CN1345798A - Novel human Cdc2 related protein kinase, its coding sequence and use - Google Patents

Abstract

The present invention provides a novel human Cdc2 related protein kinase -human hPFTAIRE1 protein, polynucleotide for coding human hPFTAIRE1 protein and method for producing this human hPETAIRE1 protein by using recombinatino technology. Said inventino also discloses the application of polynucleotide coding this human hPFTAIRE1 protein. Said invention also discloses the application of said human hPFTAIRE1 protein and nucleic acid sequence.

Description

New human Cdc 2 related protein kinase, its encoding sequence and purposes

The invention belongs to biotechnology and medical field, specifically, the present invention relates to the polynucleotide of new coding human Cdc 2 related protein kinase-hPFTAIRE1, and the polypeptide of this polynucleotide encoding.The invention still further relates to the purposes and the preparation of these polynucleotide and polypeptide.

Protein kinase in the propagation of cell, differentiation and tumour take place, have crucial effects (Funk, J.O., 1999, Anticancer Research, 19:4772-4780).Cdc2 and Cdc2 related protein kinase play a crucial role in cell cycle regulating and signal transduction path.Cdc2 at first finds in fission yeast, it at cell from G1 phase enter the S phase (Nurse, P.et al., 1981, Nature 292:558-560.) and G2 enter the M phase (Nurse, P.et al. in late period, 1976, Mol.Gen.Genet.146:167-178) play crucial effect in the process.

Many and Cdc2 albumen height homologous protein kinase is found in succession, and these albumen have been formed Cdc2 related protein kinase (Cdc2 related protein kinase) family.Many Cdc2 related protein kinases can with the combination of corresponding cyclin molecule, and this combination is necessary (Nigg, E.A., 1995 of their functionally activies, Bio.Essays.17.471-479), so be also referred to as CDK (Cyclin-dependent kinases).In high vertebrates, have been found that Cdc2 (CDK1) and CDK2 can complementary yeast in the disappearance of Cdc2/Cdc28, illustrate they undertaken the Core Feature of cell cycle regulating (Meyerson, M., et al.1992.EMBO J., 11:2909-2917).Some other Cdc2 related protein kinase is directly not relevant with cell fission, but in cell cycle regulating, undertake some subsidiary functions, or participate in other cell processes, such as: the CDK7-CyclinH-Matl complex body has CAK activity (CDK the activationkinase) (Nigg that phosphorylation activates other CDK, E.A., 1996, Curt.Opin.Cell Biol.8:312-17); Express in the refreshing system of CDK5 maincenter after mitotic division, and combine with p35 and regulate and control neurocyte and break up (Oshima, T., et al., 1996, Proc.Natl.Acad.Sci.USA 93:11173-78).

Also have the cyclin of portion C dc2 related protein kinase correspondence not find so far, be called " orphan CDK ", comprise PCTAIRE, PFTAIRE, KKIAIRE etc.Three homolgous molecules of PCTAIRE are cloned in human, mouse and rat, and wherein PCTAIRE1 has participated in differentiation (Besset, V.et al., 1999, the Cell Growth﹠amp of neurone and androgone after the mitotic division; Differentiation 10:173-181).Also be cloned into two PFTAIRE in the mouse, present their in reductional cell division and neurocyte differentiation, work (Lazzaro, M.A.et al., 1997, the J.Neurochemistry 69:348-364 of studies show that; Besset, V.etal., 1998, Molecular Reproduction and Development, 50:18-29).

Research shows that Cdc2 and related protein kinase thereof and some disease are closely related, and therefore for treatment and preventive means are provided effectively, therefore, this area presses for the new new human Cdc 2 related protein kinase of exploitation.

The purpose of this invention is to provide a kind of new human Cdc 2 related protein kinase-people PFTAIRE1 albumen 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 hPFTAIRE1 polypeptide is provided, this polypeptide is the people source, it comprises: polypeptide or its conservative property variation polypeptide or its active fragments or its reactive derivative with SEQ ID NO:2 aminoacid sequence.Preferably, this polypeptide is the polypeptide with SEQ ID NO:2 aminoacid sequence.

In a second aspect of the present invention, the polynucleotide of isolating these polypeptide of coding are provided, these polynucleotide comprise a nucleotide sequence, and this nucleotide sequence is shown at least 80% homogeny with a kind of nucleotides sequence that is selected from down group: (a) polynucleotide of the above-mentioned people hPFTAIRE1 polypeptide of coding; (b) with polynucleotide (a) complementary polynucleotide.Preferably, this polynucleotide encoding has the polypeptide of aminoacid sequence shown in the SEQ ID NO:2.More preferably, the sequence of these polynucleotide is the sequences with 1-1410 position among the SEQ ID NO:1.

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

In a fourth aspect of the present invention, provide preparation to have the method for the polypeptide of people hPFTAIRE1 protein-active, this method comprises: (a) under the proteic condition of suitable expressing human hPFTAIRE1, cultivate the above-mentioned host cell that is transformed or transduce; (b) from culture, isolate polypeptide with people hPFTAIRE1 protein-active.

In a fifth aspect of the present invention, provide and above-mentioned people hPFTAIRE1 polypeptid specificity bonded antibody.The nucleic acid molecule that can be used for detecting also is provided, and it contains a successive 10-1410 Nucleotide in the above-mentioned polynucleotide.

In a sixth aspect of the present invention, the compound of simulation, promotion, antagonism people hPFTAIRE1 polypeptide active is provided, and the compound that suppresses people hPFTAIRE1 polypeptide expression.The method of screening and/or prepare these compounds also is provided.Preferably, this compound is encoding sequence or its segmental antisense sequences of people hPFTAIRE1 polypeptide.

In a seventh aspect of the present invention, provide and whether had the proteic method of people hPFTAIRE1 in the test sample, it comprises: sample is contacted with the proteic specific antibody of people hPFTAIRE1, observe whether form antibody complex, formed antibody complex and just represented to exist in the sample people hPFTAIRE1 albumen.

In a eighth aspect of the present invention, a kind of disease relevant with people hPFTAIRE1 polypeptide unconventionality expression or method of disease susceptibility of detecting is provided, this method comprises: whether have sudden change in the nucleotide sequence of detection coding said polypeptide.

In a ninth aspect of the present invention, provide the purposes of polypeptide of the present invention and encoding sequence.Polypeptide for example of the present invention can be used to screen the agonist that promotes people hPFTAIRE1 polypeptide active, and perhaps screening suppresses the antagonist of people hPFTAIRE1 polypeptide active or is used to the peptide finger print identification.The proteic encoding sequence of people hPFTAIRE1 of the present invention or its fragment can be used as primer and be used for pcr amplification reaction, perhaps are used for hybridization as probe, perhaps are used to make gene chip or microarray.

In a tenth aspect of the present invention, a kind of pharmaceutical composition is provided, it contains people hPFTAIRE1 polypeptide of the present invention or its agonist, antagonist and the pharmaceutically acceptable carrier of safe and effective amount.These pharmaceutical compositions can be treated illnesss such as tumour, neural system and cardiovascular diseases.

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

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.

Figure 1A is the cDNA sequence of people hPFTAIRE1 of the present invention, and wherein non-coding sequence is represented with lowercase, and encoding sequence is represented with capitalization.

Figure 1A is the cDNA sequence of people hPFTAIRE1 of the present invention, and wherein non-coding sequence is represented with lowercase, and encoding sequence represents that with capitalization 11 subdomains of prediction mark with underscore.

Figure 1B has shown the structure of hPFTAIRE1 gene and coding region thereof.Initial and the termination in ATG and TGA presentation code district, NLS represents nuclear localization signal, and ATP represents the ATP binding sequence, and S/T represents serine threonine phosphorylation activity site.0.65kbPFT probe is used for Northern hybridization.

Fig. 2 A has shown that the amino acid identity of hPFTAIRE1 and other Cdc2 related protein kinase compares.Three phosphorylation sites mark with an asterisk, and the PSTAIRE primitive marks with two-wire.Fig. 2 B has shown the evolutionary tree analysis of hPFTAIRE1 and other Cdc2 related protein kinase.

Fig. 3 has shown the location of hPFTAIRE1 gene on human No. 7 karyomit(e).

Fig. 4 has shown the Northern hybridization analysis of hPFTAIRE1 gene, and the 0.65kbPFT probe is used for hybridization, and beta-actin cDNA is with comparing.

Fig. 5 has shown hPFTAIRE1 in intracellular location, and wherein nucleus dyes with DAPI, and the pEGFP-C2 plasmid is with comparing.

In the present invention, term " people hPFTAIRE1 albumen ", " people hPFTAIRE1 polypeptide " or " human Cdc 2 related protein kinase hPFTAIRE1 " are used interchangeably, all refer to the to have human Cdc 2 related protein kinase hPFTAIRE1 aminoacid sequence albumen or the polypeptide of (SEQ ID NO:2).They comprise the human Cdc 2 related protein kinase hPFTAIRE1 that contains or do not contain initial methionine.

As used herein, " isolating " is 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 people hPFTAIRE1 albumen or polypeptide " is meant that people hPFTAIRE1 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 people hPFTAIREI albumen of standard.Basically pure polypeptide can produce single master tape on non-reduced polyacrylamide gel.The purity of hPFTAIRE1 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 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 the proteic fragment of people hPFTAIRE1, derivative and analogue.As used herein, term " fragment ", " derivative " are meant with " analogue " and keep identical biological function of natural human hPFTAIRE1 albumen of the present invention or active polypeptide basically.Polypeptide fragment of the present invention, derivative or analogue 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 transformation period, polyoxyethylene glycol for example) merges formed polypeptide, or (iv) additional aminoacid sequence is fused to this peptide sequence and the polypeptide that forms (as leader sequence or secretion sequence or be used for the sequence or the proteinogen sequence of this polypeptide of purifying, or with the fusion rotein of the segmental formation of antigen I gG).According to the instruction of this paper, these fragments, derivative and analogue belong to the known scope of those skilled in the art.

In the present invention, term " people hPFTAIRE1 polypeptide " refers to have the SEQ ID NO.2 polypeptide of sequence of people hPFTAIRE1 protein-active.This term also comprises having and variant form people hPFTAIRE1 albumen 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 at C-terminal and/or N-terminal and (be generally in 20, preferably being in 10, more preferably is in 5) amino acid.For example, in the art, when replacing, can not change proteinic function usually with the close or similar amino acid of performance.Again such as, add one or several amino acid at C-terminal and/or N-terminal and also can not change proteinic function usually.This term also comprises proteic active fragments of people hPFTAIRE1 and reactive derivative.

The variant form of this polypeptide comprises: homologous sequence, conservative property varient, allelic variant, natural mutation, induced mutation body, under high or low tight degree condition can with the coded albumen of the DNA of people hPFTAIRE1 DNA hybridization and the polypeptide or the albumen that utilize the antiserum(antisera) of anti-people hPFTAIRE1 polypeptide to obtain.The present invention also provides other polypeptide, as comprises people hPFTAIRE1 polypeptide or its segmental fusion rotein (fusion rotein shown in SEQ IDNO:3).Except the polypeptide of total length almost, the present invention has also comprised the soluble fragments of people hPFTAIRE1 polypeptide.Usually, this fragment have people hPFTAIRE1 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 analogue of people hPFTAIRE1 albumen or polypeptide.The difference of these analogues and natural human hPFTAIRE1 polypeptide can be the difference on the aminoacid sequence, also can be the difference that does not influence on the modified forms of sequence, perhaps haves both at the same time.These polypeptide comprise natural or the inductive genetic variant.The induce variation body can obtain by various technology, as by radiation or be exposed to mutagenic compound and produce random mutagenesis, also can pass through site-directed mutagenesis method or the biological technology of other known moleculars.Analogue also comprises having the analogue that is different from the amino acid whose residue of natural L-(as D-amino acid), and has non-natural analogue that exist or synthetic amino acid (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.

(the not changing primary structure usually) form of modification comprises: the chemically derived form such as the acetylize or 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 as those in the synthetic and processing of polypeptide or further.This modification can be carried out glycosylated enzyme (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 (as Tyrosine O-phosphate, 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, " people hPFTAIRE1 albumen conservative property variation polypeptide " refers to compare with the aminoacid 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 property variation polypeptide preferably carry out the amino acid replacement according to table 1 and produce.

Table 1

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 shown in the SEQ ID NO:1 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:l.

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 sequence hybridization and two sequences between have at least 50%, preferably at least 70%, more preferably at least 80%, 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 90%, be more preferably 95% 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 the proteic polynucleotide of separation coding people hPFTAIRE1.

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

People hPFTAIRE1 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 obtain the dna sequence dna of code book invention albumen (or its fragment, or derivatives thereof) by chemosynthesis.This dna sequence dna can be introduced in various existing dna moleculars as known in the art (or as carrier) and the cell then.In addition, also can will suddenly change and introduce in the protein sequence of the present invention by chemosynthesis.

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 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 people hPFTAIRE1 albumen coded 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 hPFTAIRE1 polypeptide of reorganization.In general following steps are arranged:

(1). with the polynucleotide (or varient) of coding of the present invention people hPFTAIRE1 polypeptide, or with the recombinant expression vector that contains these polynucleotide proper host cell that transforms or transduce;

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

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

Among the present invention, people hPFTAIRE1 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 people hPFTAIRE1 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, 293 cells 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.Another kind method 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.

The extracellular can be expressed or be secreted into to recombinant polypeptide in the above methods 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.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 people hPFTAIRE1 albumen or the polypeptide of reorganization are of use in many ways.These purposes include, but is not limited to: the direct disease due to the low or forfeiture and be used to screen and promote or antibody, polypeptide or other part of antagonism people hPFTAIRE1 protein function as pharmacological agent people hPFTAIRE1 protein function.The peptide molecule that can suppress or stimulate people hPFTAIRE1 protein function that can be used for seeking therapeutic value with the recombinant human hPFTAIRE1 protein screening peptide library of expressing.Because the hPFTAIRE1 gene can be transcribed the long mRNA into about 6kb, expression level difference in people's various tissues, it is higher to be in the tissue of differentiation state expression amount at cell, shows that this expression of gene is relevant with people's growth course (especially the human nervous system grows).

On the other hand, the present invention also comprises people hPFTAIRE1 DNA or the polypeptide of its fragment coding has specific polyclonal antibody and monoclonal antibody, especially monoclonal antibody.Here, " specificity " is meant that antibody capable is incorporated into people hPFTAIRE1 gene product or fragment.Preferably, refer to that those can combine with people hPFTAIRE1 gene product or fragment but nonrecognition and be incorporated into the antibody of other irrelevant antigen molecule.Among the present invention antibody comprise those can in conjunction with and suppress the proteic molecule of people hPFTAIRE1, comprise that also those do not influence the antibody of people hPFTAIRE1 protein function.The present invention also comprise those can with modify or without the people hPFTAIRE1 gene product bonded antibody of modified forms.

The present invention not only comprises complete mono-clonal or polyclonal antibody, but also comprises having immunocompetent antibody fragment, as Fab ' or (Fab) ₂Fragment; Heavy chain of antibody; Light chain of antibody; Genetically engineered strand Fv molecule (people such as Ladner, U.S. Patent No. 4,946,778); Or chimeric antibody, as have the murine antibody binding specificity but still keep antibody from people's antibody moiety.

Antibody of the present invention can be prepared by the known various technology of those skilled in that art.For example, the people hPFTAIRE1 gene product of purifying or its have antigenic fragment, can be applied to animal to induce the generation of polyclonal antibody.Similarly, expressing human hPFTAIRE1 albumen or its have antigenic segmental cell and can be used to immune animal and produce antibody.Antibody of the present invention also can be monoclonal antibody.This type of monoclonal antibody can utilize hybridoma technology to prepare that (see people such as Kohler, Nature 256; 495,1975; People such as Kohler, people .Eur.J.Immunol.6:292 such as Eur.J.Immunol.6:511.1976:Kohler, 1976; People such as Hammerling, In Monoclonal Antibodies and T Cell Hybridomas, Elsevier, N.Y., 1981).Antibody of the present invention comprises the antibody that can block people hPFTAIRE1 protein function and the antibody that does not influence people hPFTAIRE1 protein function.Each antibody-like of the present invention can utilize the fragment or the functional zone of people hPFTAIRE1 gene product, obtains by the routine immunization technology.These fragments or functional zone can utilize recombinant methods or utilize Peptide synthesizer synthetic.Can come immune animal and produce with the gene product of producing in the prokaryotic cell prokaryocyte (for example E.Coli) with the unmodified form bonded antibody of people hPFTAIRE1 gene product; With posttranslational modification form bonded antibody (as the albumen or the polypeptide of glycosylation or phosphorylation), can come immune animal and obtain with the gene product that produces in the eukaryotic cell (for example yeast or insect cell).

The proteic antibody of anti-people hPFTAIRE1 can be used in the immunohistochemistry technology, detects the people hPFTAIRE1 albumen in the biopsy specimen.In addition, with the also available labelled with radioisotope of the protein bound monoclonal antibody of people hPFTAIRE1, 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 among the present invention can be used for treating or prevention and the relevant disease of people hPFTAIRE1 albumen.The antibody that gives suitable dosage can stimulate or block proteic generation of people hPFTAIRE1 or activity.

Antibody also can be used for being designed to the immunotoxin at a certain privileged sites in the body.As the monoclonal antibody of people hPFTAIRE1 albumen 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 cell (for example lymph-node cell etc.) of people hPFTAIRE1 protein positive.

The production of polyclonal antibody can choose hPFTAIRE1 albumen 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.

Utilize albumen of the present invention,, can filter out with people hPFTAIRE1 albumen interactional material takes place, as acceptor, inhibitor, agonist or antagonist etc. by various conventional screening methods.

Albumen of the present invention and antibody thereof, inhibitor, agonist, antagonist or acceptor etc. when using (administration) in treatment, can provide different effects.Usually, can these materials are formulated in nontoxic, inert and the pharmaceutically acceptable aqueous carrier medium, wherein pH is about 5-8 usually, and preferably pH is about 6-8, although the pH value can be with being changed to some extent by preparation Substance Properties and illness to be treated.The pharmaceutical composition for preparing can carry out administration by conventional route, comprising (but being not limited to): intramuscular, intraperitoneal, intravenously, subcutaneous, intracutaneous or topical.

Polypeptide of the present invention can be directly used in disease treatment, for example, is used for the treatment of nervous system development disease aspect.When using inventor hPFTAIRE1 albumen, also can use other relevant therapeutical agents simultaneously.

The present invention also provides a kind of pharmaceutical composition, and it contains hPFTAIRE1 polypeptide of the present invention or its agonist, antagonist and the pharmaceutically acceptable carrier or the vehicle of safe and effective amount.This class carrier comprises (but being not limited to): salt solution, damping fluid, glucose, water, glycerine, ethanol and combination thereof.Pharmaceutical preparation should be complementary with administering mode.Pharmaceutical composition of the present invention can be made into the injection form, for example is prepared by ordinary method with the physiological saline or the aqueous solution that contains glucose and other assistant agents.Pharmaceutical composition such as tablet and capsule can be prepared by ordinary method.Pharmaceutical composition such as injection, solution, tablet and capsule should be made under aseptic condition.The dosage of activeconstituents is the treatment significant quantity, for example every day about 1 microgram/kg body weight-Yue 5 mg/kg body weight.In addition, polypeptide of the present invention also can use with the other treatment agent.

When making pharmaceutical composition, be that the people hPFTAIRE1 albumen of safe and effective amount or its antagonist, agonist are applied to Mammals, wherein this safe and effective amount is usually at least about 10 micrograms/kg body weight, and in most of the cases be no more than about 8 mg/kg body weight, preferably this dosage is about 10 micrograms/kg body weight-Yue 1 mg/kg body weight.Certainly, concrete dosage also should be considered factors such as route of administration, patient health situation, and these all are within the skilled practitioners skill.

The proteic polynucleotide of people hPFTAIRE1 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 proteic expression of people hPFTAIRE1 of the proteic nothing expression of people hPFTAIRE1 or unusual/non-activity.The people hPFTAIRE1 albumen that the gene therapy vector (as virus vector) of reorganization can be designed to express variation is to suppress endogenic people hPFTAIRE1 protein-active.Deriving from viral expression vector such as retrovirus, adenovirus, adeno-associated virus (AAV), hsv, parvovirus etc. can be used for the hPFTAIRE1 transgenosis to cell.The method that structure carries the recombinant viral vector of hPFTAIRE1 gene is found in existing document (Sambrook, et al.).Recombinant human hPFTAIRE1 gene can be packaged in the liposome in addition, and then is transferred in the cell.

Suppress the oligonucleotide (comprising sense-rna and DNA) of people hPFTAIRE1 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.

Can be incorporated into the rondom polypeptide storehouse that solid formation forms by the various amino acid that may make up by screening with the protein bound peptide molecule of people hPFTAIRE1 obtains.During screening, must carry out mark to people hPFTAIRE1 protein molecular.

The invention still further relates to the diagnostic testing process of quantitative and detection and localization people hPFTAIRE1 protein level.These tests are known in the art, and comprise that FISH measures and radioimmunoassay.The people hPFTAIRE1 protein level that is detected in the test can be with laying down a definition the importance of people hPFTAIRE1 albumen in various diseases and be used to the disease of diagnosing people hPFTAIRE1 albumen to work.

Whether having the proteic method of people hPFTAIRE1 in a kind of detection test sample is to utilize the proteic specific antibody of people hPFTAIRE1 to detect, and it comprises: sample is contacted with people hPFTAIRE1 protein specific antibody; Observe whether form antibody complex, formed antibody complex and just represented to exist in the sample people hPFTAIRE1 albumen.

The proteic polynucleotide of people hPFTAIRE1 can be used for the diagnosis and the treatment of people hPFTAIRE1 protein related diseases.Aspect diagnosis, the proteic polynucleotide of people hPFTAIRE1 can be used for detecting the proteic expression of people hPFTAIRE1 people hPFTAIRE1 abnormal exprssion whether or under morbid state.Can be used for the hybridization of biopsy specimen to judge the proteic abnormal expression of people hPFTAIRE1 as the hPFTAIRE1DNA sequence.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.The special primer of personnel selection hPFTAIRE1 albumen carries out RNA-polymerase chain reaction (RT-PCR) amplification in vitro and also can detect the proteic transcription product of people hPFTAIRE1.

The sudden change that detects the hPFTAIRE1 gene also can be used for the disease of diagnosing people hPFTAIRE1 albumen relevant.The form of people hPFTAIRE1 protein mutation comprises that the point mutation compared with normal wild type hPFTAIRE1 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).In brief, prepare PCR primer (preferred 15-35bp), sequence can be positioned on the karyomit(e) according to the proteic cDNA of inventor hPFTAIRE1.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.

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.

In an example of the present invention, a kind of isolating polynucleotide are provided, its coding has the polypeptide of aminoacid sequence shown in the SEQ ID NO:2.Polynucleotide of the present invention are isolated from human Hela cell cDNA library.Its sequence is shown in Fig. 1 and SEQ ID NO:1, the polynucleotide sequence total length that it comprises is 2043 bases, its open reading frame is positioned at the 1-1410 position of SEQ ID NO:1, and the coding total length is 469 amino acid (not comprising terminator codon) people hPFTAIRE1 albumen (SEQ ID NO:2).This albumen has the constitutional features of serine threonine protein kinase, and this albumen and mouse PFTAIRE1 homology are the highest, reach 95%, and its about 300 amino acid in middle part and Cdc2 have 53% homology.The hPFTAIRE1 gene can be transcribed the long mRNA into about 6kb, expression level difference in the various tissues of people, expression amount is higher in tissues such as brain, pancreas, kidney, the heart, testis and ovary, then lower at expression amounts such as other tissue such as placenta, lung, liver, skeletal muscle, prostate gland, small intestine, colon and peripheral blood leucocyte, in spleen and thymus gland, be difficult to detect its expression.Utilize the GFP fluorescin as indicator protein, measure and learn that hPFTAIRE1 albumen is positioned in the cytoplasm.According to closely linked principle, hPFTAIRE1 is positioned on the karyomit(e) 7q21.The clone of hPFTAIRE1 gene provides new approach for detecting and treat human nervous system's heteroplasia, thereby has great application prospect.

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 people hPFTAIRE1 albumen cDNA

1. 1 material

Restriction enzyme, the T4 dna ligase is available from U.S. Gibco BRL company; The PCR test kit, mRNA extracting and purifying test kit, AMV reversed transcriptive enzyme and PCR test kit are available from U.S. Promega company; The hybridization nylon membrane, isotopic compound [ ³⁵S] dATP and [32P] dATP be available from U.S. Ampharmacia company; The radioactive probe labelling kit is available from U.S. Gibco BRL company; 5-bromo-4-chloro-3-indoles-galactoside (X-gal) and sec.-propyl-α-thiogalactoside (IPTG) are available from Gibco BRL company; HelacDNA lgt10 library is available from Clontech company; T-vector is available from Promega company.

XL-1-Blue, C600 are commercially available intestinal bacteria.

1. the extraction of 2. laryngeal carcinoma tissue mRNA and cDNA first chain is synthetic

After 0.1g laryngeal carcinoma tissue is fully ground through liquid nitrogen freezing and in mortar, according to the operation instruction of the PolyAtract 1000kit of Promega company, extracting and purifying laryngeal carcinoma tissue PolyA (+) mRNA.With 21merOligo-dT is primer, and laryngeal carcinoma tissue mRNA is a template, carries out the building-up reactions of cDNA first chain.

1. 3 design of primers and degenerate pcr

Protein kinase 300 the amino acid whose conservative catalyst structure domains of all having an appointment wherein can be divided into 11 the most conservative subdomains (Hanks, S.K.et al., 1998, Science 241:42-52) again.Because the conservative property of this structure and sequence height makes the degenerate pcr method be widely used in the screening of new kinase molecule.The inventor according to aminoacid sequence HRKI (L) KP of conservative subdomain VIB of Cdc2 protein kinase family and IX and DL (I, V) WSV (A, C) GCI has synthesized the degeneracy nucleotide primer that two length are respectively 17mer and 23mer:

17 poly-primers	5′-CA(C/T)(A/C)G(A/C/G/T)GA(C/T)(A/C/G/T)T(A/C/G/T)AA(A/G)CC-3′
		23 poly-primers	5′-AT(A/G)CA(A/C/G/T)CC(A/T/G/C)(A/C/G/T)(A/C)(A/C/G/T)GACCA (A/C/G/T)A(A/C/G/T)(A/G)TC-3′

With people's tissue cDNA is that template is carried out degenerate pcr.Concrete operations are: getting the above-mentioned laryngeal carcinoma tissue cDNA first chain synthetic end reaction liquid of 2ml is template, carries out the degenerate pcr reaction, and reaction system is 50ml:10 * PCR damping fluid 5ml, 25mM MgCl ₂4ml, 10mM dNTP1ml, template 2ul, Taq enzyme 0.5ml; 94 ℃ of 30S, 42 ℃ of 1min, 72 ℃ of 1min, totally 30 circulations.The result has obtained the PCR product of about 200bp.

1. 4 PCR product cloning and sequencings

The PCR product detects a band about 200bp through agarose electrophoresis, conforms to expection.This band reclaims rear clone in the T-easy vector of Promega company, obtains many independent clonings.Use the sequencing kit of USB company, 85 clones have been measured, relatively finding the back with the GenBank database, wherein 7 clones' dna sequence dna and mouse PFTAIRE-1 gene respective segments have 94% homology, this section sequence was not reported in the GenBank database, belonged to new kinase gene segment.Selected one of them gene fragment YT1 to do further to analyze.

1. the radio-labeling of 5 probes

With YT1 is the template label probe, according to the random primer probe mark test kit working instructions operation of Gibco BRL company.Probe behind mark Sephadex G-25 column separating purification.

1. 6cDNA library screening and full-length gene are cloned

The full-length gene of YT1 correspondence is cloned in the Hela cDNA gt10 library of selection Clontech company.The C600 bacterium that spends the night is inoculated among the LB+MM (LB+0.2% maltose+10mmol/L MgSO4) and is cultured to A600=0.3～0.6, as the host bacterium of library screening.Measure the library titre, according to suitable extent of dilution bed board, making the dull and stereotyped plaque number of every 15cm diameter is about 30000.When plaque length was transferred to phage DNA on the nylon membrane to suitable when size, with 0.7J/cm ²Power carry out UV-crosslinked.Under the stringent condition of 42oC, hybridize.The hybridization spot obtains single positive colony by the dilution separation.By restriction map and Southern hybridization analysis, determine and probe homologous dna segment, these segment subclones in the pBSK carrier and measure its sequence, are carried out the multistep subclone by similar way, obtain the complete sequence of gene.

With gene segment YT1 is probe screening people Hela cell cdna library, finally be separated to the cDNA sequence (Figure 1A) of a 2043kb, contain the complete open reading frame of 1410bp, infer one 469 amino acid whose protein of its coding according to sequence, the PFTAIRE-1 albumen of relatively finding it and mouse with the GenBank database has the highest homology, reach 95%, therefore with this unnamed gene behaviour PFRAIRE-1 (hPFTAIRE1), the GenBank sequence accession number of this gene is AF119833 (because of applying for a patent, being in the secret stage before the application submits to).

The proteic structural analysis of embodiment 2 hPFTAIRE1

The open reading frame of gene, the protein sequence translation, the restriction enzyme site analysis, secondary structure predictions etc. utilize DNASTAR software to help to analyze; The homology comparison query is finished by the Blaster Search network service of NCBI; Protein Motif and Subcellular Localization prediction are finished in the on-line analysis of Prosite website.

Protein sequence from the derivation of hPFTAIRE1 gene nucleotide series, at its 135th to 430 about 300 amino acid whose sections, have necessary 11 the conservative subdomains of Cdc2 related protein kinase (Figure 1A), comprising ATP-binding site LGEGSYATVYKGKSK VNWKLVALK (141-164), Ser/Thr protein kinase activity site ILHRDLKPQNLLI (252-264) (Figure 1B).

From the homology evolutionary analysis, hPFTAIRE1 and Cdc2 related protein kinase enzyme family have very high homology (Fig. 2 A), in addition, last three the important phosphorylation regulation and control residue Thr14 of Cdc2, Tyr15, Thr161 (Norbury, C.et al., 1991, EMBO J 10:3321-3329) also is present in the corresponding site among the hPFTAIRE1, be respectively Ser145, Tyr146 and Ser289.But in Cdc2 and other CDK, in hPFTAIRE1, change into PFTAIRE (Fig. 2 B) with the important primitive PSTAIRE of cyclin bonded.Similarly change is not rarely seen in other Cdc2 related protein kinases yet, as PCTAIRE and KKLAIRE etc.Up to the present PFTAIRE, the cyclin of PCTAIRE etc. does not also find.

HPFTAIRE1 has two sections non-conserved regions, and first section is positioned at the N end, about 130 amino acid, and second section is positioned at the C end, about 40 amino acid, they and other protein kinases all do not have homology.It is highly hydrophilic and flexible that Computer Analysis shows that N holds non-conserved regions to have, and have the nuclear localization signal (NLS) of two suppositions, is respectively PEDKKVR (66-72aa) and PKVRRHS (113-119aa).

In sum, people hPFTAIRE1 albumen is a new human Cdc 2 related protein kinase that does not appear in the newspapers.

Embodiment 3

The chromosomal localization of hPFTAIRE1 gene

By use Stanford radiation hybrid G3 panel test kit carry out new gene the chromosomal localization analysis (Waiter, M.A.et al., 1993, Trends Genet., 1993,9:352-356).This test kit has 83 pipe people's quasi-lymphocytes and hamster cell hybridization clone's chromosomal DNA, almost contained whole human genome, according to the close linkage principle, can utilize the inquiry of PCR reaction bonded internet database that Human genome is navigated on the concrete map.

The inventor respectively designs two upstream and downstream primers of about 200 apart according to the strong section of specificity on the new gene DNA sequence.5 '-ATATGAAGGGAATCATGGATC-3 ' and 5 '-GACGCTGTGCTGCAGAGG GAA-3 '; With above-mentioned 83 pipe DNA is template, carries out 30 cycle P CR, and reaction conditions is: 95 ℃ of 30S, 60 ℃ of 45S, 74 ℃ of 30S.For fear of false positive and false negative, every group of PCR reaction carried out 3 times altogether.The template numbering of positive band appears in statistics, the gained result is sent the homepage of stanford human genome center (SHGC), Principle of Statistics according to 2 maximum value probability analysiss, the result is analyzed, obtain the LOD value greater than 6.0 with the tight chain lock SHGC of gene location site, find pairing chromosomal region band thus.

The result proves that hPFTAIRE1 and STS G36723 close linkage are positioned human chromosome 7q21 (Fig. 3).Have a plurality of important function of gene in karyomit(e) 7q21 district, as CDK6, genes such as EPO, the generation of CDK6 gene product participation tumour (Funk, J.O., 1999, Anticancer Research, 19:4772-4780).The EPO gene participates in vascularization.

Embodiment 4

Expression and the distribution of hPFTAIRE1 gene in people's tissue

The Hybond membrane that contains 16 kinds of tissue mRNA of people is available from Clontech company, be used for Northern hybridization with 5 of hPFTAIRE1 gene coding region ' end dna sequence dna (1-655bp) as probe, utilize quick hybridization liquid to hybridize (Clontech), the concrete steps by specification carries out.

With the Northern hybridization technique expression and the Determination of distribution result of this gene in the various tissues of people shown, the hPFTAIRE1 gene can be transcribed the long mRNA into about 6kb, expression level difference in various tissues, expression amount is very high in tissues such as brain, pancreas, kidney, the heart, testis and ovary, expression amount is then lower in other tissue such as placenta, lung, liver, skeletal muscle, prostate gland, small intestine, colon and peripheral blood leucocyte, is difficult to detect its expression (Fig. 4) in spleen and thymus gland.This explanation hPFTAIRE1 expression of gene is relevant with the differentiation of cell.

Embodiment 5

HPFTAIRE1 albumen is in intracellular location

The C that hPFTAIRE1 is blended in GFP holds, and as indicator protein, detects this albumen in intracellular location with the GFP fluorescin.Method is as follows: the EcoRI and the XhoI site of the reading frame of hPFTAIRE1 gene being inserted pEGFP-C2 obtain the pEGFP-PFT fusion expression plasmid, utilize the calcium phosphate precipitation method that this plasmid is imported the Hela cell, cultivate after 48 hours, under fluorescent microscope, observe and take pictures, nucleus is with DAPI dyeing (Sambrook, J.etal., 1989, Molecular Cloning:A laboratory Manual.2nd edition, Cold SpringHarbor Laboratory Press, Cold Sprong Harbor, NY.)

The result shows that hPFTAIRE1 albumen is positioned (Fig. 5) in the cell oar.

Embodiment 6: with the proteic encoding sequence of RT-PCR method clone ZZ

As embodiment 1, with PolyAtract 1000 test kits (pressing the manufacturers instruction operation) extracting laryngeal carcinoma tissue PolyA (+) mRNA of Promega company.With 21mer Oligo-dT is primer, and laryngeal carcinoma tissue mRNA is a template, carries out the building-up reactions of cDNA first chain.

The used primer of pcr amplification is as follows:

Forward primer: 5 '-ATGTGTGACCTCATTGAGCCGCA-3 ' (SEQ ID NO:3),

Reverse primer 5 '-TCAGTGCTTGCTGTTTGATAGAC-3 ' (SEQ ID NO:4).

The PCR reaction volume is 50ml, wherein contain reverse transcription template 10ml, 0.4mM primer, 0.2mM dNTP and 1U ExTaq archaeal dna polymerase (Takara Inc.), the amplification parameter be 94 ℃ 30 seconds, 60 ℃ 30 seconds, 72 ℃ 45 seconds, capable 2% agarose gel electrophoresis of PCR product is tentatively confirmed after 25 circulations.The dna sequence analysis result shows that the coding region of the DNA sequences encoding of this PCR product and the 1-1410 position shown in the SEQ ID NO:1 is identical.Recombinant expressed and the purifying of embodiment 7 people hPFTAIRE1 albumen

Amplified production with embodiment 6 is a template, with PCR method is that hPFTAIRE1 full length gene coding region adds BamH1 and EcoRI joint, then it being cloned between the BamH1 and EcoRI site of intestinal bacteria GST fusion expression vector pGEX4T1 multiple clone site, is that the host bacterium carries out GST C end amalgamation and expression with the B21 bacterial strain.

Behind the abduction delivering, e. coli total protein can detect the expression product band of a tangible about 80Kda through SDS-PAGE gel electrophoresis separation and coomassie brilliant blue staining.Consider that fusogenic peptide GST molecular weight is 26Kda, gained expression product molecular weight conforms to the hPFTAIRE1 molecular weight of albumen 53Kda of expection.The generation of embodiment 8 anti-people hPFTAIRE1 protein antibodies

The recombinant human hPFTAIRE1 albumen that obtains among the embodiment 7 is used for immune animal to produce antibody, and concrete grammar is as follows.Recombinant molecule is standby after separating with chromatography.Also available SDS-PAGE gel electrophoresis separates, electrophoretic band downcut from gel, and with isopyknic complete Freund ' s adjuvant emulsion.Albumen with 50-100 μ g/0.2ml emulsification carries out peritoneal injection to mouse.After 14 days,, mouse is carried out peritoneal injection with booster immunization with the dosage of 50-100 μ g/0.2ml with the same antigen of non-complete Freund ' s adjuvant emulsion.Carried out booster immunization one time every 14 days, carry out at least three times.The sero-fast specific reaction that obtains is active to be assessed in the ability of external precipitation people hPFTAIRE1 protein gene translation product with it.Found that antibody can combine with albumen of the present invention specifically.

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:

(ii) denomination of invention: new human Cdc 2 related protein kinase, its encoding sequence and purposes

(iii) sequence number: the information of 4 (2) SEQ ID NO:1:

(i) sequence signature:

(A) length: 1410bp

(B) type: nucleic acid

(C) chain: two strands

(D) topological framework: linearity

(ii) molecule type: cDNA

( xi ) ：SEQ ID NO：1：ATGTGTGACC TCATTGAGCC GCAGCCGGCC GAGAAGATCG GCAAGATGAA GAAGTTGCGG 60AGAACTTTGT CGGAGAGTTT CAGTCGCATT GCTTTGAAGA AAGATGACAC CACCTTTGAT 120GAGATATGTG TCACAAAGAT GTCTACACGG AACTGCCAGG GAATGGACTC AGTGATCAAA 180CCCCTGGACA CAATTCCTGA GGATAAAAAA GTCAGAGTTC AGAGGACACA GAGCACTTTT 240GACCCATTTG AGAAACCAGC TAATCAAGTA AAGAGGGTGC ATTCTGAGAA CAATGCTTGC 300ATTAACTTTA AGACCTCCTC CACTGGCAAA GAGTCACCTA AAGTTAGGCG GCACTCCAGC 360CCCAGCTCGC CAACAAGTCC CAAATTTGGA AAAGCTGACT CATATGAAAA GCTGGAAAAA 420CTAGGGGAAG GATCTTATGC TACAGTATAC AAAGGGAAAA GCAAGGTAAA TTGGAAGTTG 480GTAGCTCTGA AGGTGATCAG GCTGCAGGAA GAAGAAGGGA CACCTTTCAC AGCTATCAGG 540GAAGCTTCTC TTTTAAAAGG ACTAAAACAT GCTAACATAG TGCTACTTCA TGACATCATC 600CATACCAAGG AGACGCTGAC ACTTGTGTTT GAATATGTGC ACACTGATTT ATGTCAGTAC 660ATGGACAAGC ACCCTGGGGG GCTGCATCCA GATAATGTGA AGTTGTTTTT ATTTCAGTTG 720CTGCGAGGTC TGTCTTACAT CCACCAGCGT TATATTTTGC ACAGAGACCT GAAACCACAG 780AACCTTCTGA TCAGTGACAC GGGGGAGTTA AAGCTGGCAG ATTTCGGTCT TGCAAGAGCA 840AAATCCGTCC CTAGCCACAC ATACTCCAAC GAAGTGGTTA CCTTGTGGTA CAGACCTCCA 900GATGTCCTTC TAGGCTCAAC AGAATATTCC ACCTGCCTTG ACATGTGGGG AGTAGGTTGC 960ATCTTTGTTG AAATGATCCA AGGAGTTGCT GCTTTTCCAG GAATGAAAGA CATTCAGGAT 1020CAACTTGAAC GAATATTTCT GGTTCTTGGA ACACCAAATG AGGACACATG GCCTGGAGTT 1080CATTCTTTAC CACATTTTAA GCCAGAACGC TTTACCCTGT ACAGCTCTAA AAACCTTAGA 1140CAAGCATGGA ATAAGCTCAG CTATGTGAAC CATGCAGAGG ACCTGGCCTC CAAGCTCCTA 1200CAATGTTCCC CAAAGAACAG ACTGTCGGCA CAGGCTGCCT TGAGCCACGA GTATTTTAGT 1260GACCTGCCGC CACGGCTATG GGAACTCACC GACATGTCTT CTATTTTTAC TGTCCCAAAT 1320GTGAGATTGC AACCAGAAGC TGGAGAAAGC ATGCGGGCCT TTGGGAAAAA CAATAGTTAT 1380GGCAAAAGTC TATCAAACAG CAAGCACTGA 1410 ( 2 ) SEQ ID NO：2： ( i ) ：

(A) length: 469 amino acid

(B) type: amino acid

( D ) ： ( ii ) ： ( xi ) ：SEQ ID NO：2：MCDLIEPQPA EKIGKMKKLR RTLSESFSRI ALKKDDTTFD EICVTKMSTR 50NCQGMDSVIK PLDTIPEDKK VRVQRTQSTF DPFEKPANQV KRVHSENNAC 100INFKTSSTGK ESPKVRRHSS PSSPTSPKFG KADSYEKLEK LGEGSYATVY 150KGKSKVNWKL VALKVIRLQE EEGTPFTAIR EASLLKGLKH ANIVLLHDII 200HTKETLTLVF EYVHTDLCQY MDKHPGGLHP DNVKLFLFQL LRGLSYIHQR 250YILHRDLKPQ NLLISDTGEL KLADFGLARA KSVPSHTYSN EVVTLWYRPP 300DVLLGSTEYS TCLDMWGVGC IFVEMIQGVA AFPGMKDIQD QLERIFLVLG 350TPNEDTWPGV HSLPHFKPER FTLYSSKNLR QAWNKLSYVN HAEDLASKLL 400QCSPKNRLSA QAALSHEYFS DLPPRLWELT DMSSIFTVPN VRLQPEAGES 450MRAFGKNNSY GKSLSNSKH* 469 ( 2 ) SEQ ID NO：3 ( i )

(A) length: 23 bases

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity is molecule type (ii): oligonucleotide (xi) sequence description: information (i) sequence signature of SEQ ID NO:3:ATGTGTGACC TCATTGAGCC GCA 23 (2) SEQ ID NO:4

(A) length: 23 bases

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity is molecule type (ii): oligonucleotide (xi) sequence description: SEQ ID NO:4:TCAGTGCTTG CTGTTTGATA GAC 23

Claims (10)

1. an isolating people hPFTAIRE1 polypeptide is characterized in that it comprises: polypeptide or its conservative property variation polypeptide or its active fragments or its reactive derivative with SEQ ID NO:2 aminoacid sequence.

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

3. isolating polynucleotide is characterized in that, it comprises a nucleotide sequence, and this nucleotide sequence is shown at least 80% homogeny with a kind of nucleotides sequence that is selected from down group:

(a) coding is as the polynucleotide of polypeptide as described in claim 1 and 2;

(b) with polynucleotide (a) complementary polynucleotide.

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

5. polynucleotide as claimed in claim 3 is characterized in that, the sequence of these polynucleotide has 1-1410 position among the SEQ IDNO:1.

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 contains the described carrier of claim 6.

8. preparation method with polypeptide of people hPFTAIRE1 protein-active is characterized in that this method comprises:

(a) under the proteic condition of suitable expressing human hPFTAIRE1, cultivate the described host cell of claim 7;

(b) from culture, isolate polypeptide with people hPFTAIRE1 protein-active.

9. energy and the described people hPFTAIRE1 of claim 1 protein-specific bonded antibody.

10. 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.

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