CN1603341A - Human calcium ion / calmodulin deopendent protein kinase II inhibitory protein alpha, its coded sequence and use - Google Patents

Abstract

This invention relates to a Calcium/Calmodulin-Dependent Protein Kinase II Inhibitor alpha, CaM-KIIN alpha. Method of coding polynucleotide of the albumen molecule and generating the albumen molecule by recombination technology are provided in this invention. The Calcium/Calmodulin-Dependent Protein Kinase II Inhibitor alpha, CaM-KIIN alpha and use of the code sequence is also disclosed. It also demonstrates the activity of CaM-KIIN alpha can combine and restrain Calcium/Calmodulin-Dependent Protein Kinase II. The diagnosis and curing tactics on disease by this albumen molecule is also disclosed, especially to cancer curing.

Description

Human calcium ion/calmodulin dependent protein kinase II repressible protein α, its encoding sequence and purposes

Technical field

The invention belongs to biotechnology and medical field, specifically, the present invention relates to new coding human calcium ion/calmodulin dependent protein kinase II repressible protein α (Calcium/Calmodulin-DependentProtein Kinase II Inhibitor α, abbreviate " CaM-KIIN α " as) polynucleotide of polypeptide, and the polypeptide of this polynucleotide encoding.The invention still further relates to the purposes and the preparation of these polynucleotide and polypeptide.The invention still further relates to the inhibition activity of CaM-KIIN α cell growth.CaM-KIIN α of the present invention is a soluble proteins in a kind of new cell relevant with cell proliferation, growth and cell cycle signal.

Background technology

Calcium ion/calmodulin dependent protein kinase II (CaMKII) is a kind of multi-functional serine/threonine protein kitase, is an important component part of signal transducting system in the cell, is Ca2 ⁺/ CaM regulates a main member in the protein family.CaMK II is one of important target enzyme of CaM, and many enzymes or protein can be as its substrates.CaMKII is by α, beta, gamma, and four kinds of subunits of δ form more than the ten kind of isozyme of encoding.Nearly all subunit all includes catalytic domain, regulatory region and land, and regulatory region is again by self inhibitory area (281-309), calmodulin land (296-311) and autophosphorylation site (Threonine 286,305,306; Serine 314) forms.

The molecular weight of CaMKII is 00-700Kd approximately for this reason, and subunit molecular is 50-62kd.The CaMKII activation needs Ca ²⁺The startup of/CaM, Ca ²⁺/ CaM is attached to the calmodulin land, removed self inhibitory area the inhibition of activation point is made enzyme activation, and behind this enzyme autophosphorylation, its activity promptly loses Ca ²⁺The dependency of/CaM.

CaMKII mainly is present in nervous tissue, can account for 2% (as the hippocampal gyrus of mouse) of total protein concentration in some zone.In addition, in the tissues such as brush border of mammalian skeleton flesh, heart, lung, liver, pancreas, retina, kidney, parathyroid gland, mammary gland, uterus, testis, small intestine, all can detect.The subunit of CaMKII distributes different because of tissue, and α and β subunit only are present in nervous tissue, and γ and delta-subunit can be present in other tissue except that brain.Along with going deep into that structure, tissue distribution and the biological function of CaMKII are studied, it is found that CaMKII all brings into play critical function in the various biological incident.It is regulating neuronic activity, the contraction of muscle, and the control of cell cycle, the secretion of cell, the repairing of dna damage, the metabolism of carbohydrate, there is important biological action aspects such as expression of gene.

The natural endogenous repressible protein of report only has calcium ion/calmodulin dependent protein kinase ii repressible protein CaM-KIIN β, the CaM-KIIN α in rat cerebral cell source and the endogenous repressible protein CaM-KIIN β in people source at present.CaMKII inhibitor KN-62, the KN-93 of chemosynthesis be by with the interaction of CaM binding site, stop combining of CaM and CaMKII, suppress the activity of CaMKII specifically, make the CaMKII can not autophosphorylation and be not activated.Endogenous CaMKII repressible protein CaM-KIIN has high specific and suppresses the active ability of CaMKII, and experimental results show that endogenous CaMKII repressible protein CaM-KIIN can only with the activatory CaMKII effect of mutually combining.According to there being bibliographical information CaMKII inhibitor can cause the damage of DNA, the change that cyclin is expressed stops cell to enter the s phase, dies thereby cause cell to take place to transfer.The CaMKII inhibitor can strengthen the expression of P53, and cell is arrested in the G2 phase.This specific specificity suppresses the active ability of CaMKII and points out these materials may participate in promoting the accent of tumour cell to die and downright bad link.

Research shows that calcium ion/calmodulin dependent protein kinase II repressible protein is relevant with multiple vital movement.Therefore, significant for the calcium ion/calmodulin dependent protein kinase II repressible protein of diagnosing and the therapeutic purpose research and development is new.Yet, but still do not have finder's calcium ion/calmodulin dependent protein kinase II repressible protein α so far.

Summary of the invention

The purpose of this invention is to provide a kind of new human calcium ion/calmodulin dependent protein kinase II repressible protein α (CaM-KIIN α albumen) with and fragment, analogue and derivative.

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

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

The present invention has found novel human calcium ion/calmodulin dependent protein kinase II repressible protein α CaM-KIIN α through extensive and deep research, and homologous protein and people CaM-KIIN β that it and known rat are originated have high homology.Similar to the effect of chemical inhibitor pair cell, people source CaM-KIIN α can check cell proliferation, has cell growth inhibiting activity.Therefore, CaM-KIIN α is the same with other inhibitor can be by specificity in conjunction with CaMKII, stop the phosphorylation of CaMKII to activate, regulate and control multiple physiology and pathology activity, play a significant role, and may be worth having important development and application aspect the immunodiagnosis in a plurality of fields such as anti-infective, anti-inflammatory response, antitumor and nerve growth reparation, immunologic function adjusting and the immunotherapy.

In a first aspect of the present invention, novel isolated CaM-KIIN α albumen is provided, this polypeptide is the people source, and it comprises: polypeptide or its conservative property variation polypeptide or its active fragments (fragment that especially has 41-58 position inhibition conserved regions) or its reactive derivative with SEQ ID NO:2 aminoacid sequence.

Preferably, this polypeptide is selected from down group:

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

(b) SEQ ID NO:2 aminoacid sequence process is one or more (as 1-20,1-10 more preferably) replacement, disappearance or the interpolation of amino-acid residue form, and have combination and suppress calcium ion/active function of calmodulin dependent protein kinase ii by (a) polypeptides derived.

More preferably, this polypeptide is the polypeptide with SEQ ID NO:2 aminoacid sequence.

In a second aspect of the present invention, the polynucleotide of isolating these polypeptide of coding are provided, these polynucleotide comprise a nucleotide sequence, and this nucleotide sequence is shown at least 80% homogeny with a kind of nucleotides sequence that is selected from down group: (a) polynucleotide of the above-mentioned people CaM-KIIN α of coding; (b) with polynucleotide (a) complementary polynucleotide.Preferably, this polynucleotide encoding has the polypeptide of aminoacid sequence shown in the SEQ ID NO:2.More preferably, the sequence of these polynucleotide is be selected from down group a kind of: the sequence that (a) has 401-634 position among the SEQ ID NO:1; (b) has the sequence of 1-860 position among the SEQ ID NO:1.

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

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

In a fifth aspect of the present invention, provide and above-mentioned people CaM-KIIN α protein-specific bonded antibody.The nucleic acid molecule that can be used for detecting also is provided, and it contains a successive 15-860 Nucleotide in the above-mentioned polynucleotide.

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

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

In a eighth aspect of the present invention, provide a kind of the detection to express the relevant disease or the method for disease susceptibility with people CaM-KIIN α abnormal protein, this method comprises: whether have sudden change in the nucleotide sequence of detection coding said polypeptide.

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

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

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

Description of drawings

Following accompanying drawing is used to illustrate specific embodiments of the present invention, and is not used in qualification by the scope of the invention that claims defined.

Fig. 1 is the cDNA sequence of people CaM-KIIN α of the present invention, and wherein non-coding sequence is represented with lowercase, and encoding sequence is with capitalizing conventional letter representation.

Fig. 2 is the proteic full length amino acid sequence of people CaM-KIIN α of the present invention.

Fig. 3 be people CaM-KIIN α albumen of the present invention and rat originate other calcium ions/calmodulin dependent protein kinase ii repressible protein amino acid sequence homology relatively.The top sequence is people CaM-KIIN α, and the below sequence is the CaM-KIIN albumen in rat source.Dash area is a concensus sequence, and frame portion is a similar sequences.

Fig. 4 is a people CaM-KIIN α RT-PCR expression analysis of the present invention, and prompting CaM-KIIN alpha expression is in some tumour cell; In the maturing dendritic cell in human peripheral blood mononuclear cell source and the reactivation process that stimulated by KLH, has the certain expression pattern.Wherein " Marker " represents the molecular weight standard thing.

Fig. 5 be under the condition of people CaM-KIIN α of the present invention high-calcium ionic in born of the same parents with the CaMKII detection that mutually combines.

Fig. 6 is the inhibition test of people CaM-KIIN α of the present invention to the CaMKII enzymic activity, and prompting has the activity of inhibition.

Embodiment

In the present invention, term " CaM-KIIN α albumen ", " CaM-KIIN α albumen " or " calcium ion/calmodulin dependent protein kinase II repressible protein α " are used interchangeably, and all refer to have the albumen or the polypeptide of human calcium ion/calmodulin dependent protein kinase II repressible protein α CaM-KIIN alpha amino acid sequence (SEQ ID NO:2).They comprise the calcium ion/calmodulin dependent protein kinase II repressible protein α CaM-KIIN α that contains or do not contain initial methionine.

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

As used herein, " isolating CaM-KIIN α albumen or polypeptide " is meant that CaM-KIIN α albumen is substantially free of natural relative other albumen, lipid, carbohydrate or other material.Those skilled in the art can use the purified technology of protein purifying CaM-KIIN α albumen of standard.Basically pure polypeptide can produce single master tape on non-reduced polyacrylamide gel.

Polypeptide of the present invention can be recombinant polypeptide, natural polypeptides, synthetic polypeptide, preferred recombinant polypeptide.Polypeptide of the present invention can be the product of natural purifying, or the product of chemosynthesis, or uses recombinant technology to produce from protokaryon or eucaryon host (for example, bacterium, yeast, higher plant, insect and mammalian cell).The host used according to the recombinant production scheme, polypeptide of the present invention can be glycosylated, maybe can be nonglycosylated.Polypeptide of the present invention also can comprise or not comprise initial methionine residues.

The present invention also comprises the proteic fragment of people CaM-KIIN α, derivative and analogue.As used herein, term " fragment ", " derivative " are meant biological function or the active polypeptide that keeps natural human CaM-KIIN α albumen of the present invention identical basically with " analogue ".Polypeptide fragment of the present invention, derivative or analogue can be that (i) has one or more conservative or substituted polypeptide of non-conservation amino-acid residue (preferred conservative amino acid residue), and the amino-acid residue of such replacement can be also can not encoded by genetic code, or (ii) in one or more amino-acid residues, has a polypeptide of substituted radical, or (iii) mature polypeptide and another compound (such as the compound that prolongs the polypeptide transformation period, polyoxyethylene glycol for example) merges formed polypeptide, or (iv) additional aminoacid sequence is fused to this peptide sequence and the polypeptide that forms (as leader sequence or secretion sequence or be used for the sequence or the proteinogen sequence of this polypeptide of purifying, or with the fusion rotein of the segmental formation of antigen I gG).According to the instruction of this paper, these fragments, derivative and analogue belong to the known scope of those skilled in the art.

In the present invention, term " people CaM-KIIN α albumen " refers to have the SEQID NO.2 polypeptide of sequence of people CaM-KIIN α protein-active.This term also comprises having and people CaM-KIIN α albumen variant form identical function, SEQ IDNO.2 sequence.These variant forms comprise (but being not limited to): several (are generally 1-20, more preferably 1-10,1-5 best) amino acid whose disappearance, insertion and/or replacement, and add one or several at C-terminal and/or N-terminal and (be generally in 20, preferably being in 10, more preferably is in 5) amino acid.For example, in the art, when replacing, can not change proteinic function usually with the close or similar amino acid of performance.Again such as, add one or several amino acid at C-terminal and/or N-terminal and also can not change proteinic function usually.This term also comprises proteic active fragments of people CaM-KIIN α 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 coded albumen of the DNA of people CaM-KIIN α DNA hybridization and polypeptide or the albumen that utilizes the proteic antiserum(antisera) of anti-people CaM-KIIN α to obtain.The present invention also provides other polypeptide, as comprises people CaM-KIIN α albumen or its segmental fusion rotein.Except the polypeptide of total length almost, the present invention has also comprised the proteic soluble fragments of people CaM-KIIN α.Usually, this fragment have people CaM-KIIN α protein sequence at least about 10 continuous amino acids, usually at least about 30 continuous amino acids, preferably at least about 50 continuous amino acids, best at least about 70 continuous amino acids.

Invention also provides the analogue of people CaM-KIIN α albumen or polypeptide.These analogues and the proteic difference of natural human CaM-KIIN α can be the difference on the aminoacid sequence, also can be the difference that does not influence on the modified forms of sequence, perhaps have both at the same time.These polypeptide comprise natural or the inductive genetic variant.The induce variation body can obtain by various technology, as by radiation or be exposed to mutagenic compound and produce random mutagenesis, also can pass through site-directed mutagenesis method or the biological technology of other known moleculars.Analogue also comprises having the analogue that is different from the amino acid whose residue of natural L-(as D-amino acid), and has non-natural analogue that exist or synthetic amino acid (as β, gamma-amino acid).Should be understood that polypeptide of the present invention is not limited to the above-mentioned representational polypeptide that exemplifies.

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

In the present invention, " people CaM-KIIN α albumen conservative property variation polypeptide " refers to compare with the aminoacid sequence of SEQ ID NO:2, has 10 at the most, preferably at the most 8, more preferably at the most 5,3 amino acid is replaced by similar performance or close amino acid and is formed polypeptide at the most best.These conservative property variation polypeptide preferably carry out the amino acid replacement according to table 1 and produce.

Table 1

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

Polynucleotide of the present invention can be dna form or rna form.Dna form comprises the DNA of cDNA, genomic dna or synthetic.DNA can be strand or double-stranded.DNA can be coding strand or noncoding strand.The coding region sequence of encoding mature polypeptide can be identical with the coding region sequence shown in the SEQ ID NO:1 or the varient of degeneracy.As used herein, " varient of degeneracy " is meant that in the present invention coding has the protein of SEQ ID NO:2, but with the differentiated nucleotide sequence of coding region sequence shown in the SEQ ID NO:1.

The polynucleotide of the mature polypeptide of coding SEQ ID NO:2 comprise: the encoding sequence of an encoding mature polypeptide; The encoding sequence of mature polypeptide and various additional code sequence; Encoding sequence of mature polypeptide (with optional additional code sequence) and non-coding sequence.

Term " polynucleotide of coded polypeptide " can be the polynucleotide that comprise this polypeptide of encoding, and also can be the polynucleotide that also comprise additional code and/or non-coding sequence.

The invention still further relates to the varient of above-mentioned polynucleotide, its coding has the polypeptide of identical aminoacid sequence or fragment, analogue and the derivative of polypeptide with the present invention.The varient of these polynucleotide can be the allelic variant of natural generation or the varient that non-natural takes place.These nucleotide diversity bodies comprise and replace varient, deletion mutation body and insert varient.As known in the art, allelic variant is the replacement form of polynucleotide, and it may be replacement, disappearance or the insertion of one or more Nucleotide, but can be from not changing the function of its encoded polypeptides in fact.

The invention still further relates to and above-mentioned sequence hybridization and two sequences between have at least 50%, preferably at least 70%, the polynucleotide of at least 80% homogeny more preferably.The present invention be more particularly directed under stringent condition and the interfertile polynucleotide of polynucleotide of the present invention.In the present invention, " stringent condition " is meant: (1) than hybridization under low ionic strength and the comparatively high temps and wash-out, as 0.2 * SSC, and 0.1%SDS, 60 ℃; Or (2) hybridization the time is added with denaturing agent, as 50% (v/v) methane amide, 0.1% calf serum/0.1%Ficoll, 42 ℃ etc.; Or (3) only at the homogeny between the two sequences at least more than 90%, be more preferably 95% and just hybridize when above.And the polypeptide of interfertile polynucleotide encoding has identical biological function and activity with the mature polypeptide shown in the SEQ ID NO:2.

The invention still further relates to nucleic acid fragment with above-mentioned sequence hybridization.As used herein, the length of " nucleic acid fragment " contains 15 Nucleotide at least, better is at least 30 Nucleotide, is more preferably at least 50 Nucleotide, preferably more than at least 100 Nucleotide.Nucleic acid fragment can be used for the amplification technique (as PCR) of nucleic acid to determine and/or the proteic polynucleotide of separation coding CaM-KIIN α.

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

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

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

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

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

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

The present invention also relates to comprise the carrier of polynucleotide of the present invention, and the host cell that produces through genetically engineered with carrier of the present invention or CaM-KIIN α albumen coded sequence, and the method that produces polypeptide of the present invention through recombinant technology.

Recombinant DNA technology (Science, 1984 by routine; 224:1431), can utilize polymerized nucleoside acid sequence of the present invention to can be used to express or produce the CaM-KIIN α albumen of reorganization.In general following steps are arranged:

(1). with the coding people proteic polynucleotide of CaM-KIIN α of the present invention (or varient), or transform or the transduction proper host cell with the recombinant expression vector that contains these polynucleotide;

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

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

Among the present invention, people CaM-KIIN α polynucleotide sequence can be inserted in the recombinant expression vector.Term " recombinant expression vector " refers to that bacterial plasmid well known in the art, phage, yeast plasmid, vegetable cell virus, mammalian cell virus are as adenovirus, retrovirus or other carriers.The carrier of Shi Yonging includes but not limited in the present invention: and the expression vector based on T7 of in bacterium, expressing (Rosenberg, et al.Gene, 1987,56:125); The pMSXND expression vector of in mammalian cell, expressing (Lee and Nathans, J Bio Chem.263:3521,1988) and at the carrier that derives from baculovirus of expressed in insect cells.In a word, as long as can duplicate in host and stablize, any plasmid and carrier can be used.A key character of expression vector is to contain replication orgin, promotor, marker gene and translation controlling elements usually.

Method well-known to those having ordinary skill in the art can be used to make up and contains people CaM-KIIN α DNA sequences encoding and suitable transcribing/the translate expression vector of control signal.These methods comprise (Sambroook, et al.Molecular Cloning, a LaboratoryManual, cold Spring Harbor Laboratory.New York, 1989) such as extracorporeal recombinant DNA technology, DNA synthetic technology, the interior recombinant technologys of body.Described dna sequence dna can effectively be connected on the suitable promotor in the expression vector, and is synthetic to instruct mRNA.The representative example of these promotors has: colibacillary lac or trp promotor; Lambda particles phage PL promotor; Eukaryotic promoter comprises LTRs and some other known may command gene expression promoter in protokaryon or eukaryotic cell or its virus of CMV immediate early promoter, HSV thymidine kinase promoter, early stage and late period SV40 promotor, retrovirus.Expression vector also comprises ribosome bind site and the transcription terminator that translation initiation is used.

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

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

Host cell can be a prokaryotic cell prokaryocyte, as bacterial cell; Or eukaryotic cell such as low, as yeast cell; Or higher eucaryotic cells, as mammalian cell.Representative example has: intestinal bacteria, streptomyces; The bacterial cell of Salmonella typhimurium; Fungal cell such as yeast; Vegetable cell; The insect cell of fruit bat S2 or Sf9; The zooblast of CHO, COS, 293 cells or Bowes melanoma cells etc.

When polynucleotide of the present invention are expressed in higher eucaryotic cells, be enhanced if will make to transcribe when in carrier, inserting enhancer sequence.Enhanser is the cis acting factor of DNA, and nearly 10 to 300 base pairs act on promotor transcribing with enhancing gene usually.

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

Can carry out with routine techniques well known to those skilled in the art with the recombinant DNA transformed host cell.When the host was prokaryotic organism such as intestinal bacteria, the competent cell that can absorb DNA can be handled with the CaCl2 method in exponential growth after date results, and used step is well-known in this area.Another kind method is to use MgCl2.If desired, transforming also the method for available electroporation carries out.When the host is an eukaryote, can select following DNA transfection method for use: coprecipitation of calcium phosphate method, conventional mechanical method such as microinjection, electroporation, liposome packing etc.

The transformant that obtains can be cultivated with ordinary method, expresses the polypeptide of coded by said gene of the present invention.According to used host cell, used substratum can be selected from various conventional substratum in the cultivation.Under the condition that is suitable for the host cell growth, cultivate.After host cell grows into suitable cell density, induce the promotor of selection with suitable method (as temperature transition or chemical induction), cell is cultivated for some time again.

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

The people CaM-KIIN α albumen or the polypeptide of reorganization are of use in many ways.These purposes include, but is not limited to: the direct disease due to the low or forfeiture and be used to screen and promote or antibody, polypeptide or other part of antagonism CaM-KIIN α protein function as pharmacological agent CaM-KIIN α protein function.The peptide molecule that can suppress or stimulate people CaM-KIIN α protein function that can be used for seeking therapeutic value with the recombinant human CaM-KIIN α protein screening peptide library of expressing.

On the other hand, the present invention also comprises people CaM-KIIN α DNA or the polypeptide of its fragment coding has specific polyclonal antibody and monoclonal antibody, especially monoclonal antibody.Here, " specificity " is meant that antibody capable is incorporated into people CaM-KIIN α gene product or fragment.Preferably, refer to that those can combine with people CaM-KIIN α gene product or fragment but nonrecognition and be incorporated into the antibody of other irrelevant antigen molecule.Among the present invention antibody comprise those can in conjunction with and suppress the proteic molecule of people CaM-KIIN α, comprise that also those do not influence the antibody of people CaM-KIIN α protein function.The present invention also comprise those can with modify or without the people CaM-KIIN α 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; Or chimeric antibody, as have the murine antibody binding specificity but still keep antibody from people's antibody moiety.

Antibody of the present invention can be prepared by the known various technology of those skilled in that art.For example, the people CaM-KIIN α gene product of purifying or its have antigenic fragment, can be applied to animal to induce the generation of polyclonal antibody.Similarly, expressing human CaM-KIIN α albumen or its have antigenic segmental cell and can be used to immune animal and produce antibody.Monoclonal antibody of the present invention can utilize hybridoma technology to prepare.Antibody of the present invention comprises the antibody that can block people CaM-KIIN α protein function and the antibody that does not influence people CaM-KIIN α protein function.Each antibody-like of the present invention can utilize the fragment or the functional zone of people CaM-KIIN α gene product, obtains by the routine immunization technology.These fragments or functional zone can utilize recombinant methods or utilize Peptide synthesizer synthetic.Can come immune animal and produce with the gene product of producing in the prokaryotic cell prokaryocyte (for example E.Coli) with the unmodified form bonded antibody of people CaM-KIIN α gene product; With posttranslational modification form bonded antibody (as the albumen or the polypeptide of glycosylation or phosphorylation), can come immune animal and obtain with the gene product that produces in the eukaryotic cell (for example yeast or insect cell).

The proteic antibody of anti-people CaM-KIIN α can be used in the immunohistochemistry technology, detects the people CaM-KIIN α albumen in the biopsy specimen.

Antibody among the present invention can be used for treating or prevention and the relevant disease of people CaM-KIIN α albumen.The antibody that gives suitable dosage can stimulate or block proteic generation of people CaM-KIIN α or activity.

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

The production of polyclonal antibody can choose CaM-KIIN α albumen or polypeptide immune animal, as rabbit, mouse, rat etc.Multiple adjuvant can be used for the enhancing immunity reaction, includes but not limited to freund's adjuvant etc.

Utilize albumen of the present invention,, can filter out with CaM-KIIN α albumen interactional material takes place, as part, inhibitor, agonist or antagonist etc. by various conventional screening methods.

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

Polypeptide of the present invention can be directly used in disease treatment, for example, is used for the treatment of aspects such as tumour.When using CaM-KIIN α albumen of the present invention, also can use the other treatment agent simultaneously, as IFN-α, IFN-β, TNF-α, TNF-β etc.

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

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

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

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

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

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

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

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

The proteic polynucleotide of CaM-KIIN α can be used for the diagnosis and the treatment of CaM-KIIN α protein related diseases.Aspect diagnosis, the proteic polynucleotide of CaM-KIIN α can be used for detecting the proteic expression of CaM-KIIN α CaM-KIIN α abnormal exprssion whether or under morbid state.Can be used for the hybridization of biopsy specimen to judge the proteic abnormal expression of CaM-KIIN α as CaM-KIIN α dna sequence dna.Hybridization technique comprises the Southern blotting, Northern blotting, in situ hybridization etc.These technological methods all are disclosed mature technologies, and relevant test kit all can obtain from commercial channels.Part or all of polynucleotide of the present invention can be used as probe stationary on microarray (microarray) or DNA chip (being called " gene chip " again), is used for analyzing the differential expression analysis and the gene diagnosis of tissue gene.Carry out RNA-polymerase chain reaction (RT-PCR) amplification in vitro with the special primer of CaM-KIIN α albumen and also can detect the proteic transcription product of CaM-KIIN α.

The sudden change that detects CaM-KIIN α gene also can be used for the disease of diagnosing CaM-KIIN α albumen relevant.The form of CaM-KIIN α protein mutation comprises that the point mutation compared with normal wild type CaM-KIIN α dna sequence dna, transposition, disappearance, reorganization and other are any unusual etc.Available existing technology such as Southern blotting, dna sequence analysis, PCR and in situ hybridization detect sudden change.In addition, sudden change might influence proteic expression, therefore can judge indirectly that with Northern blotting, Western blotting gene has or not sudden change.

Sequence of the present invention identifies it also is valuable to karyomit(e).In brief, the proteic cDNA of CaM-KIIN α prepares PCR primer (preferred 15-35bp) according to the present invention, sequence can be positioned on the karyomit(e).Then, these primers are used for the somatocyte hybrid cell that the PCR screening contains each bar human chromosome.Have only those hybrid cells that contain corresponding to the people's gene of primer can produce the fragment of amplification.

In case sequence is positioned to chromosome position accurately, the physical location of this sequence on karyomit(e) just can be associated with the gene map data.These data for example are found in, V.Mckusick, Mendelian Inheritancein Man (can by with the online acquisition of Johns Hopkins University Welch Medical Library).Can pass through linkage analysis then, determine gene and navigated to relation between the disease on the chromosomal region already.

In an example of the present invention, a kind of isolating polynucleotide are provided, its coding has the polypeptide of aminoacid sequence shown in the SEQ ID NO:2.Polynucleotide of the present invention are isolated from human dendritic cell cDNA library.Its sequence is shown in SEQ ID NO:1, and the polynucleotide sequence total length that it comprises is 860 bases, and its open reading frame is positioned at the 401-634 position, and the coding total length is 78 amino acid whose people CaM-KIIN α albumen (SEQ IDNO:2).This CaM-KIIN α albumen belongs to calcium ion/calmodulin dependent protein kinase ii repressible protein family molecule, calcium ion/calmodulin dependent protein kinase ii repressible protein α (CaM-KIIN α) and calcium ion/calmodulin dependent protein kinase ii repressible protein β (CaM-KIIN β) aminoacid sequence height homology with the rat source, wherein can be up to 98% with CaM-KIIN α consistence and similarity, with CaM-KIIN β consistence be 66%, similarity then is 79%.People CaM-KIIN alpha protein molecular weight is 8552.62Da, iso-electric point 5.22.RT-PCR analysis revealed CaM-KIIN α has particular expression in some tumour cell HeLa cell S3, MCF7, Reh and K-562 clone.The Northern engram analysis shows all expression in brain, cerebellum, hippocampus, tissue, the research prompting of having carried out, CaM-KIIN α may be the same with other inhibitor can be by specificity in conjunction with CaMKII, stop the phosphorylation of CaMKII to activate, regulate and control multiple physiology and pathology activity, play a significant role, and may be worth having important development and application aspect the immunodiagnosis in a plurality of fields such as anti-infective, anti-inflammatory response, antitumor and nerve growth reparation, immunologic function adjusting and the immunotherapy.Therefore, CaM-KIIN α albumen or its relevant antagonist, agonist etc. can be diseases such as treatment tumour, inflammation, neural system and cardiovascular diseases new immunodiagnosis and targeted therapy approach are provided, thereby have great application prospect.

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

Embodiment 1

The clone of people CaM-KIIN α cDNA

Extract the total RNA of human dendritic cell with Trizol reagent (Life Technologies company).Then, from total RNA, separate poly (A) mRNA.Poly (A) mRNA after reverse transcription forms cDNA, is cloned test kit (available from Gibco) with SuperScriptII cDNA fragment orientation is inserted on the multiple clone site of carrier, transform DH5 α bacterium and form the cDNA plasmid library.Measure random choose clone's 5 ' terminal sequence with dideoxy method.CDNA sequence and the existing public dna sequence data storehouse measured are compared, found that a cDNA clone's dna sequence dna is new full-length cDNA.By synthetic a series of primers the contained dna sequence dna of new clone is carried out two-way mensuration.Computer Analysis shows that cloning contained full-length cDNA is a new cDNA sequence (shown in SEQ ID NO:1), the new protein (shown in SEQ ID NO:2) of encoding.This protein is named as human calcium ion/calmodulin dependent protein kinase II repressible protein α (Calcium/Calmodulin-Dependent Protein Kinase II Inhibitor α, CaM-KIIN α), its encoding gene called after human calcium ion/calmodulin dependent protein kinase II repressible protein α gene, i.e. CaM-KIIN α gene.

Sequence SEQ ID NO:1 total length is 860 bp (Fig. 1 and SEQ ID NO:1), comprises 5 ' the end non-coding region of 400bp and 3 ' the end non-coding region of 223bp, and coding contains 78 amino acid whose polypeptide (Fig. 2 and SEQ IDNO:2).Calculating not in theory, the molecular weight of glycosylated ripe molecule is about 8552.62Da, iso-electric point 5.22.

They are different with known for the BLAST analysis revealed, calcium ion/calmodulin dependent protein kinase ii repressible protein CaM-KIIN α height homology with the rat source, consistence and similarity can be up to 98% (Fig. 3), calcium ion/calmodulin dependent protein kinase ii repressible protein CaM-KIIN β the consistence in rat source is 66%, and similarity then is 79% (Fig. 3).This shows that it belongs to calcium ion/calmodulin dependent protein kinase ii repressible protein family molecule.

Embodiment 2

Obtain the analysis of people's CaM-KIIN α encoding sequence and pair cell expression with the RT-PCR method

(1) obtains people CaM-KIIN α encoding sequence with the RT-PCR method

Extract the total RNA of dendritic cell that is in the corresponding clone of logarithmic phase, human peripheral blood mononuclear cell and stimulates the human peripheral blood mononuclear cell of different time to originate through LPS with Trizol reagent, get 5 μ g cell total rnas and mix, carry out reverse transcription with 1 μ g Oligo-dT12-18.The reverse transcription system is 20 μ l, adds 80 μ l ddH2O after reaction finishes and dilutes.The used primer of pcr amplification CaM-KIIN α is as follows: adopted primer 5 ' CCG AGG ATG TGA GTCCTG CTC (SEQ ID NO:3) is arranged, antisense primer 5 ' GAG GAG CCC AGA GCC TTC TC (SEQ ID NO:4), simultaneously with β-actin as positive control.The PCR reaction volume is 50 μ l, wherein contain reverse transcription template 10 μ l, 0.5mM primer, 0.2mM dNTP and 1U rTaq archaeal dna polymerase (Takara Inc.), the amplification parameter be 94 ℃ 20 seconds, 59 ℃ 30 seconds, 72 ℃ 40 seconds, capable 2% agarose gel electrophoresis of PCR product is tentatively confirmed after 30 circulations.The dna sequence analysis result shows that the DNA sequences encoding of this PCR product and the 127-840 shown in the SEQ ID NO:1 are identical.

The result shows, people CaM-KIIN α mRNA and has certain expression pattern (Fig. 4) in the reactivation process that stimulated by KLH under the different maturity states of dendritic cell in human peripheral blood mononuclear cell source.

(2) with of the analysis of RT-PCR method to CaM-KIIN α cell expressing

Get different tissues and cell, carry out RT-PCR, obtain people's CaM-KIIN α RT-PCR expression analysis collection of illustrative plates (Fig. 4) by above-mentioned same reaction conditions.Results suggest people CaM-KIIN α mRNA also is expressed in some solid tumor cell, as breast cancer cell MCF-7, glioma cell A172, cervical cancer cell HeLa, liver cancer cell SMMC7721, prostate cancer cell PC-3, lung cell A549; It is in the tumour cell that people CaM-KIIN α mRNA also is expressed in some hematopoiesis, as monocytic leukemia cell THP-1, group chronic myeloid leukemia cell U937, T lymphoma cell Jurkat, B lymphoma cell Raji and Daudi.Do not reach and in peripheral blood T, B cell and monocyte, colon cancer cell LoVo, ovarian cancer cell CaoV-3, marrow series leukemia cell HL-60 and the NB4 of fresh separated and acute lymphoblastic leukemia cell Reh, see Table.

Embodiment 3

People CaM-KIIN α is recombinant expressed

In this embodiment, be template with the RT-PCR amplified production that obtains among the embodiment 2,5 ' and 3 ' the PCR Oligonucleolide primers held following with sequence increases, and obtains people CaM-KIIN α full length coding region DNA as inserting fragment.

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

5’-AC?GGA?TCC?ATG?TCG?GAG?GTG?CTG?CCC?T-3’(SEQ?ID?NO：5)

This primer contains the restriction enzyme site of BamH I restriction enzyme, is whole encoding sequences of people CaM-KIIN α after this restriction enzyme site;

3 ' end primer sequence is:

5’-TC?GAA?TTC?TTA?GAC?ACC?AGG?AGG?TGC?CT-3’(SEQ?ID?NO：6)

This primer contains whole encoding sequences of restriction enzyme site, translation termination and the people CaM-KIIN α of EcoR I restriction enzyme.

RT-PCR amplified production with acquisition among the embodiment 2 is a template, and 5 ' and 3 ' the PCR Oligonucleolide primers held following with sequence increases, and obtains the individual amino acid whose DNA of coding people CaM-KIIN α N end 1-41 as the insertion fragment.

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

5’-AC?GGA?TCC?ATG?TCG?GAG?GTG?CTG?CCC?T-3’(SEQ?ID?NO：5)

This primer contains the restriction enzyme site of BamH I restriction enzyme, is the encoding sequence of people CaM-KIIN α after this restriction enzyme site;

3 ' end primer sequence is:

5’-TC?GAA?TTC?TTA?GTT?CTG?CCC?GGC?GCC?GA-3’(SEQ?ID?NO：7)

This primer contains the restriction enzyme site of EcoR I restriction enzyme and the 41 amino acids encoding sequence before of people CaM-KIIN α.

With the PCR product purification that obtains after EcoR I-BamH I enzyme cut and recombinate according to a conventional method with protokaryon GST fusion expression plasmid pGEX-4T3 (Amersham-Pharmacia company) again and be converted into competence e. coli bl21 (DE3), the picking positive colony is identified back purifying and order-checking (377 type sequenators of ABI company, the BigDyeTerminator test kit, PE company).Confirm through order-checking, inserted designed people CaM-KIIN α total length or 1-41 amino acids encoding sequence.The carrier of correct sequence is called pGEX-4T-people CaM-KIIN α and pGEX-4T-people CaM-KIIN α (1-41).

Choosing the positive BL21 clone who expresses CaM-KIIN α total length or 1-41 amino acids is inoculated in 100ml2 * YTA substratum, 37 ℃ of 300rpm shaking culture 12-15hr, 2 * YTA the substratum that is diluted in preheating at 1: 10 continues shaking culture 1.5hr, add behind the 100mM IPTG to 0.1mM 30 ℃ and induce 2-6hr, 5,4 ℃ of centrifugal 10min of 000g remove supernatant, put and use 50ml 1 * PBS (0.14M NaCl on ice, 2.7mM KCl, 10.1mMNa ₂HPO ₄, 1.8mM KH ₂PO ₄PH7.3) resuspended, add 20%Triton X-100 to 1% jog 30min again after ultrasonic (B.Braun Labsonic U) fragmentation, then 12,4 ℃ of centrifugal 10min of 000g, supernatant with 0.8 μ m membrane filtration after, cross 1ml 50% glutathione S epharose 4B chromatography column, behind 1 * PBS thorough washing, add 500ul gsh elution buffer (10mM gsh, 50mM Tris-HCl, pH 8.0) room temperature leaves standstill after 30 minutes and collects elutriant, repeat wash-out 2-3 time, obtain the GST-CaM-KIIN alpha fusion protein, or GST-CaM-KIIN α (1-41) fusion rotein.

Embodiment 4: the generation of anti-people CaM-KIIN Alpha antibodies

The recombinant protein total length people CaM-KIIN α that obtains among the embodiment 3 is used for immune animal to produce antibody, and concrete grammar is as follows.Recombinant molecule is standby after separating with chromatography.Also available SDS-PAGE gel electrophoresis separates, electrophoretic band downcut from gel, and with isopyknic complete Freund ' s adjuvant emulsion.Albumen with 50-100 μ g/0.2ml emulsification carries out peritoneal injection to mouse.After 14 days,, mouse is carried out peritoneal injection with booster immunization with the dosage of 50-100 μ g/0.2ml with the same antigen of non-complete Freund ' s adjuvant emulsion.Carried out booster immunization one time every 14 days, carry out at least three times.The sero-fast specific reaction that obtains is active to be assessed in the ability of external precipitation people CaM-KIIN α gene translation product with it.Found that antibody can combine with albumen of the present invention specifically.

Embodiment 5: people CaM-KIIN α Construction of eukaryotic

In this embodiment, be template with the RT-PCR amplified production that obtains among the embodiment 2,5 ' and 3 ' the PCR Oligonucleolide primers held following with sequence increases, and obtains people CaM-KIIN α full length coding region DNA as inserting fragment.The PCR reaction parameter be 95 ℃ 15 seconds, 58 ℃ 30 seconds, 72 ℃ 30 seconds, 20 circulations were extended 10 minutes for back 72 ℃,

Upstream primer is 5 '-(5 ' end contains the BamHI site to AC GAA TTC ATG TCG GAG GTG CTG CCC T-3 ', and the initial code of people CaM-KIIN α coding region) (SEQ ID NO:8), downstream primer be 5 '-TGAA GCT TAC ACC AGG AGG TGC CTT G-3 ' (5 ' end contains Kpn I site) (SEQ ID NO:9).

Behind the PCR product purification directly and pGEM-T carrier (Promega) recombinate according to a conventional method and be converted into competence bacterium DH5 α.Picking white clone identifies, also order-checking (sequencing primer is T7 and SP6) of purifying.With the purified rear clone of BamH I-Kpn I endonuclease bamhi of correct sequence to expression vector pcDNA3.1/Myc-His (-) B (Invitrogen company).After enzyme was cut the evaluation positive colony, recon was designated as pcDNA-people CaM-KIIN α.Confirm through order-checking, inserted people CaM-KIIN α encoding sequence.

Embodiment 6: the interaction of people CaM-KIIN α and CaMKII

Eukaryotic expression plasmid DNA with people CaM-KIIN α constructed among the embodiment 5 utilizes LipofectAMINE reagent (Invitrogen) to carry out the gene transfection of eukaryotic cell NIH 3T3, contrasts as irrelevant with pcDNA3.1/Myc-His (-) B plasmid vector.After the transfection 48 hours, add final concentration 5nmol/ml ionomycin and handle after 5 minutes collecting cell (5 * 10 ⁶Cell/sample), wash one time with PBS after, extract reagent (PIERCE) with the T-PER tissue protein and extract the cell whole protein, measure protein concentration with BCA method (PIERCE).After each sample room protein concentration was adjusted to unanimity, 200 μ g protein samples were with the Ni-NTA-Beads immunoprecipitation, and immunoprecipitate carries out the Western trace with anti-CaMKII antibody (Santa Cruz) and detects mutually combining of people CaM-KIIN α and CaMKII.

The Western trace detects: 6 times of sample-loading buffer (100mmol/L Tris-HCL of per 20 μ l and 4 μ l, 200mmol/L DTT, 4%SDS, 0.2% tetrabromophenol sulfonphthalein, 20% glycerine, PH6.8) mixing, after 100 ℃ of water-baths were boiled 5 minutes, row 12%SDS-PAGE electrophoresis moved on to the protein transduction in the polyacrylamide on the nitrocellulose filter in 4 ℃ with the 100V constant voltage subsequently, ponceau dyeing and label orientation mark the Marker position.4 hours (the TBST solution of 10% skim-milk) of room temperature blocking-up, be diluted in the skim-milk solution by 1: 1000 rabbit source ERK, MEK1/2 be how anti-, after 2 hours, wash 3 times each 10 minutes in room temperature reaction with TBST (the TBS solution of 0.05%Tween20) with cellulose nitrate film.In addition corresponding two of horseradish peroxidase-labeled anti-then, incubated at room 60 minutes, TBST gives a baby a bath on the third day after its birth time, each 10 minutes, A liquid in the Western trace luciferase assay reagent and B liquid is mixed with equal-volume, and incubated at room dried after 1 minute, press mold, exposure is developed.

The result shows, handles through ionomycin and improves Ca in the born of the same parents ²⁺After the concentration, can detecting existing of CaMKII in Ni-NTA-Beads sedimentary people CaM-KIIN α and the interaction protein thereof, confirmer CaM-KIIN α can mutually combine with the CaMKII of active state (Fig. 5).

Embodiment 7: people CaM-KIIN α is to the restraining effect of CaMKII enzymic activity

Utilize the people CaM-KIIN α total length recombinant expressed among the embodiment 3 or the recombinant protein of 1-41 amino acids, set up CaMKII enzymic activity detection architecture (New England Biolab company), concrete testing sequence is according to the test kit specification sheets.The variation of the phosphorylation level by CaMKII effect substrate reflects the influence of CaMKIIN to the CaMKII enzymic activity.

The result shows, the total length people CaM-KIIN alpha fusion protein of different concns all can suppress the phosphorylation of CaMKII to substrate, and lacks people CaM-KIIN α (1-41) fusion rotein of 41-58 amino acids inhibition conserved regions and the activity that contrast GST albumen all can not suppress CaMKII.Prompter CaM-KIIN α can suppress the kinase activity (Fig. 6) of CaMKII by its inhibition conserved regions.

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

＜110〉Immunology Inst., No.2 Military Medical Univ.

＜120〉human calcium ion/calmodulin dependent protein kinase II repressible protein α, its encoding sequence and purposes

<130>033707

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<170>PatentIn?version?3.1

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＜213〉homo sapiens (Homo sapiens)

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<400>1

cccgcccggc?acccggggtg?cgccctcctc?ggtcccgcgc?cctccgggct?cgcagggacg?????60

tctcctccct?cccggctcgc?ggccccgccc?ggcccggccc?ccgcccagag?ccccagcgcg????120

ccgaggatgt?gagtcctgct?cgcctctggc?ggagcagcag?ccactcgcgc?gcggagccgg????180

agcgcagcgc?agcgcagccg?cgggcgctct?ccgggccgct?cgcgcgagtg?ccgcgctctt????240

gccctagcgg?cgtcccccgg?cctctcgccg?gcgccaccgc?cgcagcagcc?cgcgggccgt????300

ccccggccgg?ccgcccccgg?ccccagcgcc?gctgaccctg?tccgccgcgg?gcggggacgc????360

gggcggagga?ggcgccgcgg?cggagccccc?ggacgcgacc?atg?tcg?gag?gtg?ctg??????415

Met?Ser?Glu?Val?Leu

1???????????????5

ccc?tac?ggc?gac?gag?aag?ctg?agc?ccc?tac?ggc?gac?ggc?ggc?gac?gtg??????463

Pro?Tyr?Gly?Asp?Glu?Lys?Leu?Ser?Pro?Tyr?Gly?Asp?Gly?Gly?Asp?Val

10??????????????????15??????????????????20

ggc?cag?atc?ttc?tcc?tgc?cgc?ctg?cag?gac?acc?aac?aac?ttc?ttc?ggc??????511

Gly?Gln?Ile?Phe?Ser?Cys?Arg?Leu?Gln?Asp?Thr?Asn?Asn?Phe?Phe?Gly

25?????????????????30??????????????????35

gcc?ggg?cag?aac?aag?cgg?ccg?ccc?aag?ctg?ggc?cag?atc?ggc?cgg?agc??????559

Ala?Gly?Gln?Asn?Lys?Arg?Pro?Pro?Lys?Leu?Gly?Gln?Ile?Gly?Arg?Ser

40??????????????????45?????????????????50

aag?cgg?gtt?gtt?att?gaa?gat?gat?agg?att?gat?gac?gtg?ctg?aaa?aat??????607

Lys?Arg?Val?Val?Ile?Glu?Asp?Asp?Arg?Ile?Asp?Asp?Val?Leu?Lys?Asn

55??????????????????60?????????????????65

atg?acc?gac?aag?gca?cct?cct?ggt?gtc?taactccccc?aaagacaatg????????????654

Met?Thr?Asp?Lys?Ala?Pro?Pro?Gly?Val

70??????????????????75

agttaaggga?gagaataaga?acggcggtaa?cagttattgg?caaaaagcat?gaaaagagaa????714

agcactttga?aatttattac?tagcttgcta?cccacgatga?aatcaacaac?ctgtatctgg????774

tatcaggccg?ggagacagat?gaggcgagag?gaggaggagg?aggaggagaa?ggctctgggc????834

tcctctgcaa?aaaaaaaaaa?aaaaaa?????????????????????????????????????????860

<210>2

<211>78

<212>PRT

＜213〉homo sapiens (Homo sapiens)

<400>2

Met?Ser?Glu?Val?Leu?Pro?Tyr?Gly?Asp?Glu?Lys?Leu?Ser?Pro?Tyr?Gly

1???????????????5??????????????????10??????????????????15

Asp?Gly?Gly?Asp?Val?Gly?Gln?Ile?Phe?Ser?Cys?Arg?Leu?Gln?Asp?Thr

20?????????????????25??????????????????30

Asn?Asn?Phe?Phe?Gly?Ala?Gly?Gln?Asn?Lys?Arg?Pro?Pro?Lys?Leu?Gly

35?????????????????40??????????????????45

Gln?Ile?Gly?Arg?Ser?Lys?Arg?Val?Val?Ile?Glu?Asp?Asp?Arg?Ile?Asp

50?????????????????55??????????????????60

Asp?Val?Leu?Lys?Asn?Met?Thr?Asp?Lys?Ala?Pro?Pro?Gly?Val

65?????????????????70??????????????????75

<210>3

<211>21

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

＜223〉primer

<400>3

ccgaggatgt?gagtcctgct?c???????????????????????????????????????????????21

<210>4

<211>20

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

＜223〉primer

<400>4

gaggagccca?gagccttctc?????????????????????????????????????????????????20

<210>5

<211>27

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

＜223〉primer

<400>5

acggatccat?gtcggaggtg?ctgccct?????????????????????????????????????????27

<210>6

<211>28

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

＜223〉primer

<400>6

tcgaattctt?agacaccagg?aggtgcct????????????????????????????????????????28

<210>7

<211>28

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

＜223〉primer

<400>7

tcgaattctt?agttctgccc?ggcgccga?????????????????????????????????????28

<210>8

<211>27

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

＜223〉primer

<400>8

acgaattcat?gtcggaggtg?ctgccct????????????????????????????????????????27

<210>9

<211>26

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

＜223〉primer

<400>9

tgaagcttac?accaggaggt?gccttg?????????????????????????????????????????26

Claims (10)

1. an isolating people CaM-KIIN α albumen is characterized in that, it contains polypeptide or its conservative property variation polypeptide or its active fragments or its reactive derivative of SEQ ID NO:2 aminoacid sequence.

2. polypeptide as claimed in claim 1 is characterized in that, this polypeptide is selected from down group:

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

(b) SEQ ID NO:2 aminoacid sequence is formed through replacement, disappearance or the interpolation of one or more amino-acid residues, and have combination and suppress calcium ion/active function of calmodulin dependent protein kinase ii by (a) polypeptides derived.

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

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

(b) with polynucleotide (a) complementary polynucleotide.

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

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

(a) has the sequence of 401-634 position among the SEQ ID NO:1;

(b) has the sequence of 1-860 position among the SEQ ID NO:1.

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

7. a genetically engineered host cell is characterized in that, it contains the described carrier of claim 6.

8. proteic preparation method of people CaM-KIIN α is characterized in that this method comprises:

(a) under conditions suitable for the expression, cultivate the described host cell of claim 7;

(b) from culture, isolate people CaM-KIIN α albumen.

9. energy and the described people CaM-KIIN of claim 1 α protein-specific bonded antibody.

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

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