CN101173003A

CN101173003A - Agkistrodon venom prothrombin activator, its coding sequence and use

Info

Publication number: CN101173003A
Application number: CNA2006101179230A
Authority: CN
Inventors: 刘小龙; 焦浩漭; 朱洪; 周元聪
Original assignee: Shanghai Institutes for Biological Sciences SIBS of CAS
Current assignee: Shanghai Institutes for Biological Sciences SIBS of CAS
Priority date: 2006-11-03
Filing date: 2006-11-03
Publication date: 2008-05-07

Abstract

The invention provides new thrombinogen activator-agkistrodon acutus snake venom pallas pit viper thrombinogen activator (that is thrombinogen activator DaPA for short), and a method of coding polynucleotide of the thrombinogen activator DaPA and producing the thrombinogen activator DaPA through a recombination technique. The thrombinogen activator DaPA of the invention can activate the thrombinogen, thereby being used as styptic in the clinic.

Description

Ahylysantinfarctase thrombase activator, its encoding sequence and purposes

Technical field

Thrombogen activator DaPA) and the polynucleotide of coding thrombogen activator DaPA the invention belongs to biotechnology and medical field, specifically, the present invention relates to new thrombogen activator-Ahylysantinfarctase thrombase activator and (be called for short:.The invention still further relates to these polynucleotide and proteinic method for making and purposes, and the composition that contains this thrombogen activator DaPA.

Background technology

The activation of thrombogen is key step in the coagulation of blood process, and thrombogen activates into zymoplasm by factor Xa in blood coagulation system usually, and the latter makes Parenogen become fibrinogen, thereby finishes hemagglutinative function.

The present known activator that can make thrombogen activate into zymoplasm that exists equally in the snake venom in multiple source, the molecular weight of the snake poison blood coagulation zymoexcitator of having reported all more than 40KD, is the proteolytic enzyme of Multidomain.

In view of the thrombogen activator has purposes such as clinical hemostasis, therefore, this area presses for the new thrombogen activator of exploitation.

Summary of the invention

The purpose of this invention is to provide a kind of new thrombogen activator-DaPA albumen with and fragment, analogue and derivative.

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

Another object of the present invention provides the purposes of producing these method of protein and this protein and encoding sequence.

In a first aspect of the present invention, a kind of isolating thrombogen activator DaPA is provided, it comprises: protein or its conservative property variant protein matter or its active fragments or its reactive derivative with SEQ ID NO:2 aminoacid sequence.

Preferably, this protein is selected from down group:

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

(b) replacement, disappearance or the interpolation of SEQ ID NO:2 aminoacid sequence through one or more (as 1-10, preferably 1-8) amino-acid residue formed, and have activate thrombogen generate zymoplasm function by (a) polypeptides derived.

More preferably, described albumen is the protein of aminoacid sequence shown in SEQ ID NO:2.

In a second aspect of the present invention, a kind of isolating polynucleotide are provided, the above-mentioned DaPA albumen of its coding.

Preferably, described polynucleotide polynucleotide encoding has the protein of aminoacid sequence shown in the SEQ ID NO:2.More preferably, these polynucleotide contain the sequence of 1-678 position among the SEQ ID NO:1.

In a third aspect of the present invention, a kind of carrier is provided, it contains the proteic polynucleotide of above-mentioned encoding D aPA.And transformed by this carrier or the host cell of transduceing or directly transformed by above-mentioned polynucleotide or the host cell of transduction.

In a fourth aspect of the present invention, a kind of DaPA of the present invention is provided proteinic preparation method, this method comprises step:

(a) under expression condition, cultivate above-mentioned host cell, thereby express DaPA albumen;

(c) from culture, isolate DaPA protein.

The present invention also provides the method for preparing the thrombogen activator from ahylysantinfarctase.

In a fifth aspect of the present invention, provide a kind of can with above-mentioned thrombogen activator DaPA specificity bonded antibody.

In a sixth aspect of the present invention, a kind of pharmaceutical composition is provided, it contains (as 0.001-99.9wt%, more preferably 0.01-99wt%) above-mentioned thrombogen activator DaPA and pharmaceutically acceptable carrier of safe and effective amount.

Aspect the of the present invention the 7th, DaPA of the present invention is provided proteinic purposes, it is used to prepare the hematostatic medicine.

Description of drawings

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

Fig. 1 has shown polynucleotide and the aminoacid sequence (SEQ ID NO:1 and 2) of Ahylysantinfarctase thrombase activator DaPA of the present invention.

Fig. 2 has shown the product hydrolysis zymoplasm chromophoric substrate Chromozym TH after the Ahylysantinfarctase thrombase activator activates thrombogen.

Fig. 3 has shown the recalcification time result.Wherein, A is a measurement result in the plastics tubing, and B is a measurement result in the Glass tubing.

Fig. 4 has shown the APTT result.

Embodiment

Separation and purification is to a kind of protein that can activate thrombogen from the snake venom of agkistrodon acutus (Agkistrodon acutus) for the inventor, and the only about 25KD of molecular weight is the metalloprotease in a kind of single structure territory.Experiment shows, this thrombogen activator DaPA acts on the P of thrombin of beef on former respectively ¹⁶⁶-L ¹⁶⁷, A ²⁶⁹-A ²⁷⁰And R ³²³-I ³²⁴Peptide bond makes thrombogen activate into zymoplasm.

Particularly, the inventor has at first separated the snake poison blood coagulation zymoexcitator that a kind of molecular weight is 25KD from ahylysantinfarctase, measured its one section aminoacid sequence of N-end, and synthesized corresponding primer on this basis, having separated relevant mRNA then from agkistrodon acutus poison gland is template, obtain cDNA through reverse transcription, with PCR method amplification with cloned snake poison blood coagulation zymoexcitator gene, and measured gene order, study its zymologic property and activation thereof and coagulated the former mechanism of action and external pharmacologically active.

In addition, the present invention has also prepared the derivative of thrombogen activator DaPA and has measured its activity.

In the present invention, term " thrombogen activator DaPA ", " DaPA albumen " or " DaPA polypeptide " are used interchangeably, and all refer to have basically the polypeptide or the protein of thrombogen activator DaPA aminoacid sequence (SEQ IDNO:2).They comprise the thrombogen activator DaPA that contains or do not contain initial methionine.These terms also comprise the thrombogen activator DaPA that contains or do not contain signal peptide.

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

As used herein, " isolating DaPA polypeptide protein " is substantially free of natural relative other albumen, lipid, carbohydrate or other material.Those skilled in the art can go out thrombogen activator DaPA with purified technology of protein (especially FPLC) separation and purification of standard.

Protein of the present invention can be recombinant protein, natural protein, synthetic protein, preferred recombinant protein.Protein 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, protein of the present invention can be glycosylated, maybe can be nonglycosylated.Protein of the present invention also can comprise or not comprise initial methionine residues.

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

In the present invention, term " thrombogen activator DaPA " refers to have the protein of the active SEQ ID of thrombogen activator DaPA NO.2 sequence.This term also comprises having and variant form thrombogen activator DaPA identical function, SEQ ID NO.2 sequence.These variant forms comprise (but being not limited to): one or more (it is individual to be generally 1-50, preferably 1-30, more preferably 1-20,1-10 best) amino acid whose disappearance, insertion and/or replacement, and (being generally in 20, preferably is in 10 to add one or several at C-terminal and/or N-terminal, more preferably be in 5) amino-acid residue as, in the art, when replacing, can not change proteinic function usually with the close or similar amino-acid residue of performance.Again such as, add one or several amino-acid residues at C-terminal and/or N-terminal and also can not change proteinic function.This term also comprises active fragments and the reactive derivative of thrombogen activator DaPA.

This proteinic variant form comprises: homologous sequence, conservative property varient, allelic variant, natural mutation, induced mutation body, under high or low tight degree condition can with the coded protein of the DNA of DaPA DNA hybridization and the polypeptide or the protein that utilize the antiserum(antisera) of antithrombin activator DaPA to obtain.The present invention also provides other protein, as comprises thrombogen activator DaPA or its segmental fusion rotein.Except whole length protein almost, the present invention has also comprised the soluble fragments of thrombogen activator DaPA sequence.Usually, this fragment have thrombogen activator DaPA sequence at least about 10 continuous amino acid residues, usually at least about 30 continuous amino acid residues, preferably at least about 50 continuous amino acid residues, more preferably at least about 80 continuous amino acid residues, best at least about 100 continuous amino acid residues.

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

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

In the present invention, " DaPA conservative property variant protein matter " refers to compare with the aminoacid sequence of SEQ ID N0:2, has 10 at the most, preferably at the most 8, more preferably at the most 5,3 amino acid is replaced by similar performance or close amino-acid residue and is formed protein at the most best.These conservative property variant protein matter are preferably carried out the amino acid replacement according to table 1 and are produced.

Table 1

Initial residue	Representational replacement	The preferred replacement
Initial residue	Representational replacement	The preferred replacement	Ala(A)	Val；Leu；Ile	Val
Arg(R)	Lys；Gln；Asn	Lys	Ala(A)	Val；Leu；Ile	Val
Arg(R)	Lys；Gln；Asn	Lys	Asn(N)	Gln；His；Lys；Arg	Gln
Asp(D)	Glu	Glu	Asn(N)	Gln；His；Lys；Arg	Gln
Asp(D)	Glu	Glu	Cys(C)	Ser	Ser
Gln(Q)	Asn	Asn	Cys(C)	Ser	Ser

Glu(E)	Asp	Asp
Glu(E)	Asp	Asp	Gly(G)	Pro；Ala	Ala
His(H)	Asn；Gln；Lys；Arg	Arg	Gly(G)	Pro；Ala	Ala
His(H)	Asn；Gln；Lys；Arg	Arg	Ile(I)	Leu；Val；Met；Ala；Phe	Leu
Leu(L)	Ile；Val；Met；Ala；Phe	Ile	Ile(I)	Leu；Val；Met；Ala；Phe	Leu
Leu(L)	Ile；Val；Met；Ala；Phe	Ile	Lys(K)	Arg；Gln；Asn	Arg
Met(M)	Leu；Phe；Ile	Leu	Lys(K)	Arg；Gln；Asn	Arg
Met(M)	Leu；Phe；Ile	Leu	Phe(F)	Leu；Val；Ile；Ala；Tyr	Leu
Pro(P)	Ala	Ala	Phe(F)	Leu；Val；Ile；Ala；Tyr	Leu
Pro(P)	Ala	Ala	Ser(S)	Thr	Thr
Thr(T)	Ser	Ser	Ser(S)	Thr	Thr
Thr(T)	Ser	Ser	Trp(W)	Tyr；Phe	Tyr
Tyr(Y)	Trp；Phe；Thr；Ser	Phe	Trp(W)	Tyr；Phe	Tyr
Tyr(Y)	Trp；Phe；Thr；Ser	Phe	Val(V)	Ile；Leu；Met；Phe；Ala	Leu

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

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

Term " polynucleotide of coded protein " can be to comprise these proteinic polynucleotide of coding, also can be the polynucleotide that also comprise additional code and/or non-coding sequence.

The invention still further relates to the varient of above-mentioned polynucleotide, it is encoded polypeptide or proteinic fragment, analogue and the derivative of identical aminoacid sequence 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 protein matter in fact.

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

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

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

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

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

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

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

Use method (Saiki, the et al.Science 1985 of round pcr DNA amplification/RNA; 230:1350-1354) be optimized for acquisition gene of the present invention.When particularly being difficult to obtain the cDNA of total length from the library, can preferably use RACE method (the terminal rapid amplifying method of RACE-cDNA), the primer that is used for PCR can suitably be selected according to sequence information of the present invention disclosed herein, and available ordinary method is synthetic.Available ordinary method is as the DNA/RNA fragment by gel electrophoresis separation and purifying amplification.

The present invention also relates to comprise the carrier of polynucleotide of the present invention, and the host cell that produces through genetically engineered with carrier of the present invention or thrombogen activator DaPA encoding sequence, and produce method of protein of the present invention through recombinant technology.

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

(1). with the polynucleotide (or varient) of coding thrombogen activator DaPA of the present invention, or transform or the transduction proper host cell with the recombinant expression vector that contains these polynucleotide;

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

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

Among the present invention, thrombogen activator DaPA polynucleotide sequence can be inserted in the recombinant expression vector.Term " recombinant expression vector " refers to that bacterial plasmid well known in the art, phage, yeast plasmid, vegetable cell virus, mammalian cell virus are as adenovirus, retrovirus or other carriers.The carrier of Shi Yonging includes but not limited in the present invention: the expression vector based on T7 of expressing in bacterium; The pMSXND expression vector of in mammalian cell, expressing and at the carrier that derives from baculovirus of expressed in insect cells.In a word, as long as can duplicate in host and stablize, any plasmid and carrier can be used.A key character of expression vector is to contain replication orgin, promotor, marker gene and translation controlling elements usually.

Method well-known to those having ordinary skill in the art can be used to make up and contains thrombogen activator DaPA DNA sequences encoding and suitable transcribing/the translate expression vector of control signal.These methods comprise extracorporeal recombinant DNA technology, DNA synthetic technology, the interior recombinant technology of body etc.Described dna sequence dna can effectively be connected on the suitable promotor in the expression vector, and is synthetic to instruct mRNA.The representative example of these promotors has: colibacillary lac or trp promotor; Lambda particles phage P _LPromotor; Eukaryotic promoter comprises LTRs and some other known may command gene expression promoter in protokaryon or eukaryotic cell or its virus of CMV immediate early promoter, HSV thymidine kinase promoter, early stage and late period SV40 promotor, retrovirus.Expression vector also comprises ribosome bind site and the transcription terminator that translation initiation is used.

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

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

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

When polynucleotide of the present invention are expressed in higher eucaryotic cells, be enhanced if will make to transcribe when in carrier, inserting enhancer sequence.Enhanser is the cis acting factor of DNA, and nearly 10 to 300 base pairs act on promotor transcribing with enhancing gene usually.Can for example be included in the SV40 enhanser of 100 to 270 base pairs of replication origin side in late period one, at the polyoma enhanser of replication origin side in late period one and adenovirus enhanser etc.

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

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

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

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

The DaPA protein of reorganization is of use in many ways.These purposes include, but is not limited to: thus be used to activate the thrombogen hemostasis and be used to screen antibody, protein or other part that promotes or resist the DaPA protein function.The protein molecule that can suppress or stimulate thrombogen activator DaPA function that can be used for seeking therapeutic value with the recombination prothrombin activated activator DaPA screening protein library of expressing.

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

The present invention not only comprises complete mono-clonal or polyclonal antibody, but also comprises having immunocompetent antibody fragment, as Fab ' or (Fab) ₂Fragment; Heavy chain of antibody; Light chain of antibody; Genetically engineered strand Fv molecule; Or chimeric antibody, as have the murine antibody binding specificity but still keep antibody from people's antibody moiety.

Antibody of the present invention can be prepared by the known various technology of those skilled in that art.For example, the thrombogen activator DaPA of purifying or its have antigenic fragment, can be applied to animal to induce the generation of polyclonal antibody.Similarly, expressing thrombogen activator DaPA or its has antigenic segmental cell and can be used to immune animal and produce antibody.Antibody of the present invention also can be monoclonal antibody.This type of monoclonal antibody can utilize hybridoma technology to prepare.Each antibody-like of the present invention can utilize fragment or the functional zone of thrombogen activator DaPA, obtains by the routine immunization technology.These fragments or functional zone can utilize recombinant methods or utilize protein synthesizer synthetic.Can come immune animal and produce with the gene product of producing in the prokaryotic cell prokaryocyte (for example E.Coli) with the unmodified form bonded antibody of thrombogen activator DaPA; With posttranslational modification form bonded antibody (as the albumen or the polypeptide of glycosylation or phosphorylation), can come immune animal and obtain with the gene product that produces in the eukaryotic cell (for example yeast or insect cell).

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

Thrombogen activator DaPA of the present invention and antibody, inhibitor, agonist or antagonist etc. when using (administration) in treatment, can provide different effects.Usually, can these materials are formulated in nontoxic, inert and the pharmaceutically acceptable aqueous carrier medium, wherein pH is about 6-8 usually, and preferably pH is about 7-8, although the pH value can be with being changed to some extent by preparation Substance Properties and illness to be treated.The pharmaceutical composition for preparing can carry out administration by conventional route, comprising (but being not limited to): intramuscular, intravenously or topical.

Protein of the present invention can be directly used in hemostasis clinically.When using thrombogen activator DaPA of the present invention, also can use the other treatment agent simultaneously, as is known haemostatic medicament etc.

The present invention also provides a kind of pharmaceutical composition, and it contains thrombogen activator DaPA of the present invention and the pharmaceutically acceptable carrier or the vehicle of safe and effective amount.This class carrier comprises (but being not limited to): salt solution, damping fluid, glucose, water, glycerine, ethanol and combination thereof.Pharmaceutical preparation should be complementary with administering mode.Pharmaceutical composition of the present invention can be made into the injection form, for example is prepared by ordinary method with the physiological saline or the aqueous solution that contains glucose and other assistant agents.Pharmaceutical composition such as tablet and capsule can be prepared by ordinary method.Pharmaceutical composition such as injection, solution, tablet and capsule should be made under aseptic condition.The dosage of activeconstituents is the treatment significant quantity, for example every day about 0.1 nanogram/kg body weight-Yue 0.01 mg/kg body weight.In addition, protein of the present invention also can use with the other treatment agent.

When making pharmaceutical composition, be that thrombogen activator DaPA with safe and effective amount is applied to Mammals, wherein this safe and effective amount is usually at least about 1 microgram/sky, and in most of the cases be no more than about 0.01 mg/kg body weight, preferably this dosage is about 1 microgram/sky-Yue 0.002 mg/kg body weight.Certainly, concrete dosage also should be considered factors such as route of administration, patient health situation, and these all are within the skilled practitioners skill.

The method that whether has thrombogen activator DaPA in a kind of test sample is to utilize the specific antibody of thrombogen activator DaPA to detect, and it comprises: sample is contacted with thrombogen activator DaPA specific antibody; Observe whether form antibody complex, formed antibody complex and just represented to exist in the sample thrombogen activator DaPA.

Major advantage of the present invention is: separation and purification is to the new protein that can activate thrombogen from the snake venom of agkistrodon acutus first, and the only about 25KD of its molecular weight is the metalloprotease in a kind of single structure territory.This proteolytic enzyme directly acts in the blood coagulation waterfall reaction reaction than the downstream, thereby it is faster to take effect, and is convenient to first aid and uses.Its molecular weight is less relatively, thereby has lower immunogenicity.Agkistrodon acutus (Agkistrodon) source is abundanter simultaneously.Thereby has a great application prospect.

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

The snake poison blood coagulation zymoexcitator of embodiment 1 separation and purification from ahylysantinfarctase

In the present embodiment, adopted the snake venom of agkistrodon acutus, in present embodiment and following examples, unless dated especially, otherwise refer to above this pallas pit viper.

Get ahylysantinfarctase, after the dissolving of pH7.6Tris-HCl damping fluid, centrifugal, get DEAE-SephoroseCL-6B post separation on the supernatant, to have the active component of thrombogen activator separates by FPLC MonoQ post again, just obtain the snake poison blood coagulation zymoexcitator of the single band of SDS-PAGE, recording its apparent molecular weight is 25KD.

This thrombogen activator is named as thrombogen activator DaPA.

The mensuration of 10 aminoacid sequences of embodiment 2 Ahylysantinfarctase thrombase activator N end

With the snake poison blood coagulation zymoexcitator sample of purifying, on commercially available PE491 protein determination instrument,, record following N terminal sequence: STEFQRYMEI through 10 circulations.

Embodiment 3 primer design and synthetic

N terminal sequence and snake class codon-bias design upstream degenerated primer according to the Ahylysantinfarctase thrombase activator that records among the embodiment 2.

The primer of designed upstream oligonucleotide is:

5′-CG AGTACTTC(A/G/C/T)AC(A/G/C/T)GA(A/G)TT(C/T)CA(A/G)AG-3′(SEQID NO：3)

Downstream primer is: oligodT (18),

The synthetic trust Shanghai of primer is given birth to worker biotech firm and is finished.

The extraction of embodiment 4 total RNA

Get ahylysantinfarctase and in liquid nitrogen, pulverize, give birth to total RNA extraction agent box specification sheets institute of worker bio-engineering corporation support method by Shanghai then and extract the total RNA of poison gland.

Embodiment 5 RT-PCR amplification snake poison blood coagulation zymoexcitator gene

With the total RNA of Ahylysantinfarctase thrombase activator that makes among the embodiment 4 is template, gives birth to the worker MMLV cDNA of the biotech firm first chain synthetic agent box reverse transcription with Shanghai, is undertaken by process specifications.

Utilizing synthetic primer among the embodiment 3, is template with the cDNA of above-mentioned gained, carries out pcr amplification, and the total reaction system is 50 μ l, adds sterilization ddH ₂O 35 μ l, 10 * PCR damping fluid, 5 μ l, dNTP 4 μ l, cDNAl μ l, upstream primer 2 μ l, oligo (dT) ₁₈2 μ l, Taq enzyme 1 μ l, reaction conditions is: 94 ℃ of sex change 5min of elder generation enter circulation, 94 ℃ of sex change 1min then; 42 ℃ of annealing 1min; 72 ℃ are extended 1.5min, carry out 5 circulations after, changing annealing temperature is 40 ℃, carries out 30 circulations, at last at 72 ℃ of insulation 10min.The PCR product is got 5 μ l and is carried out 1% agarose electrophoresis inspection.

The result shows that amplification has obtained the dna fragmentation of a treaty 1000bp.With the chloroform extracting, ethanol sedimentation reclaims with this DNA product, is dissolved in the aqua sterilisa of 30 μ l standby.

The gene clone of embodiment 6 snake poison blood coagulation zymoexcitators

Get the PCR recovery product 4 μ l that obtain among the embodiment 5 and be connected transformed into escherichia coli DH with pMD18-T carrier (available from Takara company) 1 μ l _16B, through blue hickie screening, enzyme is cut with PCR and is identified, obtains positive colony.

The evaluation of embodiment 7 Ahylysantinfarctase thrombase activator genes

The positive colony that obtains among the embodiment 6 is entrusted and is gone up the order-checking of sea base health biotech company, and gene order and encoded protein matter sequence are seen shown in Fig. 1 and the SEQ ID NO:1-2.This full length gene 849bp, the long 681bp of reading frame wherein, 226 amino acid of encoding, from the aminoacid sequence of snake poison blood coagulation zymoexcitator genes encoding, to analyze through computer software Vector NTI, this proteinic molecular weight is 25354.5D, its iso-electric point is 4.82, is an acidic protein.

Embodiment 8 Ahylysantinfarctase thrombase activators activate the determination of activity of thrombogen

(1) scleroproein coagulation

Get Fibrinogen and be dissolved in pH7.6 in right amount, 0.04mmol/LTris-HCl in the damping fluid, be made into 0.8% fibrinogen solution, in addition more respectively compound concentration be the prothrombin solution of lmg/ml and the snake poison blood coagulation zymoexcitator solution that concentration is 50 μ g/ml, getting internal diameter during mensuration is 1cm, 3 in the test tube of long 10cm, each adds the 0.2ml fibrinogen solution, put in 37 ℃ of water-baths and be incubated 5min, in these 3 test tubes, add A then) 0.1ml prothrombin solution+0.1ml damping fluid, B) 0.1ml snake poison blood coagulation zymoexcitator solution+0.1ml damping fluid, C)) 0.1ml prothrombin solution+0.1ml snake poison blood coagulation zymoexcitator solution (being pre-mixed the back) 37 ℃ of water bath heat preservation half hours, shake up immediately, as seen the result has only produced scleroproein in the C pipe and has condensed, and A pipe and B manage and all do not condense.

(2) chromophoric substrate assay method

The chromophoric substrate Chromozym TH (Cbz-Gly-Pro-Arg-pNA) that gets zymoplasm is a small amount of, be dissolved in pH7.6,0.04mmol/L in the Tris-HCl damping fluid, be made into the substrate solution that concentration is 1mmol/L, in addition more respectively compound concentration be the prothrombin solution of 10mg/ml and the snake poison blood coagulation zymoexcitator solution that concentration is 500 μ g/ml, survey is got 0.1ml prothrombin solution and 0.1ml snake poison blood coagulation zymoexcitator solution (Prothrombin+DaPA) and 0.8ml pH7.6 before living, 0.04mmol/LTris-HCl mix, and at room temperature pre-incubation 8min, survey and get substrate solution 10 μ l and damping fluid 80 μ l when living, add above-mentioned mixing solutions 10 μ l, change in 405nm record absorbancy, contrast is respectively and only adds thrombogen (Prothrombin) solution, or only adds snake poison blood coagulation zymoexcitator (DaPA) solution.The results are shown in Figure 2, show thrombogen after the snake poison blood coagulation zymoexcitator activates, the chromophoric substrate ChromozyTH of energy hydrolytically condensable hemase contrasts no any variation.

The mensuration of embodiment 9, the external pharmacologically active of Ahylysantinfarctase thrombase activator

(1) recalcification time

Get rabbit arterial blood, add the 3.8% Trisodium Citrate anti-freezing of 1/10 volume, the centrifugal 20min of room temperature 3000g, supernatant liquor are the rabbit platelet poor plasma.Get 0.1 μ l rabbit platelet poor plasma, the snake poison blood coagulation zymoexcitator sample of 0.1 μ l different concns (is dissolved in 0.02mmol/L, pH7.4 Tris-HCl, 0.15mol/LNaCl, 0.05mol/LCaCl ₂In), mixing behind 37 ℃ of water-bath 2min, record is from mixing the time that the start-stop scleroproein occurs, 0.02mol/L pH7.4 Tris-Hcl (0.15mol/LNaCl, 0.05mol/LCaCl ₂) as negative control, place plastic centrifuge tube and glass test tube to measure the rabbit platelet poor plasma respectively, measure for every kind and repeat 3 times, average.

The results are shown in Figure 3, Fig. 3 A is a measurement result in the plastics tubing, and Fig. 3 B is a measurement result in the Glass tubing, and there is notable difference in the recalcification time that records in plastics tubing and Glass tubing, and this is because glass tube walls can promote the reaction of blood coagulation waterfall.

(2) activated partial thromboplastin time (APTT) is measured

Get healthy premenopausal volunteers venous blood, add the 3.8% Trisodium Citrate anti-freezing of 1/10 volume, the centrifugal 20min of room temperature 3000g, supernatant behaviour platelet poor plasma, use the full-automatic thrombocyte of Sysmex CA-530 type to coagulate instrument, the sampling system follow procedure is provided with draws people's platelet poor plasma, iodine fat and rabbit kephalin mixed diluting liquid, Ca ⁺⁺Each 50 μ l is with the Ca of the snake poison blood coagulation zymoexcitator that contains different concns ⁺⁺Replace contained Ca in the coagulo meter reagent bottle ⁺⁺, measure twice and average.The results are shown in Figure 4.

Above-mentioned two kinds of method measurement results all show the continuous increase along with snake poison blood coagulation zymoexcitator sample size, the activated partial thromboplastin time of the recalcification time of rabbit platelet poor plasma and people's platelet poor plasma (APTT) shortens gradually, and this result has shown that also the snake poison blood coagulation zymoexcitator can promote hemagglutination.

Embodiment 10, snake poison blood coagulation zymoexcitator activate the product analysis of thrombogen

Thrombin of beef former with snake poison blood coagulation zymoexcitator DaPA the reaction product of each time point in the presence of the PMSF with carry out SDS-PAGE after 5 * loading damping fluid mixes, cut each main band after changeing pvdf membrane, deliver order-checking, according to the thrombogen hydrolysate of sequencing result and each main band correspondence of band molecular weight analyse, determine that at last the snake poison blood coagulation zymoexcitator activates the former cleavage site of thrombin of beef and is respectively P ¹⁶⁶-L ¹⁶⁷, A ²⁶⁹-A ²⁷⁰And R ³²³- ³²⁴The I peptide is strong.

Embodiment 11 thrombogen activator DaPA's is recombinant expressed

In this embodiment, the translation district of DaPA gene is recombinant expressed in prokaryotic vector pET-40b (+) (Invitrogen company).

(this carrier carries disulfide isomerase gene according to secreted expression carrier pET-40b (+), Dsbc) and the DaPA gene sequence characteristic, pET-40b (+) carrier is digested with restriction enzyme ScaI and EcoRI, the clone digests with ScaI and EcoRI after having the pMD-18T plasmid of DaPA gene to cut glue recovery band with PstI and EcoRI enzymic digestion again, and cut glue respectively and reclaim, get 1 μ l carrier recovery solution and in 10 μ l systems, be connected with the T4 ligase enzyme with 5 μ lDaPA gene fragments and spend the night.Connect product full dose transformed into escherichia coli DH5 α, cultivate on the agar plate that contains kantlex (Kanamycin), picking colony jolting in the test tube that contains the 3mlLB substratum is spent the night, and the extraction plasmid carries out enzyme and cuts evaluation.Positive colony is after PCR identifies, transformed competence colibacillus cell BL21 (DE3) delivers Bo Ya company sequence verification.

The clone there is the pET-40b plasmid transformed competence colibacillus cell BL21 (DE3) of DaPA gene, is inoculated in the fresh 200mL LB substratum, be cultured to OD at 37 ℃ of violent joltings (per minute 200 change more than) ₆₀₀Be about 0.6, the IPTG that adds final concentration and be 1mmol/L was 30 ℃ of abduction deliverings 5 hours.Then, centrifugal 10 minutes of 4 ℃ of 5000g collect and express bacterium, with its be suspended in precooling on ice 20mL 1 * NTA0buffer (0.5mol/LNaCl, 10% glycerine, 20mmol/L Tris-HCl, pH8.0) in, add PMSF to final concentration 1mmol/L.Place carrying out ultrasonic bacteria breaking on the ice bath, 15, centrifugal 30 minutes of 4 ℃ of 000g collect supernatant, carry out affinity chromatography, and whole chromatography process is all carried out under 4 ℃ of constant temperature.Get 1～2mL Ni ²⁺-NTA affinity chromatography resin (every milliliter of adsorbable about 10mg 6 * His fusion rotein of resin) in advance at 4 ℃ with the NTA0buffer balance that contains 1mmol/L PMSF, bacterium cracking supernatant is left and taken 60 μ l samples after the unconcerned filtration of 0.45 μ m filter, drawing wherein, 20 μ l are used for the SDS-PAGE analysis, the slow post of crossing of remaining supernatant adsorbs, flow velocity is not higher than 15ml/h, collects the absorption back effluent liquid and the 20 μ l that take a sample and is used for the SDS-PAGE analysis; NTA0 buffer with 10 times of column volumes washes post, and flow velocity 30ml/h collects the elutriant and the 20 μ l that take a sample and is used for the SDS-PAGE analysis.NTA20 buffer, NTA60 buffer, NTA200 buffer, NTA800 buffer with each 5 times of column volume (comprise 20mmol/L imidazoles, 60mmol/L imidazoles, 200mmol/L imidazoles, 800mmol/L imidazoles respectively on the NTA0buffer basis, convert with the NTA800 buffer of preparation in advance and NTA0 buffer and to make desired concn) and Stripbuffer (100mmol/L EDTA, 0.5mol/L NaCl, 20mmol/L Tris-HCl) washes post successively, the control flow velocity is 15ml/h, collect elutriant respectively with the 1.5ml plastic centrifuge tube, measure the OD of each pipe ₂₈₀, OD from per step ₂₈₀Sampling 20 μ l carry out the SDS-PAGE analysis in each the highest pipe, determine Dsbc-DaPA fusion rotein place.

The result shows that the protein band molecular weight is 43kD in the NTA200 elutriant, and is suitable with Dsbc-DaPA fusion rotein desired value.Western identifies and shows that polyclonal antibody and this albumen at DaPA have obvious immunological cross-reaction.

Adopt conventional dividing method, DaPA albumen is separated from the Dsbc-DaPA fusion rotein, obtain activated DaPA albumen.

Press embodiment 8 described methods (scleroproein coagulation and chromophoric substrate assay method), the activity of the DaPA albumen that mensuration is obtained and the activation thrombogen of described Dsbc-DaPA fusion rotein, result show that DaPA albumen and described Dsbc-DaPA fusion rotein that present embodiment obtains are close with isolating natural DaPA protein-active from snake venom.

Embodiment 12 carries histidine-tagged DaPA the derive preparation and the activity identification of polypeptide

Adopt conventional method, hold the encoding sequence that adds corresponding to 6his, give expression to the N end with conventional DNA recombination method then and carry 6 histidine-tagged DaPA albumen at 5 ' of SEQ ID NO:1.Separate polypeptide thereby the DaPA that has 6His of acquisition purifying derives with the Ni affinity column for expression product.

Press embodiment 8 described methods (scleroproein coagulation), measure the derive activity of activation thrombogen of polypeptide of the DaPA obtained.The result shows, the derive activity of activation thrombogen of polypeptide of this DaPA is identical with isolating natural DaPA protein-active from snake venom.

Embodiment 13 contains the pharmaceutical composition of thrombogen activator DaPA

By following prescription, use the ordinary method pharmaceutical compositions:

DaPA albumen (extraction or recombinant expressed)	5 micrograms
DaPA albumen (extraction or recombinant expressed)	5 micrograms	Dextran
	1 milligram	Dextran
	1 milligram	100 mmole phosphoric acid buffers	1 milliliter

All quote in this application as a reference at all documents that the present invention mentions, just quoted as a reference separately as each piece document.Should be understood that in addition those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims institute restricted portion equally.

Sequence table

＜110〉Shanghai Inst. of Life Science, CAS

＜120〉Ahylysantinfarctase thrombase activator, its encoding sequence and purposes

<130>064452

<160>3

<170>PatentIn version 3.1

<210>1

<211>1205

<212>DNA

＜213〉point kiss abdomen snake (Agkistrodon acutus)

<220>

<221>CDS

<222>(1)..(678)

<223>

<400>1

tct act gaa ttt cag aga tac atg gag att gtt att gtt gtg gac cat 48

Ser Thr Glu Phe Gln Arg Tyr Met Glu Ile Val Ile Val Val Asp His

1 5 10 15

tca atg gtc aaa aaa tac aat ggc gat tca gat aag ata aaa gca tgg 96

Ser Met Val Lys Lys Tyr Asn Gly Asp Ser Asp Lys Ile Lys Ala Trp

20 25 30

gta tat gaa atg atc aac act ata acg gag agt tac agc tat ttg tat 144

Val Tyr Glu Met Ile Asn Thr Ile Thr Glu Ser Tyr Ser Tyr Leu Tyr

35 40 45

atc gat ata ata ttg tct ggc ttg gaa att tgg tcc gag aaa gat ttg 192

Ile Asp Ile Ile Leu Ser Gly Leu Glu Ile Trp Ser Glu Lys Asp Leu

50 55 60

att aat gtg gag aca tca gcg gaa aat act ttg aaa tca ttt gga gaa 240

Ile Asn Val Glu Thr Ser Ala Glu Asn Thr Leu Lys Ser Phe Gly Glu

65 70 75 80

tgg aga gcg aaa gat ttg att cat cgc ata agt cat gat aat gct cag 288

Trp Arg Ala Lys Asp Leu Ile His Arg Ile Ser His Asp Asn Ala Gln

85 90 95

tta ctc acg gcc act gac ttc gat gga cca act ata gga ttg gct tac 336

Leu Leu Thr Ala Thr Asp Phe Asp Gly Pro Thr Ile Gly Leu Ala Tyr

100 105 110

gtg gcc agc atg tgt gac cca aag cgt tct gta gga gtt gtt cag gat 384

Val Ala Ser Met Cys Asp Pro Lys Arg Ser Val Gly Val Val Gln Asp

115 120 125

cat agc tca gta aat cgt ttg gtt gcc att acc ctg gcc cat gag atg 432

His Ser Ser Val Asn Arg Leu Val Ala Ile Thr Leu Ala His Glu Met

130 135 140

gct cat aat ctg ggc gtt cgt cat gac gaa ggt tcc tgt tct tgt ggt 480

Ala His Asn Leu Gly Val Arg His Asp Glu Gly Ser Cys Ser Cys Gly

145 150 155 160

agt ggt tac aca tgc atc atg tct cct gtg ata aac tct gaa gtt atc 528

Ser Gly Tyr Thr Cys Ile Met Ser Pro Val Ile Asn Ser Glu Val Ile

165 170 175

aaa tat ttc agc gat tgt agt tat atc caa tgt cgg gag tat ata tcg 576

Lys Tyr Phe Ser Asp Cys Ser Tyr Ile Gln Cys Arg Glu Tyr Ile Ser

180 185 190

aag gag aac cca cct tgc att ctc aat aaa ccc ttg aga aca gat act 624

Lys Glu Asn Pro Pro Cys Ile Leu Asn Lys Pro Leu Arg Thr Asp Thr

195 200 205

gtt tca act cca gtt tct gga aat gaa ctt ttg gag gcg gaa aaa gat 672

Val Ser Thr Pro Val Ser Gly Asn Glu Leu Leu Glu Ala Glu Lys Asp

210 215 220

tat gac tgagactctt ctgcagtctg cggcaacggg cattgtgttg atgtgactac 728

Tyr Asp

225

agcctactaa tcaactgctg gcttctctca gatttgattt tggagatcct tcttccagaa 788

ggtttcactt ccctcaagtc caaagagatc catctgcctg cattctacta gtaaatcact 848

cttagctttc gtatggaatc taaattccgc aatatttctt ctccatattt aatctgttta 908

ccttttgctg taatcaaacc ttttccccgc cacaaagctc catgggcatg gacaacacac 968

caagggctta tttgctgtca aagaaaagaa atggccattt taccatttgc caaaagcaca 1028

tttaatgcaa caagttctgc ctttttgagc tggtgtattc aaagtcaatg cttcctctcc 1088

caaaatttca tgctggcttt tccaagatgt ggctgcttcc atcaataaac acactattct 1148

cattcgaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaa 1205

<210>2

<211>226

<212>PRT

＜213〉point kiss abdomen snake (Agkistrodon acutus)

<400>2

Ser Thr Glu Phe Gln Arg Tyr Met Glu Ile Val Ile Val Val Asp His

1 5 10 15

Ser Met Val Lys Lys Tyr Asn Gly Asp Ser Asp Lys Ile Lys Ala Trp

20 25 30

Val Tyr Glu Met Ile Asn Thr Ile Thr Glu Ser Tyr Ser Tyr Leu Tyr

35 40 45

Ile Asp Ile Ile Leu Ser Gly Leu Glu Ile Trp Ser Glu Lys Asp Leu

50 55 60

Ile Asn Val Glu Thr Ser Ala Glu Asn Thr Leu Lys Ser Phe Gly Glu

65 70 75 80

Trp Arg Ala Lys Asp Leu Ile His Arg Ile Ser His Asp Asn Ala Gln

85 90 95

Leu Leu Thr Ala Thr Asp Phe Asp Gly Pro Thr Ile Gly Leu Ala Tyr

100 105 110

Val Ala Ser Met Cys Asp Pro Lys Arg Ser Val Gly Val Val Gln Asp

115 120 125

His Ser Ser Val Asn Arg Leu Val Ala Ile Thr Leu Ala His Glu Met

130 135 140

Ala His Asn Leu Gly Val Arg His Asp Glu Gly Ser Cys Ser Cys Gly

145 150 155 160

Ser Gly Tyr Thr Cys Ile Met Ser Pro Val Ile Asn Ser Glu Val Ile

165 170 175

Lys Tyr Phe Ser Asp Cys Ser Tyr Ile Gln Cys Arg Glu Tyr Ile Ser

180 185 190

Lys Glu Asn Pro Pro Cys Ile Leu Asn Lys Pro Leu Arg Thr Asp Thr

195 200 205

Val Ser Thr Pro Val Ser Gly Asn Glu Leu Leu Glu Ala Glu Lys Asp

210 215 220

Tyr Asp

225

<210>3

<211>25

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

＜223〉primer

<220>

<221>misc_feature

<222>(11)..(11)

＜223〉n=a, t, c or g

<220>

<221>misc_feature

<222>(14)..(14)

＜223〉n=a, t, c or g

<400>3

cgagtacttc nacngartty carag 25

Claims

1. an isolating protein is characterized in that, this protein is selected from down group:

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

(b) SEQ ID NO:2 aminoacid sequence is formed through replacement, disappearance or the interpolation of 1-10 amino-acid residue, and have the function that activates thrombogen generation zymoplasm by (a) polypeptides derived.

2. protein as claimed in claim 1 is characterized in that, described proteinic aminoacid sequence is shown in SEQ ID NO:2.

3. isolating polynucleotide is characterized in that, the described albumen of its coding claim 1.

4. polynucleotide as claimed in claim 3 is characterized in that this polynucleotide encoding has the protein of aminoacid sequence shown in the SEQ IDNO:2.

5. polynucleotide as claimed in claim 3 is characterized in that, these polynucleotide contain the sequence of 1-678 position among the SEQ ID NO:1.

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

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

8. described proteinic preparation method of claim 1 is characterized in that this method comprises step:

(a) under expression condition, cultivate the described host cell of claim 7, thereby express the described protein of claim 1;

(c) from culture, isolate the described protein of claim 1.

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

10. the described proteinic purposes of claim 1 is characterized in that, is used to prepare the hematostatic medicine.