CN1297928A - Human EF-chiral calcium binding protein 96 containing ATP/GTP binding structure domain as one new kind of polypeptide and polynucleotides encoding this polypeptide - Google Patents

Abstract

The present invention discloses a novel polypeptide, human EF-chiral calcium binding protein 96 containing ATP/GTP binding structure domain, polynucleotides encoding this polypeptide and DNA recombination process to produce the polypeptide. The present invention also discloses the method of applying the polypeptide in treating various diseases, such as malignant tumor, nosohemia, HIV infection, immunological diseases and inflammations. The present invention also discloses the antagonist resisting the polypeptide and its treatment effect. The present invention also discloses the application of the polynucleotides encoding this polypeptide.

Description

A kind of new polypeptide-human contains the EF-hand-type calcium binding protein 96 of ATP/GTP binding domains and the polynucleotide of this peptide species of coding

The invention belongs to biological technical field, specifically, the invention describes the EF-hand-type calcium binding protein 96 that a kind of new polypeptide-human contains the ATP/GTP binding domains, and the polynucleotide sequence of this polypeptide of encoding.The invention still further relates to the preparation method and the application of these polynucleotide and polypeptide.

The variation of calcium ion concn has caused cell response widely in the enchylema.Intracellular calcium binding protein is to regulate the key molecule that conducts in the calcium signal by enzyme reaction or albumen and protein-interacting.In them some acted in the cell cycle, or acted on cytodifferentiation.After second signal molecule (as inositoltriphosphoric acid) stimulation, they are discharged into the tenuigenin on every side from endoplasmic reticulum momently.Similar physiological process is broken and is also taken place during the chromatid of mitosis anaphase separates at nuclear membrane.

Many calcium binding proteins all belong to same evolution family, and contain common calcium binding domains, i.e. an EF-hand-type structural domain.These albumen have constituted very big protein family, i.e. an EF-hand-type calcium binding protein family.The member of this protein family distributes very extensive, and these albumen all play an important role in cell growth and atomization, is regulating and control cell cycle and is controlling the metabolic processes of organism.

EF-hand-type structural domain comprises the ring that 12 amino-acid residue is formed, and this ring stretches to both sides by the alpha helical region territory of 12 amino-acid residues, forms the angle that becomes 90 degree approximately.In the EF-hand ring, calcium ion is incorporated in the conformation of a pentagonal bipyramid shape.Relate to 6 residues of bonded 1,3,5,7,9 and 12.Going up conservative Glu or Asp for 12 provides and two Sauerstoffatoms of Ca coordinate (bidentate part).

The distribution of EF-hand-type calcium binding protein in this family is more, and such calcium binding protein has following conservative consensus sequence fragment: [LIVMFYW] (2)-X (2)-[LK]-D-X (3)-[DN]-X (3)-[DNSG]-[FY]-X-[ES]-[FYVC]-X (2)-[LIVMFS]-[LIVMF]; All EF-hand-type calcium binding proteins all have this consensus sequence fragment, but not do not contain this sequence fragment in the albumen of EF-hand-type.This sequence fragment includes the high affine zone of EF-hand-type, but this site does not possess calcium in conjunction with feature.It is not albumen and site, calcium bonded center, but is regulating the affinity interaction of albumen and calcium ion in vivo.

ATP or GTP binding motif be various ATP/GTP conjugated protein in ubiquitous structural domain, some albumen that connect ATP or GTP all more or less contain this conservative motif sequence, and this motif has conservative characteristic sequential fragments: [AG]-X (4)-G-K-[ST] (wherein X is an arbitrary amino acid residue).What guard the most among these motif is the glycine rich region, and this zone forms a flexible ring between β line and α spiral.This crooked ring usually and a certain phosphate group reaction on the Nucleotide, this motif sequence is commonly called " A " consensus sequence or P ring.This motif is responsible for combining with ATP, GTP in vivo usually, for albumen performance biological function provides required energy and stabilize proteins structure in vivo, assists albumen to finish its normal physiologic function.Its abnormal expression will directly cause some abnormal exprssion and dysfunction, thereby cause various relevant diseases, as grow generation of disorder, tumour and cancer etc.

The EF-hand-type calcium binding protein 96 that new people of the present invention contains the ATP/GTP binding domains also contains the conservative EF-hand-type consensus sequence fragment of EF-hand-type calcium binding protein, simultaneously, also contain the ATP/GTP binding domains, thereby, the newcomer of its behaviour calcium binding protein family, and have similar biological function to other member of this protein family.This albumen is being regulated and control the growth and the differentiation of cell in vivo, and the metabolic processes of regulating organism.Its abnormal expression will cause various relevant growth disorders, metabolism disorder disease and some neural diseases of each tissue.Because people's EF-hand-type calcium binding protein 96 albumen of containing the ATP/GTP binding domains plays an important role in the body critical function as mentioned above, and believe and relate to a large amount of albumen in these regulate processes, thereby need always in this area to identify that the people of more these processes of participation contains the EF-hand-type calcium binding protein 96 albumen of ATP/GTP binding domains, particularly identify this proteic aminoacid sequence.The separation that the new person contains the EF-hand-type calcium binding protein 96 protein coding gene of ATP/GTP binding domains also provides the foundation for determining the effect of this albumen under healthy and morbid state.This albumen may constitute the basis of exploitation medical diagnosis on disease and/or curative, and it is very important therefore separating its coding DNA.

An object of the present invention is to provide EF-hand-type calcium binding protein 96 that isolating new polypeptide-human contains the ATP/GTP binding domains with and fragment, analogue and derivative.

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

Another object of the present invention provides and contains the recombinant vectors of polynucleotide that the coding people contains the EF-hand-type calcium binding protein 96 of ATP/GTP binding domains.

Another object of the present invention provides and contains the genetically engineered host cell of polynucleotide that the coding people contains the EF-hand-type calcium binding protein 96 of ATP/GTP binding domains.

Another object of the present invention provides the method that the people contains the EF-hand-type calcium binding protein 96 of ATP/GTP binding domains of producing.

Another object of the present invention provides the antibody that contains the EF-hand-type calcium binding protein 96 of ATP/GTP binding domains at polypeptide-human of the present invention.

Another object of the present invention has provided simulated compound, antagonist, agonist, the inhibitor that contains the EF-hand-type calcium binding protein 96 of ATP/GTP binding domains at polypeptide-human of the present invention.

Another object of the present invention provides the method that diagnoses and treatment and people contain the unusual relevant disease of the EF-hand-type calcium binding protein 96 of ATP/GTP binding domains.

The present invention relates to a kind of isolated polypeptide, this polypeptide is the people source, and it comprises: polypeptide or its examples of conservative variations, bioactive fragment or derivative with SEQ ID No.2 aminoacid sequence.Preferably, this polypeptide is the polypeptide with SEQ ID NO:2 aminoacid sequence.

The invention still further relates to a kind of isolating polynucleotide, it comprises a kind of nucleotide sequence or its variant that is selected from down group:

(a) coding has the polynucleotide of the polypeptide of SEQ ID No.2 aminoacid sequence;

(b) with polynucleotide (a) complementary polynucleotide;

(c) with (a) or polynucleotide sequence (b) have the polynucleotide of at least 70% homogeny.

More preferably, the sequence of these polynucleotide is be selected from down group a kind of: the sequence that (a) has 117-2192 position among the SEQ ID NO:1; (b) has the sequence of 1-3181 position among the SEQ ID NO:1.

The present invention relates to a kind of carrier that contains polynucleotide of the present invention, particularly expression vector in addition; The host cell that this carrier of a kind of usefulness is genetically engineered comprises the host cell of conversion, transduction or transfection; A kind of method for preparing polypeptide of the present invention of cultivating described host cell and reclaiming expression product that comprises.

The invention still further relates to a kind of can with polypeptid specificity bonded antibody of the present invention.

The invention still further relates to a kind of simulation, activation, antagonism of screening or suppress the method for compound that the people contains the EF-hand-type calcium binding protein 96 protein-active of ATP/GTP binding domains, it comprises and utilizes polypeptide of the present invention.The invention still further relates to the compound that obtains with this method.

The invention still further relates to a kind of vitro detection and express the relevant disease or the method for disease susceptibility with the EF-hand-type calcium binding protein 96 abnormal protein that the people contains the ATP/GTP binding domains, comprise the sudden change in polypeptide described in the detection of biological sample or its coded polynucleotide sequence, perhaps the amount or the biological activity of polypeptide of the present invention in the detection of biological sample.

The present invention also relates to a kind of pharmaceutical composition, it contains polypeptide of the present invention or its stand-in, activator, antagonist or inhibitor and pharmaceutically acceptable carrier.

The invention still further relates to polypeptide of the present invention and/or polynucleotide and be used for the treatment of the metabolism disorder disease of growing disorders, each tissue and some neural diseases or other because the people contains the purposes of medicine of the EF-hand-type calcium binding protein 96 disease that abnormal expression causes of ATP/GTP binding domains in preparation.

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

The following term of using in this specification sheets and claims has following implication unless stated otherwise: " nucleotide sequence " is meant oligonucleotide, Nucleotide or polynucleotide and fragment or part, also can refer to genome or synthetic DNA or RNA, they can be strand or two strands, represent sense strand or antisense strand.Similarly, term " aminoacid sequence " is meant oligopeptides, peptide, polypeptide or protein sequence and fragment or part.When " aminoacid sequence " among the present invention related to a kind of aminoacid sequence of naturally occurring protein molecule, this " polypeptide " or " protein " did not mean that aminoacid sequence are restricted to the complete natural amino acid relevant with described protein molecule.

Protein or polynucleotide " variant " are meant a kind of polynucleotide sequence that has the aminoacid sequence of one or more amino acid or Nucleotide change or encode it.Described change can comprise disappearance, insertion or the replacement of amino acid in aminoacid sequence or the nucleotide sequence or Nucleotide.Variant can have " conservative property " and change, and wherein the amino acid of Ti Huaning has structure or the chemical property similar with original acid, as replacing Isoleucine with leucine.Variant also can have non-conservation and change, as replacing glycine with tryptophane.

" disappearance " is meant the disappearance of in aminoacid sequence or nucleotide sequence one or more amino acid or Nucleotide.

" insertion " or " interpolation " is meant that the change in aminoacid sequence or nucleotide sequence causes comparing the increase of one or more amino acid or Nucleotide with naturally occurring molecule." replacement " is meant by different amino acid or Nucleotide and replaces one or more amino acid or Nucleotide.

" biological activity " is meant the protein of structure, regulation and control or biochemical function with natural molecule.Similarly, term " immunologic competence " be meant natural, reorganization or synthetic protein and fragment thereof in suitable animal or cell, induce specific immune response and with specific antibody bonded ability.

" agonist " is meant when when EF-hand-type calcium binding protein 96 that the people contains the ATP/GTP binding domains combines, thereby a kind of this protein that causes changes the molecule of regulating this protein active.Agonist can comprise that protein, nucleic acid, carbohydrate or any other can contain the molecule of the EF-hand-type calcium binding protein 96 of ATP/GTP binding domains in conjunction with the people.

" antagonist " or " inhibition " be meant when when EF-hand-type calcium binding protein 96 that the people contains the ATP/GTP binding domains combines, a kind of seal or the mediator contains the biologic activity of EF-hand-type calcium binding protein 96 of ATP/GTP binding domains or the molecule of immunologic competence.Antagonist and inhibition can comprise that protein, nucleic acid, carbohydrate or any other can contain the molecule of the EF-hand-type calcium binding protein 96 of ATP/GTP binding domains in conjunction with the people.

" adjusting " is meant that the function that the people contains the EF-hand-type calcium binding protein 96 of ATP/GTP binding domains changes, and comprises the change of any other biological property, function or the immune property of the rising of protein active or reduction, the change of binding characteristic and the EF-hand-type calcium binding protein 96 that the people contains the ATP/GTP binding domains.

" pure basically " is meant and is substantially free of natural relative other albumen, lipid, carbohydrate or other material.Those skilled in the art can contain the EF-hand-type calcium binding protein 96 of ATP/GTP binding domains with the purified technology of protein purifying people of standard.Basically the EF-hand-type calcium binding protein 96 that pure people contains the ATP/GTP binding domains can produce single master tape on the irreducibility polyacrylamide gel.The people contains the purity available amino end acid sequence analysis of the EF-hand-type calcium binding protein 96 polypeptide of ATP/GTP binding domains.

" complementary " or " complementation " is meant under salt concn that allows and temperature condition by the natural combination of the polynucleotide of base pairing.For example, sequence " C-T-G-A " can combine with complementary sequence " G-A-C-T ".Complementation between two single chain molecules can be part or whole.Complementary degree between the nucleic acid chains has a significant effect for efficient of hybridizing between the nucleic acid chains and intensity.

" homology " is meant the complementary degree, can be portion homologous or complete homology." portion homologous " is meant a kind of part complementary sequence, and it can partly suppress the hybridization of complete complementary sequence and target nucleic acid at least.The inhibition of this hybridization can detect by hybridizing (Southern trace or Northern trace etc.) under the condition that reduces in the severity degree.Basically homologous sequence or hybridization probe can compete and suppress complete homologous sequence and target sequence the condition that reduces of severity degree under combine.This does not mean the conditions permit non-specific binding that the severity degree reduces because the conditional request two sequences that the severity degree reduces mutual be combined into specificity or selectivity interacts.

" homogeny percentage " be meant two or more amino acid or nucleotide sequence relatively in the same or analogous percentage of sequence.The available electron method is measured the homogeny percentage, as passing through MEGALIGN program (Lasergene softwarepackage, DNASTAR, Inc., Madison Wis.).The MEGALIGN program can compare two or more sequences (Higgins, D.G. and P.M.Sharp (1988) Gene73:237-244) according to diverse ways such as Cluster method.The Cluster method is organized the series arrangement cluster by checking the distance between all pairings with each.Then with each bunch with in pairs or become set of dispense.Homogeny percentage between two aminoacid sequences such as sequence A and the sequence B calculates by following formula:

The residue number of mating between sequence A and the sequence B

100

Interval residue number in the residue number-sequence B of interval in the residue number-sequence A of sequence A

Also can be by the Cluster method or with the homogeny percentage (Hein J., (1990) Methods in emzumology 183:625-645) between known method in this area such as the Jotun Hein mensuration nucleotide sequence.

" similarity " is meant the degree that the identical or conservative property of corresponding position amino-acid residue when arranging contrast between the aminoacid sequence replaces.For example be used for amino acid that conservative property replaces, electronegative amino acid can comprise aspartic acid and L-glutamic acid; Positively charged amino acid can comprise Methionin and arginine; Having uncharged head group has similar hydrophilic amino acid can comprise leucine, Isoleucine and Xie Ansuan; Glycine and L-Ala; L-asparagine and glutamine; Serine and Threonine; Phenylalanine and tyrosine.

" antisense " is meant and specific DNA or RNA sequence complementary nucleotide sequence." antisense strand " is meant and " sense strand " complementary nucleic acid chains.

" derivative " is meant HFP or encodes its chemical modification object of nucleic acid.This chemical modification object can be with alkyl, acyl group or the amino hydrogen atom of replacing.The nucleic acid derivative codified keeps the polypeptide of the main biological characteristics of natural molecule.

" antibody " is meant complete antibody molecule and fragment thereof, and as Fa, F (ab '), and Fv, it can specificity contains the antigenic determinant of the EF-hand-type calcium binding protein 96 of ATP/GTP binding domains in conjunction with the people." humanized antibody " is meant that the aminoacid sequence in non-antigen binding domain territory is replaced and becomes more similar to people's antibody, but still keep original in active antibody." isolating " speech refers to material is shifted out among its original environment (for example, if spontaneous its natural surroundings that just refers to).Such as it is exactly not to be separated that spontaneous polynucleotide or polypeptide are present in the Live Animals, but same polynucleotide or polypeptide with some or all in natural system with it the material of coexistence separately be exactly isolating.Such polynucleotide may be the parts of a certain carrier, the part that also possible such polynucleotide or polypeptide are a certain composition.Since carrier or composition are not the compositions of its natural surroundings, they remain isolating.

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

As used herein, " isolating people contains the EF-hand-type calcium binding protein 96 of ATP/GTP binding domains " is meant that the EF-hand-type calcium binding protein 96 that the people contains the ATP/GTP binding domains is substantially free of natural relative other albumen, lipid, carbohydrate or other material.Those skilled in the art can contain the EF-hand-type calcium binding protein 96 of ATP/GTP binding domains with the purified technology of protein purifying people of standard.Basically pure polypeptide can produce single master tape on non-reduced polyacrylamide gel.The people contains the purity of the EF-hand-type calcium binding protein 96 polypeptide of ATP/GTP binding domains can use amino acid sequence analysis.

The invention provides the EF-hand-type calcium binding protein 96 that a kind of new polypeptide-human contains the ATP/GTP binding domains, it is made up of the aminoacid sequence shown in the SEQ ID NO:2 basically.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 comprises that also the people contains fragment, derivative and the analogue of the EF-hand-type calcium binding protein 96 of ATP/GTP binding domains.As used herein, term " fragment ", " derivative " are meant identical biological function or the active polypeptide of EF-hand-type calcium binding protein 96 that keeps people of the present invention to contain the ATP/GTP binding domains basically with " analogue ".The fragment of polypeptide of the present invention, derivative or analogue can be: (I) is a kind of like this, wherein one or more amino-acid residues are replaced by conservative or non-conservative amino acid residues (preferably conservative amino acid residues), and the amino acid that replaces can be also can not encoded by genetic codon; Perhaps (II) is a kind of like this, and certain group on wherein one or more amino-acid residues is replaced by other group and comprises substituting group; Perhaps (III) is a kind of like this, and wherein mature polypeptide and another kind of compound (such as the compound that prolongs the polypeptide transformation period, for example polyoxyethylene glycol) merge; Perhaps (IV) is a kind of like this, wherein additional aminoacid sequence is integrated into mature polypeptide and the peptide sequence that forms (as leader sequence or secretion sequence or be used for the sequence or the proteinogen sequence of this polypeptide of purifying) by the elaboration of this paper, such fragment, derivative and analogue are considered within those skilled in the art's ken.

The invention provides isolating nucleic acid (polynucleotide), substantially the polynucleotide that have a polypeptide of SEQ ID NO:2 aminoacid sequence by coding are formed.Polynucleotide sequence of the present invention comprises the nucleotide sequence of SEQ ID NO:1.Polynucleotide of the present invention are to find from the cDNA library of people's fetal brain tissue.The polynucleotide sequence total length that it comprises is 2381 bases, its open reading frame (N1-N2) 37 amino acid of having encoded.This polypeptide has the characteristic sequence of (motif), and deducibility goes out the EF-hand-type calcium binding protein 96 that this people contains the ATP/GTP binding domains and has { the 26S Proteasome Structure and Function of motif} representative.

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 " are meant that in the present invention coding has protein or the polypeptide 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 that has only 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 " is meant polynucleotide that comprise this polypeptide of encoding and the polynucleotide that comprise additional code and/or non-coding sequence.

The invention still further relates to the varient of foregoing description polynucleotide, its coding has the polypeptide of identical aminoacid sequence or segment, 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 the polynucleotide (have at least 50% between two sequences, preferably have 70% homogeny) of sequence hybridization described above.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 adds and to use denaturing agent, as 50% (v/v) methane amide, 0.1% calf serum/0.1%Ficoll, 42 ℃ etc.; Or (3) only at the homogeny between the two sequences at least more than 95%, be more preferably 97% and just hybridize when above.And the polypeptide of interfertile polynucleotide encoding has identical biological function and activity with the mature polypeptide shown in the SEQ ID NO:2.

The invention still further relates to nucleic acid fragment with sequence hybridization described above.As used herein, the length of " nucleic acid fragment " contain 10 Nucleotide at least, better are 20-30 Nucleotide at least, are more preferably 50-60 Nucleotide at least, preferably more than at least 100 Nucleotide.The amplification technique (as PCR) that nucleic acid fragment also can be used for nucleic acid is to determine and/or to separate the polynucleotide that the coding people contains the EF-hand-type calcium binding protein 96 of ATP/GTP binding domains.

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

The special polynucleotide sequence that coding people of the present invention contains the EF-hand-type calcium binding protein 96 of ATP/GTP binding domains can obtain with several different methods.For example, separate polynucleotide with hybridization technique well known in the art.These technology including, but not limited to: 1) with probe and genome or the hybridization of cDNA library to detect homologous polynucleotide sequence and 2) antibody screening of expression library to be to detect the polynucleotide passage of the clone with common structure feature.

Sequence dna fragment of the present invention also can obtain with following method: 1) separate double chain DNA sequence from genomic dna; 2) the chemical synthesising DNA sequence is to obtain the double-stranded DNA of described polypeptide.

In the above-mentioned method of mentioning, isolation of genomic DNA is least commonly used.The direct chemical of dna sequence dna is synthetic to be the method for often selecting for use.The more frequent method of selecting for use is the separation of cDNA sequence.The standard method that separates interested cDNA is from the donorcells separating mRNA of this gene of high expression level and carries out reverse transcription, forms plasmid or phage cDNA library.Extract the existing multiple proven technique of method of mRNA, test kit also can obtain (Qiagene) from commercial channels.And the construction cDNA library also is usual method (Sambrook, et al., MolecularCloning, A Laboratory Manual, Cold Spring Harbor Laboratory.New York, 1989).Also can obtain the cDNA library of commercial offers, as the different cDNA library of Clontech company.When being used in combination the polymeric enzyme reaction technology, even few expression product also can be cloned.

Available ordinary method is screened gene of the present invention from these cDNA libraries.These methods include, but is not limited to: (1) DNA-DNA or DNA-RNA hybridization; (2) appearance of marker gene function or forfeiture; (3) measure the level of transcript that the people contains the EF-hand-type calcium binding protein 96 of ATP/GTP binding domains; (4), detect the protein product of genetic expression by immunological technique or mensuration biologic activity.Aforesaid method can singly be used, but also several different methods combined utilization.

In (1) kind method, hybridizing used probe is and any a part of homology of polynucleotide of the present invention that at least 10 Nucleotide of its length better are at least 30 Nucleotide, are more preferably at least 50 Nucleotide, preferably at least 100 Nucleotide.In addition, within 2000 Nucleotide, preferable is within 1000 Nucleotide to the length of probe usually.Probe used herein is the dna sequence dna of chemosynthesis on the basis of gene order information of the present invention normally.Gene of the present invention itself or fragment are certainly as probe.The mark of dna probe can be used radio isotope, fluorescein or enzyme (as alkaline phosphatase) etc.

In (4) kind method, the detection people contains the protein product of the EF-hand-type calcium binding protein 96 genetic expression of ATP/GTP binding domains can use immunological technique such as Western blotting, radioimmunoprecipitation, enzyme-linked immunosorbent assay (ELISA) etc.

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 from the library, obtain the cDNA of total length, 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 polynucleotide sequence information of the present invention disclosed herein, and available ordinary method is synthetic.Available ordinary method is as the DNA/RNA fragment by gel electrophoresis separation and purifying amplification.

The gene of the present invention that obtains as mentioned above, perhaps the polynucleotide sequence of various dna fragmentations etc. can with ordinary method such as dideoxy chain termination (Sanger et al.PNAS, 1977,74:5463-5467) measure.This class polynucleotide sequence is measured also available commercial sequencing kit etc.In order to obtain the cDNA sequence of total length, order-checking need be carried out repeatedly.Sometimes need to measure a plurality of clones' cDNA sequence, just can be spliced into the cDNA sequence of total length.

The present invention also relates to comprise the carrier of polynucleotide of the present invention, and with carrier of the present invention or directly personnel selection contain the host cell that the EF-hand-type calcium binding protein 96 encoding sequence of ATP/GTP binding domains produces through genetically engineered, and the method that produces polypeptide of the present invention through recombinant technology.

Among the present invention, the polynucleotide sequence that the coding people contains the EF-hand-type calcium binding protein 96 of ATP/GTP binding domains can be inserted in the carrier, contains the recombinant vectors of polynucleotide of the present invention with formation.Term " carrier " 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 carrier.The carrier of Shi Yonging includes but not limited in the present invention: and the expression vector based on the T7 promotor 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 may be used to make up recombinant expression vector.A key character of expression vector is to contain replication origin, promotor, marker gene and translational control element usually.

Method well-known to those having ordinary skill in the art can be used to make up and contain the coding people and contain the dna sequence dna of EF-hand-type calcium binding protein 96 of ATP/GTP binding domains and the expression vector of suitable transcribing/translational control element.These methods comprise (Sambroook, et al.Molecular Cloning, a Laboratory Manual, cold Spring Harbor Laboratory.NewYork, 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; The PL promotor of lambda particles phage; Eukaryotic promoter comprises LTRs and some other known may command gene expression promoter in prokaryotic cell prokaryocyte 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 that translation initiation is used and transcription terminator etc.Inserting enhancer sequence in carrier will make its transcribing in higher eucaryotic cells be enhanced.Enhanser is the cis acting factor that DNA expresses, and nearly 10 to 300 base pairs act on promotor transcribing with enhancing gene usually.Can for example be included in the SV40 enhanser of 100 to 270 base pairs of replication origin side in late period one, at the polyoma enhanser of replication origin side in late period one and adenovirus enhanser etc.

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 etc.

Persons skilled in the art all know how to select appropriate carriers/transcriptional regulatory element (as promotor, enhanser etc.) and selected marker.

Among the present invention, the coding people contains the polynucleotide of EF-hand-type calcium binding protein 96 of ATP/GTP binding domains or the recombinant vectors that contains these polynucleotide can transform or transduce into host cell, contains the genetically engineered host cell of these polynucleotide or recombinant vectors with formation.Term " host cell " refers to 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; Bacterial cell such as Salmonella typhimurium; Fungal cell such as yeast; Vegetable cell; Insect cell such as fruit bat S2 or Sf9; Zooblast such as CHO, COS or Bowes melanoma cells etc.

Can carry out with routine techniques well known to those skilled in the art with dna sequence dna of the present invention or the recombinant vectors transformed host cell that contains described dna sequence dna.When the host was prokaryotic organism such as intestinal bacteria, the competent cell that can absorb DNA can be used CaCl in exponential growth after date results ₂Method is handled, and used step is well-known in this area.Alternative is to use MgCl ₂If desired, transforming also the method for available electroporation carries out.When the host is an eukaryote, can select following DNA transfection method for use: coprecipitation of calcium phosphate method, perhaps conventional mechanical method such as microinjection, electroporation, liposome packing etc.

By the recombinant DNA technology of routine, utilize polynucleotide sequence of the present invention to can be used to express or the people that produces reorganization contains EF-hand-type calcium binding protein 96 (Science, 1984 of ATP/GTP binding domains; 224:1431).In general following steps are arranged:

(1). everybody contains the polynucleotide (or varient) of the EF-hand-type calcium binding protein 96 of ATP/GTP binding domains with coding of the present invention, or transforms or the transduction proper host cell with the recombinant expression vector that contains these polynucleotide;

(2). in suitable medium, cultivate host cell;

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

In step (2), 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.

In step (3), recombinant polypeptide can wrap and be expressed or be secreted into the extracellular 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.These methods include, but are not limited to: conventional renaturation is handled, protein precipitant is handled (salt analysis method), centrifugal, the broken bacterium of infiltration, the combination of ultrasonication, 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 antagonist of polypeptide of the present invention and this polypeptide, agonist and inhibitor can be directly used in disease treatment, for example, can treat and grow disorders, the metabolism disorder disease of respectively organizing and some neural diseases etc.

EF-hand-type calcium binding protein family is a very big super family of albumen, contains numerous family members, as calmodulin, calcyphosine etc.The member of the super family of calsequestrin is all contained a calcium binding motif, is called as " EF-hand-type " structural domain.The member of this family plays an important role in cell growth and atomization, is regulating and control cell cycle and is controlling the metabolic processes of organism.It all plays important regulatory role in important physiological processs such as Muscle contraction, glycogen degraded, neurotransmitter release, thereby, these proteic abnormal expressions will cause various and neurotransmitter to discharge relevant nervous system disorders, as: nervous system development disorder disease, nervous system degenerative disease, neuromuscular disease and neural skin syndromes etc.

This shows that the abnormal expression that people of the present invention contains the EF-hand-type calcium binding protein 96 of ATP/GTP binding domains will produce especially nervous system disorders of various diseases, these diseases include but not limited to:

Intracranial spaceoccupying lesion: all kinds of tumours such as neuroastrocytoma, oligodendroglioma, microglia knurl, ependymoma, satellite cell knurl, meningioma, neurofibroma, pituitary adenoma, intracranial granuloma;

Nervous system degenerative disease: senile dementia, Parkinson's disease, chorea, dysthymia disorders, amnesia, Heng Yandun disease, epilepsy, migraine, dementia, multiple sclerosis;

Neuromuscular disease: myasthenia gravis, spinal muscular atrophy, the false hypertrophy of flesh, Duchenne muscular dystrophy, myotonic dystrophy, musculartone disease, slow dyskinesia, myodystonia,

Neural skin syndromes: neurofibromatosis, tuberous sclerosis, encephalotrigeminal angiomatosis, ataxia telangiectasia etc.

Psychotic disorder: schizophrenia, dysthymia disorders, paranoia, anxiety disorder, obsession, phobia, neural decline etc.

Diseases of spinal cord: acute myelitis, compressive myelopathy

Peripheral nerve disease: trigeminal neuralgia, facioplegia, bulbar paralysis, sciatica, Green-barre syndrome, neurofibroma, Scs knurl etc.

Cerebro-vascular diseases: transient ischemic attack, cerebral infarction, hematencephalon, subarachnoid hemorrhage etc.

Neural growth disorder disease: dysrhaphia such as spina bifida, anencephalia, brain (meninx) bulging, cranium fissure, neurocele tumour; The brain development deformity is as hole deformity of brain, full forebrain, hydranencephaly; Neurone barrier to migration such as gyrus form unusually; Other deformity is as aqueduct deformity, cerebellar hypoplasia, Down syndromes, spinal cord deformity, congenital hydrocephalus, congenital nuclei of cranial nerves underdevelopment syndromes.

Because intracellular calcium binding protein is to regulate the key molecule that conducts in the calcium signal by enzyme reaction or albumen and protein-interacting.Break and the signal effect that EF-hand-type calcium binding protein also takes place during the chromatid of mitosis anaphase separates at nuclear membrane.Therefore, the abnormal expression of EF-hand-type calcium binding protein may produce the unusual or various tumours of fetal development.

This shows that the abnormal expression that people of the present invention contains the EF-hand-type calcium binding protein 96 of ATP/GTP binding domains also will produce the unusual or various tumours of fetal development, these diseases include but not limited to:

Grow disorders: congenital miscarriage, cleft palate, prosopoanoschisis, the neck capsule, the neck fistula, deficiency of skeletal limb, the limbs dysdifferentiation, alimentary canal locking or narrow, ileal diverticulum, umbilical fistula, congenital umbillical bernia, congenital aganglionic megacolon, larynx tracheostenosis or locking, tracheo esophageal fistula, hyaline membrane disease, congenital pulmonary cyst, atelectasis, polycystic kidney, ectopical kidney, horse tellurium kidney, double ureter, urachal fistula, latent, congenital inguinal hernia, duplex uterus, vaginal atresia, hypospadia, hermaphroditism, atrial septal defect, ventricular septal defect, arterial trunk is separated unusual, aorta or pulmonic stenosis, pulmonic stenosis, patent ductus arteriosus, neural tube defect, congenital hydrocephalus, iridocoloboma, congenital cataract, congenital glaucoma or cataract, microphthalmia, congenital deafness, aural deformity, Down syndromes etc.

The tumour of various tissues: cancer of the stomach, liver cancer, lung cancer, the esophageal carcinoma, mammary cancer, leukemia, lymphoma, thyroid tumor, hysteromyoma, neuroblastoma, astrocytoma, ependymoma, glioma, colorectal carcinoma, malignant histocytosis, melanoma, teratoma, sarcoma, adrenal carcinoma, bladder cancer, osteocarcinoma, osteosarcoma, myelomatosis, bone marrow cancer, the cancer of the brain, uterus carcinoma, carcinoma of endometrium, carcinoma of gallbladder, colorectal carcinoma, thymus neoplasms nasal cavity and tumor of sinus of nose, nasopharyngeal carcinoma, laryngocarcinoma, tracheal neoplasm, mesothelioma of pleura, fibroma, fibrosarcoma, lipoma, liposarcoma, leiomyoma etc.

The abnormal expression that people of the present invention contains the EF-hand-type calcium binding protein 96 of ATP/GTP binding domains also will produce some heredity, blood disease and disease of immune system etc.

The antagonist of polypeptide of the present invention and this polypeptide, agonist and inhibitor can be directly used in disease treatment, for example, can treat especially nervous system disorders of various diseases, the fetal development disorders, various tumours, some heredity, blood disease and disease of immune system etc.

The present invention also provides SCREENED COMPOUND to identify the method that improves (agonist) or check the medicament of the EF-hand-type calcium binding protein 96 that (antagonist) people contains the ATP/GTP binding domains.Agonist improves EF-hand-type calcium binding protein 96 that the people contains ATP/GTP binding domains biological function such as stimulate cellular proliferation, and antagonist prevention disorder such as the various cancer relevant with cell hyperproliferation with treatment.For example, can be in the presence of medicine, the film preparation that mammalian cell or expressing human is contained the EF-hand-type calcium binding protein 96 of ATP/GTP binding domains is cultivated with the EF-hand-type calcium binding protein 96 that the people of mark contains the ATP/GTP binding domains.Measure the medicine raising then or check this interactional ability.

The antagonist that the people contains the EF-hand-type calcium binding protein 96 of ATP/GTP binding domains comprises antibody, compound, acceptor disappearance thing and the analogue etc. that filter out.The antagonist that the people contains the EF-hand-type calcium binding protein 96 of ATP/GTP binding domains can combine and eliminate its function with the EF-hand-type calcium binding protein 96 that the people contains the ATP/GTP binding domains, or suppress the generation of this polypeptide, or combine with the avtive spot of this polypeptide and to make this polypeptide can not bring into play biological function.

In screening during as the compound of antagonist, the EF-hand-type calcium binding protein 96 that the people can be contained the ATP/GTP binding domains adds during bioanalysis measures, and the people is contained between the EF-hand-type calcium binding protein 96 of ATP/GTP binding domains and its acceptor interactional influence determines whether compound is antagonist by measuring compound.With the same quadrat method of above-mentioned SCREENED COMPOUND, can filter out the acceptor disappearance thing and the analogue of antagonist action.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 EF-hand-type calcium binding protein 96 bonded peptide molecule that the people contains the ATP/GTP binding domains obtains.During screening, generally tackle the EF-hand-type calcium binding protein 96 molecule that the people contains the ATP/GTP binding domains and carry out mark.

The invention provides and use polypeptide, and fragment, derivative, analogue or their cell are as the method for antigen with production antibody.These antibody can be polyclonal antibody or monoclonal antibody.The present invention also provides the antibody that contains the EF-hand-type calcium binding protein 96 antigenic determinant of ATP/GTP binding domains at the people.These antibody include, but is not limited to: the fragment that polyclonal antibody, monoclonal antibody, chimeric antibody, single-chain antibody, Fab fragment and Fab expression library produce.

The production of polyclonal antibody can choose contain the ATP/GTP binding domains EF-hand-type calcium binding protein 96 direct injection immune animal (as rabbit, mouse, rat etc.) method obtains, and multiple adjuvant can be used for the enhancing immunity reaction, includes but not limited to freund's adjuvant etc.The technology of monoclonal antibody that the preparation people contains the EF-hand-type calcium binding protein 96 of ATP/GTP binding domains includes but not limited to hybridoma technology (Kohler and Milstein.Nature, 1975,256:495-497), three knurl technology, people B-quadroma technology, EBV-hybridoma technology etc.With the variable region bonded chimeric antibody in human constant region and inhuman source can with existing technology production (Morrison et al, PNAS, 1985,81:6851).And the technology of existing manufacture order chain antibody (U.S.PatNo.4946778) also can be used for producing the single-chain antibody that anti-people contains the EF-hand-type calcium binding protein 96 of ATP/GTP binding domains.

The antibody that anti-people contains the EF-hand-type calcium binding protein 96 of ATP/GTP binding domains can be used in the immunohistochemistry technology, and the people in the detection biopsy specimen contains the EF-hand-type calcium binding protein 96 of ATP/GTP binding domains.

Contain the also available labelled with radioisotope of EF-hand-type calcium binding protein 96 bonded monoclonal antibody of ATP/GTP binding domains with the people, inject in the body and can follow the tracks of its position and distribution.This radiolabeled antibody can be used as a kind of atraumatic diagnostic method and is used for the location of tumour cell and has judged whether transfer.

Antibody also can be used for designing the immunotoxin at a certain privileged sites in the body.Contain as the people ATP/GTP binding domains EF-hand-type calcium binding protein 96 high-affinity monoclonal antibody 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, attack the amino of antibody, by the exchange of disulfide linkage, toxin is incorporated on the antibody, this hybrid antibody can be used for killing the EF-hand-type calcium binding protein 96 positive cells that the people contains the ATP/GTP binding domains.

Antibody among the present invention can be used for treating or prevention and people contain the relevant disease of EF-hand-type calcium binding protein 96 of ATP/GTP binding domains.The antibody that gives suitable dosage can stimulate or block generation or the activity that the people contains the EF-hand-type calcium binding protein 96 of ATP/GTP binding domains.

The invention still further relates to quantitatively and the detection and localization people contains the diagnostic testing process of the EF-hand-type calcium binding protein 96 level of ATP/GTP binding domains.These tests are known in the art, and comprise that FISH measures and radioimmunoassay.The people who is detected in the test contains the EF-hand-type calcium binding protein 96 level of ATP/GTP binding domains, can be with the disease that the people that lays down a definition contains the importance of EF-hand-type calcium binding protein 96 in various diseases of ATP/GTP binding domains and the EF-hand-type calcium binding protein 96 that is used to diagnose the people to contain the ATP/GTP binding domains works.

Polypeptide of the present invention also can be used as the peptide spectrum analysis, for example, the polypeptide available physical, chemistry or enzyme carry out the specificity cutting, and carries out the two-dimentional or three-dimensional gel electrophoresis analysis of one dimension, be more preferably and carry out mass spectroscopy.

The polynucleotide that the coding people contains the EF-hand-type calcium binding protein 96 of ATP/GTP binding domains also can be used for multiple therapeutic purpose.Gene therapy technology can be used for treating because the people contains that the nothing of the EF-hand-type calcium binding protein 96 of ATP/GTP binding domains is expressed or cell proliferation, growth or the metabolic disturbance of unusual/non-activity due to expressing.The gene therapy vector (as virus vector) of reorganization can be designed for the EF-hand-type calcium binding protein 96 that the people who expresses variation contains the ATP/GTP binding domains, to suppress the EF-hand-type calcium binding protein 96 activity that endogenic people contains the ATP/GTP binding domains.For example, the EF-hand-type calcium binding protein 96 that a kind of people of variation contains the ATP/GTP binding domains can be the EF-hand-type calcium binding protein 96 that people shortening, that lacked signal conduction function territory contains the ATP/GTP binding domains, though can combine with the substrate in downstream, lack signaling activity.Therefore the gene therapy vector of reorganization can be used for treating the people and contains the EF-hand-type calcium binding protein 96 expression of ATP/GTP binding domains or the disease of active caused by abnormal.Deriving from the polynucleotide that the expression vector of virus such as retrovirus, adenovirus, adeno-associated virus (AAV), hsv, parvovirus etc. can be used for the coding people is contained the EF-hand-type calcium binding protein 96 of ATP/GTP binding domains is transferred in the cell.The method of recombinant viral vector that structure carries the polynucleotide of the EF-hand-type calcium binding protein 96 that the coding people contains the ATP/GTP binding domains is found in existing document (Sambrook, et al.).The reorganization coding people polynucleotide that contain the EF-hand-type calcium binding protein 96 of ATP/GTP binding domains can be packaged in the liposome and be transferred in the cell in addition.

Polynucleotide import 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.

Suppress the people contain the ATP/GTP binding domains EF-hand-type calcium binding protein 96 mRNA oligonucleotide (comprising sense-rna and DNA) 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.

The polynucleotide that the coding people contains the EF-hand-type calcium binding protein 96 of ATP/GTP binding domains can be used for containing with the people diagnosis of relative disease of the EF-hand-type calcium binding protein 96 of ATP/GTP binding domains.The polynucleotide that the coding people contains the EF-hand-type calcium binding protein 96 of ATP/GTP binding domains can be used for detecting the unconventionality expression that expression people whether or under morbid state that the people contains the EF-hand-type calcium binding protein 96 of ATP/GTP binding domains contains the EF-hand-type calcium binding protein 96 of ATP/GTP binding domains.As the dna sequence dna that the people that encodes contains the EF-hand-type calcium binding protein 96 of ATP/GTP binding domains can be used for biopsy specimen is hybridized to judge that the people contains the expression situation of the EF-hand-type calcium binding protein 96 of ATP/GTP binding domains.Hybridization technique comprises the Southern blotting, Northern blotting, in situ hybridization etc.These technological methods all are disclosed mature technologies, and relevant test kit all can obtain from commercial channels.Part or all of polynucleotide of the present invention can be used as probe stationary on microarray (Microarray) or DNA chip (being called " gene chip " again), is used for analyzing the differential expression analysis and the gene diagnosis of tissue gene.The special primer of EF-hand-type calcium binding protein 96 that personnel selection contains the ATP/GTP binding domains carries out RNA-polymerase chain reaction (RT-PCR) amplification in vitro and also can detect the transcription product that the people contains the EF-hand-type calcium binding protein 96 of ATP/GTP binding domains.

Detect sudden change that the people contains the EF-hand-type calcium binding protein 96 gene of ATP/GTP binding domains and also can be used for the relevant disease of EF-hand-type calcium binding protein 96 of diagnosing the people to contain the ATP/GTP binding domains.The form that the people contains the EF-hand-type calcium binding protein 96 sudden change of ATP/GTP binding domains comprises that to contain point mutation that the EF-hand-type calcium binding protein 96 dna sequence dna of ATP/GTP binding domains compares, transposition, disappearance, reorganization and other any unusual etc. with the normal wild type people.Available existing technology such as Southern blotting, dna sequence analysis, PCR and in situ hybridization detect sudden change.In addition, sudden change might influence proteic expression, therefore can judge indirectly that with Northern blotting, Western blotting gene has or not sudden change.

Sequence of the present invention identifies it also is valuable to karyomit(e).This sequence can be specifically at certain bar human chromosome particular location and and can with its hybridization.At present, need to identify the concrete site of each gene on the karyomit(e).Now, have only chromosomal marker thing seldom to can be used for the marker chromosomes position based on actual sequence data (repetition polymorphism).According to the present invention, for these sequences are associated with disease related gene, its important the first step is positioned these dna sequence dnas on the karyomit(e) exactly.

In brief, prepare PCR primer (preferred 15-35bp), sequence can be positioned on the karyomit(e) according to cDNA.Then, these primers are used for the somatocyte hybrid cell that the PCR screening contains each bar human chromosome.Have only those hybrid cells that contain corresponding to the people's gene of primer can produce the fragment of amplification.

The PCR localization method of somatocyte hybrid cell is that DNA is navigated to concrete chromosomal quick method.Use Oligonucleolide primers of the present invention,, can utilize one group to realize inferior location from specific chromosomal fragment or a large amount of genomic clone by similar approach.Other the similar strategy that can be used for chromosomal localization comprises in situ hybridization, uses the karyomit(e) prescreen and the hybridization preliminary election of the airflow classification of mark, thereby makes up the special cDNA storehouse of karyomit(e).

The cDNA clone is carried out fluorescence in situ hybridization (FISH) with Metaphase Chromosome, can in a step, accurately carry out chromosomal localization.The summary of this technology is referring to Verma etc., Human Chromosomes:a Manualof Basic Techniques, Pergamon Press, New York (1988).

In case sequence is positioned to chromosome position accurately, the physical location of this sequence on karyomit(e) just can be associated with the gene map data.These data for example are found in, V.Mckusick, MendelianInheritance in 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.

Then, need to measure ill and not cDNA between diseased individuals or genome sequence difference.If observe certain sudden change in some or all of diseased individuals, and this sudden change is not observed in any normal individual, then this sudden change may be the cause of disease of disease.More ill and diseased individuals not is usually directed at first seek the variation of structure in the karyomit(e), as from the horizontal visible of karyomit(e) or use based on detectable disappearance of the PCR of cDNA sequence or transposition.Resolving power according to present physical mapping and assignment of genes gene mapping technology, being accurately positioned to the cDNA of the chromosomal region relevant with disease, can be a kind of (the supposing that 1 megabasse mapping resolving power and every 20kb are corresponding to a gene) between 50 to 500 potential Disease-causing genes.

Polypeptide of the present invention, polynucleotide and stand-in thereof, agonist, antagonist and inhibitor and suitable pharmaceutical carrier combination back can be used.These carriers can be water, glucose, ethanol, salt, damping fluid, glycerine and their combination.Composition comprises the polypeptide or the antagonist of safe and effective amount and carrier and the vehicle that does not influence effect of drugs.These compositions can be used as medicine and are used for disease treatment.

The present invention also provides medicine box or the test kit that contains one or more containers, and one or more medicinal compositions compositions of the present invention are housed in the container.With these containers, can have by the given indicative prompting of government authorities of making, using or selling medicine or biological products, the government authorities that this prompting reflects production, uses or sells permits it to use on human body.In addition, polypeptide of the present invention can be used in combination with other treatment compound.

Pharmaceutical composition can be with mode administration easily, as by in part, intravenously, intraperitoneal, intramuscular, subcutaneous, the nose or the route of administration of intracutaneous.The EF-hand-type calcium binding protein 96 that the people contains the ATP/GTP binding domains comes administration with the amount that treats and/or prevents concrete indication effectively.Amount and dosage range that the people who is applied to the patient contains the EF-hand-type calcium binding protein 96 of ATP/GTP binding domains will depend on many factors, as administering mode, person's to be treated healthiness condition and diagnostician's judgement.

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 contains the polyacrylamide gel electrophoresis figure (SDS-PAGE) of the EF-hand-type calcium binding protein 96 of ATP/GTP binding domains for isolating people.96kDa is proteinic molecular weight.The arrow indication is isolated protein band.

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 people contains the clone of the EF-hand-type calcium binding protein 96 of ATP/GTP binding domains

Extract the total RNA of people's tire brain with guanidinium isothiocyanate/phenol/chloroform single stage method.From total RNA, separate poly (A) mRNA with Quik mRNA Isolation Kit (Qiegene company product).2ug poly (A) mRNA forms cDNA through reverse transcription.CDNA fragment orientation is inserted on the multiple clone site of pBSK (+) carrier (Clontech company product) with Smart cDNA clone's test kit (available from Clontech), transforms DH5 α, bacterium forms the cDNA library.Measure all clones' 5 ' and 3 ' terminal sequence with Dyeterminate cycle reaction sequencing kit (Perkin-Elmer company product) and ABI 377 automatic sequencers (Perkin-Elmer company).CDNA sequence and the existing public dna sequence data storehouse (Genebank) measured are compared, found that the cDNA sequence of one of them clone 0952B08 is new DNA.By synthetic a series of primers the contained insertion cDNA fragment of this clone is carried out two-way mensuration.The result shows, the contained full-length cDNA of 0952B08 clone is 3181bp (shown in Seq ID NO:1), from 117bp to 2192bp the open reading frame (ORF) of a 2076bp, the new protein (shown in Seq ID NO:2) of encoding arranged.We are with this clone's called after pBS-0952B08, and the name of encoded protein matter is behaved and contained the EF-hand-type calcium binding protein 96 of ATP/GTP binding domains.Embodiment 2: the gene that contains the EF-hand-type calcium binding protein 96 of ATP/GTP binding domains with RT-PCR method clones coding people

Total RNA is a template with fetus brain cell, is that primer carries out the synthetic cDNA of reverse transcription reaction with oligo-dT, with behind the test kit purifying of Qiagene, carries out pcr amplification with following primer:

Primerl：5’-GGCGGCGGGCCCCGGCGGCCGAAG-3’(SEQ?ID?NO：3)

Primer2：5’-AGTACACAGCAGTTATTTATTTAA-3’(SEQ?ID?NO：4)

Primerl is the forward sequence that begins of lbp that is positioned at the 5 ' end of SEQ ID NO:1;

Primer2 be SEQ ID NO:1 in 3 ' end reverse sequence.

The condition of amplified reaction: in the reaction volume of 50 μ l, contain 50mmol/L KCl, 10mmol/L Tris-Cl, (pH8.5), 1.5mmol/L MgCl2,200 μ mol/L dNTP, 10pmol primer, the Taq archaeal dna polymerase of 1U (Clontech company product).Go up by 25 cycles of following conditioned response at PE9600 type DNA thermal cycler (Perkin-Elmer company): 94 ℃ of 30sec; 55 ℃ of 30sec; 72 ℃ of 2min.When RT-PCR, establish the blank negative contrast of positive contrast of β-actin and template simultaneously.Amplified production is connected to (Invitrogen company product) on the pCR carrier with the test kit purifying of QIAGEN company with TA clone test kit.The dna sequence analysis result shows that the dna sequence dna of PCR product and the 1-318lbp shown in the SEQ ID NO:1 are identical.Embodiment 3:Northern blotting analyst contains the EF-hand-type calcium binding protein 96 expression of gene of ATP/GTP binding domains:

Extract total RNA[Anal.Biochem 1987,162,156-159 with single stage method].This method comprises acid guanidine thiocyanate phenol-chloroform extracting.Promptly use 4M guanidinium isothiocyanate-25mM Trisodium Citrate, 0.2M sodium acetate (pH4.0) carries out homogenate to tissue, adds the phenol of 1 times of volume and the chloroform-primary isoamyl alcohol (49: 1) of 1/5 volume, and is centrifugal after mixing.The sucking-off aqueous phase layer adds Virahol (0.8 volume) and with the centrifugal RNA precipitation that obtains of mixture.With RNA precipitation 70% washing with alcohol that obtains, dry and soluble in water.With 20 μ g RNA, on 1.2% sepharose that contains 20mM 3-(N-morpholino) propanesulfonic acid (pH7.0)-5mM sodium acetate-1mM EDTA-2.2M formaldehyde, carry out electrophoresis.Be transferred on the nitrocellulose filter then.The dna probe for preparing the 32p-mark with α-32p dATP by random priming.Used dna probe is the EF-hand-type calcium binding protein 96 coding region sequence (117bp to 2192bp) that the people of pcr amplification shown in Figure 1 contains the ATP/GTP binding domains.(about 2 * 106cpm/ml) spend the night in 42 ℃ of hybridization in a solution with the nitrocellulose filter that has shifted RNA, and this solution comprises 50% methane amide-25mM KH2P04 (pH7.4)-5 * SSC-5 * Denhardt ' s solution and 200 μ g/ml salmon sperm DNAs with the probe of 32P-mark.After the hybridization, filter membrane is washed 30min in 55 ℃ in 1 * SSC-0.1%SDS.Then, analyze with quantitative with Phosphor Imager.Embodiment 4: recombinant human contains vivoexpression, separation and the purifying of the EF-hand-type calcium binding protein 96 of ATP/GTP binding domains

According to SEQ ID NO:1 and coding region sequence shown in Figure 1, design a pair of specificity amplification primer, sequence is as follows:

Primer3：5’-CATCCATGGATGAAGAAAGACGTGCGGATCCTGC-3’(Seq?ID?No：5)

Primer4：5’-CATGAATTCTCATCGCTGTTTCAATAATGCTTTG-3’(Seq?ID?No：6)

5 ' end of these two sections primers contains NcoI and EcoRI restriction enzyme site respectively, be respectively the encoding sequence of target gene 5 ' end and 3 ' end thereafter, NcoI and EcoRI restriction enzyme site are corresponding to expression vector plasmid pET-28b (+) (Novagen company product, Cat.No.69865.3) the selectivity restriction enzyme site on.With the pBS-0952B08 plasmid that contains the total length goal gene is template, carries out the PCR reaction.The PCR reaction conditions is: contain pBS-0952B08 plasmid 10pg, primer Primer-3 and Primer-4 among the cumulative volume 50 μ l and be respectively 10pmol, Advantage polymerase Mix (Clontech company product) 1 μ l.Loop parameter: 94 ℃ of 20s, 60 ℃ of 30s, 68 ℃ of 2min, totally 25 circulations.Respectively amplified production and plasmid pET-28 (+) are carried out double digestion with NcoI and EcoRI, reclaim big fragment respectively, and connect with the T4 ligase enzyme.Connect product and transform, after the dull and stereotyped overnight incubation of the LB that contains kantlex (final concentration 30 μ g/ml), use the colony polymerase chain reaction (PCR) method screening positive clone, and check order with the big enterobacterial DH5 of Calcium Chloride Method α.Select the correct positive colony of sequence (pET-0952B08) with Calcium Chloride Method with recombinant plasmid transformed e. coli bl21 (DE3) plySs (Novagen company product).In the LB liquid nutrient medium that contains kantlex (final concentration 30 μ g/ml), host bacterium BL21 (pET-0952B08) is cultured to logarithmic phase at 37 ℃, adds IPTG to final concentration lmmol/L, continues to cultivate 5 hours.Centrifugal collection thalline, through the broken bacterium of ultrasonic wave, centrifugal collection supernatant, with carrying out chromatography with 6 Histidines (6His-Tag) bonded affinity column His.Bind Quick Cartridge (Novagen company product), the target protein people who has obtained purifying contains the EF-hand-type calcium binding protein 96 of ATP/GTP binding domains.Through the SDS-PAGE electrophoresis, obtain a single band (Fig. 2) at the 96kDa place.This band is transferred on the pvdf membrane carries out the n terminal amino acid sequential analysis with the Edams hydrolysis method, 15 amino acid of N-end hold 15 amino-acid residues identical with the N-shown in the SEQ ID NO:2 as a result.Embodiment 5 anti-people contain the EF-hand-type calcium binding protein 96 production of antibodies of ATP/GTP binding domains

The specific polypeptide of EF-hand-type calcium binding protein 96 that contains the ATP/GTP binding domains with the synthetic following people of Peptide synthesizer (PE company product):

NH ₂-Met-Lys-Lys-Asp-Val-Arg-Ile-Leu-Leu-Val-Gly-Glu-Pro-Arg-Val-COOH

(SEQ?ID?NO：7)。Form compoundly with hemocyanin and bovine serum albumin coupling this polypeptide respectively, method is referring to Avrameas, et al.Immunochemistry, 1969; 6:43.Add the complete Freund's adjuvant immunizing rabbit with the above-mentioned hemocyanin polypeptide complex of 4mg, add the incomplete Freund's adjuvant booster immunization once with the hemocyanin polypeptide complex again after 15 days.Employing is done the titre that ELISA measures antibody in the rabbit anteserum through the titer plate of 15 μ g/ml bovine serum albumin polypeptide complex bag quilts.From the rabbit anteserum of antibody positive, separate total IgG with albumin A-Sepharose.Polypeptide is incorporated on the Sepharose4B post of cyanogen bromide-activated, from total IgG, separates anti-peptide antibody with affinity chromatography.The EF-hand-type calcium binding protein 96 that immuno-precipitation proof antibody purified can be specifically with the people contains the ATP/GTP binding domains combines.Embodiment 6: polynucleotide passage of the present invention is as the application of hybridization probe

Picking out suitable oligonucleotide fragment from polynucleotide of the present invention is of use in many ways as hybridization probe, as can whether containing polynucleotide sequence of the present invention and detect the homologous polynucleotide sequence to identify it with the healthy tissues of different sources or the genome or the hybridization of cDNA library of pathological tissue with this probe, whether further also available this probe in detecting polynucleotide sequence of the present invention or the expression of its homologous polynucleotide sequence in healthy tissues or pathological tissue cell be unusual.

The purpose of present embodiment is to pick out suitable oligonucleotide fragment as hybridization probe from polynucleotide SEQ ID NO:1 of the present invention, and identifies in some tissues whether contain polynucleotide sequence of the present invention or its homologous polynucleotide sequence with the filter hybridization method.The filter hybridization method comprises dot blotting, Southern blotting, Northern blotting and copy method etc., and they all are that polynucleotide sample to be measured is fixed on use essentially identical step crossover in back on the filter membrane.These identical steps are: the filter membrane of having fixed sample at first carries out prehybridization with the hybridization buffer that does not contain probe, so that nonspecific combining site suppressed by vector of sample and synthetic polymer institute are saturated on the filter membrane.Prehybridization solution is contained the hybridization buffer replacement of label probe then, and insulation makes probe and target nucleic acid hybridization.After the hybridization step, the probe in the hybridization is not removed by a series of film steps of washing.Present embodiment utilizes the film condition of washing (as than low salt concn and higher temperature) of higher-strength, so that hybrid context reduces and only keep the signal of high specificity.The probe that present embodiment is selected for use comprises two classes: first kind probe be fully with the identical or complementary oligonucleotide fragment of polynucleotide SEQ ID NO:1 of the present invention; The second class probe is part and the identical or complementary oligonucleotide fragment of polynucleotide SEQ ID NO:1 of the present invention.Present embodiment selects for use dot blotting that sample is fixed on the filter membrane, higher-strength wash under the film condition, the hybridization specificity of first kind probe and sample is the strongest and kept.One, probe selects for use

From polynucleotide SEQ ID NO:1 of the present invention, select oligonucleotide fragment as hybridization probe, the several aspects that should follow following principle and need to consider: 1, the probe size preferable range is a 18-50 Nucleotide; 2, GC content is 30%-70%, and surpassing then, non-specific hybridization increases; 3, probe interior should not have complementary region; 4, what meet above condition can be used as the primary election probe, further do the computer sequential analysis then, comprise this primary election probe is carried out homology relatively with its source sequence area (being SEQ ID NO:1) and other known genome sequence and complementary district thereof respectively, if with the homology in non-target molecule zone greater than 85% or there are 15 continuous bases of surpassing identical, then this primary election probe is general just should not use; 5, whether the primary election probe finally is chosen to be the probe of actual application value also should further be determined by experiment.Select and synthetic following two probes after finishing the analysis of above each side: probe 1 (probel) belongs to first kind probe, with complete homology of gene fragment or the complementation (41Nt) of SEQ ID NO:1

5′-AGAAAGACGTGCGGATCCTGCTGGTGGGAGAACCTAGAGTTGGG-3′ (SEQ?ID?NO：8)

Probe 2 (probe2) belongs to the second class probe, is equivalent to gene fragment or its complementary segmental replacement mutant nucleotide sequence (41Nt) of SEQ ID NO:1:

5′-AGAATCGCGTGCGGATCCTGATCGTGGGAGAACCTAATCGTGGG-3′ (SEQ?I?D?NO：9)

Other unlisted common agents and the compound method thereof relevant with following concrete experimental procedure please refer to document: DNA PROBES G.H.Keller; M.M.Manak; Stockton Press, 1989 (USA) and molecular cloning laboratory manual books more commonly used are as works such as " molecular cloning experiment guide " (second edition in 1998) [U.S.] Sa nurse Brooker, Science Press.

Specimen preparation: 1, from fresh or frozen tissue, extract DNA

Step: 1) the fresh or fresh normal liver tissue that thaws is put into the plate that is immersed on ice and fills phosphate buffered saline buffer (PBS).With scissors or scalpel tissue is cut into small pieces.Should keep organizing moistening in the operation.2) with 1000g centrifugal chopper tissue 10 minutes.3) with cooled homogenate damping fluid (0.25mol/L sucrose; 25mmol/LTris-HCl, pH7.5; 25mmol/LnaCl; 25mmol/L MgCl ₂) precipitation that suspends (approximately 10ml/g).4) 4 ℃ with electric homogenizer homogenate tissue suspension at full speed, until tissue by broken fully.5) 1000g is centrifugal 10 minutes.6) with re-suspended cell precipitation (the initial tissue sample of every 0.1g adds 1-5ml), centrifugal 10 minutes again with 1000g.7) with the resuspended precipitation of lysis buffer (the initial tissue sample of every 0.1g adds 1ml), connect following phenol extraction process then.2, the phenol extraction process of DNA

Step: 1) wash cell, centrifugal 10 minutes of 1000g with the cold PBS of 1-10ml.2) with the sedimentary cell (1 * 10 of cold cell pyrolysis liquid resuspension ⁸The minimum of applications 100ul lysis buffer of cell/ml).3) adding SDS is 1% to final concentration, if before re-suspended cell SDS is directly joined in the cell precipitation, cell may form big agglomerate and be difficult to fragmentation, and the overall yield that reduces.This point is in extracting＞10 ⁷Especially severe during cell.4) add Proteinase K to final concentration 200ug/ml.5) 50 ℃ of insulation reaction 1 hour or 37 ℃ gently jolting spend the night.6) use equal-volume phenol: chloroform: differently defend alcohol (25: 24: 1) extracting, in little centrifuge tube centrifugal 10 minutes.Two corresponding clear separation, otherwise carry out centrifugal again.7) water is transferred to new pipe.8) use the equal-volume chloroform: primary isoamyl alcohol (24: 1) extracting, centrifugal 10 minutes.9) water that will contain DNA is transferred to new pipe.Carry out purifying and the ethanol sedimentation of DNA then.3, the purifying of DNA and ethanol sedimentation

Step: 1) 1/10 volume 2mol/L sodium-acetate and 2 times of cold 100% ethanol of volume are added in the dna solution mixing.-20 ℃ of placements 1 hour or to spending the night.2) centrifugal 10 minutes.3) careful sucking-off or pour out ethanol.4) with 70% cold ethanol 500ul washing precipitation, centrifugal 5 minutes.5) careful sucking-off or pour out ethanol.With the cold washing with alcohol precipitation of 500ul, centrifugal 5 minutes.6) careful sucking-off or pour out ethanol is inverted on thieving paper then residual ethanol is flow to end.Dry air 10-15 minute, so that the volatilization of surperficial ethanol.Note not making the precipitation complete drying, otherwise dissolve again than difficulty.7) with small volume TE or the resuspended DNA precipitation of water.The low speed vortex vibrates or uses the dropper pressure-vaccum, increases TE gradually simultaneously, is mixed to DNA and fully dissolves, every 1-5 * 10 ⁶Cell extracted approximately adds 1ul.

Below the 8-13 step only be used for must removing when depolluting, otherwise can directly carry out the 14th step.8) RNA enzyme A is added in the dna solution, final concentration is 100ug/ml, and 37 ℃ are incubated 30 minutes.9) add SDS and Proteinase K, final concentration is respectively 0.5% and 100ug/ml.37 ℃ are incubated 30 minutes.10) use isopyknic phenol: chloroform: primary isoamyl alcohol (2 5: 24: 1) extractive reaction liquid, centrifugal 10 minutes.11) carefully shift out water, use isopyknic chloroform: primary isoamyl alcohol (24: 1) extracting again, centrifugal 10 minutes.12) carefully shift out water, add 1/10 volume 2mol/L sodium-acetate and the cold ethanol of 2.5 volumes, mixing put-20 ℃ 1 hour.13) with 70% ethanol and 100% washing with alcohol precipitation, dry air, resuspended nucleic acid, process is with the 3-6 step.14) measure A ₂₆₀And A ₂₈₀To detect purity and the productive rate of DNA.15) deposit in-20 ℃ after the packing.The preparation of sample film:

1) get 4 * 2 suitably nitrocellulose filters (NC film) of size, mark point sample position and sample number thereon gently with pencil, each probe needs two NC films, so that wash film with high strength condition and strength condition respectively in the experimental procedure of back.

2) draw and contrast respectively 15 microlitres, put on the sample film, dry at room temperature.

3) place infiltration that 0.1mol/LNaOH is arranged, last 5 minute of filter paper (twice) of 1.5mol/LNaCl, drying to place to soak into has 0.5mol/L Tris-HCl (pH7.0), last 5 minute of filter paper (twice) of 3mol/LNaCl, dries.

4) be sandwiched in the clean filter paper, wrap, 60-80 ℃ of vacuum-drying 2 hours with aluminium foil.

The mark of probe

1) 3 μ lProbe (0.10D/10 μ 1) add 2 μ lKinase damping fluids, 8-10uCi γ- ³²P-dATP+2UKinase is to add to final volume 20 μ l.

2) 37 ℃ are incubated 2 hours.

3) add the tetrabromophenol sulfonphthalein indicator (BPB) of 1/5 volume.

4) cross Sephadex G-50 post.

5) to having ³²Before washing out, p-Probe begins to collect first peak (available Monitor monitoring).

6) 5/pipe, collect the 10-15 pipe.

7) monitor isotopic weight with liquid glimmer instrument

8) merge and to be required preparation behind the collection liquid of first peak ³²P-Probe (second peak for free γ- ³²P-dATP).

Prehybridization

The sample film is placed plastics bag, and adding 3-10mg prehybridization solution (10 * Denhardt ' s; 6 * SSC, 0.1mg/mlCT DNA (calf thymus DNA).), seal sack after, 68 ℃ of water-baths were shaken 2 hours.

Hybridization

Plastics bag is cut off one jiao, adds the probe prepare, seal sack after, 42 ℃ of water-baths are shaken and are spent the night.

Wash film: high strength is washed film:

1) good sample film has been hybridized in taking-up.

2) 2 * SSC among the 0.1%SDS, washes 15 minutes (2 times) for 40 ℃.

3) 0.1 * SSC among the 0.1%SDS, washes 15 minutes (2 times) for 40 ℃.

4) 0.1 * SSC among the 0.1%SDS, washes 30 minutes (2 times) for 55 ℃, and room temperature is dried.Low strength is washed film:

1) good sample film has been hybridized in taking-up.

2) 2 * SSC among the 0.1%SDS, washes 15 minutes (2 times) for 37 ℃.

3) 0.1 * SSC among the 0.1%SDS, washes 15 minutes (2 times) for 37 ℃.

4) 0.1 * SSC among the 0.1%SDS, washes 15 minutes (2 times) for 40 ℃, and room temperature is dried.

X-light autography:

-70 ℃, X-light autography (the compressing tablet time decides according to hybridization spot radioactivity is strong and weak).

Experimental result:

Adopt low strength to wash the hybrid experiment that the film condition is carried out, more than four strong and weak not obviously differences of probe hybridization spot radioactivity; And adopting low strength to wash the hybrid experiment that the film condition is carried out, the hybridization spot radioactive intensity of probe 1 obviously is better than the radioactive intensity of other three probe hybridization spots.Thereby available probe 1 is qualitative and analyze existence and the differential expression of polynucleotide of the present invention in different tissues quantitatively.

Sequence table

(1) general information:

(ⅱ) denomination of invention: the people contains the EF-hand-type calcium binding protein 96 and the encoding sequence thereof of ATP/GTP binding domains

(ⅲ) sequence number: 9

(2) information of SEQ ID NO:1:

(ⅰ) sequence signature:

(A) length: 3181bp

(B) type: nucleic acid

(C) chain: two strands

(D) topological framework: linearity

(ⅱ) molecule type: cDNA

( ⅹⅰ ) ：SEQ ID NO：1： 1 GGCGGCGGGCCCCGGCGGCCGAAGAGGCTGGCAGGTGGCGCCGTGGGGTGGGTGCTCCTG 61 GTGAGAGGAGTCCACTCCGTGCGTGCGGGCGGAGGCCGGCCCCCGAGAGCCGCGACATGA121 AGAAAGACGTGCGGATCCTGCTGGTGGGAGAACCTAGAGTTGGGAAGACATCACTGATTA181 TGTCTCTGGTCAGTGAAGAATTTCCAGAAGAGGTTCCTCCCCGGGCAGAAGAAATCACCA241 TTCCAGCTGATGTCACCCCAGAGAGAGTTCCAACACACATTGTAGATTACTCAGAAGCAG301 AACAGAGTGATGAACAACTTCATCAAGAAATATCTCAGGTTAATGTCATCTGTATAGTGT361 ATGCCGTTAACAACAAGCATTCTATTGATAAGGTAACAAGTCGATGGATTCCTCTCATAA421 ATGAAAGAACAGACAAAGACAGCAGGCTGCCTTTAATATTGGTTGGGAACAAATCTGATC481 TGGTGGAATATAGTAGTATGGAGACCATCCTTCCTATTATGAACCAGTATACAGAAATAG541 AAACCTGTGTGGAGTGTTCAGCGAAAAACCTGAAGAACATATCAGAGCTCTTTTATTACG601 CACAGAAAGCTGTTCTTCATCCTACAGGGCCCCTGTACTGCCCAGAGGAGAAGGAGATGA661 AACCAGCTTGTATAAAAGCCCTTACTCGTATATTTAAAATATCTGATCAAGATAATGATG721 GTACTCTCAATGATGCTGAACTCAACTTCTTTCAGAGGATTTGTTTCAACACTCCATTAG781 CTCCTCAAGCTCTGGAGGATGTCAAGAATGTAGTCAGAAAACATATAAGTGATGGTGTGG841 CTGACAGTGGGTTGACCCTGAAAGGTTTTCTCTTTTTACACACACTTTTTATCCAGAGAG 901 GGAGACACGAAACTACTTGGACTGTGCTTCGACGATTTGGTTATGATGATGACCTGGATT 961 TGACACCTGAATATTTGTTCCCCCTGCTGAAAATACCTCCTGATTGCACTACTGAATTAA1021 ATCATCATGCATATTTATTTCTCCAAAGCACCTTTGACAAGCATGATTTGGATAGAGACT1081 GTGCTTTGTCACCTGATGAGCTTAAAGATTTATTTAAAGTTTTCCCTTACATACCTTGGG1141 GGCCAGATGTGAATAACACAGTTTGTACCAATGAAAGAGGCTGGATAACCTACCAGGGAT1201 TCCTTTCCCAGTGGACGCTCACGACTTATTTAGATGTACAGCGGTGCCTGGAATATTTGG1261 GCTATCTAGGCTATTCAATATTGACTGAGCAAGAGTCTCAAGCTTCAGCTGTTACAGTGA1321 CAAGAGATAAAAAGATAGACCTGCAGAAAAAACAAACTCAAAGAAATGTGTTCAGATGTA1381 ATGTAATTGGAGTGAAAAACTGTGGGAAAAGTGGAGTTCTTCAGGCTCTTCTTGGAAGAA1441 ACTTAATGAGGCAGAAGAAAATTCGTGAAGATCATAAATCCTACTATGCGATTAACACTG1501 TTTATGTATATGGACAAGAGAAATACTTGTTGTTGCATGATATCTCAGAATCGGAATTTC1561 TAACTGAAGCTGAAATCATTTGTGATGTTGTATGCCTGGTATATGATGTCAGCAATCCCA1621 AATCCTTTGAATACTGTGCCAGGATTTTTAAGCAACACTTTATGGACAGCAGAATACCTT1681 GCTTAATCGTAGCTGCAAAGTCAGACCTGCATGAAGTTAAACAAGAATACAGTATTTCAC1741 CTACTGATTTCTGCAGGAAACACAAAATGCCTCCACCACAAGCCTTCACTTGCAATACTG1801 CTGATGCCCCCAGTAAGGATATCTTTGTTAAATTGACAACAATGGCCATGTATCCAGAGG1861 ATCATTACAGAGACAGACTCTCCCGAGACATGGGCCACACTGATAGAATAGAGAATTTGA1921 GAAAAATCTGGGTCTTTCTAAAAACTGCTTTCCATGCCCGGTTACGCTGTATGTGCACCT1981 GCAACAGGTGTACATTTTGCATCTGTCAGAACTTCCTCAACTCAGACTTGCTGCAATCTG2041 TAAAGAACAAAATCTTCACTGCAGTTCTTAACAGGCACGTGACACAAGCTGACCTCAAGA2101 GCTCCACGTTTTGGCTTCGAGCAAGTTTTGGTGCTACTGTTTTTGCAGTTTTGGGCTTTG2161 CTATGTACAAAGCATTATTGAAACAGCGATGATATAAAAAGAAATACTGTCCCTACCAAA2221 AACAAATACTTTTATGTACATTCTGAATGCTTTAAGTTCTGCTAGAATTATTGAGATATT2281 TATACATGCAGAGTTACTTTATTAATATTTGTAATTCATGCATAAGAGTATTTTAATGAT2341 AGTTATAACTGCAGTATTGGCTAGCATATGGAAAGAAAACAGCTAACAGCCAAACTAAAA2401 TGGCTAAATTCCAGAGGCCAAAAGGGAATATTTTGTAAATATATGTACATATTCAGGCAA2461 GATATGGTCTCCCAAGCTGAGTTCTAGAAATGATGTTTCTAGACATTTCTAAGTGGTATT2521 GTTAGTGCTCACTTGGCTCACTCTTCTAGGTTTAAGTTAGCCCAGAGATTGTATTTACTC2581 ATGGATCACTTTATTTATTTCACATTTACTCAGAATGATCCTTTGGGTTCTATAAGGACA2641 TAAGGTACAATTTGCCATTGTCTCTCCATTTTTAAAAACATACAAGTCAGTGTCAGCTTA2701 CCAACATGACATTTTTTCAGTCAGTTGTGGTAGGCCAGCCTTGAAGCCATCGCACAGTCT2761 AGAAACTTGTGTAGCTGAGTGTGCAGCTCACCTTTAAGGGTGAAGTTAGGTAAAAGCAAT2821 TAGCAGAGGCGTTATCTATGTGATTATGTTGCTTCCTTGTCAGTATGTTGAATTTTATAG2881 CCCTTTCAATGAAATAAAAAAAAATTTGTATATTACCAATGTTTTTAGTTTAAATAAAGA2941 GTCACCCTTACTACTGTTGAATTTCATCCCAAGTGTAAATCATTCTATAATGGCTGTGTC3001 TGTTATAGTATATTACAGTAACTGCATGTGTCACCAAGTGTTCTATATCAGGCTAGGATA3061 ACCTAGAGGCAGTAATTTTTTAAATGATAAAATAAATCTAATGAATATAAACTCTCATGA3121 TAAACCTATTTTTTCCATCATCAGCCTTTTCAAGTATTTAAATAAATAACTGCTGTGTAC3181 T

(3) information of SEQ ID NO:2: (ⅰ) sequence signature:

(A) length: 691 amino acid

(B) type: amino acid

( D ) ： ( ⅱ ) ： ( ⅹⅰ ) ：SEQ ID NO：2： 1 Met Lys Lys Asp Val Arg Ile Leu Leu Val Gly Glu Pro Arg Val 16 Gly Lys Thr Ser Leu Ile Met Ser Leu Val Ser Glu Glu Phe Pro 31 Glu Glu Val Pro Pro Arg Ala Glu Glu Ile Thr Ile Pro Ala Asp 46 Val Thr Pro Glu Arg Val Pro Thr His Ile Val Asp Tyr Ser Glu 61 Ala Glu Gln Ser Asp Glu Gln Leu His Gln Glu Ile Ser Gln Val 76 Asn Val Ile Cys Ile Val Tyr Ala Val Asn Asn Lys His Ser Ile 91 Asp Lys Val Thr Ser Arg Trp Ile Pro Leu Ile Asn Glu Arg Thr106 Asp Lys Asp Ser Arg Leu Pro Leu Ile Leu Val Gly Asn Lys Ser121 Asp Leu Val Glu Tyr Ser Ser Met Glu Thr Ile Leu Pro Ile Met136 Asn Gln Tyr Thr Glu Ile Glu Thr Cys Val Glu Cys Ser Ala Lys151 Ash Leu Lys Asn Ile Ser Glu Leu Phe Tyr Tyr Ala Gln Lys Ala166 Val Leu His Pro Thr Gly Pro Leu Tyr Cys Pro Glu Glu Lys Glu181 Met Lys Pro Ala Cys Ile Lys Ala Leu Thr Arg Ile Phe Lys Ile196 Ser Asp Gln Asp Ash Asp Gly Thr Leu Asn Asp Ala Glu Leu Asn211 Phe Phe Gln Arg Ile Cys Phe Asn Thr Pro Leu Ala Pro Gln Ala226 Leu Glu Asp Val Lys Ash Val Val Arg Lys His Ile Ser Asp Gly241 Val Ala Asp Ser Gly Leu Thr Leu Lys Gly Phe Leu Phe Leu His256 Thr Leu Phe Ile Gln Arg Gly Arg His Glu Thr Thr Trp Thr Val271 Leu Arg Arg Phe Gly Tyr Asp Asp Asp Leu Asp Leu Thr Pro Glu286 Tyr Leu Phe Pro Leu Leu Lys Ile Pro Pro Asp Cys Thr Thr Glu301 Leu Asn His His Ala Tyr Leu Phe Leu Gln Ser Thr Phe Asp Lys316 His Asp Leu Asp Arg Asp Cys Ala Leu Ser Pro Asp Glu Leu Lys331 Asp Leu Phe Lys Val Phe Pro Tyr Ile Pro Trp Gly Pro Asp Val346 Asn Asn Thr Val Cys Thr Asn Glu Arg Gly Trp lle Thr Tyr Gln361 Gly Phe Leu Ser Gln Trp Thr Leu Thr Thr Tyr Leu Asp Val Gln376 Arg Cys Leu Glu Tyr Leu Gly Tyr Leu Gly Tyr Ser Ile Leu Thr391 Glu Gln Glu Ser Gln Ala Ser Ala Val Thr Val Thr Arg Asp Lys406 Lys Ile Asp Leu Gln Lys Lys Gln Thr Gln Arg Asn Val Phe Arg421 Cys Asn Val lle Gly Val Lys Asn Cys Gly Lys Ser Gly Val Leu436 Gln Ala Leu Leu Gly Arg Asn Leu Met Arg Gln Lys Lys Ile Arg451 Glu Asp His Lys Ser Tyr Tyr Ala Ile Asn Thr Val Tyr Val Tyr466 Gly Gln Glu Lys Tyr Leu Leu Leu His Asp Ile Ser Glu Ser Glu481 Phe Leu Thr Glu Ala Glu Ile Ile Cys Asp Val Val Cys Leu Val496 Tyr Asp Val Ser Asn Pro Lys Ser Phe Glu Tyr Cys Ala Arg Ile511 Phe Lys Gln His Phe Met Asp Ser Arg Ile Pro Cys Leu lle Val526 Ala Ala Lys Ser Asp Leu His Glu Val Lys Gln Glu Tyr Ser Ile541 Ser Pro Thr Asp Phe Cys Arg Lys His Lys Met Pro Pro Pro Gln556 Ala Phe Thr Cys Asn Thr Ala Asp Ala Pro Ser Lys Asp Ile Phe571 Val Lys Leu Thr Thr Met Ala Met Tyr Pro Glu Asp His Tyr Arg586 Asp Arg Leu Ser Arg Asp Met Gly His Thr Asp Arg Ile Glu Asn601 Leu Arg Lys Ile Trp Val Phe Leu Lys Thr Ala Phe His Ala Arg616 Leu Arg Cys Met Cys Thr Cys Asn Arg Cys Thr Phe Cys Ile Cys631 Gln Asn Phe Leu Asn Ser Asp Leu Leu Gln Ser Val Lys Asn Lys646 Ile Phe Thr Ala Val Leu Asn Arg His Val Thr Gln Ala Asp Leu661 Lys Ser Ser Thr Phe Trp Leu Arg Ala Ser Phe Gly Ala Thr Val676 Phe Ala Val Leu Gly Phe Ala Met Tyr Lys Ala Leu Leu Lys Gln691 Arg

(4) information of SEQ ID NO:3 (ⅰ) sequence signature

(A) length: 24 bases

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linear (ⅱ) molecule type: oligonucleotide (ⅹ ⅰ) sequence description: information (ⅰ) sequence signature of SEQ ID NO:3:GGCGGCGGGCCCCGGCGGCCGAAG 24 (5) SEQ ID NO:4

(A) length: 24 bases

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linear (ⅱ) molecule type: oligonucleotide (ⅹ ⅰ) sequence description: information (ⅰ) sequence signature of SEQ ID NO:4:AGTACACAGCAGTTATTTATTTAA 24 (6) SEQ ID NO:5

(A) length: 34 bases

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linear (ⅱ) molecule type: oligonucleotide (ⅹ ⅰ) sequence description: information (ⅰ) sequence signature of SEQ ID NO:5:CATCCATGGATGAAGAAAGACGTGCGGATCCTGC 34 (7) SEQ ID NO:6

(A) length: 34 bases

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linear (ⅱ) molecule type: oligonucleotide (ⅹ ⅰ) sequence description: the information of SEQ ID NO:6:CATGAATTCTCATCGCTGTTTCAATAATGCTTTG 34 (8) SEQ ID NO:7: (ⅰ) sequence signature:

(A) length: 15 amino acid

(B) type: amino acid

(D) topological framework: linear (ⅱ) molecule type: polypeptide (ⅹ ⅰ) sequence description: information (ⅰ) sequence signature of SEQ ID NO:7:Met-Lys-Lys-Asp-Val-Arg-Ile-Leu-Leu-Val-Gly-Glu-Pro-Arg-Val 15 (9) SEQ ID NO:8

(A) length: 41 bases

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linear (ⅱ) molecule type: oligonucleotide (ⅹ ⅰ) sequence description: information (ⅰ) sequence signature of SEQ ID NO:8:AGAAAGACGTGCGGATCCTGCTGGTGGGAGAACCTAGAGTTGGG 41 (10) SEQ ID NO:9

(A) length: 41 bases

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linear (ⅱ) molecule type: oligonucleotide (ⅹ ⅰ) sequence description: SEQ ID NO:9:AGAATCGCGTGCGGATCCTGATCGTGGGAGAACCTAATCGTGGG 41

Claims (18)

1, a kind of isolated polypeptide-people contains the EF-hand-type calcium binding protein 96 of ATP/GTP binding domains, it is characterized in that it includes: the polypeptide of the aminoacid sequence shown in the SEQ ID NO:2 or active fragments, analogue or the derivative of its polypeptide.

2, polypeptide as claimed in claim 1, the aminoacid sequence that it is characterized in that described polypeptide, analogue or derivative has the homogeny with the aminoacid sequence at least 95% shown in the SEQ ID NO:2.

3, polypeptide as claimed in claim 2 is characterized in that it comprises the polypeptide with the aminoacid sequence shown in the SEQ ID NO:2.

4, a kind of isolating polynucleotide, it is characterized in that described polynucleotide comprise be selected from down the group in a kind of:

(a) coding has the polypeptide of aminoacid sequence shown in the SEQ ID NO:2 or its fragment, analogue, derivative

Polynucleotide;

(b) with polynucleotide (a) complementary polynucleotide; Or

(c) with (a) or the polynucleotide of at least 70% homogeny (b) are arranged.

5, polynucleotide as claimed in claim 4 is characterized in that described polynucleotide comprise coding and have SEQ ID

The polynucleotide of aminoacid sequence shown in the NO:2.

6, polynucleotide as claimed in claim 4, the sequence that it is characterized in that described polynucleotide include the sequence of 1-3181 position among the sequence of 117-2192 position among the SEQ ID NO:1 or the SEQ ID NO:1.

7, a kind of recombinant vectors that contains exogenous polynucleotide is characterized in that it is the recombinant vectors that is formed by the described polynucleotide of arbitrary claim among the claim 4-6 and plasmid, virus or vehicle expression vector establishment.

8, a kind of genetically engineered host cell that contains exogenous polynucleotide is characterized in that it is to be selected from following a kind of host cell:

(a) host cell that transforms or transduce with the described recombinant vectors of claim 7; Or

(b) host cell that transforms or transduce with the described polynucleotide of the arbitrary claim among the claim 4-6.

9, a kind of people of having contains the preparation method of the active polypeptide of EF-hand-type calcium binding protein 96 of ATP/GTP binding domains, it is characterized in that described method comprises:

(a) contain under the EF-hand-type calcium binding protein 96 condition of ATP/GTP binding domains in expressing human, cultivate the described through engineering approaches host cell of claim 8;

(b) from culture, isolate and have the active polypeptide of EF-hand-type calcium binding protein 96 that the people contains the ATP/GTP binding domains.

10, a kind of can with polypeptide bonded antibody, it is characterized in that described antibody is the EF-hand-type calcium binding protein 96 specificity bonded antibody that can contain the ATP/GTP binding domains with the people.

11, the compound of an analoglike or adjusting polypeptide active or expression is characterized in that they are simulation, promotion, antagonism or suppress the active compound that the people contains the EF-hand-type calcium binding protein 96 of ATP/GTP binding domains.

12, compound as claimed in claim 11 is characterized in that it is the polynucleotide sequence shown in the SEQ ID NO:1 or its segmental antisense sequences.

13, the described application of compound of a kind of claim 11, it is characterized in that described compound be used for EF-hand-type calcium binding protein 96 that the mediator contains the ATP/GTP binding domains in vivo, the method for external activity.

14, a kind of disease relevant or method of disease susceptibility of detecting with the described polypeptide of arbitrary claim among the claim 1-3, it is characterized in that it comprises the described polypeptide expression amount that detects, perhaps detect the activity of described polypeptide, perhaps detect and cause described expression of polypeptides amount or active unusual nucleotide diversity in the polynucleotide.

15, as the application of polypeptide as described in the arbitrary claim among the claim 1-3, it is characterized in that it is applied to screen stand-in, the agonist that the people contains the EF-hand-type calcium binding protein 96 of ATP/GTP binding domains, antagonist or inhibitor; Perhaps be used for the peptide finger print identification.

16, as the application of the described nucleic acid molecule of arbitrary claim among the claim 4-6, it is characterized in that it is used for nucleic acid amplification reaction as primer, perhaps be used for hybridization, perhaps be used to make gene chip or microarray as probe.

17,, it is characterized in that forming the pharmaceutical composition that contains the relevant unusually disease of the EF-hand-type calcium binding protein 96 of ATP/GTP binding domains as diagnosis or treatment and people with safe and effective dosage and pharmaceutically acceptable carrier with described polypeptide, polynucleotide or its stand-in, agonist, antagonist or inhibitor as the described polypeptide of arbitrary claim, polynucleotide or application of compound in claim 1-6 and 11.

18, the described polypeptide of arbitrary claim, polynucleotide or the application of compound among the claim 1-6 and 11, it is characterized in that being used for the treatment of as malignant tumour with described polypeptide, polynucleotide or compound, hemopathy, the medicine of HIV infection and immunological disease and all kinds of inflammation.

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