce. The induce variation body can obtain by various technology, as by radiation or be exposed to mutagens and produce random mutagenesis, also can pass through direct mutagenesis method or the biological technology of other known moleculars. Analog also comprises having the analog that is different from the amino acid whose residue of natural L-(such as D-amino acid), and the analog with that non-natural exists or synthetic amino acid (such as β, gamma-amino acid). Should be understood that polypeptide of the present invention is not limited to the above-mentioned representational polypeptide that exemplifies.

(usually the not changing primary structure) form of modification comprises: chemically derived form such as acetylation or the carboxylated of the polypeptide that body is interior or external. Modification also comprises glycosylation, carries out glycosylation modified and polypeptide that produce in the procedure of processing such as those in the synthetic and processing of polypeptide or further. This modification can be carried out glycosylated enzyme (such as mammiferous glycosylase or deglycosylating enzyme) and finishes by polypeptide is exposed to. Modified forms also comprises have the phosphorylated amino acid residue sequence of (such as phosphotyrosine, phosphoserine, phosphothreonine). Thereby also comprise the polypeptide that has been improved its anti-proteolysis performance or optimized solubility property by modifying.

In the present invention, " human NIP 2 associated protein conservative variation polypeptide " refers to compare with the amino acid sequence of SEQ ID NO:2, has 10 at the most, preferably at the most 8, more preferably at the most 5,3 amino acid is replaced by similar performance or close amino acid and is formed polypeptide at the most best. These conservative variation polypeptide preferably carry out amino acid substitution according to Table A and produce.

Table A

Initial residue	Representational replacement	The preferred replacement
			Ala(A)	Val；Leu；Ile	Val
Arg(R)	Lys；Gln；Asn	Lys
			Asn(N)	Gln；His；Lys；Arg	Gln
Asp(D)	Glu	Glu
			Cys(C)	Ser	Ser
Gln(Q)	Asn	Asn
			Glu(E)	Asp	Asp
Gly(G)	Pro；Ala	Ala
			His(H)	Asn；Gln；Lys；Arg	Arg
Ile(I)	Leu；Val；Met；Ala；Phe	Leu
			Leu(L)	Ile；Val；Met；Ala；Phe	Ile
Lys(K)	Arg；Gln；Asn	Arg
			Met(M)	Leu；Phe；Ile	Leu
Phe(F)	Leu；Val；Ile；Ala；Tyr	Leu
			Pro(P)	Ala	Ala
Ser(S)	Thr	Thr
			Thr(T)	Ser	Ser
Trp(W)	Tyr；Phe	Tyr
			Tyr(Y)	Trp；Phe；Thr；Ser	Phe
Val(V)	Ile；Leu；Met；Phe；Ala	Leu

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 (618-1445 position) shown in the SEQ ID NO:3 or the varient of degeneracy.As used herein, " varient of degeneracy " is meant that in the present invention coding has the protein of SEQ ID NO:2, but with the differentiated nucleotide sequence of coding region sequence shown in the SEQ ID NO:3.

The polynucleotide of the mature polypeptide of coding SEQ ID NO:2 comprise: the encoding sequence of an encoding 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 " can be the polynucleotide that comprise this polypeptide of encoding, and also can be the polynucleotide that also comprise additional code and/or non-coding sequence.

The invention still further relates to the varient of above-mentioned polynucleotide, its coding has the polypeptide of identical aminoacid sequence or fragment, analogue and the derivative of polypeptide with the present invention.The varient of these polynucleotide can be the allelic variant of natural generation or the varient that non-natural takes place.These nucleotide diversity bodies comprise and replace varient, deletion mutation body and insert varient.As known in the art, allelic variant is 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 above-mentioned nucleic acid array hybridizing and two sequences between have at least 60%, preferably at least 80%, more preferably at least 85%, the polynucleotide of at least 90% homogeny best.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 is added with 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 above-mentioned sequence hybridization.As used herein, the length of " nucleic acid fragment " contains 15 Nucleotide at least, better is at least 30 Nucleotide, is more preferably at least 50 Nucleotide, preferably more than at least 100 Nucleotide.Nucleic acid fragment can be used for the amplification technique (as PCR) of nucleic acid to determine and/or to separate the polynucleotide of coding human NIP 2 associated protein.

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

Dna sequence dna of the present invention can obtain with several method.For example, with hybridization technique DNA isolation 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 homology nucleotide sequence and 2) antibody screening of expression library to be to detect the dna fragmentation of the clone with common structure feature.

The specific DNA fragment sequence of coding human NIP 2 associated protein produces also and 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 required polypeptide.

In the above-mentioned method of mentioning, isolation of genomic DNA is least commonly used.When the whole aminoacid sequence of the polypeptide product of needs was known, the direct chemical of dna sequence dna is synthetic to be the method for often selecting for use.When if required amino acid whose whole sequence is not known, the direct chemical of dna sequence dna is synthetic to be impossible, and the method for 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., Molecular Cloning, A Laboratory Manual, ColdSpring 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) function of marker gene occurs or forfeiture; (3) level of the transcript of mensuration human NIP 2 associated protein; (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 15 Nucleotide of its length better are at least 30 Nucleotide, are more preferably at least 50 Nucleotide, preferably at least 100 Nucleotide.In addition, the length of probe within 2kb, preferably is within the 1kb usually.Probe used herein is the dna sequence dna of chemosynthesis on the basis of gene DNA sequence 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 NIP 2 associated protein genetic expression and can use immunological technique such as Western blotting, radioimmunoprecipitation, enzyme-linked immunosorbent assay (ELISA) etc.

Use method (Saiki, the et al.Science 1985 of round pcr DNA amplification/RNA; 230:1350-1354) be optimized for acquisition gene of the present invention.When particularly being difficult to obtain the cDNA of total length from the library, 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 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 available ordinary method of mensuration of the nucleotide sequence of various dna fragmentations etc. such as dideoxy chain termination (Sanger et al.PNAS, 1977,74:5463-5467).This class nucleotide sequencing is available commercial sequencing kit etc. also.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 the host cell that produces through genetically engineered with carrier of the present invention or human NIP 2 associated protein encoding sequence, and the method that produces polypeptide of the present invention through recombinant technology.

Recombinant DNA technology (Science, 1984 by routine; 224:1431), can utilize polymerized nucleoside acid sequence of the present invention to can be used to express or produce the human NIP 2 associated protein polypeptide of reorganization.In general following steps are arranged:

(1). with the polynucleotide (or varient) of coding human NIP 2 associated protein of the present invention, or transform or the transduction proper host cell with the recombinant expression vector that contains these polynucleotide;

(2). the host cell of in suitable medium, cultivating;

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

Among the present invention, the human NIP 2 associated protein polynucleotide sequence can be inserted in the recombinant expression vector.Term " recombinant expression vector " 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 carriers.The carrier of Shi Yonging includes but not limited in the present invention: and the expression vector based on T7 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 can be used.A key character of expression vector is to contain replication orgin, promotor, marker gene and translation controlling elements usually.

Method well-known to those having ordinary skill in the art can be used to make up and contains human NIP 2 associated protein DNA sequences encoding and suitable transcribing/the translate expression vector of control signal.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; Lambda particles phage P _LPromotor; Eukaryotic promoter comprises LTRs and some other known may command gene expression promoter in protokaryon 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 and the transcription terminator that translation initiation is used.

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.

Comprise the carrier of above-mentioned suitable dna sequence dna and suitable promotor or control sequence, can be used to transform appropriate host cell, so that it can marking protein.

Host cell can be a 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; The bacterial cell of Salmonella typhimurium; Fungal cell such as yeast; Vegetable cell; The insect cell of fruit bat S2 or Sf9; The zooblast of CHO, COS or Bowes melanoma cells etc.

When polynucleotide of the present invention are expressed in higher eucaryotic cells, be enhanced if will make to transcribe when in carrier, inserting enhancer sequence.Enhanser is the cis acting factor of DNA, 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.

Persons skilled in the art all know how to select appropriate carriers, promotor, enhanser and host cell.

Can carry out with routine techniques well known to those skilled in the art with the recombinant DNA transformed host cell.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, conventional mechanical method such as microinjection, electroporation, liposome packing etc.

The transformant that obtains can be cultivated with ordinary method, expresses the polypeptide of coded by said gene of the present invention.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.

Recombinant polypeptide in the above methods can wrap by in cell, extracellular or on cytolemma, express or be secreted into the extracellular.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.The example of these methods includes, but are not limited to: conventional renaturation handles, with protein precipitant handle (salt analysis method), centrifugal, the broken bacterium of infiltration, superly handle, the combination of 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 human NIP 2 associated protein or the polypeptide of reorganization are of use in many ways.These purposes include, but is not limited to: directly as the disease due to pharmacological agent human NIP 2 associated protein hypofunction or the forfeiture (in particular for suppressing tumor growth, in particular for treatment liver cancer) and be used to screen antibody, polypeptide or other part that promotes or resist the human NIP 2 associated protein function.For example, antibody can be used for activating or suppressing the function of human NIP 2 associated protein.The peptide molecule that can suppress or stimulate the human NIP 2 associated protein function that can be used for seeking therapeutic value with the reorganization human NIP 2 associated protein screening peptide library of expressing.

The present invention also provides screening of medicaments to identify the method that improves (agonist) or check the medicament of (antagonist) human NIP 2 associated protein.Agonist improves human NIP 2 associated protein 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 human NIP 2 associated protein be cultivated with the human NIP 2 associated protein of mark.Measure the medicine raising then or check this interactional ability.

The antagonist of human NIP 2 associated protein comprises antibody, compound, acceptor disappearance thing and the analogue etc. that filter out.The antagonist of human NIP 2 associated protein can combine and eliminate its function with human NIP 2 associated protein, or suppresses the generation of human NIP 2 associated protein, or combines with the avtive spot of polypeptide and to make polypeptide can not bring into play biological function.The antagonist of human NIP 2 associated protein can be used for therepic use.

In screening during as the compound of antagonist, human NIP 2 associated protein can be added during bioanalysis measures, determine by the interaction of measuring between compounds affect human NIP 2 associated protein and its acceptor 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.

Polypeptide of the present invention can be directly used in disease treatment, for example, and various malignant tumours and cellular abnormality propagation etc.

Polypeptide of the present invention, and fragment, derivative, analogue or their cell can be used as antigen to produce antibody.These antibody can be polyclone or monoclonal antibody.Polyclonal antibody can obtain by the method with this polypeptide direct injection animal.The technology of preparation monoclonal antibody comprises hybridoma technology, three knurl technology, people B-quadroma technology, EBV-hybridoma technology etc.

Can be with polypeptide of the present invention and antagonist and suitable pharmaceutical carrier combination back use.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 NIP 2 associated protein 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 NIP 2 associated protein will depend on many factors, as administering mode, person's to be treated healthiness condition and diagnostician's judgement.

The polynucleotide of human NIP 2 associated protein 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 expression of the human NIP 2 associated protein of the nothing expression of human NIP 2 associated protein or unusual/non-activity.The human NIP 2 associated protein that the gene therapy vector (as virus vector) of reorganization can be designed to express variation is to suppress endogenic human NIP 2 associated protein activity.For example, a kind of human NIP 2 associated protein of variation can be the human NIP 2 associated protein 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 NIP 2 associated protein 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 human NIP 2 associated protein transgenosis to cell.The method that makes up the recombinant viral vector of carrier NIP2 related protein gene is found in existing document (Sambrook, et al.).Recombinant human NIP2 related protein gene can be packaged in the liposome and be transferred in the cell in addition.

Suppress the oligonucleotide (comprising sense-rna and DNA) of human NIP 2 associated protein 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.

Polynucleotide imports 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.

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 carry out the two-dimentional or three-dimensional gel electrophoresis analysis of one dimension.

The present invention also provides the antibody at the human NIP 2 associated protein 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 antibody of anti-human NIP 2 associated protein can be used in the immunohistochemistry technology, detects the human NIP 2 associated protein in the biopsy specimen.

With the also available labelled with radioisotope of human NIP 2 associated protein 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.

The disease that antibody among the present invention can be used for treating or prevention is relevant with human NIP 2 associated protein.The antibody that gives suitable dosage can stimulate or block the generation or the activity of human NIP 2 associated protein.

Antibody also can be used for designing the immunotoxin at a certain privileged sites in the body.As the monoclonal antibody of human NIP 2 associated protein 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 NIP 2 associated protein positive cells.

Available human NIP 2 associated protein of the production of polyclonal antibody or polypeptide immune animal, as rabbit, mouse, rat etc.Multiple adjuvant can be used for the enhancing immunity reaction, includes but not limited to freund's adjuvant etc.

The human NIP 2 associated protein monoclonal antibody can with hybridoma technology production (Kohler and Milstein.Nature, 1975,256:495-497).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 NIP 2 associated protein.

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 NIP 2 associated protein bonded peptide molecule obtains.During screening, must carry out mark to the human NIP 2 associated protein molecule.

The invention still further relates to the diagnostic testing process of quantitative and detection and localization human NIP 2 associated protein level.These tests are known in the art, and comprise that FISH measures and radioimmunoassay.The human NIP 2 associated protein level that is detected in the test can be with laying down a definition the importance of human NIP 2 associated protein in various diseases and be used to the disease of diagnosing human NIP 2 associated protein to work.

The polynucleotide of human NIP 2 associated protein can be used for the diagnosis and the treatment of human NIP 2 associated protein relative disease.Aspect diagnosis, the unconventionality expression of the expression that the polynucleotide of human NIP 2 associated protein can be used for detecting human NIP 2 associated protein human NIP 2 associated protein whether or under morbid state.Can be used for the hybridization of biopsy specimen to judge the abnormal expression of human NIP 2 associated protein as the human NIP 2 associated protein dna sequence dna.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 NIP 2 associated protein with the special primer of human NIP 2 associated protein.

The sudden change that detects the human NIP 2 associated protein gene also can be used for the disease of diagnosing human NIP 2 associated protein relevant.The form of human NIP 2 associated protein sudden change comprises that the point mutation compared with normal wild type human NIP 2 associated protein 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 Inheritance inMan (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.

Human NIP 2 associated protein Nucleotide full length sequence of the present invention or its fragment can obtain with the method for pcr amplification method, recombination method or synthetic usually.For the pcr amplification method, can be disclosed according to the present invention about nucleotide sequence, especially open reading frame sequence designs primer, and with commercially available cDNA storehouse or by the prepared cDNA storehouse of ordinary method well known by persons skilled in the art as template, amplification and must relevant sequence.When sequence is longer, usually needs to carry out twice or pcr amplification repeatedly, and then the fragment that each time amplifies is stitched together by proper order.

In case obtained relevant sequence, just can obtain relevant sequence in large quantity with recombination method.This normally is cloned into carrier with it, changes cell again over to, separates obtaining relevant sequence then from the host cell after the propagation by ordinary method.

In addition, also the method for available synthetic is synthesized relevant sequence, especially fragment length more in short-term.Usually, by first synthetic a plurality of small segments, and then connect and to obtain the very long fragment of sequence.

At present, can be fully come the dna sequence dna of code book invention albumen (or its fragment, or derivatives thereof) by chemosynthesis.This dna sequence dna can be introduced then in the various dna moleculars (as carrier) and cell in this area.In addition, also can will suddenly change and introduce in the protein sequence of the present invention by chemosynthesis.

In one embodiment of the invention, from the human placenta cDNA library, by to the external DNA transfections screening of liver cancer cell 7721, separate, and by 5 '-RACE and full clone technology obtain the full length cDNA clone of NIP2 associated protein.In addition, Northern is hybridized proof, and NIP2 related protein gene of the present invention has expression in the heart, brain, placenta, lung, liver, muscle, pancreas, do not have to express in the kidney or a little less than.

In view of NIP2 AP has the effect of anticancer growth, so it not only has the regulating cell division and promotes the effect of cancer cell-apoptosis, and has the using value to treating malignant tumor and diagnosis.In addition, because human NIP 2 associated protein of the present invention has the natural acid sequence that is derived from the people, therefore compare with the albumen of the same clan that derives from other species, estimate to have higher active and/or lower side effect (for example in the intravital immunogenicity of people lower or do not have) being applied to man-hour.

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

NIP 2 AP cDNA clone's separation reaches the growth-inhibiting to human liver cancer cell

Get the placenta tissue at 3,6,10 monthly ages, (GIBCO BRL company) extracts total RNA by manufacturer's specification sheets with Trizol reagent, extracts mRNA with the mRNA test kit (Pharmacia company) of purifying.Make up the cDNA library of above-mentioned mRNA with pCMV-script TMXRcDNA library construction test kit (Stratagene company).Wherein ThermoScript II is used MMLV-RT-Superscript II (GIBCO BRL) instead, and reverse transcription reaction carries out at 42 ℃.Transform XL 10-Gold competent cell, obtained 1 * 10 ⁶The cDNA library of cfu/ μ g cDNA titre.The first round is picking cDNA clone at random, is probe with high abundance cDNA clone with the cDNA clone who has proved cancer inhibitor cell growth function thereafter, screening by hybridization cDNA library, weak positive and negative clone of picking.With Qiagen 96 orifice plate plasmid extraction test kits, carry out the extraction of plasmid DNA by shop instruction.Plasmid DNA and empty carrier transfection simultaneously hepatoma cell line 7721.After the 100ngDNA alcohol precipitation drying, add 6 μ l H ₂Transfection is treated in the O dissolving.Add 0.74 μ l liposome and 9.3 μ l serum-free mediums in every part of DNA sample, behind the mixing, room temperature was placed 10 minutes.Add 150 μ l serum-free mediums in every pipe, divide equally and add 3 holes and grow in 7721 cells of 96 orifice plates, placed 2 hours for 37 ℃, every hole adds 50 μ l serum-free mediums again, 37 ℃ 24 hours.Every hole is changed 100 μ l and is trained liquid entirely, 37 ℃ 24 hours, change the full training liquid 100 μ l that contain G418,37 ℃ 24～48 hours, the limit is observed, the training liquid that G418 concentration does not wait is changed on the limit.After about 2～3 times, there is the clone to form up to the microscopy cell, counting.Found that cDNA clone PP753 has the function (it is 50,26,31 that the empty carrier clone forms number, and clone PP753 is 0,0,0) that suppresses the clone and form

To finding after the analysis of cDNA cloned sequence that gene is still imperfect, adopt the Clontech SMART RACEcDNA of company amplification kit (Cat.No.K1811-1), gene specific primer is 5 ' ATTCAGCCTTCCGTCCCAGCAACTG 3 ' and 5 ' CCCAGCAGATCAGTTTCCCAGCCTC 3 ', by specification is operated, obtain full-length clone, called after NIP 2 AP.

The sequential analysis of embodiment 2:NIP2 AP cDNA

The NIP2 AP cDNA total length that embodiment 1 obtains is 2360 Nucleotide, contains the individual Nucleotide of reading frame 725 (618-1445 position), the albumen of being made up of 275 amino acid (not comprising terminator codon) of encoding.

A: nucleotide sequence: (SEQ ID NO:1) length: 2360bp

1 ACGCGGGGGA GCTACAACAG CTGAGACAGA AAAGAGGTAA GGAAGTGTTG

51 GGGGCTGGGA CAACCAGCTC CCCGACAACT CCTAGGTGTT TAAAGAAGGA

101 GGCAGGAAGA CTTGTGAAGA TGGGAACTAT ACAAGAGGCA GGAAAAAAGA

151 CAGATGTTGG GTGGAGCAAC TGCTACAGAG GTAAATATGA TTAACTTTAC

201 ATTCCATCTT TCGTCTGCTC CCAAACTTAA CAGCAGGTAA TCTGCTTCTA

251 GCAAGTGGTG AAGGTAAGAG AAGCATCTGT ATAGGAGGCA AGAGATCTGA

301 GTCCTTTTGA AGGCCTATCC TCTGCTCTGT ATCTCAATTA CTGTTCTTCA

351 TTTCAATTAT TCTTACCTAC TATTCAGTTC CCTTGATCTT TTCTTCTTGG

401 GGGCTGTCTT AGGGTCAGGG AGATTGCAGA AGCACCAGAA CTAGGAGCAG

451 CCCTGAGACA TGGGGAGTTG GAGCTGAAGG AGGAATGGCA GGATGAAGAA

501 TTCCCTAGAT TGCTTCCTGA GGAGGCTGGC ACTTCTGAAG ATCCTGAAGA

551 CCCTAAAGGA GATTCACAGG CAGCTGCAGG TACCCCCAGC ACTTTAGCCC

601 TGTGTGGCCA GCGCCCCATG CGCAAGCGTC TTTCTGCCCC AGAGTTGCGG

651 CTGAGTCTGA CTAAGGGGCC TGGAAATGAT GGAGCTTCAC CCACCCAGTC

701 TGCACCTTCC TCTCCTGATG GCAGTTCTGA CCTGGAGATA GACGAATTGG

751 AGACACCTTC AGACTCGGAG CAGCTGGACA GTGGACATGA ATTTGAATGG

801 GAAGATGAAC TACCCCGGGC AGAGGGTCTG GGCACCAGTG AGACAGCTGA

851 AAGGCTGGGC CGAGGTTGTA TGTGGGATGT GACTGGAGAA GATGGACATC

901 ACTGGAGGGT GTTCCGAATG GGACCACGGG AGCAGCGCGT AGACATGACT

951 GTCATTGAGC CCTATAAGAA AGTCCTGTCT CATGGAGGTT ACCACGGTGA

1001 TGGCCTCAAT GCTGTCATCC TTTTTGCTTC CTGTTATCTA CCCAGAAGCA

1051 GCATCCCCAA CTACACCTAT GTCATGGAAC ACTTGTTTAG GTATATGGTG

1101 GGAACTCTGG AGCTGCTAGT AGCTGAAAAT TACCTGCTTG TTCATTTGAG

1151 TGGAGGCACA AGCAGGGCCC AAGTTCCACC TCTAAGCTGG ATACGTCAGT

1201 GTTACCGTAC CCTGGATCGG CGGCTACGGA AAAACCTGCG AGCCCTGGTG

1251 GTTGTCCATG CTACATGGTA TGTGAAAGCA TTTCTGGCAC TGCTTCGGCC

1301 CTTCATCAGT TCCAAATTCA CACGAAAAAT CCGTTTTCTG GACAGCCTGG

1351 GGGAGCTGGC CCAACTCATA TCCCTGGATC AAGTCCACAT CCCTGAAGCT

1401 GTCAGACAGC TGGACCGGGA TCTCCATGGC TCAGGAGGGA CATAGCACAG

1451 GACTGGATAA AGGGCCTTAG AACCAGTTAG TGATCTGCCT ACACCTGAAT

1501 CCCTGAAACA TCTGAACTGT TTTGTAAATC ATCTTATCCC CAACCTCAGT

1551 ACCACCGGAT CTTCACTTCT CAGTGGGATT TTGTCCTTTG CATGACCCTA

1601 CTTTCAGCAG GGCTTTGAGG CTGGGAAACT GATCTGCTGG GTGACTTTCA

1651 TTCCAGTTGC TGGGACGGAA GGCTGAATGT AGGGTCATTT TGTATGGGAT

1701 ATGCAGAGCT CTGGGTTTTT TTGTTTTTTG TTTTTTGAGA CGGAGTCTCG

1751 CTCTGTCGCC AGGCTGGAGT GCAGTGGTGC GATCTCGGCT CACTGCAACC

1801 TCCGCCTCCC GGTTCAAGCC ATTCTTCTGC CTCAGCCTCA GCGAGTAGCT

1851 GAGACTACCG GCGCACGCCA CCACGCCCAG CTAATTGTTT TGTATTTTTA

1901 GTAGAGACGG GGTTTCACCA TGTTGGCCAG GATGGTCTCG ATCTCTTGAC

1951 CTCGTGATCT GCCCGCCTCG GCCTCCCAAA GTGCCGAGAT TACAGGTGTG

2001 AGCCACCGCG CTCGGCCAGA GCTCTGGGTT TTAATCCTAC TTTAGCTGTA

2051 GAACCTTGGG CAAATAACTT CATCTTTTTG GGTCCTAATT TTCCTCCTGT

2101 CTAACTGGAG GGACTGTGAT CCTTCCACCT TGGATGGAAG AGGCTTACCT

2151 GACAGCCAGC CTGCCTGCTG GGAGCCAGGA GAGTCAGCTC ATTAAATCTT

2201 GAAGAACCGC TGTATGCCCT TTTTCCTTCT AAGTCATGTC TGCTGCCTGT

2251 GAGCCTGGGA AGGAGTGCTT TCAAAACCTG TATTTTTGCC CTCTTTAGTA

2301 AATTTAGAAC TGTAGAGGCT AATAAACTGC GATGAGACAT TTAAAAAAAA

2351 AAAAAAAAAA

B: aminoacid sequence: (SEQ ID NO:2) length: 275 amino acid

1 MRKRLSAPEL RLSLTKGPGN DGASPTQSAP SSPDGSSDLE IDELETPSDS

51 EQLDSGHEFE WEDELPRAEG LGTSETAERL GRGCMWDVTG EDGHHWRVFR

101 MGPREQRVDM TVIEPYKKVL SHGGYHGDGL NAVILFASCY LPRSSIPNYT

151 YVMEHLFRYM VGTLELLVAE NYLLVHLSGG TSRAQVPPLS WIRQCYRTLD

201 RRLRKNLRAL VVVHATWYVK AFLALLRPFI SSKFTRKIRF LDSLGELAQL

251 ISLDQVHIPE AVRQLDRDLH GSGGT

C: composite sequence: NIP 2 AP (SEQ ID NO:3)

Start code: 618 ATG stop coding: 1445 TAG

Protein molecular weight: 30972.46

1 AC GCG GGG GAG CTA CAA CAG CTG AGA CAG AAA AGA GGT AAG GAA GTG 47

48 TTG GGG GCT GGG ACA ACC AGC TCC CCG ACA ACT CCT AGG TGT TTA AAG 95

96 AAG GAG GCA GGA AGA CTT GTG AAG ATG GGA ACT ATA CAA GAG GCA GGA 143

144 AAA AAG ACA GAT GTT GGG TGG AGC AAC TGC TAC AGA GGT AAA TAT GAT 191

192 TAA CTT TAC ATT CCA TCT TTC GTC TGC TCC CAA ACT TAA CAG CAG GTA 239

240 ATC TGC TTC TAG CAA GTG GTG AAG GTA AGA GAA GCA TCT GTA TAG GAG 287

288 GCA AGA GAT CTG AGT CCT TTT GAA GGC CTA TCC TCT GCT CTG TAT CTC 335

336 AAT TAC TGT TCT TCA TTT CAA TTA TTC TTA CCT ACT ATT CAG TTC CCT 383

384 TGA TCT TTT CTT CTT GGG GGC TGT CTT AGG GTC AGG GAG ATT GCA GAA 431

432 GCA CCA GAA CTA GGA GCA GCC CTG AGA CAT GGG GAG TTG GAG CTG AAG 479

480 GAG GAA TGG CAG GAT GAA GAA TTC CCT AGA TTG CTT CCT GAG GAG GCT 527

528 GGC ACT TCT GAA GAT CCT GAA GAC CCT AAA GGA GAT TCA CAG GCA GCT 575

576 GCA GGT ACC CCC AGC ACT TTA GCC CTG TGT GGC CAG CGC CCC ATG CGC 623

1 Met Arg 2

624 AAG CGT CTT TCT GCC CCA GAG TTG CGG CTG AGT CTG ACT AAG GGG CCT 671

3 Lys Arg Leu Ser Ala Pro Glu Leu Arg Leu Ser Leu Thr Lys Gly Pro 18

672 GGA AAT GAT GGA GCT TCA CCC ACC CAG TCT GCA CCT TCC TCT CCT GAT 719

19 Gly Asn Asp Gly Ala Ser Pro Thr Gln Ser Ala Pro Ser Ser Pro Asp 34

720 GGC AGT TCT GAC CTG GAG ATA GAC GAA TTG GAG ACA CCT TCA GAC TCG 767

35 Gly Ser Ser Asp Leu Glu Ile Asp Glu Leu Glu Thr Pro Ser Asp Ser 50

768 GAG CAG CTG GAC AGT GGA CAT GAA TTT GAA TGG GAA GAT GAA CTA CCC 815

51 Glu Gln Leu Asp Ser Gly His Glu Phe Glu Trp Glu Asp Glu Leu Pro 66

816 CGG GCA GAG GGT CTG GGC ACC AGT GAG ACA GCT GAA AGG CTG GGC CGA 863

67 Arg Ala Glu Gly Leu Gly Thr Ser Glu Thr Ala Glu Arg Leu Gly Arg 82

864 GGT TGT ATG TGG GAT GTG ACT GGA GAA GAT GGA CAT CAC TGG AGG GTG 911

83 Gly Cys Met Trp Asp Val Thr Gly Glu Asp Gly His His Trp Arg Val 98

912 TTC CGA ATG GGA CCA CGG GAG CAG CGC GTA GAC ATG ACT GTC ATT GAG 959

99 Phe Arg Met Gly Pro Arg Glu Gln Arg Val Asp Met Thr Val Ile Glu 114

960 CCC TAT AAG AAA GTC CTG TCT CAT GGA GGT TAC CAC GGT GAT GGC CTC 1007

115 Pro Tyr Lys Lys Val Leu Ser His Gly Gly Tyr His Gly Asp Gly Leu 130

1008 AAT GCT GTC ATC CTT TTT GCT TCC TGT TAT CTA CCC AGA AGC AGC ATC 1055

131 Asn Ala Val Ile Leu Phe Ala Ser Cys Tyr Leu Pro Arg Ser Ser Ile 146

1056 CCC AAC TAC ACC TAT GTC ATG GAA CAC TTG TTT AGG TAT ATG GTG GGA 1103

147 Pro Asn Tyr Thr Tyr Val Met Glu His Leu Phe Arg Tyr Met Val Gly 162

1104 ACT CTG GAG CTG CTA GTA GCT GAA AAT TAC CTG CTT GTT CAT TTG AGT 1151

163 Thr Leu Glu Leu Leu Val Ala Glu Asn Tyr Leu Leu Val His Leu Ser 178

1152 GGA GGC ACA AGC AGG GCC CAA GTT CCA CCT CTA AGC TGG ATA CGT CAG 1199

179 Gly Gly Thr Ser Arg Ala Gln Val Pro Pro Leu Ser Trp Ile Arg Gln 194

1200 TGT TAC CGT ACC CTG GAT CGG CGG CTA CGG AAA AAC CTG CGA GCC CTG 1247

195 Cys Tyr Arg Thr Leu Asp Arg Arg Leu Arg Lys Asn Leu Arg Ala Leu 210

1248 GTG GTT GTC CAT GCT ACA TGG TAT GTG AAA GCA TTT CTG GCA CTG CTT 1295

211 Val Val Val His Ala Thr Trp Tyr Val Lys Ala Phe Leu Ala Leu Leu 226

1296 CGG CCC TTC ATC AGT TCC AAA TTC ACA CGA AAA ATC CGT TTT CTG GAC 1343

227 Arg Pro Phe Ile Ser Ser Lys Phe Thr Arg Lys Ile Arg Phe Leu Asp 242

1344 AGC CTG GGG GAG CTG GCC CAA CTC ATA TCC CTG GAT CAA GTC CAC ATC 1391

243 Ser Leu Gly Glu Leu Ala Gln Leu Ile Ser Leu Asp Gln Val His Ile 258

1392 CCT GAA GCT GTC AGA CAG CTG GAC CGG GAT GTC CAT GGC TCA GGA GGG 1439

259 Pro Glu Ala Val Arg Gln Leu Asp Arg Asp Leu His Gly Ser Gly Gly 274

1440 ACA TAG CAC AGG ACT GGA TAA AGG GCC TTA GAA CCA GTT AGT GAT CTG 1487

275 Thr *** 276

1488 CCT ACA CCT GAA TCC CTG AAA CAT CTG AAC TGT TTT GTA AAT CAT CTT 1535

1536 ATC CCC AAC CTC AGT ACC ACC GGA TCT TCA CTT CTC AGT GGG ATT TTG 1583

1584 TCC TTT GCA TGA CCC TAC TTT CAG CAG GGC TTT GAG GCT GGG AAA CTG 1631

1632 ATC TGC TGG GTG ACT TTC ATT CCA GTT GCT GGG ACG GAA GGC TGA ATG 1679

1680 TAG GGT CAT TTT GTA TGG GAT ATG CAG AGC TCT GGG TTT TTT TGT TTT 1727

1728 TTG TTT TTT GAG ACG GAG TCT CGC TCT GTC GCC AGG CTG GAG TGC AGT 1775

1776 GGT GCG ATC TCG GCT CAC TGC AAC CTC CGC CTC CCG GTT CAA GCC ATT 1823

1824 CTT CTG CCT CAG CCT CAG CGA GTA GCT GAG ACT ACC GGC GCA CGC CAC 1871

1872 CAC GCC CAG CTA ATT GTT TTG TAT TTT TAG TAG AGA CGG GGT TTC ACC 1919

1920 ATG TTG GCC AGG ATG GTC TCG ATC TCT TGA CCT CGT GAT CTG CCC GCC 1967

1968 TCG GCC TCC CAA AGT GCC GAG ATT ACA GGT GTG AGC CAC CGC GCT CGG 2015

2016 CCA GAG CTC TGG GTT TTA ATC CTA CTT TAG CTG TAG AAC CTT GGG CAA 2063

2064 ATA ACT TCA TCT TTT TGG GTC CTA ATT TTC CTC CTG TCT AAC TGG AGG 2111

2112 GAC TGT GAT CCT TCC ACC TTG GAT GGA AGA GGC TTA CCT GAC AGC CAG 2159

2160 CCT GCC TGC TGG GAG CCA GGA GAG TCA GCT CAT TAA ATC TTG AAG AAC 2207

2208 CGC TGT ATG CCC TTT TTC CTT CTA AGT CAT GTC TGC TGC CTG TGA GCC 2255

2256 TGG GAA GGA GTG CTT TCA AAA CCT GTA TTT TTG CCC TCT TTA GTA AAT 2303

2304 TTA GAA CTG TAG AGG CTA ATA AAC TGC GAT GAG ACA TTT AAA AAA AAA 2351

2352 AAA AAA AAA 2360

D. homology relatively

NIP2 AP cDNA clone of the present invention contains full length cDNA sequence.

(1) the nucleotide sequence homology relatively

There is not homology.

(2) comparison of aminoacid sequence and genes involved

Query=PP753 (275 amino acid)

>gi|6093506|sp|Q12982|NIP2_HUMAN BCL2/ADENOVIRUS E1B 19-KD

PROTEIN-INTERACTING PROTEIN 2

Length=314

Score value=287bits (726), predicated value=3e-82

Homogeny=131/275 (47%), similarity=204/275 (73%), breach=22/275 (8%)

Query：1 MRKRLSAPELRLSLTKGPGNDGASPTQSAPSSPDGSSDLEIDELETPSDSEQLDSGHEFE 60

+RK+L AP++ L+L G+ D S ++++D L+TPS++ +EFE

Sbjct：49 VRKKLMAPDISLTLDPSDGS-------VLSDDLDESGEIDLDGLDTPSENS-----NEFE 96

Query：61 WEDELPRAEGLGTSETAERLGRGCMWDVTG----EDGHHWRVFRMGPREQRVDMTVIEPY 116

WED+LP+ + T E + +G + + T EDG WR+FR+G ++ RVDM IEPY

Sbjct：97 WEDDLPKPK------TTEVIRKGSITEYTAAEEKEDGRRWRMFRIGEQDHRVDMKAIEPY 150

Query：117 KKVLSHGGYHGDGLNAVILFASCYLPRSSIPNYTYVMEHLFRYMVGTLELLVAENYLLVH 176

KKV+SHGGY+GDGLNA+++FA C++P SS PNY Y+M++LF+Y++GTLELLVAENY++V+

Sbjct：151 KKVISHGGYYGDGLNAIVVFAVCFMPESSQPNYRYLMDNLFKYVIGTLELLVAENYMIVY 210

Query：177 LSGGTSRAQVPPLSWIRQCYRTLDRRLRKNLRALVVVHATWYVKAFLALLRPFISSKFTR 236

L+G T+R ++P L W+R+CY+ +DRRLRKNL++L++VH +W+++ LA+ RPFISSKF++

Sbjct：211 LNGATTRRKMPSLGWLRKCYQQIDRRLRKNLKSLIIVHPSWFIRTLLAVTRPFISSKFSQ 270

Query：237 KIRFLDSLGELAQLISLDQVHIPEAVRQLDRDLHG 271

KIR++ +L ELA+L+ ++ V IPE ++Q+D++L+G

Sbjct：271 KIRYVFNLAELAELVPMEYVGIPECIKQVDQELNG 305

E: the motif analysis of aminoacid sequence

The proteic aminoacid sequence of NIP2 AP is carried out the motif analysis, find that the 126..269 position is: Nip21Nip2 W02b12.8 GTP enzyme activation Rho breach, the relevant Small GTPases region of activation (Protein Nip21Nip2 W02b12.8 GTPase activating Rho gap Rho-related Small GTPase Activator) of Rho

1..132 is: NIP2L NIP2 O54940 (1), Q12982 (1)

Embodiment 3:NIP 2 AP are the mRNA express spectra in people's tissue

With NIP 2 AP genes is probe, hybridizes in multiple tissue mRNA diaphragm (Clontech).The result as shown in Figure 1.The result shows that NIP 2 AP have expression in the heart, brain, placenta, lung, liver, muscle, pancreas, do not have to express in the kidney or a little less than.

Embodiment 4:NIP 2 AP are in the expression of external translating system

(1) with NIP 2 AP gene clones in the expression vector that contains the T7 promotor

The a pair of special primer of design NIP 2 AP gene open reading frames (ORF); 5 ' end primer contains NCOI enzyme point of contact, and 3 ' end primer contains HindIII enzyme point of contact, is template with NIP 2 AP cloned plasmids or RT-PCT amplified production; pcr amplified fragment, product is cut through NcoI and HindIII enzyme.The used expression vector of present embodiment is p22450 (this carrier is Chinese Academy of Sciences's Shanghai biotechnology center construction), contains the T7 promotor.Carrier is also cut through NcoI and HindIII enzyme, is connected with the PCR endonuclease bamhi, sets up expression plasmid pT7X-NIP 2 AP.

(2) external translation

(Promega Cat#L1130), and utilizes Transcend to adopt E.coli T7 S30 Extract System for Circular DNA ^TMColorimetric Translation Detection System (Promema, Cat.#L5070), vivoexpression INP 2 AP.

1. set up external translation reaction system:

Plasmid DNA: 1-4 μ g

Complete amino acid tRNA mixed solution: 5 μ l

There is not amino acid whose S30 premix: 20 μ l

Transcend ^TM tRNA ^*： 1μl

T7 S30 cell pyrolysis liquid: 15 μ l

The nuclease free sterilized water adds to final concentration: 50 μ l

^*Transcend ^TMTRNA is that ε-NH2 is by biotin labeled Lys-tRNA.

37 ℃ were reacted 1-2 hour.

2. the analysis of external translation product:

The SDS-PAGE electrophoretic analysis: 15% separation gel, 4% concentrates glue.

Electrotransfer is to PVDF:100V, 60 minutes.

Utilize Streptavidin-Alkaline Phosphatase (streptavidin-alkaline phosphatase lipase) in conjunction with the translation albumen that contains vitamin H, and by Western Blue Stablized Substrate for AlkalinePhosphatase (alkaline phosphatase lipase immobilization staining agent) colour developing.

The result shows that positive control sample CA7 fusion protein expression plasmid has protein band at the 39KD place, and NIP 2AP expression plasmid pT7X-NIP 2 AP have protein band at the 41KD place, shows that NIP 2 AP have obtained expression (Fig. 2) in the vivoexpression system.

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

Sequence table

(1) general information:

(i) applicant: Shanghai Inst. of Tumor

(ii) denomination of invention: human NIP 2 associated protein and encoding sequence and uses thereof

(iii) sequence number: 3

(2) information of SEQ ID NO.1

(i) sequence signature:

(A) length: 2360bp

(B) type: Nucleotide

(C) chain: two strands

(D) topological framework: linearity

(ii) molecule type: Nucleotide

(ix) sequence description: SEQ ID NO.1:

ACGCGGGGGA GCTACAACAG CTGAGACAGA AAAGAGGTAA GGAAGTGTTG GGGGCTGGGA 60

CAACCAGCTC CCCGACAACT CCTAGGTGTT TAAAGAAGGA GGCAGGAAGA CTTGTGAAGA 120

TGGGAACTAT ACAAGAGGCA GGAAAAAAGA CAGATGTTGG GTGGAGCAAC TGCTACAGAG 180

GTAAATATGA TTAACTTTAC ATTCCATCTT TCGTCTGCTC CCAAACTTAA CAGCAGGTAA 240

TCTGCTTCTA GCAAGTGGTG AAGGTAAGAG AAGCATCTGT ATAGGAGGCA AGAGATCTGA 300

GTCCTTTTGA AGGCCTATCC TCTGCTCTGT ATCTCAATTA CTGTTCTTCA TTTCAATTAT 360

TCTTACCTAC TATTCAGTTC CCTTGATCTT TTCTTCTTGG GGGCTGTCTT AGGGTCAGGG 420

AGATTGCAGA AGCACCAGAA CTAGGAGCAG CCCTGAGACA TGGGGAGTTG GAGCTGAAGG 480

AGGAATGGCA GGATGAAGAA TTCCCTAGAT TGCTTCCTGA GGAGGCTGGC ACTTCTGAAG 540

ATCCTGAAGA CCCTAAAGGA GATTCACAGG CAGCTGCAGG TACCCCCAGC ACTTTAGCCC 600

TGTGTGGCCA GCGCCCCATG CGCAAGCGTC TTTCTGCCCC AGAGTTGCGG CTGAGTCTGA 660

CTAAGGGGCC TGGAAATGAT GGAGCTTCAC CCACCCAGTC TGCACCTTCC TCTCCTGATG 720

GCAGTTCTGA CCTGGAGATA GACGAATTGG AGACACCTTC AGACTCGGAG CAGCTGGACA 780

GTGGACATGA ATTTGAATGG GAAGATGAAC TACCCCGGGC AGAGGGTCTG GGCACCAGTG 840

AGACAGCTGA AAGGCTGGGC CGAGGTTGTA TGTGGGATGT GACTGGAGAA GATGGACATC 900

ACTGGAGGGT GTTCCGAATG GGACCACGGG AGCAGCGCGT AGACATGACT GTCATTGAGC 960

CCTATAAGAA AGTCCTGTCT CATGGAGGTT ACCACGGTGA TGGCCTCAAT GCTGTCATCC 1020

TTTTTGCTTC CTGTTATCTA CCCAGAAGCA GCATCCCCAA CTACACCTAT GTCATGGAAC 1080

ACTTGTTTAG GTATATGGTG GGAACTCTGG AGCTGCTAGT AGCTGAAAAT TACCTGCTTG 1140

TTCATTTGAG TGGAGGCACA AGCAGGGCCC AAGTTCCACC TCTAAGCTGG ATACGTCAGT 1200

GTTACCGTAC CCTGGATCGG CGGCTACGGA AAAACCTGCG AGCCCTGGTG GTTGTCCATG 1260

CTACATGGTA TGTGAAAGCA TTTCTGGCAC TGCTTCGGCC CTTCATCAGT TCCAAATTCA 1320

CACGAAAAAT CCGTTTTCTG GACAGCCTGG GGGAGCTGGC CCAACTCATA TCCCTGGATC 1380

AAGTCCACAT CCCTGAAGCT GTCAGACAGC TGGACCGGGA TCTCCATGGC TCAGGAGGGA 1440

CATAGCACAG GACTGGATAA AGGGCCTTAG AACCAGTTAG TGATCTGCCT ACACCTGAAT 1500

CCCTGAAACA TCTGAACTGT TTTGTAAATC ATCTTATCCC CAACCTCAGT ACCACCGGAT 1560

CTTCACTTCT CAGTGGGATT TTGTCCTTTG CATGACCCTA CTTTCAGCAG GGCTTTGAGG 1620

CTGGGAAACT GATCTGCTGG GTGACTTTCA TTCCAGTTGC TGGGACGGAA GGCTGAATGT 1680

AGGGTCATTT TGTATGGGAT ATGCAGAGCT CTGGGTTTTT TTGTTTTTTG TTTTTTGAGA 1740

CGGAGTCTCG CTCTGTCGCC AGGCTGGAGT GCAGTGGTGC GATCTCGGCT CACTGCAACC 1800

TCCGCCTCCC GGTTCAAGCC ATTCTTCTGC CTCAGCCTCA GCGAGTAGCT GAGACTACCG 1860

GCGCACGCCA CCACGCCCAG CTAATTGTTT TGTATTTTTA GTAGAGACGG GGTTTCACCA 1920

TGTTGGCCAG GATGGTCTCG ATCTCTTGAC CTCGTGATCT GCCCGCCTCG GCCTCCCAAA 1980

GTGCCGAGAT TACAGGTGTG AGCCACCGCG CTCGGCCAGA GCTCTGGGTT TTAATCCTAC 2040

TTTAGCTGTA GAACCTTGGG CAAATAACTT CATCTTTTTG GGTCCTAATT TTCCTCCTGT 2100

CTAACTGGAG GGACTGTGAT CCTTCCACCT TGGATGGAAG AGGCTTACCT GACAGCCAGC 2160

CTGCCTGCTG GGAGCCAGGA GAGTCAGCTC ATTAAATCTT GAAGAACCGC TGTATGCCCT 2220

TTTTCCTTCT AAGTCATGTC TGCTGCCTGT GAGCCTGGGA AGGAGTGCTT TCAAAACCTG 2280

TATTTTTGCC CTCTTTAGTA AATTTAGAAC TGTAGAGGCT AATAAACTGC GATGAGACAT 2340

TTAAAAAAAA AAAAAAAAAA 2360

(2) information of SEQ ID NO.2

(i) sequence signature:

(A) length: 275 amino acid

(B) type: amino acid

(C) chain: strand

(D) topological framework: linearity

(ii) molecule type: polypeptide

(ix) sequence description: SEQ ID NO.2:

MRKRLSAPEL RLSLTKGPGN DGASPTQSAP SSPDGSSDLE IDELETPSDS 50

EQLDSGHEFE WEDELPRAEG LGTSETAERL GRGCMWDVTG EDGHHWRVFR 100

MGPREQRVDM TVIEPYKKVL SHGGYHGDGL NAVILFASCY LPRSSIPNYT 150

YVMEHLFRYM VGTLELLVAE NYLLVHLSGG TSRAQVPPLS WIRQCYRTLD 200

RRLRKNLRAL VVVHATWYVK AFLALLRPFI SSKFTRKIRF LDSLGELAQL 250

ISLDQVHIPE AVRQLDRDLH GSGGT 275

(2) information of SEQ ID NO.3

(i) sequence signature:

(A) length: 2360bp

(B) type: Nucleotide

(C) chain: two strands

(D) topological framework: linearity

(ii) molecule type: Nucleotide

(ix) sequence description: SEQ ID NO.3:

1 AC GCG GGG GAG CTA CAA CAG CTG AGA CAG AAA AGA GGT AAG GAA GTG 47

48 TTG GGG GCT GGG ACA ACC AGC TCC CCG ACA ACT CCT AGG TGT TTA AAG 95

96 AAG GAG GCA GGA AGA CTT GTG AAG ATG GGA ACT ATA CAA GAG GCA GGA 143

144 AAA AAG ACA GAT GTT GGG TGG AGC AAC TGC TAC AGA GGT AAA TAT GAT 191

192 TAA CTT TAC ATT CCA TCT TTC GTC TGC TCC CAA ACT TAA CAG CAG GTA 239

240 ATC TGC TTC TAG CAA GTG GTG AAG GTA AGA GAA GCA TCT GTA TAG GAG 287

288 GCA AGA GAT CTG AGT CCT TTT GAA GGC CTA TCC TCT GCT CTG TAT CTC 335

336 AAT TAC TGT TCT TCA TTT CAA TTA TTC TTA CCT ACT ATT CAG TTC CCT 383

384 TGA TCT TTT CTT CTT GGG GGC TGT CTT AGG GTC AGG GAG ATT GCA GAA 431

432 GCA CCA GAA CTA GGA GCA GCC CTG AGA CAT GGG GAG TTG GAG CTG AAG 479

480 GAG GAA TGG CAG GAT GAA GAA TTC CCT AGA TTG CTT CCT GAG GAG GCT 527

528 GGC ACT TCT GAA GAT CCT GAA GAC CCT AAA GGA GAT TCA CAG GCA GCT 575

576 GCA GGT ACC CCC AGC ACT TTA GCC CTG TGT GGC CAG CGC CCC ATG CGC 623

1 Met Arg 2

624 AAG CGT CTT TCT GCC CCA GAG TTG CGG CTG AGT CTG ACT AAG GGG CCT 671

3 Lys Arg Leu Ser Ala Pro Glu Leu Arg Leu Ser Leu Thr Lys Gly Pro 18

672 GGA AAT GAT GGA GCT TCA CCC ACC CAG TCT GCA CCT TCC TCT CCT GAT 719

19 Gly Asn Asp Gly Ala Ser Pro Thr Gln Ser Ala Pro Ser Ser Pro Asp 34

720 GGC AGT TCT GAC CTG GAG ATA GAC GAA TTG GAG ACA CCT TCA GAC TCG 767

35 Gly Ser Ser Asp Leu Glu Ile Asp Glu Leu Glu Thr Pro Ser Asp Ser 50

768 GAG CAG CTG GAC AGT GGA CAT GAA TTT GAA TGG GAA GAT GAA CTA CCC 815

51 Glu Gln Leu Asp Ser Gly His Glu Phe Glu Trp Glu Asp Glu Leu Pro 66

816 CGG GCA GAG GGT CTG GGC ACC AGT GAG ACA GCT GAA AGG CTG GGC CGA 863

67 Arg Ala Glu Gly Leu Gly Thr Ser Glu Thr Ala Glu Arg Leu Gly Arg 82

864 GGT TGT ATG TGG GAT GTG ACT GGA GAA GAT GGA CAT CAC TGG AGG GTG 911

83 Gly Cys Met Trp Asp Val Thr Gly Glu Asp Gly His His Trp Arg Val 98

912 TTC CGA ATG GGA CCA CGG GAG CAG CGC GTA GAC ATG ACT GTC ATT GAG 959

99 Phe Arg Met Gly Pro Arg Glu Gln Arg Val Asp Met Thr Val Ile Glu 114

960 CCC TAT AAG AAA GTC CTG TCT CAT GGA GGT TAC CAC GGT GAT GGC CTC 1007

115 Pro Tyr Lys Lys Val Leu Ser His Gly Gly Tyr His Gly Asp Gly Leu 130

1008 AAT GCT GTC ATC CTT TTT GCT TCC TGT TAT CTA CCC AGA AGC AGC ATC 1055

131 Asn Ala Val Ile Leu Phe Ala Ser Cys Tyr Leu Pro Arg Ser Ser Ile 146

1056 CCC AAC TAC ACC TAT GTC ATG GAA CAC TTG TTT AGG TAT ATG GTG GGA 1103

147 Pro Asn Tyr Thr Tyr Val Met Glu His Leu Phe Arg Tyr Met Val Gly 162

1104 ACT CTG GAG CTG CTA GTA GCT GAA AAT TAC CTG CTT GTT CAT TTG AGT 1151

163 Thr Leu Glu Leu Leu Val Ala Glu Asn Tyr Leu Leu Val His Leu Ser 178

1152 GGA GGC ACA AGC AGG GCC CAA GTT CCA CCT CTA AGC TGG ATA CGT CAG 1199

179 Gly Gly Thr Ser Arg Ala Gln Val Pro Pro Leu Ser Trp Ile Arg Gln 194

1200 TGT TAC CGT ACC CTG GAT CGG CGG CTA CGG AAA AAC CTG CGA GCC CTG 1247

195 Cys Tyr Arg Thr Leu Asp Arg Arg Leu Arg Lys Asn Leu Arg Ala Leu 210

1248 GTG GTT GTC CAT GCT ACA TGG TAT GTG AAA GCA TTT CTG GCA CTG CTT 1295

211 Val Val Val His Ala Thr Trp Tyr Val Lys Ala Phe Leu Ala Leu Leu 226

1296 CGG CCC TTC ATC AGT TCC AAA TTC ACA CGA AAA ATC CGT TTT CTG GAC 1343

227 Arg Pro Phe Ile Ser Ser Lys Phe Thr Arg Lys Ile Arg Phe Leu Asp 242

1344 AGC CTG GGG GAG CTG GCC CAA CTC ATA TCC CTG GAT CAA GTC CAC ATC 1391

243 Ser Leu Gly Glu Leu Ala Gln Leu Ile Ser Leu Asp Gln Val His Ile 258

1392 CCT GAA GCT GTC AGA CAG CTG GAC CGG GAT CTC CAT GGC TCA GGA GGG 1439

259 Pro Glu Ala Val Arg Gln Leu Asp Arg Asp Leu His Gly Ser Gly Gly 274

1440 ACA TAG CAC AGG ACT GGA TAA AGG GCC TTA GAA CCA GTT AGT GAT CTG 1487

275 Thr *** 276

1488 CCT ACA CCT GAA TCC CTG AAA CAT CTG AAC TGT TTT GTA AAT CAT CTT 1535

1536 ATC CCC AAC CTC AGT ACC ACC GGA TCT TCA CTT CTC AGT GGG ATT TTG 1583

1584 TCC TTT GCA TGA CCC TAC TTT CAG CAG GGC TTT GAG GCT GGG AAA CTG 1631

1632 ATC TGC TGG GTG ACT TTC ATT CCA GTT GCT GGG ACG GAA GGC TGA ATG 1679

1680 TAG GGT CAT TTT GTA TGG GAT ATG CAG AGC TCT GGG TTT TTT TGT TTT 1727

1728 TTG TTT TTT GAG ACG GAG TCT CGC TCT GTC GCC AGG CTG GAG TGC AGT 1775

1776 GGT GCG ATC TCG GCT CAC TGC AAC CTC CGC CTC CCG GTT CAA GCC ATT 1823

1824 CTT CTG CCT CAG CCT CAG CGA GTA GCT GAG ACT ACC GGC GCA CGC CAC 1871

1872 CAC GCC CAG CTA ATT GTT TTG TAT TTT TAG TAG AGA CGG GGT TTC ACC 1919

1920 ATG TTG GCC AGG ATG GTC TCG ATC TCT TGA CCT CGT GAT CTG CCC GCC 1967

1968 TCG GCC TCC CAA AGT GCC GAG ATT ACA GGT GTG AGC CAC CGC GCT CGG 2015

2016 CCA GAG CTC TGG GTT TTA ATC CTA CTT TAG CTG TAG AAC CTT GGG CAA 2063

2064 ATA ACT TCA TCT TTT TGG GTC CTA ATT TTC CTC CTG TCT AAC TGG AGG 2111

2112 GAC TGT GAT CCT TCC ACC TTG GAT GGA AGA GGC TTA CCT GAC AGC CAG 2159

2160 CCT GCC TGC TGG GAG CCA GGA GAG TCA GCT CAT TAA ATC TTG AAG AAC 2207

2208 CGC TGT ATG CCC TTT TTC CTT CTA AGT CAT GTC TGC TGC CTG TGA GCC 2255

2256 TGG GAA GGA GTG CTT TCA AAA CCT GTA TTT TTG CCC TCT TTA GTA AAT 2303

2304 TTA GAA CTG TAG AGG CTA ATA AAC TGC GAT GAG ACA TTT AAA AAA AAA 2351

2352 AAA AAA AAA 2360

Claims (9)

1. isolating NIP2 associated protein is characterized in that it is selected from down group:

(a) has the polypeptide of SEQ ID NO:2 aminoacid sequence;

(b) SEQ ID NO:2 aminoacid sequence is formed through disappearance, insertion or the replacement of 1-10 amino-acid residue, and have suppress hepatoma cell line 7721 clone's formation functions by (a) polypeptides derived.

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

3. isolating polynucleotide is characterized in that, it comprises a nucleotide sequence, and this nucleotide sequence is selected from down group:

(a) polynucleotide of polypeptide according to claim 1 of encoding;

(b) with polynucleotide (a) complementary polynucleotide.

4. polynucleotide as claimed in claim 3 is characterized in that the polypeptide of this polynucleotide encoding has the aminoacid sequence of SEQID NO:2.

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

Coding region sequence or the full length sequence of SEQ ID NO:3.

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

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

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

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

8. the preparation method with the active polypeptide of NIP2 associated protein is characterized in that, this method comprises:

(a) being fit to express under the condition of NIP2 associated protein, cultivate the described host cell of claim 7;

(b) from culture, isolate and have the active polypeptide of NIP2 associated protein.

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