CN1414103A

CN1414103A - Human serine/threonine protein kitase, its coding sequence, preparation method and use

Info

Publication number: CN1414103A
Application number: CN02136497A
Authority: CN
Inventors: 余龙; 唐丽莎; 吴海; 郭金虎; 叶光明
Original assignee: Fudan University
Current assignee: Fudan University
Priority date: 2002-08-14
Filing date: 2002-08-14
Publication date: 2003-04-30

Abstract

The present invention relates to a human serine/threonine kinase a (hSTKa), the cDNA sequence for the transcription of the said hSTKa, the polypeptide coded by the said nucleotide sequence, and the application and preparing process of the said polynucleotide and polypeptide.

Description

Human serine/threonine protein kitase, its encoding sequence, Preparation method and use

Technical field

The present invention relates to the genetically engineered field, particularly, the present invention relates to a kind of human gene nucleotide sequence.More particularly, the present invention relates to a kind of human serine/threonine kinase ( hUman sErine/ tHreonine kInase a, hSTKa) and the cDNA sequence of transcript hSTKc.The invention still further relates to by above-mentioned nucleotide sequence coded polypeptide, the application of these polynucleotide and polypeptide and production method

Background technology

As everyone knows, extensively exist complicated signal pipeline in the biology.Each cell from extraneous received signal, passes through the bio-chemical pathway of a series of complexity by surperficial acceptor then, and signal is delivered in the born of the same parents from plasma membrane.And proteinic phosphorylation and dephosphorylation be exactly wherein one of most important bio-chemical pathway ( Bioorg Med Chem.1997 Sep; 5 (9): 1751-1773.).

The main effect of protein kinase is the phosphorylation of catalytic proteins, comprises proteinic serine/threonine kinase (serine/threonine kinase is called for short " STK ") and proteinic Tyrosylprotein kinase two big classes generally.Protein kinase is being brought into play important function in vivo, and it mainly is by proteic phosphorylation or by combining with the small molecules that plays regulating effect in vivo, and on level after the translation albumen being brought into normal play, function regulates and control.The expression of serine/threonine protein kitase is subjected to the influence of various factorss such as glucocorticosteroid, somatomedin, cell volume and cell injury.

STK is very conservative on evolving, and especially in its catalysis language many conservative regions is arranged on its structure.N end as catalytic domain is glycine enrichment section, also has a Methionin near this enrichment section, studies show that this district is relevant with the combination of ATP.The catalytic domain center is the aspartic acid of guarding, and it and kinase whose catalytic activity are closely related.Serine/threonine protein kitase extensively is present in the various piece of organism, comprise in various tissues, organ and the serum, participate in the cell necessary numerous processes of function of bringing into normal play, as: gene transcription, translation, the biosynthesizing of the mobile and organoid of proteic secretion and cell etc.

Because the fundamental role that serine/threonine protein kitase is risen in the cell physiological activity, people are to it, and especially the research of the expressional function situation of this family member in the mankind is day by day extensive.1994, people such as Small D have cloned human stem cell STK-1 and have studied its expression in normal people's marrow, the result shows that STK-1 may be growth factor receptors (the Proc Natl Acad Sci 1994,91 (2): 459-463) of hemopoietic stem cell.1996, Chen, H., wait that the people studied human skin microvascular endotheliocyte STK-1 genome the 9th and the tenth exon with the sudden change situation.1998, people such as Li X. reported that the clone has obtained STK-2.Brodbeck, D., Nakatani, K., Masure, S. waits the people to clone and identifies the RAC-gamma serine/threonine protein kinase (Akt-3) that knows clearly (J.Biol.Chem.274 (14), 9133-9136 (1999); Biochem.Biophys.Res.Commun.257 (3), 906-910 (1999); Eur.J.Biochem.265 (1), 353-360 (1999).Calendar year 2001 Stanchi, F. wait the people when the Human genome of separation and yeast sequence similarity, also to obtain a serine/threonine protein kitase of forming by 603 amino-acid residues, the Genbank accession number is CAA07196, further prove the essential (Yeast of serine/threonine protein kitase cell growth, 18 (1), 69-80).

Yet before the present invention, still nobody disclosed human serine/threonine kinase hSTKa of the present invention and hSTKc.

Summary of the invention

One of purpose of the present invention provides a kind of polynucleotide sequence, a kind of human serine/threonine protein kitase a of this polynucleotide sequence coding ( hUman sErine/ tHreonine kInase a, hSTKa).

Two of purpose of the present invention provides a kind of polynucleotide sequence, a kind of human serine/threonine protein kitase b of this polynucleotide sequence coding ( hUman sErine/ tHreonine kInase c, hSTKc), it is that transcribing of above-mentioned hSTKa sheared this.

Three of purpose of the present invention provides a kind of albumen, this albumen be named as human serine/threonine protein kitase a ( hUman sErine/ tHreonine kInase a, hSTKa).

Four of purpose of the present invention provides a kind of albumen, this albumen be named as human serine/threonine protein kitase c ( hUman sErine/ tHreonine kInase c, hSTKc).

Five of purpose of the present invention provides a kind of recombinant technology that utilizes and produces described hSTKa and the proteic method of hSTKc.

Six of purpose of the present invention provides hSTKa and hSTKc nucleotide sequence and proteic application.

The invention provides a kind of isolated dna molecular, it comprises: coding has the nucleotide sequence of the polypeptide of people hSTK protein-active, and the nucleotides sequence among described nucleotide sequence and the SEQ ID NO.1 is shown at least 70% homology; Perhaps described nucleotide sequence can be under the moderate stringent condition with SEQ ID NO.1 in nucleotide sequence hybridization.Preferably, described sequence encoding one polypeptide, this polypeptide have the sequence shown in SEQ ID NO.2 or the SEQ ID NO.4.More preferably, this sequence has the nucleotide sequence shown in SEQ ID NO.1 or the SEQ ID NO.3.

In another aspect of this invention, provide a kind of isolating hSTK protein polypeptide, it comprises: have polypeptide or its active fragments of SEQ ID NO.2 or SEQ ID NO.4 aminoacid sequence, or its reactive derivative.Preferably, this polypeptide is to have SEQ ID NO.2 or SEQ ID NO.4 polypeptide of sequence.

In another aspect of this invention, provide a kind of carrier, it contains above-mentioned isolated DNA.

In another aspect of this invention, provide a kind of described carrier transformed host cells.

In another aspect of this invention, the method that provides a kind of generation to have the polypeptide of hSTK protein-active, this method comprises:

(a) nucleotide sequence that coding is had a polypeptide of hSTK protein-active operationally is connected in expression regulation sequence, form the hSTK protein expression vector, nucleotide sequence has at least 70% homology among described nucleotide sequence and SEQ ID NO.1 or the SEQ ID NO.3;

(b) change the expression vector in the step (a) over to host cell, form the proteic reconstitution cell of hSTK;

(c) be fit to express under the condition of hSTK protein polypeptide the reconstitution cell in the culturing step (b);

(d) isolate polypeptide with hSTK protein-active.

In the present invention, " isolating ", " purifying " or " pure substantially " DNA are meant, this DNA or fragment have been arranged in the sequence of its both sides and have separated under native state, refer to that also this DNA or fragment with under the native state follow the component of nucleic acid to separate, and separate with the protein of in cell, following it.

In the present invention, term " hSTK albumen (or polypeptide) encoding sequence " refer to the encode nucleotide sequence of polypeptide with hSTK protein-active is as 3-2009 position nucleotide sequence and degenerate sequence thereof among the SEQ ID NO.1.This degenerate sequence is meant, is arranged in the encoder block 3-2009 position Nucleotide of SEQ ID NO.1 sequence, and having one or more codons to be encoded, the degenerate codon of same amino acid replaces the back and the sequence that produces.Because the degeneracy of codon, thus with SEQ ID NO.1 in 3-2009 position nucleotide sequence homology be low to moderate about 70% the degenerate sequence described sequence of SEQ ID NO.2 of also encoding out.This term also comprises can be under the moderate stringent condition, more preferably under the height stringent condition with SEQ ID NO.1 in from the nucleotide sequence of the nucleotide sequence hybridization of Nucleotide 3-2009 position.In addition, this term also comprise with SEQ ID NO.1 in from the homology of nucleotide sequence at least 70% of Nucleotide 3-2009 position, preferably at least 80%, at least 90% nucleotide sequence more preferably.

This term also comprises encoding to have variant form with proteic, the SEQ ID NO.1 sequence of people hSTK identical function.These variant forms comprise (but being not limited to): several (are generally 1-90, preferably 1-60, more preferably 1-20,1-10 best) disappearance, insertion and/or the replacement of Nucleotide, and several (are generally in 60 to hold interpolation 5 ' and/or 3 ', preferably being in 30, more preferably is in 10, is in 5 best) Nucleotide.

In the present invention, " pure substantially " protein or polypeptide are meant that it accounts at least 20% of the total material of sample at least, preferably at least 50%, more preferably at least 80%, and at least 90% (by dry weight or weight in wet base) best.Purity can be measured with any suitable method, as measure the purity of polypeptide with column chromatography, PAGE or HPLC method.Substantially pure polypeptide is substantially free of the component of following it under the native state.

In the present invention, term " hSTK protein polypeptide " refers to have the SEQ IDNO.2 or the SEQ IDNO.4 polypeptide of sequence of hSTK protein-active.This term also comprises having and variant form people hSTK identical function, SEQ ID NO.2 or SEQ ID NO.4 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 hSTK and reactive derivative.

The variant form of this polypeptide comprises: homologous sequence, allelic variant, natural mutation, induced mutation body, under high or low rigorous degree condition can with the coded albumen of the DNA of hSTK DNA hybridization of the present invention and the polypeptide or the albumen that utilize the antiserum(antisera) of anti-hSTK polypeptide to obtain.The present invention also provides other polypeptide, as comprises hSTK polypeptide or its segmental fusion rotein.Except the polypeptide of total length almost, the present invention has also comprised the soluble fragments of hSTK polypeptide.Usually, this fragment have the hSTK 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.

The difference of the hSTK albumen of indication of the present invention or the analogue of polypeptide and natural hSTK 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.

The present invention also comprises the antisense sequences of hSTK polypeptid coding sequence.This antisense sequences can be used for suppressing the expression of hSTK in the cell.

The present invention also comprises a kind of probe molecule, and this molecule has 8-100 of hSTK polypeptid coding sequence, preferably 15-50 continuous nucleotide usually.This probe can be used for whether existing in the test sample nucleic acid molecule of the hSTK that encodes.

The present invention also comprises the method that detects the hSTK nucleotide sequence, and it comprises with above-mentioned probe and sample and hybridizing whether detection probes combination has taken place then.Preferably, this sample is the product behind the pcr amplification, and wherein the pcr amplification primer is corresponding to the encoding sequence of hSTK polypeptide, and can be positioned at the both sides or the centre of this encoding sequence.Primer length is generally 20-50 Nucleotide.

In the present invention, can select various carrier known in the art for use, as commercially available carrier.

In the present invention, term " host cell " comprises prokaryotic cell prokaryocyte and eukaryotic cell.The example of prokaryotic host cell commonly used comprises intestinal bacteria, Bacillus subtilus etc.Eukaryotic host cell commonly used comprises yeast cell, insect cell and mammalian cell.Preferably, this host cell is an eukaryotic cell, as Chinese hamster ovary celI, COS cell etc.

On the other hand, the present invention also comprises hSTK 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 hSTK gene product or fragment.Preferably, refer to that those can combine with hSTK 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 hSTK, comprise that also those do not influence the antibody of hSTK protein function.The present invention also comprise those can with modify or without the hSTK 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 hSTK gene product of purifying or its have antigenic fragment, can be applied to animal to induce the generation of polyclonal antibody.Similarly, expressing hSTK or its has 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 prepare (see people such as Kohler, Nature256; 495,1975; People such as Kohler, Eur.J.Immunol.6:511,1976; People such as Kohler, Eur.J.Immunol.6:292,1976; 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 the hSTK function and the antibody that does not influence the hSTK function.Each antibody-like of the present invention can utilize the fragment or the functional zone of hSTK 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 hSTK 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).

In the present invention, the cDNA nucleotide sequence of people hSTKa is so to obtain, and is template with people's testis λ gt10cDNA library (available from Clontech company), is primer with two pairs of oligonucleotide: A1 CGATGACATCGACGGGGAAGGAC; A2 CAACAA GCTGCTG CAGGACCTG is a forward primer; B1:CAGGTCCTCATC CTCCTGGCTCG; B2 GATGGCCTC GTGGAGGT GACATG is a reverse primer, carries out PCR.The PCR condition be 93 ℃ 4 minutes, carried out 35 circulations in 1 minute with 93 ℃ 1 minute, 64 ℃ 1 minute and 72 ℃ thereupon, last 72 ℃ were extended 5 minutes.The PCR segment that electrophoresis detection obtains, size are respectively the fragment and the impurity of about 1,000 and 1,100 base pairs.Remove impurity, improve the concentration of required nucleotide fragments, cut the fragment that obtains with the PshAI enzyme respectively, and connect, obtain the purpose fragment at last.

With people's testis λ gt10cDNA library (available from Clontech company) is template, is primer with two pairs of oligonucleotide: A1CGATGACATCG ACGGGGAAGGAC; A2 CAACAA GCTGCTG CAGGACCTG is a forward primer; B1:CA GGTCCTCATC CTCCTGGCTCG; B2 GCTAGTG TGTCTAAGGCTGCTCG is a reverse primer, carries out PCR.The PCR condition be 93 ℃ 4 minutes, carried out 35 circulations in 1 minute with 93 ℃ 1 minute, 64 ℃ 1 minute and 72 ℃ thereupon, last 72 ℃ were extended 5 minutes.The PCR segment that electrophoresis detection obtains, size are respectively the fragment and the impurity of about 1,000 and 1,300 base pairs.Remove impurity, improve the concentration of required nucleotide fragments, cut the fragment that obtains with the PshAI enzyme respectively, and connect, obtain the purpose fragment at last.

HSTKa of the present invention and hSTKc are by same DNA transcription and translation, just in transcription because the shearing connecting method difference of transcription product, so produced different albumen, but it has very big similarity on sequence and preparation method.

In Non-redundant GenBank+EMBL+DDBJ+PDB database and Non-redundant GenBank CDS ranslations+PDB+SwissProt+Spupdate+PIR database, carry out nucleic acid and albumen homology retrieval with hSTKa of the present invention and hSTKc albumen with BLAST software.Found that the serine/threonine kinase protein family member (as CAA64382, JC1446 and CAA07196) that it and some have been identified has certain homology, and have serine/threonine kinase protein family member's feature functionality territory in its structure.

In sum, hSTKa of the present invention and hSTKc belong to the serine/threonine kinase family member, common trait with this family member, can engage with cytokine by the outer immunoglobulin (Ig) spline structure of born of the same parents, and be passed in the born of the same parents by phosphate esterase active the signal that cytokine is carried of part in its born of the same parents, act on target molecule, thereby the physiological activity of pair cell and even organism is mentioned regulating and controlling effect.These physiological actions comprise the promotion tissue growth, and the growth of regulating cell and propagation activate, suppress immunocyte, and the hemopoietic cell activates inflammatory reaction, prevents virus, bacterial invasion etc.

HSTKa of the present invention and hSTKc Nucleotide or aminoacid sequence are linked to each other with pharmaceutically acceptable carrier can generate a reagent, medicine and test kit, with prevention, diagnosis and treatment because hSTKa or the undesired disease that causes of hSTKc expression.

Table 1 is that the amino acid sequence homologous of hSTKa, hSTKac and CAA07196 compares.Wherein, " ^*" represent that the amino acid on the corresponding position is identical in three sequences; ": " is though represent that amino acid its physico-chemical property inequality on the corresponding position is roughly the same in three sequences; the amino acid physico-chemical property in three sequences of ". " expression on the corresponding position has certain homogeny, does not have corresponding amino acid on the corresponding position in three sequences of " " expression.

Embodiment

Embodiment 1

The clone and the mensuration of the cDNA sequence of hSTKa

(1). primer amplification

With people's testis λ gt10cDNA library (available from Clontech company) is template, is primer with two pairs of oligonucleotide: A1CGATGACATCG ACGGGGAAGGAC; A2 CAACAA GCTGCTG CAGGACCTG is a forward primer; B1:CA GGTCCTCATC CTCCTGGCTCG; B2 GATGGCCTC GTGGAGGTGACATG is a reverse primer, carries out PCR.The PCR condition be 93 ℃ 4 minutes, carried out 35 circulations in C1 minute with 93 ℃ 1 minute, 64 ℃ 1 minute and 72 ℃ thereupon, last 72 ℃ were extended 5 minutes.The PCR segment that electrophoresis detection obtains, size are respectively the fragment and the impurity of about 1,000 and 1,100 base pairs.Remove impurity, improve the concentration of required nucleotide fragments, cut the fragment that obtains with the PshAI enzyme respectively, and connect, obtain the purpose fragment at last.

(2) order-checking of .PCR product

With pcr amplification product and pGEM-T ^TMCarrier (Promega) connects, transformed into escherichia coli JM103, extract plasmid with QIAprepPlasmid test kit (QIAGEN), carry out the mistake of orientations Lieque to inserting fragment, carry out Rapid identification and ordering with PCR to lacking son then with double-stranded nested type disappearance test kit (Pharmacia).Use SequiTherm EXCEL ^TMDna sequencing kit (Epicentre Technologies) checks order to disappearance of brachymemma successively, obtains full length cDNA sequence at last, is total to 2023bp, and detailed sequence is seen SEQ ID NO.1, and wherein open reading frame is positioned at 3-2009 position Nucleotide.

Derive the aminoacid sequence of hSTKa according to the full length cDNA sequence that obtains, totally 668 amino-acid residues, its aminoacid sequence sees SEQ ID NO.2 for details.

Embodiment 2

The expression of hSTKa in intestinal bacteria

In this embodiment,, use PCR Oligonucleolide primers to increase, obtain hSTK a cDNA as inserting fragment corresponding to 5 of this dna sequence dna ' and 3 ' end with the cDNA sequence of coding hSTKa.

5 ' Oligonucleolide primers sequence of using in the PCR reaction is:

5 '-CCCCGGATCCATGACA TCGACGGGGA AGG, this primer contains the restriction enzyme site of BamHI restriction enzyme, is the part encoding sequence of the hSTKa that begun by initiator codon after this restriction enzyme site;

3 ' end primer sequence is:

5 ' TGACAAGCTTACTTTTCG GGATAATTC, this primer contain the part encoding sequence of restriction enzyme site, translation termination and the hSTKa of HindIII restriction enzyme.

The restriction enzyme site of the restriction enzyme on the primer is corresponding to bacterial expression vector pQE-9 (Qiagen Inc., Chatsworth, the CA) restriction enzyme digestion sites on, this plasmid vector coding antibiotics resistance (Amp ^r), a bacterium replication orgin (ori), an adjustable promotor/operon of IPTG-(P/O), a ribosome bind site (RBS), a 6-histidine mark thing (6-His) and restriction enzyme cloning site.

With BamHI and HindIII digestion pQE-9 carrier and insertion fragment, will insert fragment subsequently and be connected to the pQE-9 carrier and keep open reading frame initial at bacterium RBS.Transform available from Qiagen with connecting mixture subsequently, the E.coli bacterial strain of commodity M15/rep4 by name, M15/rep4 contains the plasmid pREP4 of multiple copied, and it is expressed the lacI repressor and carries kalamycin resistance (Kan ^r).Screen transformant containing on the LB culture dish of Amp and Kan, the extracting plasmid cuts with the PshAI enzyme and to identify and insert clip size and direction, and the cDNA fragment of sequence verification hSTK has correctly been inserted carrier.

Incubated overnight (O/N) contains the positive transformant clone of required construction in the LB liquid nutrient medium of adding Amp (100 μ g/ml) and Kan (25 μ g/ml).Spend the night (O/N) culture with 1: 100-1: 250 thinning ratio dilution, be inoculated into then in the large volume substratum, culturing cell grows to 600 optical density(OD) (OD ₆₀₀) when being 0.4-0.6, add IPTG (" isopropylthio-") to final concentration be 1mM.By making lacI repressor inactivation, IPTG induces startup P/O to cause gene expression dose to improve.Continued culturing cell 3-4 hour, centrifugal subsequently (6000 * g, 20 minutes).The ultrasonic degradation inclusion body, collecting cell also is dissolved in cell precipitation in the Guanidinium hydrochloride of 6M.After the clarification, by containing under the condition that 6-His marker albumen combines closely making, with nickel-chelate column chromatography purifying dissolved hSTK from solution.With 6M Guanidinium hydrochloride (pH5.0) wash-out hSTK from post.Available several method is the sex change protein precipitation from Guanidinium hydrochloride.Perhaps use the dialysis step to remove Guanidinium hydrochloride, perhaps isolate purifying protein from nickel-chelate column, the albumen behind the purifying can be incorporated in second post, has the linear Guanidinium hydrochloride gradient of successively decreasing in this post.Protein denaturation when being attached to this post is used Guanidinium hydrochloride (pH5.0) wash-out subsequently.At last, soluble protein is dialysed to containing PBS, then protein is kept in the stock solution that final concentration is 10% (w/v) glycerine.

In addition, the amino acid of expressing proteic N end and each 10 amino acid length of C end is checked order, find consistent with the sequence of SEQ ID NO.2 with ordinary method.

Embodiment 3

The expression of hSTKa in eukaryotic cell (Chinese hamster ovary celI strain)

In this embodiment, the following Oligonucleolide primers of cDNA sequence of coding hSTKa is increased, obtain hSTKacDNA as inserting fragment.

5 ' Oligonucleolide primers sequence of using in the PCR reaction is:

5’-CCCCAAGCTTATGACA?TCGACGGGGA?AGG

This primer contains the restriction enzyme site of HindIII restriction enzyme, is the part encoding sequence of the hSTKa that begun by initiator codon after this restriction enzyme site;

3 ' end primer sequence is:

5’-TGACGGATCCACTTTTCG GGATAATTC

This primer contains the part encoding sequence of the restriction enzyme site of BamHI restriction enzyme, translation termination and hSTKa.

The restriction enzyme site of the restriction enzyme on the primer is corresponding to the restriction enzyme digestion sites on the expressing cho cell carrier pcDNA3, this plasmid vector coding antibiotics resistance (Amp ^rAnd Neo ^r), a phage replication starting point (f1 ori), a virus replication starting point (SV40 ori), T7 promotor, a viral promotors (P-CMV), a Sp6 promotor, a SV40 promotor, a SV40 tailing signal and corresponding polyA order, a BGH tailing signal and a corresponding polyA order.

With HindIII and BamHI digestion pcDNA3 carrier and insertion fragment, will insert fragment subsequently and be connected to the pcDNA3 carrier.Subsequently with connecting mixture Transformed E .coli DH5 α bacterial strain.Screen transformant containing on the LB culture dish of Amp, incubated overnight (O/N) contains the clone of required construction in the LB liquid nutrient medium of adding Amp (100 μ g/ml).The extracting plasmid cuts evaluation with the PshAI enzyme and insert clip size and direction, and the cDNA fragment of sequence verification hSTKa has correctly been inserted carrier.

The plasmid transfection Chinese hamster ovary celI is to use lipofection, carries out with Lipofectin (GiBco Life), and transfection is after 48 hours, and through the lasting G418 pressurization screening in 2-3 week, collecting cell and cell conditioned medium are measured the expressing protein enzyme activity.Remove G418, continuous passage is cultivated; To mixing the clone cell Method of Limited Dilution, select to have the cell subclone of higher protein-active.The above-mentioned positive subclone of a large amount of according to a conventional method cultivations.After 48 hours, beginning collecting cell and supernatant are with ultrasonic degradation method smudge cells.With 50mMTrisHCl (pH7.6) solution that contains 0.05%Triton is balance liquid and elutriant, uses through the SuperdexG-75 of pre-equilibration post and collects above-mentioned proteic active peak.Using 50mMTrisHCl (pH8.0) equilibrated DEAE-Sepharose post again, is that elutriant carries out gradient elution with 50mMTrisHCl (pH8.0) solution that contains 0-1MNaCl, collects above-mentioned proteic active peak.Be that dialyzate is dialysed to expressing protein solution with PBS (pH7.4) then.Last freeze-drying is preserved.

Embodiment 4

The clone and the mensuration of the cDNA sequence of hSTKc

1. primer amplification

2.PCR the order-checking of product

With pcr amplification product and pGEM-T ^TMCarrier (Promega) connects, transformed into escherichia coli JM103, extract plasmid with QIAprepPlasmid test kit (QIAGEN), carry out the mistake of orientations Lieque to inserting fragment, carry out Rapid identification and ordering with PCR to lacking son then with double-stranded nested type disappearance test kit (Pharmacia).Use SequiTherm EXCEL ^TMDna sequencing kit (Epicentre Technologies) checks order to disappearance of brachymemma successively, obtains full length cDNA sequence at last, is total to 2223bp, and detailed sequence is seen SEQ ID NO.3, and wherein open reading frame is positioned at 3-2009 position Nucleotide.

Derive the aminoacid sequence of hSTKc according to the full length cDNA sequence that obtains, totally 736 amino-acid residues, its aminoacid sequence sees SEQ ID NO.4 for details.

Embodiment 5

The expression of hSTKc in intestinal bacteria

In this embodiment,, use PCR Oligonucleolide primers to increase, obtain hSTKc cDNA as inserting fragment corresponding to 5 of this dna sequence dna ' and 3 ' end with the cDNA sequence of coding hSTKc.

5 ' Oligonucleolide primers sequence of using in the PCR reaction is:

5 '-CCCCGGATCCATGACA TCGACGGGGA AGG, this primer contains the restriction enzyme site of BamHI restriction enzyme, is the part encoding sequence of the hSTKc that begun by initiator codon after this restriction enzyme site;

3 ' end primer sequence is:

5 '-AGTGAAGCTTAG GCTGCTCGCG GCGGGCG, this primer contain the part encoding sequence of restriction enzyme site, translation termination and the hSTKc of HindIII restriction enzyme.

With BamHI and HindIII digestion pQE-9 carrier and insertion fragment, will insert fragment subsequently and be connected to the pQE-9 carrier and keep open reading frame initial at bacterium RBS.Transform available from Qiagen with connecting mixture subsequently, the E.coli bacterial strain of commodity M15/rep4 by name, M15/rep4 contains the plasmid pREP4 of multiple copied, and it is expressed the lacI repressor and carries kalamycin resistance (Kan ^r).Screen transformant containing on the LB culture dish of Amp and Kan, the extracting plasmid cuts with the PshAI enzyme and to identify and insert clip size and direction, and the cDNA fragment of sequence verification hSTKc has correctly been inserted carrier.

In addition, the amino acid of expressing proteic N end and each 10 amino acid length of C end is checked order, find consistent with the sequence of SEQ ID NO.4 with ordinary method.

Embodiment 6

The expression of hSTKc in eukaryotic cell (Chinese hamster ovary celI strain)

In this embodiment, the following Oligonucleolide primers of cDNA sequence of coding hSTKc is increased, obtain hSTKccDNA as inserting fragment.

5 ' Oligonucleolide primers sequence of using in the PCR reaction is:

5’-CCCCAAGCTTATGACA?TCGACGGGGA?AGG

This primer contains the restriction enzyme site of HindIII restriction enzyme, is the part encoding sequence of the hSTKc that begun by initiator codon after this restriction enzyme site;

3 ' end primer sequence is:

5’-AG?TGGGATCCAG?GCTGCTCGCG?GCGGGCG

This primer contains the part encoding sequence of the restriction enzyme site of BamHI restriction enzyme, translation termination and hSTKc.

The restriction enzyme site of the restriction enzyme on the primer is corresponding to the restriction enzyme digestion sites on the expressing cho cell carrier pcDNA3, this plasmid vector coding antibiotics resistance (Amp ^rAnd Neo ^r), a phage replication starting point (f1 ori), a virus replication starting point (SV40ori), T7 promotor, a viral promotors (P-CMV), a Sp6 promotor, a SV40 promotor, a SV40 tailing signal and corresponding polyA order, a BGH tailing signal and a corresponding polyA order.

With HindIII and BamHI digestion pcDNA3 carrier and insertion fragment, will insert fragment subsequently and be connected to the pcDNA3 carrier.Subsequently with connecting mixture Transformed E .coli DH5 α bacterial strain.Screen transformant containing on the LB culture dish of Amp, incubated overnight (O/N) contains the clone of required construction in the LB liquid nutrient medium of adding Amp (100 μ g/ml).The extracting plasmid cuts evaluation with the PshAI enzyme and insert clip size and direction, and the cDNA fragment of sequence verification hSTKc has correctly been inserted carrier.

The plasmid transfection Chinese hamster ovary celI is to use lipofection, carries out with Lipofectin (GiBco Life), and transfection is after 48 hours, and through the lasting G418 pressurization screening in 2-3 week, collecting cell and cell conditioned medium are measured the expressing protein enzyme activity.Remove G418, continuous passage is cultivated; To mixing the clone cell Method of Limited Dilution, select to have the cell subclone of higher protein-active.The above-mentioned positive subclone of a large amount of according to a conventional method cultivations.After 48 hours, beginning collecting cell and supernatant are with ultrasonic degradation method smudge cells.With 50mMTrisHCl (pH7.6) solution that contains 0.05%Triton is balance liquid and elutriant, uses through the SuperdexG-75 of pre-equilibration post and collects above-mentioned proteic active peak.Using 50mMTrisHCl (pH8.0) equilibrated DEAE-Sepharose post again, is that elutriant carries out gradient elution with 50mMTrisHCl (pH8.0) solution that contains 0-1M NaCl, collects above-mentioned proteic active peak.Be that dialyzate is dialysed to expressing protein solution with PBS (pH7.4) then.Last freeze-drying is preserved.

Embodiment 7

Homology relatively

In Non-redundantGenBank+EMBL+DDBJ+PDB database and Non-redundant GenBank CDSranslations+PDB+SwissProt+Spupdate+PIR database, carry out nucleic acid and albumen homology retrieval with hSTKa of the present invention and hSTKc albumen (SEQ ID NO.2 and SEQ ID NO.4) with BLAST software.Found that the serine/threonine kinase protein family member (as CAA64382, JC1446 and CAA07196) that it and some have been identified has certain homology, and have serine/threonine kinase protein family member's constitutional features in its structure.

Serine/threonine kinase protein family member's constitutional features is [LIVMFYC]-x-[HY]-x-D-[LIVMFY]-K-x (2)-N-[LIVMFYCT] (3). wherein, [LIVMFYC] is meant from LIVMFYC and wherein chooses one wantonly the amino acid, x is an arbitrary amino acid, and x (2) is any 2 amino acid.It is as follows that albumen of the present invention belongs to this structure:

HSTKa:RLEKTLGKGQTGLVKLGVHCVTCQKVAIKIVNREKLSESVLMKVEREIAI LKLIEHPHVLKLHDVYENKKYLYLVLEHVSGGELFDYLVKKGRLTPKEARKFFRQI ISALDFCHSHSICHRDLKPENLLLDEKNNIRIADFGMASLQVGDSLLETSCGSPHY ACPEVIRGEKYDGRKADVWSCGVILFALLVGALPFDDDNLRQLLEKVKRGVFHMPH FIPPDCQSLLRGMSEVDAARRLTLEHIQKHIWY (19-270 among the SEQ ID NO.2);

HSTKc:RLEKTLGKGQTGLVKLGVHCVTCQKVAIKIVNREKLSESVLMKVEREIAI LKLIEHPHVLKLHDVYENKKYLYLVLEHVSGGELFDYLVKKGRLTPKEARKFFRQI ISALDFCHSHSICHRDLKPENLLLDEKNNIRIADFGMASLQVGDSLLETSCGSPHY ACPEVIRGEKYDGRKADVWSCGVILFALLVGALPFDDDNLRQLLEKVKRGVFHMPH FIPPDCQSLLRGMSEVDAARRLTLEHIQKHIWY (19-270 among the SEQ ID NO.4) ( Htttp: //smart.embl-heidelberg.de/smart/show motifs.pl)

HSTKa of the present invention and hSTKc are used for further functional study except can be used as this family's a member, also can be used for producing fusion rotein with other albumen, such as producing fusion rotein with immunoglobulin (Ig).In addition, hSTKa of the present invention and hSTKc can also merge or exchange fragment with other members of this family, to produce new albumen, for example the N end of hSTKa of the present invention and hSTKc and the N end of mouse STK can be exchanged, to produce the albumen that new activity is higher or have new features.

At the antibody of hSTKa of the present invention and hSTKc, be used to screen other members of this family, perhaps be used for affinity purification associated protein (as other members of this family).

Embodiment 8

Preparation antibody

HSTKa of the present invention and hSTKc recombinant protein are used for immune animal to produce antibody, specific 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 hSTK gene translation product with it.

Embodiment 9

Substrate as micromatrix

Micromatrix is called the DNA chip again.(, select by using some famous biosoftwares hSTK full-length cDNA, EST or gene fragment substrate samples as micromatrix as LASERGENESOFTWARE (DNASTAR).By using ink-jet technology and a series of chemical process such as ray, chemistry, thermodynamics, mechanical means etc. that sample is fixed on the carrier,, obtain chip (Schena, M.et al. (1995) Science 270:467-470 then as on glass; AndShalon, D.et al. (1996) Genome Res.6:639-645.), the element of formation has point-like, bar shaped or the like.A typical chip contains the element of some amount usually.Behind the hybridization, the unconjugated probe of flush away detects the element of generation hybridization and the degree of reaction with scanner then.The complementarity of probe and each chip component and binding capacity all can be judged by the analysis to the image that scans.

Results of hybridization can be used for detecting hSTKa and hSTKc genovariation, sudden change and polymorphism, understands because hSTKa and hSTKc express the molecular basis of the undesired disease that causes, diagnoses the illness, and can improve and monitor the activity of related agents.

Embodiment 10:

The preparation of test kit

Test kit 1:

The primer that it contains (1) specific amplification hSTKa and hSTKc to and operation instruction.This test kit also can contain or not contain the required reagent that the mRNA reverse transcription is become cDNA.Wherein, the sequence of this Auele Specific Primer is derived from the sequence of SEQ IDNO:1.The cDNA that reverse transcription is become carries out specific amplification, whether to contain the nucleotide sequence of hSTKa and hSTKc in the test sample.This test kit also can contain and carries out other required reagent of PCR reaction, for example damping fluid etc.

In addition, preferably, at least one used primer has been crossed over two exons, because will not produce amplified production corresponding to the genome sequence of hSTKa and hSTKc this moment.

Test kit 2:

This test kit contains the specific antibody (for example polyclonal antibody of preparation among the embodiment 5, the perhaps anti-hSTKa of the hybridoma technology generation of usefulness standard and the monoclonal antibody of hSTKc) at hSTKa and hSTKc, and operation instruction.Whether this test kit is used for direct test sample and exists or lack hSTKa and hSTKc albumen.Form immunocomplex by specific immune response earlier, detect immunocomplex with routine techniques then.

Test kit 3:

This test kit contains can be specifically and the probe of the mRNA hybridization of hSTKa and hSTKc.It also can contain or not contain hybridization buffer.This test kit comes whether to exist in the test sample nucleic acid molecule of hSTKa and hSTKc by the hybridization between nucleic acid molecule and hSTKa and the hSTKc specific probe.

Test kit also can be used for detecting genovariation, sudden change and polymorphism, and detects the relevant gene of hSTK a series of and of the present invention, thereby diagnosis, the treatment of relative disease helped out.

Example 11:

Make medicine

HSTKa of the present invention and hSTKc albumen and antibody, inhibitor, antagonist or acceptor etc. can be used as medicine, can promote the wound healing after scalds and burns, cutting tissue and even organ are extractd, and prevent wound infection.With hSTKa of the present invention and hSTKc nucleic acid antisense strand or hSTKa and hSTKc antibody generate a reagent box can be used for alleviating or assisting therapy owing to hSTKa and the too high disease that causes of hSTKc expression amount, also can assist the diagnosis of tumour and cancer clinically, prevention and treatment.

Albumen of the present invention and antibody thereof, inhibitor, 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 generally about 5-8, 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, subcutaneous, intracutaneous or topical.

In addition, hSTKa of the present invention and hSTKc nucleic acid (encoding sequence or antisense sequences) can directly be introduced cell, with expression level that improves hSTKa and hSTKc or the overexpression that suppresses hSTKa and hSTKc.HSTKa of the present invention and hSTKc albumen or its active polypeptide fragment can be applied to patient, with treatment or alleviate because of hSTKa and hSTKc disappearance, no function or unusual cause related disorders arranged.In addition, can also be with carrying out relevant diagnosis or prognosis judgement based on nucleotide sequence of the present invention or antibody.

When hSTKa of the present invention and hSTKc protein polypeptide are used as medicine, the treatment effective dose 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.

Sequence table＜210〉1＜211〉2032＜212〉nucleotides＜213〉human＜220〉＜221〉coded sequence＜222〉(3) .. (2009)＜223〉＜400〉1cg atg aca tcg acg ggg aag gac ggc ggc gcg cag cac gcg cag tat, 47 Met Thr Ser Thr Gly Lys Asp Gly Gly Ala Gln His Ala Gln Tyr, 15 10 15gtt ggg ccc tac cgg ctg gag aag acg ctg ggc aag ggg cag aca ggt 95Val Gly Pro Tyr Arg Leu Glu Lys Thr Leu Gly Lys Gly Gln Thr Gly

20 25 30ctg?gtg?aag?ctg?ggg?gtt?cac?tgc?gtc?acc?tgc?cag?aag?gtg?gcc?atc 143Leu?Val?Lys?Leu?Gly?Val?His?Cys?Val?Thr?Cys?Gln?Lys?Val?Ala?Ile

35 40 45aag?atc?gtc?aac?cgt?gag?aag?ctc?agc?gag?tcg?gtg?ctg?atg?aag?gtg 191Lys?Ile?Val?Asn?Arg?Glu?Lys?Leu?Ser?Glu?Ser?Val?Leu?Met?Lys?Val

50 55 60gag?cgg?gag?atc?gcg?atc?ctg?aag?ctc?att?gag?cac?ccc?cac?gtc?cta 239Glu?Arg?Glu?Ile?Ala?Ile?Leu?Lys?Leu?Ile?Glu?His?Pro?His?Val?Leu

65 70 75aag?ctg?cac?gac?gtt?tat?gaa?aac?aaa?aaa?tat?ttg?tac?ctg?gtg?cta 287Lys?Leu?His?Asp?Val?Tyr?Glu?Asn?Lys?Lys?Tyr?Leu?Tyr?Leu?Val?Leu80 85 90 95gaa?cac?gtg?tca?ggt?ggt?gag?ctc?ttc?gac?tac?ctg?gtg?aag?aag?ggg 335Glu?His?Val?Ser?Gly?Gly?Glu?Leu?Phe?Asp?Tyr?Leu?Val?Lys?Lys?Gly

100 105 110agg?ctg?acg?cct?aag?gag?gct?cgg?aag?ttc?ttc?cgg?cag?atc?atc?tct 383Arg?Leu?Thr?Pro?Lys?Glu?Ala?Arg?Lys?Phe?Phe?Arg?Gln?Ile?Ile?Ser

115 120 125gcg?ctg?gac?ttc?tgc?cac?agc?cac?tcc?ata?tgc?cac?agg?gat?ctg?aaa 431Ala?Leu?Asp?Phe?Cys?His?Ser?His?Ser?Ile?Cys?His?Arg?Asp?Leu?Lys

130 135 140cct?gaa?aac?ctc?ctg?ctg?gac?gag?aag?aac?aac?atc?cgc?atc?gca?gac 479Pro?Glu?Asn?Leu?Leu?Leu?Asp?Glu?Lys?Asn?Asn?Ile?Arg?Ile?Ala?Asp

145 150 155ttt?ggc?atg?gcg?tcc?ctg?cag?gtt?ggc?gac?agc?ctg?ttg?gag?acc?agc 527Phe?Gly?Met?Ala?Ser?Leu?Gln?Val?Gly?Asp?Ser?Leu?Leu?Glu?Thr?Ser160 165 170 175tgt?ggg?tcc?ccc?cac?tac?gcc?tgc?ccc?gag?gtg?atc?cgg?ggg?gag?aag 575Cys?Gly?Ser?Pro?His?Tyr?Ala?Cys?Pro?Glu?Val?Ile?Arg?Gly?Glu?Lys

180 185 190tat?gac?ggc?cgg?aag?gcg?gac?gtg?tgg?agc?tgc?ggc?gtc?atc?ctg?ttc 623Tyr?Asp?Gly?Arg?Lys?Ala?Asp?Val?Trp?Ser?Cys?Gly?Val?Ile?Leu?Phe

195 200 205gcc?ttg?ctg?gtg?ggg?gct?ctg?ccc?ttc?gac?gat?gac?aac?ttg?cga?cag 671Ala?Leu?Leu?Val?Gly?Ala?Leu?Pro?Phe?Asp?Asp?Asp?Asn?Leu?Arg?Gln

210 215 220ctg?ctg?gag?aag?gtg?aag?cgg?ggc?gtg?ttc?cac?atg?ccg?cac?ttt?atc 719Leu?Leu?Glu?Lys?Val?Lys?Arg?Gly?Val?Phe?His?Met?Pro?His?Phe?Ile

225 230 235ccg?ccc?gac?tgc?cag?agt?ctg?cta?cgg?ggc?atg?agc?gag?gtg?gac?gcc 767Pro?Pro?Asp?Cys?Gln?Ser?Leu?Leu?Arg?Gly?Met?Ser?Glu?Val?Asp?Ala240 245 250 255gca?cgc?cgc?ctc?acg?cta?gag?cac?att?cag?aaa?cac?ata?tgg?tat?ata 815Ala?Arg?Arg?Leu?Thr?Leu?Glu?His?Ile?Gln?Lys?His?Ile?Trp?Tyr?Ile

260 265 270ggg?ggc?aag?aat?gag?ccc?gaa?cca?gag?cag?ccc?att?cct?cgc?aag?gtg 863Gly?Gly?Lys?Asn?Glu?Pro?Glu?Pro?Glu?Gln?Pro?Ile?Pro?Arg?Lys?Val

275 280 285cag?atc?cgc?tcg?ctg?ccc?agc?ctg?gag?gac?atc?gac?ccc?gac?gtg?ctg 911Gln?Ile?Arg?Ser?Leu?Pro?Ser?Leu?Glu?Asp?Ile?Asp?Pro?Asp?Val?Leu

290 295 300gac?agc?atg?cac?tca?ctg?ggc?tgc?ttc?cga?gac?cgc?aac?aag?ctg?ctg 959Asp?Ser?Met?His?Ser?Leu?Gly?Cys?Phe?Arg?Asp?Arg?Asn?Lys?Leu?Leu

305 310 315cag?gac?ctg?ctg?tcc?gag?gag?gag?aac?cag?gag?aag?atg?att?tac?ttc 1007Gln?Asp?Leu?Leu?Ser?Glu?Glu?Glu?Asn?Gln?Glu?Lys?Met?Ile?Tyr?Phe320 325 330 335ctc?ctc?ctg?gac?cgg?aaa?gaa?agg?tac?ccg?agc?cag?gag?gat?gag?gac 1055Leu?Leu?Leu?Asp?Arg?Lys?Glu?Arg?Tyr?Pro?Ser?Gln?Glu?Asp?Glu?Asp

340 345 350ctg?ccc?ccc?cgg?aac?gag?ata?gac?cct?ccc?cgg?aag?cgt?gtg?gac?tcc 1103Leu?Pro?Pro?Arg?Asn?Glu?Ile?Asp?Pro?Pro?Arg?Lys?Arg?Val?Asp?Ser

355 360 365ccg?atg?ctg?aac?cgg?cac?ggc?aag?cgg?cgg?cca?gaa?cgc?aaa?tcc?atg 1151Pro?Met?Leu?Asn?Arg?His?Gly?Lys?Arg?Arg?Pro?Glu?Arg?Lys?Ser?Met

370 375 380gag?gtg?ctc?agc?gtg?acg?gac?ggc?ggc?tcc?ccg?gtg?cct?gcg?cgg?cgg 1199Glu?Val?Leu?Ser?Val?Thr?Asp?Gly?Gly?Ser?Pro?Val?Pro?Ala?Arg?Arg

385 390 395gcc?att?gag?atg?gcc?cag?cac?ggc?cag?agg?tct?cgg?tcc?atc?agc?ggt 1247Ala?Ile?Glu?Met?Ala?Gln?His?Gly?Gln?Arg?Ser?Arg?Ser?Ile?Ser?Gly400 405 410 415gcc?tcc?tca?ggc?ctt?tcc?acc?agc?cca?ctc?agc?agc?ccc?cgg?gtg?acc 1295Ala?Ser?Ser?Gly?Leu?Ser?Thr?Ser?Pro?Leu?Ser?Ser?Pro?Arg?Val?Thr

420 425 430cct?cac?ccc?tca?cca?agg?ggc?agt?ccc?ctc?ccc?acc?ccc?aag?ggg?aca 1343Pro?His?Pro?Ser?Pro?Arg?Gly?Ser?Pro?Leu?Pro?Thr?Pro?Lys?Gly?Thr

435 440 445cct?gtc?cac?acg?cca?aag?gag?agc?ccg?gct?ggc?acg?ccc?aac?ccc?acg 1391Pro?Val?His?Thr?Pro?Lys?Glu?Ser?Pro?Ala?Gly?Thr?Pro?Asn?Pro?Thr

450 455 460ccc?ccg?tcc?agc?ccc?agc?gtc?gga?ggg?gtg?ccc?tgg?agg?gcg?cgg?ctc 1439Pro?Pro?Ser?Ser?Pro?Ser?Val?Gly?Gly?Val?Pro?Trp?Arg?Ala?Arg?Leu

465 470 475aac?tcc?atc?aag?aac?agc?ttt?ctg?ggc?tca?ccc?cgc?ttc?cac?cgc?cga 1487Asn?Ser?Ile?Lys?Asn?Ser?Phe?Leu?Gly?Ser?Pro?Arg?Phe?His?Arg?Arg480 485 490 495aaa?ctg?caa?gtt?ccg?acg?ccg?gag?gag?atg?tcc?aac?ctg?aca?cca?gag 1535Lys?Leu?Gln?Val?Pro?Thr?Pro?Glu?Glu?Met?Ser?Asn?Leu?Thr?Pro?Glu

500 505 510tcg?tcc?cca?gag?ctg?gcg?aag?aag?tcc?tgg?ttt?ggg?aac?ttc?atc?agc 1583Ser?Ser?Pro?Glu?Leu?Ala?Lys?Lys?Ser?Trp?Phe?Gly?Asn?Phe?Ile?Ser

515 520 525ctg?gag?aag?gag?gag?cag?atc?ttc?gtg?gtc?atc?aaa?gac?aaa?cct?ctg 1631Leu?Glu?Lys?Glu?Glu?Gln?Ile?Phe?Val?Val?Ile?Lys?Asp?Lys?Pro?Leu

530 535 540agc?tcc?atc?aag?gct?gac?atc?gtg?cac?gcc?ttc?ctg?tcg?att?ccc?agt 1679Ser?Ser?Ile?Lys?Ala?Asp?Ile?Val?His?Ala?Phe?Leu?Ser?Ile?Pro?Ser

545 550 555ctc?agc?cac?agc?gtc?atc?tcc?caa?acg?agc?ttc?cgg?gcc?gag?tac?aag 1727Leu?Ser?His?Ser?Val?Ile?Ser?Gln?Thr?Ser?Phe?Arg?Ala?Glu?Tyr?Lys560 565 570 575gcc?acg?ggg?ggg?cca?gcc?gtg?ttc?cag?aag?ccg?gtc?aag?ttc?cag?gtt 1775Ala?Thr?Gly?Gly?Pro?Ala?Val?Phe?Gln?Lys?Pro?Val?Lys?Phe?Gln?Val

580 585 590gat?atc?acc?tac?acg?gag?ggt?ggg?gag?gcg?cag?aag?gag?aac?ggc?atc 1823Asp?Ile?Thr?Tyr?Thr?Glu?Gly?Gly?Glu?Ala?Gln?Lys?Glu?Asn?Gly?Ile

595 600 605tac?tcc?gtc?acc?ttc?acc?ctg?ctc?tca?ggc?ccc?agc?cgt?cgc?ttc?aag 1871Tyr?Ser?Val?Thr?Phe?Thr?Leu?Leu?Ser?Gly?Pro?Ser?Arg?Arg?Phe?Lys

610 615 620agg?gtg?gtg?gag?acc?atc?cag?gcc?cag?ctg?ctg?agc?aca?cac?gac?ccg 1919Arg?Val?Val?Glu?Thr?Ile?Gln?Ala?Gln?Leu?Leu?Ser?Thr?His?Asp?Pro

625 630 635cct?gcg?gcc?cag?cac?ttg?tca?gac?acc?act?aac?tgt?atg?gaa?atg?atg 1967Pro?Ala?Ala?Gln?His?Leu?Ser?Asp?Thr?Thr?Asn?Cys?Met?Glu?Met?Met640 645 650 655acg?ggg?cgg?ctt?tcc?aaa?tgt?gga?att?atc?ccg?aaa?agt?taa 2009Thr?Gly?Arg?Leu?Ser?Lys?Cys?Gly?Ile?Ile?Pro?Lys?Ser

660 665catgtcacct ccacgaggcc atc 2032＜210〉2＜211〉668＜212〉amino acid＜213〉mankind＜400〉2Met Thr Ser Thr Gly Lys Asp Gly Gly Ala Gln His Ala Gln Tyr Val1 5 10 15Gly Pro Tyr Arg Leu Glu Lys Thr Leu Gly Lys Gly Gln Thr Gly Leu

20 25 30Val?Lys?Leu?Gly?Val?His?Cys?Val?Thr?Cys?Gln?Lys?Val?Ala?Ile?Lys

35 40 45Ile?Val?Asn?Arg?Glu?Lys?Leu?Ser?Glu?Ser?Val?Leu?Met?Lys?Val?Glu

50 55 60Arg?Glu?Ile?Ala?Ile?Leu?Lys?Leu?Ile?Glu?His?Pro?His?Val?Leu?Lys65 70 75 80Leu?His?Asp?Val?Tyr?Glu?Asn?Lys?Lys?Tyr?Leu?Tyr?Leu?Val?Leu?Glu

85 90 95His?Val?Ser?Gly?Gly?Glu?Leu?Phe?Asp?Tyr?Leu?Val?Lys?Lys?Gly?Arg

100 105 110Leu?Thr?Pro?Lys?Glu?Ala?Arg?Lys?Phe?Phe?Arg?Gln?Ile?Ile?Ser?Ala

115 120 125Leu?Asp?Phe?Cys?His?Ser?His?Ser?Ile?Cys?His?Arg?Asp?Leu?Lys?Pro

130 135 140Glu?Asn?Leu?Leu?Leu?Asp?Glu?Lys?Asn?Asn?Ile?Arg?Ile?Ala?Asp?Phe145 150 155 160Gly?Met?Ala?Ser?Leu?Gln?Val?Gly?Asp?Ser?Leu?Leu?Glu?Thr?Ser?Cys

165 170 175Gly?Ser?Pro?His?Tyr?Ala?Cys?Pro?Glu?Val?Ile?Arg?Gly?Glu?Lys?Tyr

180 185 190Asp?Gly?Arg?Lys?Ala?Asp?Val?Trp?Ser?Cys?Gly?Val?Ile?Leu?Phe?Ala

195 200 205Leu?Leu?Val?Gly?Ala?Leu?Pro?Phe?Asp?Asp?Asp?Asn?Leu?Arg?Gln?Leu

210 215 220Leu?Glu?Lys?Val?Lys?Arg?Gly?Val?Phe?His?Met?Pro?His?Phe?Ile?Pro225 230 235 240Pro?Asp?Cys?Gln?Ser?Leu?Leu?Arg?Gly?Met?Ser?Glu?Val?Asp?Ala?Ala

245 250 255Arg?Arg?Leu?Thr?Leu?Glu?His?Ile?Gln?Lys?His?Ile?Trp?Tyr?Ile?Gly

260 265 270Gly?Lys?Asn?Glu?Pro?Glu?Pro?Glu?Gln?Pro?Ile?Pro?Arg?Lys?Val?Gln

275 280 285Ile?Arg?Ser?Leu?Pro?Ser?Leu?Glu?Asp?Ile?Asp?Pro?Asp?Val?Leu?Asp

290 295 300Ser?Met?His?Ser?Leu?Gly?Cys?Phe?Arg?Asp?Arg?Asn?Lys?Leu?Leu?Gln305 310 315 320Asp?Leu?Leu?Ser?Glu?Glu?Glu?Asn?Gln?Glu?Lys?Met?Ile?Tyr?Phe?Leu

325 330 335Leu?Leu?Asp?Arg?Lys?Glu?Arg?Tyr?Pro?Ser?Gln?Glu?Asp?Glu?Asp?Leu

340 345 350Pro?Pro?Arg?Asn?Glu?Ile?Asp?Pro?Pro?Arg?Lys?Arg?Val?Asp?Ser?Pro

355 360 365Met?Leu?Asn?Arg?His?Gly?Lys?Arg?Arg?Pro?Glu?Arg?Lys?Ser?Met?Glu

370 375 380Val?Leu?Ser?Val?Thr?Asp?Gly?Gly?Ser?Pro?Val?Pro?Ala?Arg?Arg?Ala385 390 395 400Ile?Glu?Met?Ala?Gln?His?Gly?Gln?Arg?Ser?Arg?Ser?Ile?Ser?Gly?Ala

405 410 415Ser?Ser?Gly?Leu?Ser?Thr?Ser?Pro?Leu?Ser?Ser?Pro?Arg?Val?Thr?Pro

420 425 430His?Pro?Ser?Pro?Arg?Gly?Ser?Pro?Leu?Pro?Thr?Pro?Lys?Gly?Thr?Pro

435 440 445Val?His?Thr?Pro?Lys?Glu?Ser?Pro?Ala?Gly?Thr?Pro?Asn?Pro?Thr?Pro

450 455 460Pro?Ser?Ser?Pro?Ser?Val?Gly?Gly?Val?Pro?Trp?Arg?Ala?Arg?Leu?Asn465 470 475 480Ser?Ile?Lys?Asn?Ser?Phe?Leu?Gly?Ser?Pro?Arg?Phe?His?Arg?Arg?Lys

485 490 495Leu?Gln?Val?Pro?Thr?Pro?Glu?Glu?Met?Ser?Asn?Leu?Thr?Pro?Glu?Ser

500 505 510Ser?Pro?Glu?Leu?Ala?Lys?Lys?Ser?Trp?Phe?Gly?Asn?Phe?Ile?Ser?Leu

515 520 525Glu?Lys?Glu?Glu?Gln?Ile?Phe?Val?Val?Ile?Lys?Asp?Lys?Pro?Leu?Ser

530 535 540Ser?Ile?Lys?Ala?Asp?Ile?Val?His?Ala?Phe?Leu?Ser?Ile?Pro?Ser?Leu545 550 555 560Ser?His?Ser?Val?Ile?Ser?Gln?Thr?Ser?Phe?Arg?Ala?Glu?Tyr?Lys?Ala

565 570 575Thr?Gly?Gly?Pro?Ala?Val?Phe?Gln?Lys?Pro?Val?Lys?Phe?Gln?Val?Asp

580 585 590Ile?Thr?Tyr?Thr?Glu?Gly?Gly?Glu?Ala?Gln?Lys?Glu?Asn?Gly?Ile?Tyr

595 600 605Ser?Val?Thr?Phe?Thr?Leu?Leu?Ser?Gly?Pro?Ser?Arg?Arg?Phe?Lys?Arg

610 615 620Val?Val?Glu?Thr?Ile?Gln?Ala?Gln?Leu?Leu?Ser?Thr?His?Asp?Pro?Pro625 630 635 640Ala?Ala?Gln?His?Leu?Ser?Asp?Thr?Thr?Asn?Cys?Met?Glu?Met?Met?Thr

645 650 655Gly?Arg?Leu?Ser?Lys?Cys?Gly?Ile?Ile?Pro?Lys?Ser

660 665＜210〉3＜211〉2223＜212〉nucleotides＜213〉human＜220〉＜221〉coded sequence＜222〉(3) .. (2213)＜223〉＜400〉3cg atg aca tcg acg ggg aag gac ggc ggc gcg cag cac gcg cag tat, 47 Met Thr Ser Thr Gly Lys Asp Gly Gly Ala Gln His Ala Gln Tyr, 15 10 15gtt ggg ccc tac cgg ctg gag aag acg ctg ggc aag ggg cag aca ggt 95Val Gly Pro Tyr Arg Leu Glu Lys Thr Leu Gly Lys Gly Gln Thr Gly

625 630 635cct?gcg?gcc?cag?cac?ttg?tca?gac?acc?act?aac?tgt?atg?gaa?atg?atg 1967Pro?Ala?Ala?Gln?His?Leu?Ser?Asp?Thr?Thr?Asn?Cys?Met?Glu?Met?Met640 645 650 655acg?ggg?cgg?ctt?tcc?aaa?tgt?ggc?agc?cca?ttg?agt?aac?ttc?ttt?gac 2015Thr?Gly?Arg?Leu?Ser?Lys?Cys?Gly?Ser?Pro?Leu?Ser?Asn?Phe?Phe?Asp

660 665 670gta?att?aaa?caa?ctt?ttt?tca?gac?gag?aag?aac?ggg?cag?gcg?gcc?cag 2063Val?Ile?Lys?Gln?Leu?Phe?Ser?Asp?Glu?Lys?Asn?Gly?Gln?Ala?Ala?Gln

675 680 685gcc?ccc?agc?acg?ccc?gcc?aag?cgg?agt?gcc?cac?ggc?cca?ctc?ggt?gac 2111Ala?Pro?Ser?Thr?Pro?Ala?Lys?Arg?Ser?Ala?His?Gly?Pro?Leu?Gly?Asp

690 695 700tcc?gcg?gcc?gct?ggc?cct?ggc?ccc?gga?ggg?gac?gcc?gag?tac?cca?acg 2159Ser?Ala?Ala?Ala?Gly?Pro?Gly?Pro?Gly?Gly?Asp?Ala?Glu?Tyr?Pro?Thr

705 710 715ggc aag gac acg gcc aag atg ggc ccg ccc acc gcc cgc cgc gag cag 2207Gly Lys Asp Thr Ala Lys Met Gly Pro Pro Thr Ala Arg Arg Glu Gln720,725 730 735cct tag acacactagc 2223Pro＜210〉4＜211〉736＜212〉amino acid＜213〉mankind＜400〉4Met Thr Ser Thr Gly Lys Asp Gly Gly Ala Gln His Ala Gln Tyr Val1 5 10 15Gly Pro Tyr Arg Leu Glu Lys Thr Leu Gly Lys Gly Gln Thr Gly Leu

20 25 30Val?Lys?Leu?Gly?Val?His?Cys?Val?Thr?Cys?Gln?Lys?Val?Ala?Ile?Lys

35 40 45Ile?Val?Asn?Arg?Glu?Lys?Leu?Ser?Glu?Ser?Val?Leu?Met?Lys?Val?Glu

85 90 95His?Val?Ser?Gly?Gly?Glu?Leu?Phe?Asp?Tyr?Leu?Val?Lys?Lys?Gly?Arg

100 105 110Leu?Thr?Pro?Lys?Glu?Ala?Arg?Lys?Phe?Phe?Arg?Gln?Ile?Ile?Ser?Ala

115 120 125Leu?Asp?Phe?Cys?His?Ser?His?Ser?Ile?Cys?His?Arg?Asp?Leu?Lys?Pro

165 170 175Gly?Ser?Pro?His?Tyr?Ala?Cys?Pro?Glu?Val?Ile?Arg?Gly?Glu?Lys?Tyr

180 185 190Asp?Gly?Arg?Lys?Ala?Asp?Val?Trp?Ser?Cys?Gly?Val?Ile?Leu?Phe?Ala

195 200 205Leu?Leu?Val?Gly?Ala?Leu?Pro?Phe?Asp?Asp?Asp?Asn?Leu?Arg?Gln?Leu

245 250 255Arg?Arg?Leu?Thr?Leu?Glu?His?Ile?Gln?Lys?His?Ile?Trp?Tyr?Ile?Gly

260 265 270Gly?Lys?Asn?Glu?Pro?Glu?Pro?Glu?Gln?Pro?Ile?Pro?Arg?Lys?Val?Gln

275 280 285Ile?Arg?Ser?Leu?Pro?Ser?Leu?Glu?Asp?Ile?Asp?Pro?Asp?Val?Leu?Asp

325 330 335Leu?Leu?Asp?Arg?Lys?Glu?Arg?Tyr?Pro?Ser?Gln?Glu?Asp?Glu?Asp?Leu

340 345 350Pro?Pro?Arg?Asn?Glu?Ile?Asp?Pro?Pro?Arg?Lys?Arg?Val?Asp?Ser?Pro

355 360 365Met?Leu?Asn?Arg?His?Gly?Lys?Arg?Arg?Pro?Glu?Arg?Lys?Ser?Met?Glu

405 410 415Ser?Ser?Gly?Leu?Ser?Thr?Ser?Pro?Leu?Ser?Ser?Pro?Arg?Val?Thr?Pro

420 425 430His?Pro?Ser?Pro?Arg?Gly?Ser?Pro?Leu?Pro?Thr?Pro?Lys?Gly?Thr?Pro

435 440 445Val?His?Thr?Pro?Lys?Glu?Ser?Pro?Ala?Gly?Thr?Pro?Asn?Pro?Thr?Pro

485 490 495Leu?Gln?Val?Pro?Thr?Pro?Glu?Glu?Met?Ser?Asn?Leu?Thr?Pro?Glu?Ser

500 505 510Ser?Pro?Glu?Leu?Ala?Lys?Lys?Ser?Trp?Phe?Gly?Asn?Phe?Ile?Ser?Leu

515 520 525Glu?Lys?Glu?Glu?Gln?Ile?Phe?Val?Val?Ile?Lys?Asp?Lys?Pro?Leu?Ser

565 570 575Thr?Gly?Gly?Pro?Ala?Val?Phe?Gln?Lys?Pro?Val?Lys?Phe?Gln?Val?Asp

580 585 590Ile?Thr?Tyr?Thr?Glu?Gly?Gly?Glu?Ala?Gln?Lys?Glu?Asn?Gly?Ile?Tyr

595 600 605Ser?Val?Thr?Phe?Thr?Leu?Leu?Ser?Gly?Pro?Ser?Arg?Arg?Phe?Lys?Arg

645 650 655Gly?Arg?Leu?Ser?Lys?Cys?Gly?Ser?Pro?Leu?Ser?Asn?Phe?Phe?Asp?Val

660 665 670Ile?Lys?Gln?Leu?Phe?Ser?Asp?Glu?Lys?Asn?Gly?Gln?Ala?Ala?Gln?Ala

675 680 685Pro?Ser?Thr?Pro?Ala?Lys?Arg?Ser?Ala?His?Gly?Pro?Leu?Gly?Asp?Ser

690 695 700Ala?Ala?Ala?Gly?Pro?Gly?Pro?Gly?Gly?Asp?Ala?Glu?Tyr?Pro?Thr?Gly705 710 715 720Lys?Asp?Thr?Ala?Lys?Met?Gly?Pro?Pro?Thr?Ala?Arg?Arg?Glu?Gln?Pro

725 730 735

Table 1hSTKa MTSTGKDGGAQHAQYVGPYRLEKTLGKGQTGLVKLGVHCVTCGKVAIKIVNREKLS ESVLhSTKc MTSTGKDGGAQHAQYVGPYRLEKTLGKGQTGLVKLGVHCVTCQKVAIKIVNREKLS ESVLCAA07196------------------------------------------------------------hSTKa MKVEREIAILKLIEHPHVLKLHDVYENKKYLYLVLEHVSGGELFDYLVKKGRLTPK EARKhSTKc MKVEREIAILKLIEHPHVLKLHDVYENKKYLYLVLEHVSGGELFDYLVKKGRLTPK EARKCAA07196-----------LIEHPHVLKLHDVYENKKYLYLVLEHVSGGELF DYLVKKGRLTPKEARK

*************************************************hSTKa FFRQIISALDFCHSHSICHRDLKPENLLLDEKNNIRIADFGMASLQVGDSLLETSCGSPHhSTKc FFRQIISALDFCHSHSICHRDLKPENLLLDEKNNIRIADFGMASLQVGDSLLETSCGSPHCAA07196 FFRQIISALDFCHSHSICHRDLKPENLLLDEKNNIRIADFGMASLQVGDSLLETSCGSPH

************************************************************hSTKa YACPEVIRGEKYDGRKADVWSCGVILFALLVGALPFDDDNLRQLLEKVKRGVFHMPHFIPhSTKc YACPEVIRGEKYDGRKADVWSCGVILFALLVGALPFDDDNLRQLLEKVKRGVFHMPHFIPCAA07196 YACPEVIRGEKYDGRKADVWSCGVILFALLVGALPFDDDNLRQLLEKVKRGVFHMPHFIP

************************************************************hSTKa PDCQSLLRGMSEVDAARRLTLEHIQKHIWYIGGKNEPEPEQPIPRKVQIRSLPSLEDIDPhSTKc PDCQSLLRGMSEVDAARRLTLEHIQKHIWYIGGKNEPEPEQPIPRKVQIRSLPSLEDIDPCAA07196 PDCQSLLRGMSEVDAARRLTLEHIQKHIWYIGGKNEPEPEQPIPRKVQIRSLPSLEDIDP

************************************************************hSTKa DVLDSMHSLGCFRDRNKLLGDLLSEEENQEKMIYFLLLDRKERYPSQEDEDLPPRNEIDPhSTKc DVLDSMHSLGCFRDRNKLLQDLLSEEENQEKMIYFLLLDRKERYPSQEDEDLPPRNEIDPCAA07196 DVLDSMHSLGCFRDRNKLLQDLLSEEENQEKMIYFLLLDRKERYPSQEDEDLPPRNEIDP

************************************************************hSTKa PRKRVDSPMLNRHGKRRPERKSMEVLSVTDGGSPVPARRAIEMAQHGQRSRSISGASSGLhSTKc PRKRVDSPMLNRHGKRRPERKSMEVLSVTDGGSPVPARRAIEMAQHGQRSRSISGASSGLCAA07196 PRKRVDSPMLNRHGKRRPERKSMEVLSVTDGGSPVPARRAIEMAQHGQRSRSISGASSGL

************************************************************hSTKa STSPLSSPRVTPHPSPRGSPLPTPKGTPVHTPKESPAGTPNPTPPSSPSVGGVPWRARLNhSTKc STSPLSSPRVTPHPSPRGSPLPTPKGTPVHTPKESPAGTPNPTPPSSPSVGGVPWRARLNCAA07196 STSPLSSPRVTPHPSPRGSPLPTPKGTPVHTPKESPAGTPNPTPPSSPSVGGVPWRARLN

************************************************************hSTKa SIKNSFLGSPRFHRRKLQVPTPEEMSNLTPESSPELAKKSWFGNFISLEKEEQIFVVIKDhSTKc SIKNSFLGSPRFHRRKLQVPTPEEMSNLTPESSPELAKKSWFGNFISLEKEEQIFVVIKDCAA07196 SIKNSFLGSPRFHRRKLQVPTPEEMSNLTPESSPELAKKSWFGNFISLEKEEQIFVVIKD

************************************************************hSTKa KPLSSIKADIVHAFLSIPSLSHSVISQTSFRAEYKATGGPAVFQKPVKFQVDITYTEGGEhSTKc KPLSSIKADIVHAFLSIPSLSHSVISQTSFRAEYKATGGPAVFQKPVKFQVDITYTEGGECAA07196 KPLSSIKADIVHAFLSIPSLSHSVISQTSFRAEYKATGGPAVFQKPVKFQVDITYTEGGE

************************************************************hSTKa AQKENGIYSVTFTLLSGPSRRFKRVVETIQAQLLSTHDPPAAQHLSDTTNCMEMMTGRLShSTKc AQKENGIYSVTFTLLSGPSRRFKRVVETIQAQLLSTHDPPAAQHLSDTTNCMEMMTGRLSCAA07196 AQKENGIYSVTFTLLSGPSRRFKRVVETIQAQLLSTHDPPAAQHLSEPPPPAPGLSWGAG

**********************************************:.. ::hSTKa KCG--------IIPKS--------------------------------------------hSTKc KCGSPLSNFFDVIKQLFSDEKNGQAAQAPSTPAKRSAHGPLGDSAAAGPGPGGDAEYPTGCAA07196 LKG-------QKVATSYESSL---------------------------------------

* :hSTKa ----------------hSTKc KDTAKMGPPTARREQPCAA07196 ----------------

Claims

1. isolated dna molecular is characterized in that it comprises: coding has the nucleotide sequence of hSTK protein kinase polypeptide, and nucleotide sequence has at least 70% homology among described nucleotide sequence and the SEQ ID NO.1; Perhaps described nucleotide sequence can with nucleotide sequence hybridization among the SEQ ID NO.1.

2. dna molecular as claimed in claim 1 is characterized in that, described sequence encoding one polypeptide, and this polypeptide has the sequence shown in the SEQ ID NO.2.

3. dna molecular as claimed in claim 1 is characterized in that, this sequence is a nucleotide sequence among the SEQ ID NO.1.

4. dna molecular as claimed in claim 1 is characterized in that, described sequence encoding one polypeptide, and this polypeptide has the sequence shown in the SEQ ID NO.4.

5. dna molecular as claimed in claim 1 is characterized in that, this sequence is a nucleotide sequence among the SEQ ID NO.3.

6. the hSTK protein polypeptide that separation obtains is characterized in that, it is with SEQ ID NO.2 aminoacid sequence at least 95% homology to be arranged.

7. the hSTK protein polypeptide that separation obtains is characterized in that, it is polypeptide, its active fragments or its reactive derivative with SEQ ID NO.2 aminoacid sequence.

8. polypeptide as claimed in claim 7 is characterized in that, this polypeptide is to have SEQ ID NO.2 polypeptide of sequence.

9. polypeptide as claimed in claim 6 is characterized in that, this polypeptide is to have SEQ ID NO.4 polypeptide of sequence.

10. a carrier is characterized in that, it contains the described DNA of claim 1.

11. one kind with the described carrier transformed host cells of claim 10.

12. host cell as claimed in claim 11 is characterized in that, this cell is intestinal bacteria.

13. host cell as claimed in claim 11 is characterized in that, this cell is an eukaryotic cell.

14. a generation has the method for the polypeptide of hSTK protein-active, it is characterized in that, this method comprises:

(a) will be connected in expression regulation sequence as the described nucleotide sequence of claim 1 to 5, form the hSTK protein expression vector;

(d) isolate polypeptide with hSTK protein-active.

15. energy and the described hSTK protein polypeptide of claim 6 specificity bonded antibody.

16. a nucleic acid molecule is characterized in that, it is the antisense sequences of the described dna molecular of claim 1.

17. the application of the described dna molecular of claim 1 is characterized in that, it is used for nucleic acid amplification reaction as primer or with making gene chip; Perhaps be used to prepare medicine.

18. the application of the described protein polypeptide of claim 6 is characterized in that, it is applied to screen the agonist or the inhibitor of human serine/threonine kinase; Perhaps be used to prepare medicine.