CN1259573A - New human serine threonine protein kinase, its code sequence, prepn. and use thereof - Google Patents

Abstract

The present invention relates to new member STKS of serine/threonine protein kinase gene family. It provides cDNA coding sequence of said new serine/threonine protein kinase, polypeptide of said sequencial code, and method of utilizing recombination technology to produce said new human serine/threonine protein kinase. The present invention also provides the application of said new human serine/threonine protein kinase and its coding sequence.

Description

New human serine threonine protein kinase, its encoding sequence and method for making and purposes

The present invention relates to a kind of new polynucleotide, by the polypeptide of this polynucleotide encoding, the application of these polynucleotide and polypeptide, and the production of described polynucleotide and described polypeptide.More particularly, polypeptide of the present invention is accredited as a newcomer of serine/threonine protein kitase family by deduction.

Serine/threonine protein kitase (serine/threonine kinase, be called for short " STK ") extensively be 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.

1989, people such as Flusser G found to exist a kind of albumen, this albumen to belong to serine/threonine protein kitase family in the liver of rat, and its expression is subjected to adjusting (Mol Cell Endocrinol.1989,64 (2): 213-22) of glucocorticosteroid.1993, people such as Webster M.K. separate member's serum glucocorticosteroid adjusting protein kinase (the serum-andglucocorticoid regulated kinase that has obtained serine/threonine protein kitase family in the rat body, be called for short SGK) and clone (Mol Cell Biol.1993,13 (4): 2031-40).1997, newcomer's human serum glucocorticosteroid that people such as Waldegger S. clone in the human body has obtained serine/threonine protein kitase family is regulated protein kinase (h-SGK) (Proc Natl Acad Sci U S A.1997,94 (9): 4440-5).Recently, people such as Waldegger S. has also obtained another newcomer (Pflugers Arch.1998,436 (4): 575-80) of this family from the shark vivo clone.

But before the application, there be not openly or delivered other human serine/threonine protein kitase albumen or gene.

An object of the present invention is to provide a kind of new polynucleotide, a newcomer of this polynucleotide encoding serine/threonine protein kitase gene family, the new human serine/threonine protein kitase gene of the present invention is named as STK3.

Another object of the present invention provides a kind of new serine/threonine protein kitase protein family member, and this enzyme is named as STK3.

A further object of the present invention provides a kind of method of utilizing recombinant technology to produce described new human serine/threonine protein kitase STK3.

The invention still further relates to the application of this human serine/threonine protein kitase STK3 nucleotide sequence and polypeptide.

In one aspect of the invention, a kind of isolated dna molecular is provided, it comprises: coding has the nucleotide sequence of the polypeptide of people STK3 protein-active, shows at least 70% homology from the nucleotides sequence of Nucleotide 88-1377 position among described nucleotide sequence and the SEQ ID NO.3; Perhaps described nucleotide sequence can be under the moderate stringent condition with SEQ ID NO.3 in from the nucleotide sequence hybridization of Nucleotide 88-1377 position.Preferably, described sequence encoding one polypeptide, this polypeptide has the sequence shown in the SEQ ID NO.4.More preferably, this sequence has among the SEQ ID NO.3 nucleotide sequence from Nucleotide 88-1377 position.

In another aspect of this invention, provide a kind of isolating STK3 protein polypeptide, it comprises: have polypeptide or its conservative property variation polypeptide or its active fragments of SEQID NO.4 aminoacid sequence, or its reactive derivative.Preferably, this polypeptide is to have 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 STK3 protein-active, this method comprises:

(a) nucleotide sequence that coding is had a polypeptide of STK3 protein-active operationally is connected in expression regulation sequence, form the STK3 protein expression vector, show at least 70% homology from the nucleotides sequence of Nucleotide 88-1377 position among described nucleotide sequence and the SEQ ID NO.3;

(b) change the expression vector in the step (a) over to host cell, form the proteic reconstitution cell of STK3;

(c) be fit to express under the condition of STK3 protein polypeptide the reconstitution cell in the culturing step (b);

(d) isolate polypeptide with STK3 protein-active.

In a specific embodiments of the present invention, isolating polynucleotide total length of the present invention is 583 Nucleotide, and its detailed sequence is seen SEQ ID NO.3, and wherein open reading frame is positioned at 88-1377 position Nucleotide.

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 " STK3 albumen (or polypeptide) encoding sequence " refer to the encode nucleotide sequence of polypeptide with STK3 protein-active is as 88-1377 position nucleotide sequence and degenerate sequence thereof among the SEQ ID NO.3.This degenerate sequence is meant, is arranged in the encoder block 88-1377 position Nucleotide of SEQ ID NO.3 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.3 in 88-1377 position nucleotide sequence homology be low to moderate about 70% the degenerate sequence described sequence of SEQ ID NO.4 of also encoding out.This term also comprises can be under the moderate stringent condition, preferably under the height stringent condition with SEQ ID NO.3 in from the nucleotide sequence of the nucleotide sequence hybridization of Nucleotide 88-1377 position.In addition, this term also comprise with SEQ ID NO.3 in from the homology of nucleotide sequence at least 70% of Nucleotide 88-1377 position, preferably at least 80%, at least 90% nucleotide sequence more preferably.

This term also comprises encoding to have the variant form of open reading frame sequence among proteic, the SEQ ID NO.3 with people STK3 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 " people STK3 protein polypeptide " refers to have the SEQ ID NO.4 polypeptide of sequence of people STK3 protein-active.This term also comprises having and the variant form human serine/threonine protein kitase identical function, SEQ IDNO.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 STK3 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 STK3 DNA hybridization and the polypeptide or the albumen that utilize the antiserum(antisera) of anti-STK3 polypeptide to obtain.The present invention also provides other polypeptide, as comprises STK3 polypeptide or its segmental fusion rotein.Except the polypeptide of total length almost, the present invention also provides the soluble fragments of STK3 polypeptide.Usually, this fragment have the STK3 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 STK3 albumen or polypeptide.The difference of these analogues and natural STK3 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 can also comprise 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 STK3 conservative property variation polypeptide " refers to compare with the aminoacid sequence of SEQ ID No.4, has 10 at the most, and preferably at the most 8, more preferably 5 amino acid is replaced by similar performance or close amino acid and formed polypeptide at the most.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; Nor-leucine	Leu
			Leu(L)	Nor-leucine; 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; Nor-leucine	Leu

The present invention also comprises people STK3 polypeptid coding sequence and segmental antisense sequences thereof.This antisense sequences can be used for suppressing the expression of STK3 in the cell.

The present invention also comprises a kind of nucleic acid molecule that can be used as probe or primer, and this molecule has 8-100 of STK3 nucleotide sequence, preferably 15-50 continuous nucleotide usually.This probe can be used for whether existing in the test sample nucleic acid molecule of the STK3 that encodes.

The present invention also comprises the method that detects the STK3 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 can be positioned at the both sides or the centre of this encoding sequence corresponding to the encoding sequence of STK3 polypeptide.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.Such as, select commercially available carrier for use, the nucleotide sequence with code book invention polypeptide operationally is connected in expression regulation sequence then, can form protein expression vector.

As used herein, " operationally being connected in " refers to a kind of like this situation, and promptly some part of linear DNA sequence can influence the activity of same other parts of linear DNA sequence.For example, if signal peptide DNA as precursor expression and participate in the secretion of polypeptide, signal peptide (secretion leader sequence) DNA operationally is connected in polypeptid DNA so; If transcribing of promotor control sequence, it is operationally to be connected in encoding sequence so; When if ribosome bind site is placed in the position that can make its translation, it is operationally to be connected in encoding sequence so.Generally, " operationally being connected in " means adjoining, then means in reading frame adjacent for the secretion leader sequence.

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 people STK3 DNA or the polypeptide of its fragment coding has specific antibody, especially monoclonal antibody.Here, " specificity " is meant that antibody capable is incorporated into STK3 gene product or fragment.Preferably, refer to that those can combine with STK3 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 STK3, comprise that also those do not influence the antibody of STK3 protein function.The present invention also comprise those can with modify or without the STK3 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 STK3 gene product of purifying or its have antigenic fragment, can be applied to animal to induce the generation of polyclonal antibody.Similarly, expressing STK3 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 to prepare that (see people such as Kohler, Nature 256; 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, InMonoclonal Antibodies and T Cell Hybridomas, Elsevier, N.Y., 1981).Antibody of the present invention comprises the antibody that can block the STK3 function and the antibody that does not influence the STK3 function.Each antibody-like of the present invention can utilize the fragment or the functional zone of STK3 gene product, obtains by immunological technique.These fragments or functional zone can utilize recombinant methods or utilize Peptide synthesizer synthetic.With the unmodified form bonded antibody of STK3 gene product, can come immune animal and produce with the gene product of producing in the prokaryotic cell prokaryocyte (for example E.Coli); 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).

People STK3 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 relevant nucleotide sequence, especially open reading frame sequence designs primer, and with commercially available cDNA storehouse or the cDNA storehouse of press the currently known methods preparation as template, increase and must be about 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.

In one embodiment of the invention, polynucleotide total length of the present invention is 3019 Nucleotide, and its detailed sequence is seen SEQ ID NO.3, and wherein open reading frame is positioned at 88-1377 position Nucleotide.These polynucleotide are so to obtain, with human brain λ gtllcDNA library (available from Clontech company) is template, synthetic forward primer A1:5 '-TCCAGATGTCAGAGCATTCCTTC-3 ' and reverse primer B1:5 '-CAAGAGCACATTACGTTCAGC-3 ' carry out PCR, obtain the purpose fragment of 3019bp.Obtain the full length cDNA sequence of SEQ ID NO.3 after the order-checking.

According to homology result relatively, the serine/threonine protein kitase of nucleotide sequence of the present invention and encoded protein matter sequence and different sources has shown significant homology, therefore, this shows that it is a newcomer of serine/threonine protein kitase family, and has some critical functions of serine/threonine protein kitase family protein.

Serine/threonine protein kitase belongs to a subfamily in the protein kinase extended familys.Protein kinase is being brought into play important function in vivo, and it mainly is by proteic phosphorylation or dephosphorylation in vivo, and perhaps by combining with the small molecules that plays regulating effect, 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.Glucocorticosteroid is biological intravital a kind of important hormone, and it is controlling stable, the differentiation and the development growth process of biological tissue.Glucocorticosteroid is by stimulating the proteic expression of some important intermediate regulations to finish to these functions of biological tissue, and it is exactly one of them that the serum glucocorticosteroid is regulated protein kinase.The serum glucocorticosteroid is regulated protein kinase and has mainly been participated in some cell internal reactions relevant with glucocorticosteroid in vivo, and it may be the initial step of cell proliferation and other physiological processs relevant with the glucocorticosteroid adjusting that the activation of SGK is expressed.

In the accompanying drawings, Fig. 1 is the electrophoresis detection result of pcr amplified fragment in people's the various tissue cDNA library.Swimming lane 1 is in people's TESTIS cDNA library, and the size that is gone out by primer STK3-A and STK3-B specific amplified is about the fragment of 320bp.Molecular weight marker is that plasmid PBR322 cuts product through restriction enzyme HindIII and EcoRI bimolecular enzyme.

Fig. 2 is a primary dcreening operation positive colony collocation amplification PCR product array ordering chart.The real black line of band scale is represented the nearly total length of STK3 gene cDNA, and small arrow is represented the primer position, and primer is respectively: the special primer STK3-A, the STK3-B that design according to the N75655EST fragment; The first and second relay primer STK3-C, STK3-D.Represent the size of collocation amplified fragments in the different positive colonies and the position of the nearly total length of relative STK3 gene cDNA by the line segment that fine line or dotted line constitute, C represents the primary dcreening operation clone in clone's numbering, numeral is a serial number, and A, B, F, R represent primer STK3-A, STK3-B, λ gt10-F, λ gt10-R respectively.Solid line represents that partly this place's nucleotide sequence is from gene STK3; It is chimeric that dotted portion shows that this place of fragment nucleotide sequence takes place, from another independent basis because of.

Fig. 3 is the homology comparison diagram of the aminoacid sequence of people STK3 of the present invention and people HSGK.Wherein, identical amino acid marks with " | " between two sequences, and similar amino acid marks with " ".Similar amino acid is: A, S, T; D, E; N, Q; R, K; I, L, M, V; F, Y, W.

Fig. 4 is the homology comparison diagram of the aminoacid sequence of people STK3 of the present invention and rat RSGK.Wherein, identical amino acid marks with " | " between two sequences, and similar amino acid marks with " ".Similar amino acid is: A, S, T; D, E; N, Q; R, K; I, L, M, V; F, Y, W.

Fig. 5 is the homology comparison diagram of people STK3 of the present invention and people HSGK and three kinds of proteic catalyst structure domains of rat RSGK.Wherein, identical amino acid marks with " * " between below the sequence, and similar amino acid marks with " ".Roman character above sequence is the numbering of each subdomain.The zone of band underscore is the zone closely related and/or conservative with function.

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 normal condition, people such as Sambrook for example, molecular cloning: laboratory manual (New York:Cold Spring HarborLaboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.

Embodiment 1

The clone and the mensuration of the cDNA sequence of people STK3

1. based on the segmental acquisition of the EST of protein kinase conserved domain

In the search to GENEBANK EST Non-redundant data storehouse, we find the EST fragment that an ACCESSIONNUMBER is N75655, after utilizing PC-GENE software that it is translated into three frames reading albumen order, the albumen of the gene L01624 that the albumen of first frame order and known homology degree are the highest compares in proper order, the 28th of EST fragment and this mouse SGK gene coded protein rise, and 40% homology degree is arranged to 167 amino-acid sequences.Mouse SGK (RSGK) gene is the gene order that a supposition has encoding serine or Serineprotein kinase.In order to determine that this EST fragment whether really from a undiscovered new protein kinase gene of the mankind, has designed corresponding a pair of primer STK3-A, B at these fragment two ends, pcr amplification obtains the fragment that size is 320bp in people's TESTIS cDNA library.Fragment transforms with the T-VECTOR clone and enters intestinal bacteria JM103, and plasmid extraction shows through order-checking, and this order is in full accord with N75655.

2.PCR amplification target cDNA fragment:

Design special primer according to N75655 EST fragment: STK3-A:5 '-TCCAGATGTCAGAGCATTCC TTC-3 ' (SEQ ID NO.1), STK3-B:5 '-CAAGAGCACATTACGTTCAG C-3 ' (SEQ ID NO.2), and with above-mentioned special primer collocation, the annealing temperature that low stringent condition is the promptly lower various cDNA library of moleculess that increase, reaction system is 25ul:10 * PCR damping fluid 2.5ul, MgC12 (25mmol/L) 1.5ul, dNTP (20mmol/L) 0.25ul, each 0.25ul of special primer (25pmol/L), Taq enzyme (5Unit/L, Promega company) 0.25 and deionization ultrapure water 9ul, template is respectively various cDNA library of moleculess (carrier λ DASH CLONETECH company) 1ul, reaction conditions: 93 ℃ 3 minutes, 93 ℃ 1 minute, 60 ℃ 1 minute 30 seconds, 72 ℃ 1 minute, 35 circulations, 72 ℃ were extended 10 minutes, gained PCR product utilizes ultraviolet gel detection instrument to detect clip size through 1% agarose gel electrophoresis (the multiple day company in Shanghai), the fragment that obtains 320bp is (referring to Fig. 1, wherein molecular weight marker is that plasmid PBR322 cuts product, the ascending the 1st through restriction enzyme HindIII and EcoRI bimolecular enzyme, 3,7, the size of 12 bar segment is respectively 560bp, 940bp, 2030bp, 21200bp.)。

3. the clone of amplified fragments identifies

Finally in the amplified reaction that with people's TESTIS cDNA library is template, obtained the PCR product, through column purification test kit (Boehringer Mannheim company), (PROMEGA company) is connected transformed into escherichia coli JM103 with plasmid Pgem-T carrier, the extracting plasmid, with the plasmid double-stranded DNA is template, adopts PE/ABI company 377 automatic instrument to check order as dideoxy chain termination.

4.cDNA library screening

Above-mentioned through the sequence verification target cDNA fragment consistent with AC N75655 nucleotide sequence, the random primer labelling test kit of the MegaPrime DNA Labelling System of employing Amersham company, α-P ³²-dATP mark screens people's testis tissue cDNA library.Hybond membrane H-BOND ^TM42 ℃ of citric acid buffer salt system are all adopted in the prehybridization of N+ (Amersham company) and hybridization.

5.STK3 full-length clone

Pcr amplification obtains the fragment that a size is about 320bp in people's TESTIS cDNA library, and order-checking shows that this order is in full accord with N75655.With amplified fragments as probe, α-P ³²Behind the dATP mark, people's TESTIS cDNA library is screened, the primary dcreening operation positive colony carries out size through special primer STK3-A/B amplification to be identified, selects the further usefulness of analysis of the identical positive colony conduct of size.With special primer STK3-A, B and corresponding to the universal primer λ gt10-F of the carrier λ gt10 left and right arms in cDNA library, R carries out five pairs and is respectively STK3-A, λ gt10-F; STK3-A, λ gt10-R; STK3-B, λ gt10-F; STK3-B, λ gt10-R; The PCR collocation amplification of λ gt10-F, λ gt10-R.The amplification tabulation is analyzed, and in the STK3 length range, respectively 13,11, among 16,15,29,8,32,8, No. 5 primary dcreening operation clones, obtains the specific amplified product as shown in Figure 2.

Difference by amplimer is divided into 4 classes: (1) is by the insertion fragment of the carrier library of packing into of cDNA library left and right arms primer amplification acquisition, (2) participate in the PCR product that collocation (5 ' → 3 ' direction) obtains towards the amplification of fragment 3 ' direction by the STK3-A primer, (3) participate in the PCR product that collocation (3 ' → 5 ' direction) obtains towards the amplification of fragment 5 ' direction by the STK3-B primer, (4) by the first relay primer STK3-C (5 '-GTATACAGAGACTTGAAACCAG-3 ', SEQ IDNo.9) participate in PCR product that collocation (5 ' → 3 ' direction) obtains towards the amplification of fragment 3 ' direction.The PCR product is the same clones, and utilizes the two ends amplimer and the first relay primer STK3-C, the second relay primer STK3-D (5 '-TGCCAGTGTATTGGAGGCAGATG-3 ', SEQ ID No.10) the full nucleotide sequence mensuration of cloned sequence fragment is finished.The nearly full length nucleotide order of STK3 gene, overlapped by above-mentioned these fragments, evidence is finished through splicing.Full length sequence is 3019bp altogether, and wherein open reading frame is 88-1377.

Derive the aminoacid sequence of STK3 according to the full length cDNA sequence that obtains, totally 429 amino-acid residues, its aminoacid sequence sees SEQ ID NO.4 for details.

Embodiment 2

The Northern hybridization analysis

Random priming mark specific fragment, is hybridized by the blotting membrane (MTN Blot Clontech company) of standard method to containing 16 kinds of tissues of human body as probe among the employing embodiment 1.With phosphoglyceraldehy-de dehydrogenase (GAPDH) is contrast.

The Northern hybridization of mouse SGK gene shows have a size to be the transcript positive signal of 2.4KB.The expression of higher level is arranged at ovary, thymus gland, lung; Low but can detectedly be expressed in the multiple tissue of mouse discovery is all arranged.The transcript positive signal that in people HELA cell strain a size to be arranged also be 2.4KB, the transcriptional level road of this transcript can be activated by glucocorticoid inducible equally.Show and in the people's gene group, also have the homology nucleotide sequence similar to mouse SGK gene.People H-SGK gene has the expression of higher level at pancreas, liver, heart; There is more relatively low medium level to express at placenta, lung, skeletal muscle; Low but can detectedly be expressed in discovery is also arranged in brain and the kidney.Its transcript size is 2.6KB, and is close with SGK gene transcription thing size in the above-mentioned mouse of mentioning.In addition, more than in people's pancreas the transcript positive signal that size is 7.7KB.The STK3 gene all has expression in various degree in people's various tissues.The expression of higher level is arranged at thymus gland, colon, peripheral blood leucocyte, the heart, lung.The expression of medium level is arranged at spleen, small intestine.Have low at prostate gland, ovary, brain, large intestine, liver, skeletal muscle, kidney, pancreas but can detectedly express.Transcript has only one, and size is about 4.4KB, and is all different with people H-SGK gene transcription thing size with above-mentioned mouse SGK gene.The height of the different expression levels in different tissues also may be relevant with the genome nucleotide order of this gene 5 ' upstream in different tissues.

Embodiment 3

Homology comparison and functional study

Full-length cDNA encoding sequence and proteins encoded thereof with STK3 of the present invention, in Non-redundantGenBank+EMBL+DDBJ+PDB database and Non-redundant GenBank CDStranslations+PDB+SwissProt+Spupdate+PIR database, carry out nucleic acid and albumen homology retrieval with BLAST software.Found that people STK3 of the present invention and serine/threonine protein kitase family member have shown higher homology.As use the PCGENE software analysis, and the serum glucocorticosteroid of it and rat regulates protein kinase (HSGK)) on protein level, shown 65.3% identity and 75.1% similarity (Fig. 4); And for example, it and human serum glucocorticosteroid are regulated protein kinase (HSGK) shown 65.3% identity and 75.1% similarity (Fig. 3) on protein levels.This shows that STK3 of the present invention is a new serine/threonine kinase family member.

Particularly discover, in the aminoacid sequence of serum glucocorticosteroid adjusting protein kinase (SGK), contain a very big catalyst structure domain, approximately constitute, be broadly divided into 11 subdomains (referring to Fig. 5) by 260 amino acid.Wherein contain the conservative order that sequence A sp-Phe-Gly is the key of protein kinase catalyst structure domain among the subdomain VII; The amino-acid sequence that contains Gly-X-Gly-X-X-Gly (wherein X the is expressed as arbitrary amino acid) high conservative that protein kinase all contains among the subdomain I participates in the combination of ATP molecule; The Gly-Thr-X-X-Tyr-X-Ala-Pro-Glu sequence that contains among Asp-Leu-Lys-Pro-Glu-Asn sequence that contains among the subdomain VI and the subdomain VIII shows that the substrate of this protein kinase has specificity (Mol CellBiol.1993,13 to Serine, Threonine; 2031-2040).

STK3 albumen of the present invention is made up of 429 amino acid altogether, and it has the catalyst structure domain that 260 very big amino acid constitute equally.The identity of this zone and mouse RSGK and people HSGK is up to 76.5%, and similarity is up to 95.5% (Fig. 5).This zone of STK3 is high conservative with the proteic catalyst structure domain of SGK on some keying action sites, and the sequence fragment that wherein meets above-mentioned pattern is respectively: Asp-Phe-Gly (237-239 position among the SEQ IDNO.4); Gly-Lys-Gly-Ser-Phe-Gly (102-107 position among the SEQ ID NO.4); Asp-Leu-Lys-Pro-Glu-Asn (219-224 position among the SEQ ID NO.4); Gly-Thr-Pro-Glu-Tyr-Leu-Ala-Pro-Glu (256-264 position among the SEQ ID NO.4) (referring to Fig. 5).So STK3 albumen of the present invention and SGK albumen height correlation have more been determined in the research of active structure domain, belong to serine/threonine protein kitase family, and have the correlation function of serine/threonine protein kitase family.

Protein kinase participates in the cell necessary numerous processes of function of bringing into normal play in vivo, as: gene transcription, translation, proteic secretion is instructing albumen and specific target site to combine, regulate albumen reaction between each organoid and in cell development, the position and the quantity of control specific protein in the cell cycle in cell.Protein kinase is an important intermediate regulations thing of signal transduction path in the organism, and its to regulate control action kou mainly be to proteic phosphorylation or dephosphorylation, or finish by combining with the small molecules of regulating effect.Protein kinase is divided into two subfamilies again, and serine/threonine protein kitase promptly is one of them.It then is that (Proc.Natl.Acad.Sci.USA 1997,94 for a newcomer in the serine/threonine protein kitase subfamily that the serum glucocorticosteroid is regulated proteohormone; 4440-4445), hence one can see that, and it very important effect in vivo for they, participating in biological intravital various important vital processes.

The serum glucocorticosteroid is regulated protein kinase expression in vivo and controlled by the adjusting of serum and sugared cortex, it receives the signal from serum and glucocorticosteroid, promptly by phosphorylation and dephosphorylation to target protein, to conduct to target protein from the information that serum and glucocorticosteroid transmit, correctly finish its important physical function to regulate these albumen.It is considered to serum and glucocorticosteroid is regulated the intermediate of cytosis.Serum glucocorticosteroid adjusting protein kinase receives the signal from hormone, beginning is at cell inner expression, and target protein carried out phosphorylation, thereby regulate these albumen and finish every function in vivo, comprise: (Mol Cell Biol.1993,13 such as cell proliferation, differentiation, metabolism and various inflammatory reactions; 031-2040).

This shows that serine/threonine protein kitase directly affects the multiple vital processes such as propagation, differentiation, metabolism of organism inner cell in vivo as mediators in the middle of signal conduction important.Because SGK of STK3 of the present invention and rat and people's h-SGK belong to serine/threonine protein kitase family together, so may also have same as described above or similar function.

People STK3 of the present invention is 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, inventor STK3 can also merge with other members of this family or exchange fragment, to produce new albumen.For example the N end with inventor STK3 exchanges with RSGK of rat or the N end of people HSGK, to produce the albumen that new activity is higher or have new features.

At the antibody of inventor STK3, be used to screen other members of this family, perhaps be used for affinity purification associated protein (as other members of this family, such as HSGK).

For example, inventor STK3 nucleic acid (encoding sequence or antisense sequences) can be introduced into cell, with expression level that improves people STK3 or the overexpression that suppresses people STK3.People STK3 albumen of the present invention or its active polypeptide fragment can be applied to patient, with treatment or alleviate because of people STK3 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.

Embodiment 4

The expression of STK3 in intestinal bacteria

The PCR Oligonucleolide primers of the cDNA sequence of coding STK3 corresponding to 5 of this dna sequence dna ' and 3 ' end, personnel selection testis λ gt11cDNA library (available from Clontech company) are for template increases, with synthetic insertion fragment.

5 ' Oligonucleolide primers sequence is

5′-TTGCGGATCCATGGCCCTGAAGATTCCTG-3′(SEQ?ID?NO.5)，

This primer contains the restriction enzyme site of BamHI restriction enzyme, and what connect is 19 Nucleotide of the STK3 encoding sequence that begun by initiator codon;

3 ' Oligonucleolide primers sequence is

5’-GTTCGTCGACTCACAAAAATAAGTCTTCT-3’(SEQ?ID?NO.6)，

This primer contains the restriction enzyme site of SalI restriction enzyme, the encoding sequence of translation termination and STK3.

The restriction enzyme site of restriction enzyme 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 SalI 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, 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).The extracting plasmid is cut with the HindIII enzyme and identify to be inserted clip size and direction, and sequence verification result shows that the cDNA of STK3 inserts the fragment carrier of correctly packing into.

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 STK3 from solution.With 6M Guanidinium hydrochloride (pH5.0) wash-out STK3 from post.Available several method is the sex change protein precipitation from Guanidinium hydrochloride.At first, use the dialysis step to remove Guanidinium hydrochloride, perhaps isolated purifying protein can be incorporated in second post from nickel-chelate column, 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 with PBS, then protein is kept in the stock solution that final concentration is 10% (w/v) glycerine.

SDS-PAGE glue with 12% carries out electrophoresis, identifies that the molecular weight size of expressing protein is 49KDa.

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 5

The expression of STK3 in eukaryotic cell (Chinese hamster ovary celI strain)

In this embodiment, the cDNA sequence of coding STK3 is used PCR Oligonucleolide primers corresponding to 5 of this dna sequence dna ' and 3 ' end, personnel selection testis λ gt11cDNA library (available from Clontech company) is for template increases, with synthetic insertion fragment.

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

5′-TTGCGGATCCATGGCCCTGAAGATTCCTG-3′(SEQ?ID?NO.7)，

This primer contains the restriction enzyme site of BamHI restriction enzyme, and what connect is 19 Nucleotide of the STK3 encoding sequence that begun by initiator codon;

3 ' end primer sequence is:

5’-GTTCCTCGAGTCACAAAAATAAGTCTTCT-3’(SEQ?ID?NO.8)

This primer contains the encoding sequence of the restriction enzyme site of XhoI restriction enzyme, translation termination and STK3.

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 (fl 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 BamHI, XhoI 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, sequence verification result shows that the cDNA of STK3 inserts the fragment carrier of correctly packing into.

Plasmid transfection is to adopt lipofection, carries out with Lipofectin test kit (GiBco Life).After the transfection 48 hours, 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 50mM TrisHCl (pH7.6) solution that contains 0.05%Triton is balance liquid and elutriant, uses through the Superdex of pre-equilibration G-75 post and collects above-mentioned proteic active peak.Using 50mM TrisHCl (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.

SDS-PAGE glue with 12% carries out electrophoresis, identifies that the molecular weight size of expressing protein is 49KDa.

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

Preparation antibody

The recombinant protein that obtains in embodiment 4 and 5 is 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 STK3 gene translation product with it.Found that antibody can precipitate 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: (i) applicant: Fudan University is denomination of invention (ii): new human serine threonine protein kinase, its encoding sequence and method for making and purposes be the sequence number (iii): information (i) sequence signature of 10 (2) SEQ ID NO.1

(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.1:TCCAGATGTC AGAGCATTCC TTC 23 (2) SEQ ID NO.2

(A) length: 21 bases

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity is molecule type (ii): oligonucleotide (xi) sequence description: the information of SEQ ID NO:2:CAAGAGCACA TTACGTTCAG C 21 (2) SEQ ID NO.3: (i) sequence signature:

(A) length: 3019bp

(B) type: nucleic acid

(C) chain: strand

( D ) ： ( ii ) ：cDNA ( xi ) ：SEQ ID NO.3：TCAGTGGGAA GAAGTGAATG GTTTGTCTTC AGGAGATATG CAGAGGTTGA TAAACTTTAT 60AACACTTTAA AAAAACAGTT TCCTGCTATG GCCCTGAAGA TTCCTGCCAA GAGAATATTT 120GGTGATAATT TTGATCCAGA TTTTATTAAA CAAAGACGAG CAGGACTAAA CGAATTCATT 180CAGAACCTAG TTAGGTATCC AGAACTTTAT AACCATCCAG ATGTCAGAGC ATTCCTTCAA 240ATGGACAGTC CAAAACACCA GTCAGGTCCA TCTGAAGATG AGGATGAAAG AAGTTCTCAG 300AAGCTACACT CTACCTCACA GAACATCAAC CTGGGACCGT CTGGAAATCC TCATGCCAAA 360CCAACTGACT TTGATTTCTT AAAAGTTATT GGAAAAGGCA GCTTTGGCAA GGTTCTTCTT 420GCAAAACGGA AACTGGATGG AAAAGTTTAT GCTGTCAAAG TGTTACAGAA AAAAATAGTT 480CTCAACAGAA AAGAGCAAAA ACATATTATG GCTGAACGTA ATGTGCTCTT GAAAAATGTG 540AAACATCCGT TTTTGGTTGG ATTGCATTAT TCCTTCCAAA CAACTGAAAA GCTTTATTTT 600GTTCTGGATT TTGTTAATGG AGGGGAGCTT TTTTTCCACT TACAAAGAGA ACGGTCCTTT 660CCTGAGCACA GAGCTAGGTT TTACGCTGCT GAAATTGCTA GTGCATTGGG TTACTTACAT 720TCCATCAAAA TAGTATACAG AGACTTGAAA CCAGAAAATA TTCTTGTGGA TTCAGTAGGA 780CATGTTGTCT TAACAGATTT TGGGCTTTGT AAAGAAGGAA TTGCTATTTC TGACACCACT 840ACCACATTTT GTGGGACACC AGAGTATCTT GCACCTGAAG TAATTAGAAA ACAGCCCTAT 900GACAATACCG TAGATTGGTG GTGCCTTGGG GCTGTTCTGT ATGAAATGCT GTATGGATTG 960CCTCCTTTTT ATTGCCGAGA TGTTGCTGAA ATGTATGACA ATATCCTTCA CAAACCCCTA 1020AGTTTGAGGC CAGGAGTGAG TCTTAGAGCC TGGTCCATTC TGGAAGAACT CCTAGAAAAA 1080GACAGGCAAA ATCGACTTGG TGCCAAGGAA GACTTTCTTG AAATTCAGAA TCATCCTTTT 1140TTTGAATCAC TCAGCTGGGC TGACCTTGTA CAAAAGAAGA TTCCACCACC ATTTAATCCT 1200AATGTGGCTG GACCAGATGA TATCAGAAAC TTTGACACAG CATTTACAGA AGAAACAGTT 1260CCATATTCTG TGTGTGTATC TTCTGACTAT TCTATAGTGA ATGCCAGTGT ATTGGAGGCA 1320GATGATGCAT TCGTTGGTTT CTCTTATGCA CCTCCTTCAG AAGACTTATT TTTGTGAGCA 1380GTTGCCATAC AGAAACCATT GAGCAAAATA AGTCTATAGA TGGGACTGAA ACTTCTATTT 1440GTGTGAATAT ATTCAAATAT GTATAACTAG TGCCTCATTT TTATATGTAA TGATGAAAAC 1500TATGAAAAAA TGTATTTTCT TCTATGTGCA AGAAAAATAG GGCATTTCAA AGAGCTGTTT 1560TGATTAAAAT TTATATTCTT GTTTAATAAG CTTATTTTTA AACAATTTAA AAGCTATTAT 1620TCTTAGCATT AACCTATTTT TAAAGAAACC TTTTTGCTAT TGACTGTTTT TTCCCTCTAA 1680GTTTACACTA ACATCTACCC AAGATAGACT GTTTTTTAAC AGTCAATTTC AGTTCAGCTA 1740ACATATATTA ATACCTTTGT AACTCTTTGC TATGGCTTTT GTTATCACAC CAAAACTATG 1800CAATTGGTAC ATGGTTGTTT AAGAAGAAAC CGTATTTTTC CATGATAAAT CACTGTTTGA 1860AATATTTGGT CATGGTATGA TCGAAATGTA AAAGCATAAT TAACACATTG GCTGCTAGTT 1920AACAATTGGA ATAACTTTAT TCTGCAGATC ATTTAAGAAG TAACAGGCCG GGCGCGGGTT 1980GTCACGCCTG TAATCCCAGC ACTTTGGGAG GCTGAGGCGG GCAGATCACC TGAGGTCAGG 2040AGTTGGAGAC CAGCCTGACC AACATGGACA AACCCCGTCT CTACTAAAAA TACAAAATTG 2100GCAGGGTGTG GTGCACATGC CTATAATCCC AGCTACTTGG GAGGCTAAGG CAGGAGAATC 2160GCTTGAACCC GGGAGGCGGA GGTTGCAGTG AGCCGAGATC GCACCATTGC ACTCCTGCCT 2220GGGCAACAAG AGTGAAACTC CATCTCCAAA AAAAAAAAGG AAAAAGTAAC AAAAGGAAAT 2280TATTTGTTTT TGAAATACCA GTTCAACTTT GTGGATTATT TTTCCTCTGA AGGAAAAGAA 2340AAGGCTTAAT GGTTAGGATT TTTAAGTATT CCCAAAGATC TGAAGGGTAA TAAAATGTAC 2400TGGATTTTTT AAGGTGGTAC CAAAAATGAA TGTCTGTCAT ATATTTATAT TACAAATACA 2460TTATATTTAT GTTCTATTCA TCTTTTGAAT GTTTAGTATG CTATTAAGTC ATTCTGAATC 2520TTTGTATTTG CTTTTGCAAA TAGGTATTTC AAAGCTCTTT TCCTAACTGG TTAAGTAAAA 2580TAAAAAATTG AGCTTTCTAG AATATTTGCC TAATTGGGAA TTAAAAAGTA AAATAATAGG 2640CCAGGCATGG TGGCTCATGC CTATAAGCAC CCTGGGAGGC CGAGGCAGGC AGATTATTTG 2700AGCTCAGGAG TTTGAGACCA TCCTGGGCAA CATGGCGAAA CCCTATCTCT ACAAAAAATA 2760CAAAAATTAG CCAGACATGG TGGCACATGC CTTTAGTCCC AGCTACTCTG GAGGCTGAAG 2820TTGGAGGATG GCTTGAGCCC ACGAGATGGA AGTTGCAGTG AACTGAAATT GTGCCACTGC 2880ACTTTTCAGC CTGGGTGCCA CAGCGAGACC CTAGTTAAGA GAAAAAAAAA GTAAAAACAA 2940ATTGTGGGTC AAAGTAAATG TATACAGTTT TATTACAATG TAACAAAAGT TGAAAATCGG 3000GCCCGGAATT CAGTTTCAG 3019 ( 2 ) SEQ ID NO.4： ( i ) ：

(A) length: 429 amino acid

(B) type: amino acid

( D ) ： ( ii ) ： ( xi ) ：SEQ ID NO.4：Met Ala Leu Lys Ile Pro Ala Lys Arg Ile Phe Gly Asp Asn Phe 15Asp Pro Asp Phe Ile Lys Gln Arg Arg Ala Gly Leu Asn Glu Phe 30Ile Gln Asn Leu Val Arg Tyr Pro Glu Leu Tyr Asn His Pro Asp 45Val Arg Ala Phe Leu Gln Met Asp Ser Pro Lys His Gln Ser Gly 60Pro Ser Glu Asp Glu Asp Glu Arg Ser Ser Gln Lys Leu His Ser 75Thr Ser Gln Asn Ile Asn Leu Gly Pro Ser Gly Asn Pro His Ala 90Lys Pro Thr Asp Phe Asp Phe Leu Lys Val Ile Gly Lys Gly Ser 105Phe Gly Lys Val Leu Leu Ala Lys Arg Lys Leu Asp Gly Lys Val 120Tyr Ala Val Lys Val Leu Gln Lys Lys Ile Val Leu Asn Arg Lys 135Glu Gln Lys His Ile Met Ala Glu Arg Asn Val Leu Leu Lys Asn 150Val Lys His Pro Phe Leu Val Gly Leu His Tyr Ser Phe Gln Thr 165Thr Glu Lys Leu Tyr Phe Val Leu Asp Phe Val Asn Gly Gly Glu 180Leu Phe Phe His Leu Gln Arg Glu Arg Ser Phe Pro Glu His Arg 195Ala Arg Phe Tyr Ala Ala Glu Ile Ala Ser Ala Leu Gly Tyr Leu 210His Ser Ile Lys Ile Val Tyr Arg Asp Leu Lys Pro Glu Asn Ile 225Leu Val Asp Ser Val Gly His Val Val Leu Thr Asp Phe Gly Leu 240Cys Lys Glu Gly Ile Ala Ile Ser Asp Thr Thr Thr Thr Phe Cys 255Gly Thr Pro Glu Tyr Leu Ala Pro Glu Val Ile Arg Lys Gln Pro 270Tyr Asp Asn Thr Val Asp Trp Trp Cys Leu Gly Ala Val Leu Tyr 285Glu Met Leu Tyr Gly Leu Pro Pro Phe Tyr Cys Arg Asp Val Ala 300Glu Met Tyr Asp Asn Ile Leu His Lys Pro Leu Ser Leu Arg Pro 315Gly Val Ser Leu Arg Ala Trp Ser Ile Leu Glu Glu Leu Leu Glu 330Lys Asp Arg Gln Asn Arg Leu Gly Ala Lys Glu Asp Phe Leu Glu 345Ile Gln Asn His Pro Phe Phe Glu Ser Leu Ser Trp Ala Asp Leu 360Val Gln Lys Lys Ile Pro Pro Pro Phe Asn Pro Asn Val Ala Gly 375Pro Asp Asp Ile Arg Asn Phe Asp Thr Ala Phe Thr Glu Glu Thr 390Val Pro Tyr Ser Val Cys Val Ser Ser Asp Tyr Ser Ile Val Asn 405Ala Ser Val Leu Glu Ala Asp Asp Ala Phe Val Gly Phe Ser Tyr 420Ala Pro Pro Ser Glu Asp Leu Phe Leu 429 ( 2 ) SEQ ID NO.5 ( i )

(A) length: 29 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.5:TTGCGGATCC ATGGCCCTGA AGATTCCTG 29 (2) SEQ ID NO.6

(A) length: 29 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.6:GTTCGTCGAC TCACAAAAAT AAGTCTTCT 29 (2) SEQ ID NO.7

(A) length: 29 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.7:TTGCGGATCC ATGGCCCTGA AGATTCCTG 29 (2) SEQ ID NO.8

(A) length: 29 bases

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity is molecule type (ii): oligonucleotide (xi) sequence description: the information of SEQ ID NO.8:GTTCCTCGAG TCACAAAAAT AAGTCTTCT 29 (2) SEQ ID NO.9

(i) sequence signature

(A) length: 22 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.9:GTATACAGAG ACTTGAAACC AG 22 (2) SEQ ID NO.10

(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.10:TGCCAGTGTA TTGGAGGCAG ATG 23

Claims (14)

1. isolated dna molecular is characterized in that it comprises: coding has the nucleotide sequence of the polypeptide of people STK3 protein-active,

Show at least 70% homology from the nucleotides sequence of Nucleotide 88-1377 position among described nucleotide sequence and the SEQ ID NO.3; Perhaps

Described nucleotide sequence can be under the moderate stringent condition with SEQ ID NO.3 in from the nucleotide sequence hybridization of Nucleotide 88-1377 position.

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.4.

3. dna molecular as claimed in claim 1 is characterized in that, this sequence has among the SEQ ID NO.3 nucleotide sequence from Nucleotide 88-1377 position.

4. isolating people STK3 protein polypeptide is characterized in that it comprises: have polypeptide or its conservative property variation polypeptide or its active fragments of SEQ ID NO.4 aminoacid sequence, or its reactive derivative.

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

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

7. one kind with the described carrier transformed host cells of claim 6.

8. host cell as claimed in claim 7 is characterized in that this cell is intestinal bacteria.

9. host cell as claimed in claim 7 is characterized in that this cell is an eukaryotic cell.

10. a generation has the method for the polypeptide of people STK3 protein-active, it is characterized in that this method comprises:

(a) nucleotide sequence that coding is had a polypeptide of people STK3 protein-active operationally is connected in expression regulation sequence, form people STK3 protein expression vector, show at least 70% homology from the nucleotides sequence of Nucleotide 88-1377 position among described nucleotide sequence and the SEQ ID NO.3;

(b) change the expression vector in the step (a) over to host cell, form the proteic reconstitution cell of STK3;

(c) under the condition that is fit to expressing human STK3 protein polypeptide, the reconstitution cell in the culturing step (b);

(d) isolate polypeptide with people STK3 protein-active.

11. method as claimed in claim 10 is characterized in that, this sequence is from Nucleotide 88-1377 position among the SEQ ID NO.3.

12. energy and the described people STK3 of claim 4 protein polypeptide specificity bonded antibody.

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

14. a probe molecule is characterized in that, it contains about 8-100 continuous nucleotide in the described dna molecular of claim 1.

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