CN100357322C - Disintegrating element for poisonous snake and its coding gene and use - Google Patents

Abstract

The present invention discloses a disintegrating element, and a coding gene and applications of the disintegrating element. The present invention aims at providing a disintegrating element, a coding gene thereof, and applications of the disintegrating element to the preparation of medicine for inhibiting platelet aggregation. The disintegrating element is a protein with one of the following amino acid residue sequences: 1) SEQ ID No. 1 in a sequence table and 2) a protein with a function of inhibiting platelet aggregation, wherein the 2) protein is formed by replacing or deleting or adding one to ten amino acid residues in the amino acid residue sequence of the SEQ ID No. 1 in the sequence table. The disintegrating element capable of inhibiting platelet aggregation and the coding gene thereof provided by the present invention can probably become clinical platelet aggregation inhibitors or medicine in the protein class. Moreover, the disintegrating element capable of inhibiting platelet aggregation and the coding gene thereof can be used as ideal base substances for the medicine design of platelet inhibitors (antagonists of platelet membrane receptors) used clinically, and have significant meaning for developing platelet aggregation inhibitors with autonomous intellectual property.

Description

A kind of disintegrating element for poisonous snake and encoding gene thereof and application

Technical field

The present invention relates to animal proteinum and encoding gene thereof and application, particularly relate to a kind of disintegrating element for poisonous snake and encoding gene thereof and its application in preparation anticoagulant medicine.

Background technology

The mortality ratio that cardiovascular and cerebrovascular diseases causes is positioned at first of all kinds of diseases of China.At present, the medicine of treatment cardiovascular and cerebrovascular has anti-bolt medicine, thrombolytic drug and medicament for resisting platelet aggregation.In snake venom, just there are above-mentioned three kinds of pharmaceutical cpds, Thrombin-like enzyme as anti-bolt, the fibrinoclase of thrombolysis and the disintegrin of platelet aggregation-against etc., wherein disintegrin is isolating a kind of protein molecule in snake venom, because of it can integrate plain gaining the name by powerful antagonism, the characteristic feature of disintegrating element for poisonous snake is to contain hydrophilic aminoacid sequence Arg-Gly-Asp (RGD), be rich in cysteine residues (cysteine), molecular weight is at the small molecular protein of 5-9kDa, the mechanism of disintegrating element for poisonous snake anticoagulant is that the fibrinogen deceptor (GPIIb/IIIa) on its RGD sequence and the thrombocyte has high binding affinity, thrombocyte capable of blocking and fibrinogenic combination, thus reach the effect that suppresses platelet aggregation.Be used at present the synthesis of cyclic peptide class that blocking platelet and Fibrinogen have human mouse chimeric antibody-ReoPro (abciximab) of anti-GPIIb/IIIa in conjunction with producing the accumulative medicine, contain the RGD sequence clinically, as Eptifibatide (Eptifibatide), synthetic non-peptide derivative, as Tirofiban (tirofiban), Lamifiban (lamifiban) etc.; Therefore the at present main dependence on import of the above-mentioned three class medicines of domestic application presses for the medicament for resisting platelet aggregation with independent intellectual property right.

Summary of the invention

The purpose of this invention is to provide a kind of disintegrin and encoding gene thereof with anticoagulant effect.

Disintegrin provided by the present invention, name is called Kistin, and derive from Viperidae Crotalinae abdomen snake and belong to point kiss abdomen snake (Deinagkistrodon acutus), be the protein with one of following amino acid residue sequences:

1) the SEQ ID № in the sequence table: 1;

2) with SEQ ID № in the sequence table: 1 amino acid residue sequence is through replacement, disappearance or the interpolation of one to ten amino-acid residue and have the protein of anticoagulant effect.

SEQ ID № in the sequence table: 1 is made up of 81 amino-acid residues.

The gene (Kistin) of code book invention disintegrin also belongs to protection scope of the present invention.It is one of following nucleotide sequence:

1) SEQ ID № in the sequence table: 2 dna sequence dna;

2) under the rigorous condition of height can with SEQ ID № in the sequence table: the nucleotide sequence of the 2 dna sequence dnas hybridization that limit.

The rigorous condition of described height can be that (or 0.1 * SSC), the solution of 0.1%SDS is hybridized and washed film with 0.1 * SSPE under 65 ℃.

SEQ ID № in the sequence table: 2 by 243 based compositions, and its open reading frame is that coding has SEQ ID № in the sequence table: the protein of 1 amino acid residue sequence from 5 ' end 1-the 243rd bit base.

Contain expression carrier of the present invention, transgenic cell line and host bacterium and all belong to protection scope of the present invention.

Arbitrary segmental primer is to also within protection scope of the present invention among the amplification Kistin.

Another object of the present invention provides a kind of method of expressing above-mentioned disintegrin.

The method of the above-mentioned disintegrin of expression provided by the present invention is that the recombinant expression vector that will contain the above-mentioned disintegrin gene of encoding imports host cell, expresses obtaining disintegrin.

But described host is the protokaryon or the eukaryotic cell of arbitrary expression alien gene.

Described prokaryotic cell prokaryocyte can be colibacillus, as E.coli TB1 or E.coli DE3 etc.

Described eukaryotic cell can be mammalian cell, yeast cell and vegetable cell etc.Comprise COS-7, CHO, BHK-21 or Pichia pastoris etc.

The carrier that sets out that is used to make up the recombinant expression vector that contains the disintegrin encoding gene can be pMAL-P2X, pET32 or pQE-30 etc.

Be the carrier that sets out with pMAL-P2X, the recombinant expression vector that contains described disintegrin gene of structure is pMAL-P2X/Kistin.

When described host is colibacillus, need to add IPTG and carry out abduction delivering, add IPTG concentration be 0.1-0.5mM, be preferably 0.3mM.

Cultivation contains the substratum and the culture condition of the host cell of disintegrin encoding gene of the present invention, all can be substratum and the culture condition of cultivating the host that sets out.

The present invention also provides a kind of medicine of anticoagulant.

The medicine of anticoagulant provided by the present invention, activeconstituents are above-mentioned disintegrin.

When needing, in the medicine of above-mentioned anticoagulant, can also add one or more pharmaceutically acceptable carriers.Described carrier comprises thinner, vehicle, weighting agent, tackiness agent, wetting agent, disintegrating agent, absorption enhancer, tensio-active agent, absorption carrier of pharmaceutical field routine etc.

Medicine of the present invention can be made various ways such as injection liquid, tablet, pulvis, granula, capsule, oral liquid.The medicine of above-mentioned various formulations all can be according to the ordinary method preparation of pharmaceutical field.

The consumption of said medicine is generally 0.5-2mg disintegrin Kistin/kg body weight/day, and be 10-20 days the course of treatment.

Disintegrin Kistin and encoding gene thereof that can anticoagulant provided by the present invention, be expected to become clinical anticoagulant or protein medicaments, and can be used as clinically the desirable substrate of platelet suppressant drug (antagonist of platelet membrane acceptor) when medicinal design that uses, it is significant to have an anticoagulant of independent intellectual property right to research and development.

Description of drawings

Fig. 1 is the agarose gel electrophoresis detected result of the disintegrin gene of RT-PCR amplification

Fig. 2 cuts qualification result for the PCR and the enzyme that transform the positive colony that the disintegrin gene clone carrier is arranged

Fig. 3 is the pcr amplification detected result that contains the disintegrin gene of mutating alkali yl

Fig. 4 cuts qualification result for PCR and the enzyme of integrating plain prokaryotic expression carrier pMAL-P2X

Fig. 5 is the sequence alignment result who contains the disintegrin gene and the former disintegrin gene Kistin of mutating alkali yl

Fig. 6 is the SDS-PAGE detected result of the disintegrin of prokaryotic expression

Fig. 7 is the SDS-PAGE detected result of the disintegrin of purified prokaryotic expression

Fig. 8 for the disintegrin that adds purified prokaryotic expression after again with the graphic representation of ADP inductor inductive thrombocyte 5min aggregation rate

Fig. 9 is for adding behind the reference protein MBP graphic representation with ADP inductor inductive thrombocyte 5min aggregation rate again

Embodiment

Method therefor is ordinary method if no special instructions among the following embodiment.It is synthetic that the primer is given birth to the worker by Shanghai.

The acquisition of embodiment 1, disintegrin gene (Kistin)

1, RT-PCR amplification

Nucleotide sequence (Eur J Biochem.2000Mar according to disintegrating element for poisonous snake; 267 (5): 1359-67, Tsai IH, Wang YM, Chiang TY, Chen YL, Huang RJ, Purificat ion, cloning andsequence analyses for pro-metal loprotease-di sintegrin variants fromDeinagkistrodon acutus venom and subclassification of the small venommetalloproteases), choose the conservative property nucleotide sequence design primer of disintegrin 5 ' and 3 ' end, the total RNA of snake venom gland that belongs to agkistrodon acutus (Deinagkistrodon acutus) with the abdomen snake is a template, carries out the RT-PCR amplification, and primer sequence is as follows:

Primer 1 (upstream primer): 5 '-CCAGTTTCTGGAAATGAA-3 '

Primer 2 (downstream primer): 5 '-GGCATGGGAGTGATATCT-3 '

Extract the total RNA of snake venom gland of agkistrodon acutus (Agkistrodon acutus) (area, Nanning) and as template, under the guiding of primer 1 and primer 2, carry out the RT-PCR amplification, concrete grammar is as follows:

First round reaction system and reaction conditions are: template ribonucleic acid 2 μ g, Oligo (dT) primer 3 μ L, add DEPC water to 10 μ L, behind insulation 5min under 65 ℃, place cooled on ice rapidly, add 5 * PCR damping fluid (TAKARA company product), 4 μ L, dNTPs mixture (10mM each) 1 μ L, RNase inhibitor (TAKARA company product) 20U, ThermoScript II M-MLV (TAKARA company product) 200U then, complement to 20 μ L with DEPC water at last, 42 ℃ of insulation 1h.

Second takes turns reaction system is: first round RT-PCR product 1 μ L, 10 * PCR damping fluid (contains Mg ²⁺) 4 μ L, dNTPs (2.5mmol/L each) 4 μ L, LA-Taq enzyme (TAKARA company product) (5U/ μ L) 0.2 μ L, each 1 μ L of primer 1 and primer 2 (upstream and downstream primer) (25 μ mol/L) adds deionized water postreaction system to 50 μ L.The PCR reaction conditions is: 94 ℃ of 5min of elder generation; 94 ℃ of 30sec again, 57 ℃ of 30sec, 72 ℃ of 30sec, totally 30 circulations; Last 72 ℃ of 10min.

After reaction finishes, the RT-PCR product is carried out 1.2% agarose gel electrophoresis detect, detected result is (swimming lane 1 is Marker, and swimming lane 2 is the RT-PCR product) as shown in Figure 1, amplifies the dna fragmentation of the about 250bp of length, conforms to expected results.

2, the structure of gene clone carrier

1) connects

Reclaim the also purpose fragment of the about 250bp of length of purification step 1 acquisition, it is linked to each other with carrier pMD18-T (TaKaRa company), obtain containing the segmental cloning vector of purpose, linked system is: T ₄Dna ligase 10 * damping fluid 1 μ L, pMD18-T 1 μ L, purified amplified production 3 μ L, T ₄Dna ligase (TaKaRa company) (3U/ μ L) 1 μ L adds deionized water postreaction system to 10 μ L, and 16 ℃ connect 12h.

2) screening of conversion and positive colony

Connection product transformed into escherichia coli DH5 α competent cell (sky, Beijing is a Time Inc.) with step 1), concrete grammar is: get 5 μ L and connect product, add 100 μ L bacillus coli DH 5 alpha competent cells, behind the ice bath 1h, 42 ℃ of heat-shocked 90sec place 3min immediately in ice bath, add 900 μ L LB liquid nutrient mediums, under 37 ℃, 150rpm, at the uniform velocity shake 1h.Get 200 μ L and evenly coat on the LB resistant panel that contains 100 μ g/mL penbritins (Amp), cultivate 12-24h for 37 ℃.

3) positive colony selects and the extraction of positive plasmid

Single bacterium colony that random choose grows in the LB resistant panel is inoculated in 5mL with it and contains in the 100 μ g/mL penbritin LB liquid nutrient mediums, at the uniform velocity shakes down amplification cultivation 12-16h at 37 ℃, 225rpm.After cultivating end, plasmid rapid extraction test kit and reference reagent box specification sheets with Beijing ancient cooking vessel state biotech company extract plasmid, concrete grammar is: get above-mentioned cultivation bacterium liquid 3mL, 15, behind the centrifugal 30sec of 000rpm, abandon supernatant, add 200 μ L suspension and 10 μ L RNase A (10mg/mL) and thoroughly suspend, handle with 300 μ L lysates and 450 μ L denaturing agents again, behind the centrifugal 5min of 12000rpm, move supernatant to adsorption column, leave standstill 2min, the centrifugal 30sec of 8000rpm, with behind the washings washing adsorption column, the centrifugal 1min of 12000rpm dries remaining liq, and adsorption column is placed new centrifuge tube again, the deionized water that adds 40 μ L50 ℃ preheatings, leave standstill 1min, 12, the centrifugal 1min of 000rpm, precipitation is plasmid DNA, and-20 ℃ of preservations are standby.

4) PCR of positive colony identifies

Get the bacterium liquid of 400 μ L step 1) through amplification, get its precipitation after centrifugal, every pipe adds TE liquid 80 μ L, in boiling water, hatch 10min after, 15, the centrifugal 10min of 000rpm gets supernatant.Be template with the plasmid of step 3) extraction and the bacterium liquid supernatant that obtains with aforesaid method respectively, under the guiding of primer 1 and primer 2, identify with conventional PCR method, the pcr amplification amplification condition is: 94 ℃ of 30sec, 57 ℃ of 30sec, 72 ℃, 30sec, totally 30 circulations.After the PCR reaction finishes, get 5 μ L amplified productions and carry out the detection of 1.2% agarose gel electrophoresis, detected result is the (plasmid of swimming lane 2 for extracting as shown in Figure 2, swimming lane 3 is a thalline PCR qualification result, swimming lane 4 is a plasmid PCR qualification result, and swimming lane 5 is DL2000 DNA Marker), amplified the dna fragmentation of the about 250bp of length, conform to expected results, with its called after pMD18-T/Kistin.

5) order-checking of positive colony plasmid

The positive colony plasmid that step 4) is obtained checks order, sequencing result shows that institute's cloned genes has SEQ ID № in the sequence table: 2 dna sequence dna, SEQ ID № in the sequence table: 2 by 243 based compositions, its open reading frame is from 5 ' end 1-the 243rd bit base, coding has SEQ ID № in the sequence table: the protein of 1 amino acid residue sequence, through this unnamed gene is Kistin, the albumen called after Kistin that it is coded.Nucleotide sequence to this gene carries out the Blast compare of analysis, the result shows gene of the present invention and Gloydiussaxatilis metalloproteinase/disintegrin saxin precursor (gi|31322300), T.mucrosquamatus mRNA for metalloprotease and disintegrin (gi|467703), Gloydius halys brevicaudus platelet aggregation inhibitor (gi|3003024), Gloydius saxatilis disintegrin mRNA, partial cds (gi|31322308), the dna sequence dna that Trimeresurusflavoviridis hemorrhagic metalloprotease HR2a (gi|14595994) and Gloydiusussuriensis metalloproteinase/disintegrin ussurin precursor (gi|31322302) are limited has 90% above homology, and coding identical function protein DNA sequence.Proteic amino acid residue sequence to this coded by said gene carries out the Blast compare of analysis, the result shows the albumen and the metalloproteinase/disintegrin saxin precursor (gi|31322301) of coded by said gene of the present invention, Hemorrhagic protein-rhodostomin precursor (RHO, gi|461932), Hemorrhagicmetalloproteinase HR2a precursor (Trimerelysin II, gi|50403719), Zincmetalloproteinase jerdonin precursor (gi|48428041), Disintegrin acostatinalpha precursor (gi|48428043) and platelet aggregation disintegrin (basilicin), the aminoacid sequence that venom-Mexican West-Coast rattlesnake (gi|281138) is limited has 80% above homology, and the proteinic aminoacid sequence of coding identical function.

The structure of embodiment 2, disintegrin prokaryotic expression carrier pMAL-P2X/Kistin and the expression of target protein

One, the structure of disintegrin prokaryotic expression carrier pMAL-P2X/Kistin

1, the amplification of disintegrin gene

Redesign primer PCR amplification disintegrin gene segment, with in the primer 1 (upstream primer) from 5 ' the 3rd G of end and the 9th A respectively the people be A and C for nonsense mutation, become the codon that intestinal bacteria are had a preference for, but its coded amino acid is constant, introduce restriction enzyme Hind III recognition site and two successive terminator codons in primer 2 (downstream primer), the primer sequence that obtains is:

Primer 3 (upstream primer): 5 '-GAAGCAGGCGAAGAGTGT-3 '

Primer 4 (downstream primer): 5 '-GCTAAAGCTTCTATTAGGCATGGGAGTG-3 '

The positive colony plasmid pMD18-T/Kistin that obtains with embodiment 1 is a template, under the guiding of primer 3 and primer 4, and pcr amplification disintegrin gene Kistin, the PCR reaction system is: cloned plasmids 2 μ L, 10 * PCR damping fluid (contains Mg ²⁺) 20 μ L, dNTPs (2.5mmol/L each) 16 μ L, LA-Taq enzyme (5U/ μ L) 0.8 μ l, primer 3 (25 μ mol/L) 4 μ L, primer 4 (25 μ mol/L) 4 μ L add deionized water postreaction system to 200 μ L.The PCR reaction conditions is: 94 ℃ of 5min of elder generation; 94 ℃ of 30sec again, 57 ℃ of 30sec, 72 ℃ of 30sec, totally 30 circulations; Last 72 ℃ of 10min.After reaction finishes, pcr amplification product is carried out 1.2% agarose gel electrophoresis, (swimming lane 1 is DL-2000 DNA Marker to the result shown in 3, swimming lane 2 and swimming lane 3 are pcr amplification product), reclaim the also purpose segment of purifying 250bp with DNA purifying/recovery test kit (Beijing ancient cooking vessel state biotech company), purified product is dissolved among the 40 μ LTE, and-20 ℃ frozen standby.

2, reclaim the terminal flush end processing of product

Recovery product to step 1 carries out terminal flush end processing, reaction system and reaction conditions are: purified recovery product 26.5 μ L, 10 * damping fluid (TAKARA company), 3.6 μ L, 0.1%BSA 3.6 μ L, and 10mM dNTPs 1 μ L (application of sample is in proper order: 10 * damping fluid → 0.1%BSA → DNA).Put 70 ℃ of insulation 5min earlier, place the T that adds 1.3 μ L after 37 ℃ of insulations then ₄Archaeal dna polymerase (TAKARA company) reaction 5min, concuss on the earthquake device makes enzyme deactivation, place again on ice, behind adding equal-volume phenol/chloroform, mixing, 12,000rpm, 4 ℃ of centrifugal 10min carefully draw the superiors, the 3M sodium acetate (pH5.2) that adds 1/10 volume, 100% ethanol of 2.5 times of volumes of adding behind the mixing is in 4 ℃ of precipitation 12-24h, 12, the centrifugal 20min of 500rpm, with 70% washing with alcohol precipitation, dry back adds the deionized water of 20 μ L.

3, the Hind III enzyme of flush end PCR product is cut processing

Step 2 is carried out enzyme through the PCR of flush end processing product with restriction enzyme Hind III cut processing, the endonuclease reaction system is: flush end PCR product 20 μ L, 10 * damping fluid, 3 μ L, Hind III enzyme (TaKaRa company) 2 μ L, deionization H ₂O 5 μ L.Behind 37 ℃ of reaction 2.5h, 65 ℃ of 10min inactivators are behind adding equal-volume phenol/chloroform, mixing, 12,000rpm, 4 ℃ of centrifugal 10min, draw the superiors, the 3M sodium acetate (pH5.2) that adds 1/10 volume, 100% ethanol of 2.5 times of volumes of adding behind the mixing is in 4 ℃ of precipitation 12-24h, 12, the centrifugal 20min of 500rpm, with 70% washing with alcohol precipitation, dry back adds the deionized water of 15 μ L.

4, the amplification of pMAL-P2X plasmid vector and enzyme are cut processing

1) amplification of pMAL-P2X plasmid vector

Carrier pMAL-P2X (available from New Englang Biolabs company) is used CaCl ₂Method transformed into escherichia coli JM109 bacterial strain.The picking positive colony is inoculated in the 5mL LB liquid nutrient medium, after 37 ℃, 225rpm are cultivated 12h, extracts plasmid.

2) Xmn I of pMAL-P2X plasmid vector (available from New Englang Biolabs company) and Hind III (available from New Englang Biolabs company) enzyme are cut processing

The pMAL-P2X plasmid vector of step 1) amplification is carried out enzyme with restriction enzyme Xmn I cut processing, the enzyme system of cutting is: pMAL-P2X plasmid 25 μ L, 10 * damping fluid, 5 μ L, 0.1%BSA 0.5 μ L, Xmn I enzyme (TaKaRa company) 2.5 μ L, deionization H ₂O 17 μ L.37 ℃ of reactions 2.5h, 65 ℃ of 10min inactivators again.Enzyme is cut product carry out the detection of 1.2% agarose gel electrophoresis, reclaim 6700bp purpose band.Again it is carried out enzyme with restriction enzyme Hind III and cut, the enzyme system of cutting is: the carrier pMAL-P2X 25 μ L after Xmn I enzyme is cut processing, 10 * damping fluid, 5 μ L, Hind III enzyme 2.5 μ L, deionization H ₂O 17.5 μ L.37 ℃ of reaction 2.5h, 65 ℃ of 10 minutes inactivators again, add equal-volume phenol/chloroform after, mixing, 12,000rpm, 4 ℃ of centrifugal 10min, draw the superiors, the 3M sodium acetate that adds 1/10 volume, 100% ethanol of 2.5 times of volumes of adding behind the mixing is in 4 ℃ of precipitation 12-24h, 12, the centrifugal 20min of 500rpm, with 70% washing with alcohol precipitation, dry back adds 15 μ L deionized waters.

5, the acquisition of disintegrin prokaryotic expression carrier pMAL-P2X/Kistin

1) connects

Cut the processing intent segment and be connected with the plasmid vector pMAL-P2X that Hind III enzyme is cut processing through Hind III enzyme what step 3 obtained through Xmn I, linked system and reaction conditions are: the carrier pMAL-P2X1 μ L that cuts through enzyme, purpose fragment 5 μ L, deionization H ₂O 11 μ L, 45 ℃ of insulation 5min add following composition: 10 * T after cooled on ice ₄Dna ligase damping fluid (TAKARA company) 2 μ L, T ₄Dna ligase 1 μ L, behind 16 ℃ of reaction 12h, standby in-20 ℃ of preservations.

2) connect the conversion of product, the screening and the evaluation of positive colony

Get the connection product of 5 μ L step 1), it is used CaCl ₂After method transforms 200 μ L escherichia coli jm109 competent cells, be inoculated in the LB resistance agar plate that contains 100 μ g/ml penbritins (Amp), cultivate 12-24h for 37 ℃, random choose list bacterium colony is inoculated in the 5mL LB liquid nutrient medium, cultivates 12-24h for 37 ℃.After cultivating end, the upgrading grain, it is carried out enzyme with restriction enzyme Xmn I and Hind III cut evaluation, (swimming lane 1 is DL-2000 DNA Marker to the result as shown in Figure 4, swimming lane 2 is cut product for Xmn I and Hind III enzyme, swimming lane 3 is the PCR qualification result), cut acquisition 6700bp and the positive clone of 250bp dna fragmentation through enzyme; Do to identify that further the method that PCR identifies is: get 1 μ L bacterium liquid as the PCR reaction template, under the guiding of primer 3 and primer 4, carry out pcr amplification with the PCR method.After reaction finishes, get 5 μ L amplified productions and carry out the detection of 1.2% agarose gel electrophoresis, (swimming lane 1 is DL-2000 DNA Marker to detected result as shown in Figure 4, swimming lane 2 is cut product for Xmn I and Hind III enzyme, swimming lane 3 is the PCR product), pcr amplification conforms to expected results to the dna fragmentation of 250bp; At last acquisition positive colony plasmid is checked order, the result as shown in Figure 5, goal gene in the positive colony plasmid and disintegrin gene remove after the G of 5 ' end the 3rd bit base and the 9th bit base A nonsense mutation are A and C, all the other base sequences are all identical, the prokaryotic expression carrier that shows constructed disintegrin is correct, will contain disintegrin Prokaryotic Expression carrier called after pMAL-P2X/Kistin.

Two, the expression of disintegrin and detection thereof

1, the expression of disintegrin

Disintegrin prokaryotic expression carrier pMAL-P2X/Kistin transformed into escherichia coli TB1 competent cell with the step 1 structure, behind 16 ℃ of cultivation 12h, single bacterium colony of random choose, being inoculated in 3mL contains in the LB liquid nutrient medium of 100 μ g/mL penbritins (Amp), with 37 ℃, the 225rpm shaken overnight is cultivated; Do to increase bacterium 3h after the dilution in 1: 50 with the LB liquid nutrient medium that contains 100 μ g/mL penbritins, when OD600 reached 0.4, adding the IPTG inductor was 0.3mmol/L to final concentration, abduction delivering 6h.

2, the detection of disintegrin

After expressing end, getting thalline carries out the SDS-PAGE electrophoresis and detects, (swimming lane 1 is a pMAL-P2X abduction delivering product to the result as shown in Figure 6, swimming lane 2 is a not abduction delivering of pMAL-P2X, swimming lane 3 is protein Marker, swimming lane 4 is a not induced product of pMAL-P2X/Kistin, swimming lane 5 is a pMAL-P2X/Kistin abduction delivering product), expression has obtained the albumen of 50KD, conform to expected results, with Amylose resin affinity purification system (available from New Englang Biolabs company) method it is carried out purifying, the capable again SDS-PAGE electrophoresis of albumen to purifying detects, (swimming lane 1 is pMAL-P2X abduction delivering product purification result to the result as shown in Figure 7, swimming lane 2 is unpurified pMAL-P2X abduction delivering product, and swimming lane 3 is unpurified pMAL-P2X/Kistin abduction delivering product, and swimming lane 4 is protein Marker, swimming lane 5 is the pMAL-P2X/Kistin abduction delivering product of purifying, shows the disintegrin that obtains higher degree.

Embodiment 3, vitro inhibition platelet aggregation test

Adding final concentration in the healthy human blood platelets (307 hospitals of PLA or blood station, Beijing) is the ADP solution (Sigma company) of 33 μ M/L, the thrombocyte MA is near 100% in the 5min, with it as reference, again concentration is respectively 25,37.5, the disintegrin that the embodiment 2 of 50 μ g/mL obtains adds in the thrombocyte, after hatching 3 minutes under 37 ℃, adding concentration again is the ADP aggregation inducing agent of 33 μ M/L, detect platelet aggregation rate immediately, (ordinate zou is a platelet aggregation rate to the result as shown in Figure 8, X-coordinate is the time after the administration), after adding the agent of ADP aggregation inducing, thrombocyte 5min MA is along with the increase of disintegrin concentration obviously descends, when the disintegrin final concentration was 50 μ g/mL, platelet aggregation almost completely was suppressed; Disintegrin is replaced with MBP albumen (pMAL-P2X expression product) (preparation method is identical with the method that this reorganization disintegrin obtains) in contrast, all the other are identical with aforesaid method, (ordinate zou is a platelet aggregation rate to the result as shown in Figure 9, X-coordinate is the time after the administration), under each MBP protein concentration, thrombocyte 5min MA is all near 100%, above-mentioned experimental result is added up, statistics is as shown in table 1, shows that MBP albumen does not have the active effect of anticoagulant.

Table 1 adds behind various dose fusion rotein and the reference protein hematoblastic five minutes MAs (X ± S)

Group	Five minutes MAs of thrombocyte
					Albumen dosage (μ g/mL)	25	37.5	50
	The disintegrin that ADP+ reference protein (MBP) ADP+ expresses		1.00±0 0.65±0.04	0.97±0.04 0.37±0.04					0.97±0.05 0.01±0.02

Sequence table

<160>2

<210>1

<211>81

<212>PRT

＜213〉Viperidae Crotalinae point kiss abdomen snake (Agkistrodon acutus)

<400>1

Pro Val Ser Gly Asn Glu Leu Leu Glu Ala Gly Glu Glu Cys Asp Cys

1 5 10 15

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

20 25 30

Gln Gly Ala Gln Cys Ala Lys Gly Leu Cys Cys Asp Gln Cys Thr Phe

35 40 45

Val Lys Ala Gly Lys Ile Cys Arg Arg Ala Arg Gly Asp Asn Pro Asp

50 55 60

Asp Arg Cys Thr Gly Gln Ser Gly Asp Cys Pro Arg Tyr His Ser His

65 70 75 80

Ala

<210>2

<211>243

<212>DNA

＜213〉Viperidae Crotalinae abdomen snake belongs to point kiss abdomen snake (Deinagkistrodon acutus)

<400>2

ccagtttctg gaaatgaact tttggaggca ggagaagagt gtgactgtgg ctctcctgaa 60

aatccgtgct gcgatgctgc aacctgtaaa ctgaagcaag gagcacagtg tgcaaaagga 120

ctgtgttgtg accagtgcac atttgtaaaa gcaggaaaaa tatgccggag agcaaggggt 180

gataatccgg atgatcgctg cactggccaa tctggtgact gtcccagata tcactcccat 240

gcc 243

Claims (10)

1, a kind of disintegrin, its amino acid residue sequence are as the SEQ ID № in the sequence table: shown in 1.

2, the gene of the described disintegrin of coding claim 1.

3, gene according to claim 2 is characterized in that: its dna sequence dna is as SEQ ID № in the sequence table: shown in 2.

4, contain claim 2 or 3 described expression carrier.

5, the transgenic cell line that contains claim 2 or 3 described genes.

6, the host bacterium that contains claim 2 or 3 described genes.

7, a kind of method of expressing the described disintegrin of claim 1 is that the recombinant expression vector that will contain described disintegrin gene imports host cell, expresses obtaining disintegrin.

8, method according to claim 7 is characterized in that: the recombinant expression vector of described expression disintegrin is pMAL-P2X/Kistin.

9, according to claim 7 or 8 described methods, it is characterized in that: described host needs to add IPTG and carry out abduction delivering when the colibacillus, add IPTG concentration be 0.1-0.5mM.

10, a kind of medicine of anticoagulant, its activeconstituents are the described disintegrin of claim 1.

Images