CN1324132C

CN1324132C - Snake venom thrombin-like enzyme and its encoding gene and application

Info

Publication number: CN1324132C
Application number: CNB2004100840551A
Authority: CN
Inventors: 顾军; 陈师勇
Original assignee: Peking University
Current assignee: Peking University
Priority date: 2004-10-19
Filing date: 2004-10-19
Publication date: 2007-07-04
Anticipated expiration: 2024-10-19
Also published as: CN1763188A

Abstract

The present invention discloses a snake venom thrombin-like enzyme, an encoding gene thereof and the application thereof, which aims to provide a snake venom thrombin-like enzyme, a maturation protein of the snake venom thrombin-like enzyme, and encoding genes thereof. The present invention also provides medicine with the functions of thrombolysis and thrombosis prevention, wherein active ingredients of the medicine comprises the venom thrombin-like enzyme, the maturation protein of the snake venom thrombin-like enzyme. The snake venom thrombin-like enzyme provided by the present invention has sequence 1 disclosed in the sequence table, or is a protein obtained by the substitution, deletion and/or addition of 1 to 10 amino acid residues in sequence 1 and having the functions of thrombolysis and thrombosis prevention. The recombinant expression maturation protein of the snake venom thrombin-like enzyme has amide enzymolysis activity and arginine esterase activity of the snake venom thrombin-like enzyme. In addition, the maturation protein can specifically degrade the beta chain of fibrinogen and thrombus caused by fibrinogen, and has the efficiency of viscosity reduction and thrombolysis.

Description

A kind of snake venom thrombin-like enzyme and encoding gene thereof and application

Technical field

The present invention relates to snake venom thrombin-like enzyme and encoding gene thereof and application, particularly relate to snake venom thrombin-like enzyme, snake venom thrombin-like enzyme maturation protein and their encoding gene and with this snake venom thrombin-like enzyme or snake venom thrombin-like enzyme maturation protein be having thrombolysis and preventing the medicine of thrombosis effect of activeconstituents.

Background technology

Thrombosis is operating common complication, also is that the important factor of obstruction and pathogenic, the cause of death of multiple cardiovascular and cerebrovascular diseases take place modern intervention property postangioplasty again.The physiological thrombosis is a kind of means of hematostatic, and the pathologic thrombosis then can cause the internal organs generation dysfunction of being correlated with.At present, the thrombotic diseases of the heart, brain position has become one of the highest disease of disability rate and lethality rate, and serious threat is to human beings'health.According to statistics, the whole world has the thromboembolic states patient about 1,500 ten thousand, and the potential market of required thrombolytics can reach 2,000,000,000 dollars, thereby a lot of in the world country all is devoted to this respect drug research and exploitation.

Thrombus is the grumeleuse of a kind of nonuniformity structure of forming in the blood circulation process of blood component.Thrombus is made of thrombocyte, white corpuscle, red corpuscle and scleroproein, and the shared ratio of various compositions is because of the different differences to some extent with the blood flow condition of vascular site.Thrombosis is a complexity and physiology or pathologic process that concern is extremely wide, mainly shows as blood vessel endothelium injury, blood ingredient change and abnormal hemodynamics.Impaired and the subcutaneous collagenous tissue of endotheliocyte exposes and can cause hematoblastic adhesion and gathering.For example, von Wilebrand factor one end combines with interior collagen, and an end combines with platelet membrane platelet surface glycoprotein ibalpha (GP Ib), thereby hematoblastic adhesion takes place, and adherent thrombocyte is activated immediately, and causes hematoblastic gathering, promptly at Ca ²⁺Auxiliary down and the membranin GP IIb-IIa on two platelet membranes combine; Release reaction takes place and disengages its granular contents in activatory thrombocyte fast, as serotonin, ADP, Ca ²⁺, B-thromboglobulin, arachidonic acid etc., these releasers further promote hematoblastic adhesion again, assemble and activate, and the platelet aggregation thing is constantly increased.Simultaneously, the breakage of endotheliocyte activates a series of thrombin, and the further activation of the primosome intravascular coagulation factor, Fibrinogen generates scleroproein polypeptide A and scleroproein polypeptide B under thrombin action, two kinds of scleroproein polypeptide change into fibrin monomer (α, beta, gamma) ₂Back spontaneous polymerization becomes unsettled fibrin polymer, and unsettled fibrin polymer is at Ca ²⁺, the XIII factor effect under be cross-linked into stable fibrin polymer, the main matrix of Here it is thrombus.The scleroproein support embeds platelet thrombus, finally forms thrombocyte one fibrin clot.

In fact, thrombus is not nonvolatil structure in vivo, and it is among fibrinous continuous deposition and the dissolved running balance.Therefore, remove fibrinous precursor, can effectively prevent the former and control the development of thrombus, and strengthen the normal fibrinolytic effect of human body.

Snake venom thrombin-like enzyme has stronger thrombolysis effect in vivo, and it has the arginine ester enzymic activity, can directly act on Fibrinogen, and hydrolysis discharges fibrinopeptide 2, causes fibrinous monomer head and the tail polymerization and solidifies, and is called as Thrombin-like enzyme.But it does not activate factor I in vivo, it is crosslinked that the fibrin clot that is generated by its hydrolysis does not produce side chain, digestion to plasmin is extremely sensitive, easily removed by natural reticuloendothelial system or normal fibrinolytic effect, therefore cause that fibrinogen concentration significantly descends in the endochylema, the effect of fibre, anti-freezing falls in performance.Clinically, snake venom thrombin-like enzyme has become the active drug of control thrombotic disease.

Fibrinogen is one of important factor of decision blood viscosity, and Thrombin-like enzyme has lowered fibrinogen level in the blood plasma, thereby has reduced whole blood viscosity and plasma viscosity, has strengthened blood flow rate.Simultaneously, opposite with zymoplasm, not induced platelet cohesion and discharging of Thrombin-like enzyme, they be rich in the formed grumeleuse of hematoblastic blood plasma and do not shrink, make body can keep normal hemostatic function.

External existing Ancrod and Batroxobin snake venom antithrombotics, domestic then have dispel fine enzyme, northeast agkistrodon halys ussuriensis embolism-resisting enzyme (Ahylysantinfarctase), Jiangsu and Zhejiang Provinces Ahylysantinfarctase etc. of agkistrodon acutus venom to be used for clinical.Though the title difference of said medicine, main component is consistent, is Thrombin-like enzyme.These antithrombotics have been used for clinical treatment cerebral thrombosis, vasculitis, treatment coronary heart disease, myocardial infarction because of having unique character such as the fibre of dispelling, viscosity reduction, thrombolysis, depolymerization, human are also arranged in treatment pain caused by cancer syndromes.Think that at present embolism-resisting enzyme is that treatment vasculitis, deep phlebitis, venous thrombosis form ideal active drug.Shi Yong indication has clinically: dvt forms (complication or not concurrent pulmonary infarction), the central vein of retina thrombosis, myocardial infarction, embolism from artificial valve, priapism, sicklemia, rheumatoid arthritis, organ transplantation is repelled, the prevention of thrombosis recurrence after surgery or fibrolysis treatment, the anti-freezing of vein surgery and thrombotic prevention, extracorporeal blood dialysis, keeping of artificial heart-lung life, peripheral arterial occlusion and coronary stricture etc.Some results prove that they are the therapeutical agents that get a good chance of.

Snake venom thrombin-like enzyme mainly is present in the snake venom of the Crotalinae snake in the Viperidae, and some snake venom in the Viperinae also contains Thrombin-like enzyme.Yet, in Hydrophiidae, also there is not report to find Thrombin-like enzyme, and the snake class Thrombin-like enzyme of clinical application is for purifying from snake venom now, and output is limited, and impure snake class foreign protein that wherein contains or enzyme produce one of reason of big toxic side effect just.

Summary of the invention

The purpose of this invention is to provide a kind of snake venom thrombin-like enzyme, snake venom thrombin-like enzyme maturation protein and their encoding gene.

Snake venom thrombin-like enzyme provided by the present invention, name is called TLE, is 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 thrombolysis and prevent the protein of thrombosis effect.

Wherein, the SEQ ID № in the sequence table: 1 is made up of 265 amino-acid residues.

This snake venom thrombin-like enzyme maturation protein is the protein with one of following amino acid residue sequences:

1) the SEQ ID № in the sequence table: 3;

2) with SEQ ID № in the sequence table: 3 amino acid residue sequence is through replacement, disappearance or the interpolation of one to ten amino-acid residue and have thrombolysis and prevent the protein of thrombosis effect.

SEQ ID № in the sequence table: 3 are made up of 232 amino-acid residues, are SEQ ID №: 1 from aminoterminal 34-265 amino acids residue sequence.

The encoding gene of above-mentioned snake venom thrombin-like enzyme (TLE) also belongs to protection scope of the present invention.It can have one of following nucleotide sequence:

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

2) SEQ ID № in the code sequence tabulation: the polynucleotide of 1 protein sequence;

3) with sequence table in SEQ ID №: 2 dna sequence dnas that limit have 95% above homology, and coding has thrombolysis and prevents the protein DNA sequence of thrombosis function;

4) 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.

Wherein, the SEQ ID № in the sequence table: 2 by 827 based compositions, and its encoding sequence is that coding has SEQ ID № in the sequence table: the protein of 1 amino acid residue sequence from 5 ' end the 1st to the 795th bit base.

The encoding gene of above-mentioned snake venom thrombin-like enzyme maturation protein also belongs to protection scope of the present invention.It can have one of following nucleotide sequence:

1) SEQ ID № in the sequence table: 4 dna sequence dna;

2) SEQ ID № in the code sequence tabulation: the polynucleotide of 3 protein sequences;

3) with sequence table in SEQ ID №: 4 dna sequence dnas that limit have 95% above homology, and coding has thrombolysis and prevents the protein DNA sequence of thrombosis function;

4) under the rigorous condition of height can with SEQ ID № in the sequence table: the nucleotide sequence of the 4 dna sequence dnas hybridization that limit.

Wherein, the SEQ ID № in the sequence table: 4 by 728 based compositions, and its encoding sequence is that coding has SEQ ID № in the sequence table: the protein of 3 amino acid residue sequence from 5 ' end the 1st to the 696th bit base.

The rigorous condition of described height for hybridization back with contain 0.1 * SSPE (or 0.1 * SSC), the solution of 0.1%SDS washes film under 65 ℃.

The expression vector, clone and the engineering bacteria that contain the encoding gene of the encoding gene of snake venom thrombin-like enzyme of the present invention and maturation protein thereof all belong to protection scope of the present invention.

Increase arbitrary segmental primer in the encoding gene of the encoding gene of above-mentioned snake venom thrombin-like enzyme and maturation protein thereof to also within protection scope of the present invention.

Another object of the present invention provides a kind of method of expressing above-mentioned snake venom thrombin-like enzyme and maturation protein thereof.

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

The method of the above-mentioned snake venom thrombin-like enzyme maturation protein of expression provided by the present invention is that the recombinant expression vector that will contain above-mentioned snake venom thrombin-like enzyme maturation protein encoding gene imports host cell, expresses obtaining the snake venom thrombin-like enzyme maturation protein.

Described host can be yeast, intestinal bacteria, mammalian cell, insect cell or Bacillus subtilus etc., is preferably yeast.

Described yeast is preferably pichia pastoris (Pichia pastoris).Wherein, described pichia pastoris is preferably pichia pastoris GS115, KM7 or SMD1168.

The carrier that sets out that is used for making up described recombinant yeast expression vector can be the expression vector at above-mentioned host's expression alien gene, as pPIC9K, pPIC9, pPIC3, pHIL-D1, pA0804, pA0815 or the pPSC3K that can express in pichia pastoris (Pichia pastoris).

When described host is pichia pastoris (Pichia pastoris), need to carry out abduction delivering with methyl alcohol, the final concentration of methyl alcohol can be 1%.

Above-mentioned recombinant expression vector all can make up according to ordinary method.

Cultivation contains the substratum and the culture condition of host cell of the encoding gene of snake venom thrombin-like enzyme encoding gene of the present invention or above-mentioned snake venom thrombin-like enzyme maturation protein, all can be substratum and the culture condition of cultivating the host that sets out.

The present invention also provides a kind of medicine that has thrombolysis and prevent the thrombosis effect.

Medicine provided by the present invention, its activeconstituents are above-mentioned snake venom thrombin-like enzyme or snake venom thrombin-like enzyme maturation protein.

When needing, in said medicine, 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.

Snake venom thrombin-like enzyme provided by the present invention and maturation protein thereof can utilize genetic engineering means to express, obtain a large amount of purity height, the zymoprotein that biologic activity is high, this zymoprotein has the acid amides enzymolysis activity that snake venom thrombin-like enzyme has, the arginine ester enzymic activity, the former β chain of fibrin degradation specifically, the thrombus that causes of biodegradable fiber proteinogen also, has viscosity reduction, the effect of thrombolysis, can be used for preparing various illness such as the coronary heart disease that cause because of thrombus of clinical treatment, myocardial infarction, vasculitis, the medicine of venous thrombosis, the present invention has broad application prospects at medical field.

Description of drawings

Fig. 1 is the SDS-PAGE electrophoretogram of the nutrient solution supernatant of recombinant plasmid pPIC9k-TLE transformed bacteria

Fig. 2 is the SDS-PAGE electrophoretogram of the snake venom thrombin-like enzyme maturation protein expression product of purifying

Fig. 3 is the former active detected result of the fibrin degradation of snake venom thrombin-like enzyme maturation protein

Embodiment

Method among the following embodiment is ordinary method if no special instructions.

The acquisition of embodiment 1, TLE and snake venom thrombin-like enzyme maturation protein are expressed

1, the acquisition of TLE

May further comprise the steps:

1) isolates the poison gland tissue from the Lapemis (Lapemis curtus) and the steel gray sea snake (Hydrophiscaerulescens) in the marine site, NORTH CHINA gulf of catching, extract total RNA of two kinds of sea snake venom;

2) from total RNA of two kinds of sea snake venom, extract mRNA respectively, and be the synthetic cDNA sequence separately of template reverse transcription with mRNA;

3) two kinds of cDNA of synthetic are carried out ligation with T7 phage vector (Novagen company) respectively, by the external sea snake phage-displayed polypeptides storehouse that is packaged to be;

4) adopt the ELISA method, the phage display peptide library that step 3) makes up is screened for target molecule with Fibrinogen (fibrinogen, sigma company), and therefrom filtered out the phage clone that specific combination power is arranged with target molecule.The target phage that screens is carried out the cDNA sequence (sequence 1 in the sequence table that gene sequencing has obtained this gene, derive from Lapemis (Lapemis curtus)), analyze and find, the land snake thrombase-like gene sequence of having announced among the nucleotide sequence of this gene and the Genebank is (as Gloydius halys, Genebank number AY225505) have 81% similarity, their amino acids coding residue sequence have 59% similarity.The gene order that obtains is analyzed the back find that the 1-18 amino acids residue sequence of this zymoprotein sequence is a signal peptide sequence, is maturation protein since the 34th; Find that simultaneously this enzyme has avtive spot one Histidine avtive spot (70-75), Serine avtive spot (206-217) the aspartic acid avtive spot (118) of typical Thrombin-like enzyme.According to this gene and a plurality of affirmation that has had high similarity and this each enzyme active sites of gene of Thrombin-like enzyme sequence, obtained a snake venom thrombin-like enzyme, the cDNA sequence of this enzyme is for finding in sea snake first.

2, the structure that contains the recombinant expression vector of snake venom thrombin-like enzyme maturation protein coding gene sequence

The DNA of the target phage that obtains with step 1 is a template, primer 1:5 '- CTC

GAGAAAAGAATCATTGGAGGTTTTGAATG-3 ' (line part base is the XhoI recognition site) and primer 2:

3 '- GAATTCCarry out the PCR reaction, the cDNA sequence of amplification snake venom thrombin-like enzyme maturation protein under the guiding of CTAGGGGCAGATCACATTTG-5 ' (line part base is the EcoRI recognition site).In PCR reaction, the temperature changing process of PCR reaction is: be warming up to 94 ℃ earlier, kept 5 minutes, follow by following temperature variation program loop 28 times: be warming up to 94 ℃, kept 50 seconds, be cooled to 57 ℃, kept 1 minute, be warming up to 72 ℃, kept 1 minute; Kept the end amplified reaction 10 minutes in 72 ℃ at last.The PCR product two ends that obtained have XhoI and EcoRI restriction enzyme enzyme recognition site, with XhoI and EcoRI restricted type restriction endonuclease the PCR product is carried out the double digestion reaction after, use T ₄Dna ligase with its with cut with same enzyme enzyme after plasmid vector pPIC9k (Invitrogen company) be connected, obtain recombinant expression vector, called after pPIC9k-TLE.PPIC9k-TLE is checked order, prove and merge that to connect into the dna sequence dna of plasmid pPIC9k identical with sequence 4 in the sequence table, the recombinant expression vector pPIC9k-TLE that contains snake venom thrombin-like enzyme maturation protein coding gene sequence of structure is correct.This recombinant plasmid that builds will be expressed the 34th beginning of sequence 1 from sequence table until the snake venom thrombin-like enzyme maturation protein of sequence end (being sequence 3).

3, the expression of snake venom thrombin-like enzyme maturation protein

After recombinant expression vector pPIC9k-TLE made it linearizing with the SalI digestion with restriction enzyme, adopt the electric shock mode, linearizing carrier pPIC9k-TLE is imported among the pichia yeast bacterium GS115 (Invitrogen company), press the operational manual operation of Invitrogen company, through selective medium (1% yeast extract, 2% peptone, 2% glucose, G418 4mg/ml) cultivates screening His ⁺High expression level bacterial strain with anti-G418 (4mg/ml).Picking, is cultivated after 12-24 hour, is changed the OD that continues to be cultured to bacterium liquid in the 250mL BMGY substratum over to for 30 ℃ in 5ml YPD liquid nutrient medium at the single colony inoculation that grows on the selective medium ₆₀₀=2-3, centrifugal collection thalline is diluted to OD with no carbon source BMMY substratum with it again ₆₀₀=1 back adds 1% methyl alcohol and carries out inducing culture, and adding methyl alcohol to final concentration every 24 hours between incubation period is 1%, is cultured to 96 hours and can stops to cultivate.Centrifugal collection supernatant liquor, get 15 μ L supernatant liquors, detect with the SDS-PAGE electrophoresis, (swimming lane 1 is Marker (116KD to the result as shown in Figure 1,66KD, 45KD, 35KD, 25KD, 18KD, 14KD), swimming lane 2 is empty plasmid pPIC9k, and swimming lane 3 is the nutrient solution supernatant of recombinant plasmid pPIC9k-TLE transformed bacteria, and arrow shows the purpose band) show that the molecular weight of the target protein that recombinant bacterial strain is expressed is about 26KD under methanol induction, conform to the molecular weight of the maturation protein of snake venom thrombin-like enzyme, obtained correct expression product.

4, the purifying of snake venom thrombin-like enzyme maturation protein expression product

With the culture supernatant of step 3 through ammonium sulfate precipitation, HPCL cation seperation column (DEAD ion column, waters company.Elutriant: pH8.0 contains the Tris-HCl damping fluid of the 0.05M of NaCl 0-75%) chromatography and molecular sieve gel post (Sephacryl S100, Pharmacia company, the Tris-HCl damping fluid of the 0.05M of elutriant: pH8.0) chromatography carries out purifying, obtain highly purified snake venom thrombin-like enzyme maturation protein, (swimming lane 1 is the snake venom thrombin-like enzyme maturation protein expression product of purifying to SDS-PAGE electrophoresis detection result as shown in Figure 2, swimming lane 2 is a Marker (116KD (not shown), 66KD, 45KD, 35KD, 25KD, 18KD, 14KD), show and obtained electrophoretically pure snake venom thrombin-like enzyme maturation protein.

The enzymic activity of embodiment 2, snake venom thrombin-like enzyme maturation protein detects

1, acid amides enzymolysis determination of activity

At 37 ℃, add chromogenic substrate (N-α-p-tosyl-Gly-Pro-Arg-p-nitronilide in the 50mM phosphate buffered saline buffer (pH8.0), Sigma company) be that snake venom thrombin-like enzyme maturation protein to the concentration that 2mM and embodiment 1 obtain is 1mg/mL to concentration, behind the reaction 10min, by detecting OD ₄₀₅Variation measure the acid amides enzymolysis activity of snake venom thrombin-like enzyme maturation protein, experimental result shows snake venom thrombin-like enzyme maturation protein this chromogenic substrate of can degrading, has acid amides enzymolysis activity (1062.5 enzyme activity U/min/mg enzyme), illustrate that this snake venom thrombin-like enzyme maturation protein is similar with the Thrombin-like enzyme of other land snake, all has the acid amides enzymolysis activity, the amido linkage in can the degrade proteins polypeptide.

2, arginine ester enzyme assay

At 37 ℃, add substrate tosic acid-L-arginine methyl esters hydrochloride (TAME in the 50mM phosphate buffered saline buffer (pH8.0), Sigma company) be that snake venom thrombin-like enzyme maturation protein to the concentration that 1mM and embodiment 1 obtain is 1mg/mL to concentration, behind the reaction 5min, by detecting OD ₂₄₇Variation measure the arginine ester enzymic activity of snake venom thrombin-like enzyme maturation protein, experimental result shows snake venom thrombin-like enzyme maturation protein that embodiment 1 obtains this chromogenic substrate of can degrading, has arginine ester enzymic activity (1130 enzyme activity U/min/mg enzyme), illustrate that this snake venom thrombin-like enzyme maturation protein is similar with the Thrombin-like enzyme of other land snake, all has the arginine ester enzymic activity, the specific arginine ester bond in can the degrade proteins polypeptide.

3, the scleroproein congealing activity detects

Getting concentration is the snake venom thrombin-like enzyme maturation protein 10 μ l of 5mg/L, joining 100 μ l contains in the fibrinogenic 40mM Tris-HCl of the 4mg/ml damping fluid (pH8.0), 37 ℃ are carried out water-bath, at 0min, 5min, 30min and 1h take a sample respectively, sample is carried out the SDS-PAGE electrophoresis to observe the fibrinous phenomenon of condensing, the result is the (sample that swimming lane 1 is got for water-bath 1h as shown in Figure 3, the sample that swimming lane 2 is got for water-bath 30min, the sample that swimming lane 3 is got for water-bath 5min, the sample that swimming lane 4 is got for water-bath 0min, swimming lane 5 is Marker, show increase along with the reaction times, this snake venom thrombin-like enzyme maturation protein is the former β chain of fibrin degradation gradually, illustrate that this Thrombin-like enzyme maturation protein is similar with the Thrombin-like enzyme of other land snake, can be used as the thrombolytic drug lead compound, be used to the thrombi of degrading and forming, thereby reach the effect of thrombolysis by Fibrinogen or Fibrinogen.

Sequence table

<160>4

<210>1

<211>265

<212>PRT

＜213〉Lapemis (Lapemis curtus)

<400>1

Met?Pro?Leu?Ile?Arg?Val?Leu?Ala?Ser?Leu?Leu?Ile?Leu?Gln?Leu?Ser

1 5 10 15

Tyr?Gly?Lys?Ser?Leu?Asp?Asn?Gly?Ala?Lys?Ala?Ile?Thr?Ser?Leu?Asp

20 25 30

Arg?Ile?Ile?Gly?Gly?Phe?Glu?Cys?Asn?Pro?Ser?Glu?His?Arg?Ser?Leu

35 40 45

Val?Tyr?Leu?Tyr?Asn?Ser?Ala?Gly?Phe?Phe?Cys?Ser?Gly?Thr?Leu?Leu

50 55 60

Asn?His?Glu?Trp?Val?Leu?Thr?Ala?Ala?His?Cys?Asn?Arg?Glu?Asp?Ile

65 70 75 80

Gln?Ile?Arg?Leu?Gly?Val?His?Asn?Val?His?Val?His?Tyr?Glu?Asp?Glu

85 90 95

Gln?Ile?Arg?Val?Pro?Lys?Glu?Lys?Leu?Cys?Cys?Leu?Ser?Thr?Asn?Asn

100 105 110

Cys?Thr?Gln?Phe?Ser?Gln?Asp?Ile?Met?Leu?Ile?Arg?Leu?Asn?Ser?Pro

115 120 125

Val?Asn?Tyr?Ser?Glu?His?Ile?Ala?Pro?Leu?Ser?Leu?Pro?Ser?Asn?Pro

130 135 140

Pro?Ser?Met?Gly?Ser?Val?Cys?Cys?Val?Met?Gly?Trp?Gly?Thr?Ile?Thr

145 150 155 160

Ser?Pro?Glu?Val?Thr?Tyr?Pro?Glu?Val?Pro?His?Cys?Val?Asp?Ile?Asn

165 170 175

Ile?Leu?His?Ile?Pro?Val?Cys?Gln?Ala?Ala?Tyr?Pro?Thr?Met?Ser?Gly

180 185 190

Lys?Asn?Ile?Leu?Cys?Ala?Gly?Ile?Leu?Glu?Gly?Gly?Lys?Asp?Ser?Cys

195 200 205

Lys?Gly?Asp?Ser?Gly?Gly?Pro?Leu?Ile?Cys?Asn?Gly?Gln?Ile?Gln?Gly

210 215 220

Ile?Val?Ser?Trp?Gly?Arg?Phe?Pro?Cys?Ala?Gln?Phe?Leu?Glu?Pro?Gly

225 230 235 240

Ile?Tyr?Thr?Lys?Val?Phe?Asp?Tyr?Lys?Asp?Trp?Ile?Glu?Gly?Ile?Ile

245 250 255

Ala?Gly?Asn?Ser?Asn?Val?Ile?Cys?Pro

260 265

<210>2

<211>827

<212>DNA

＜213〉Lapemis (Lapemis curtus)

<400>2

atgcctctga?tcagagtgct?agcaagcctt?ctgatactac?agctttctta?cggtaagagt 60

ctggacaatg?gagcaaaagc?aataacatct?cttgatcgga?tcattggagg?ttttgaatgt 120

aacccaagtg?aacatcgttc?ccttgtatac?ttgtataact?ctgcagggtt?tttctgttca 180

gggaccttgc?tcaaccatga?atgggtgctc?accgctgcac?actgcaacag?ggaagatatc 240

cagataaggc?ttggtgtgca?taacgtacat?gtacactatg?aggatgagca?gataagggtc 300

ccgaaggaga?agttgtgttg?tctcagtacc?aataactgta?cccaatttag?ccaagatatc 360

atgttgatca?ggctgaacag?tcctgttaac?tatagtgaac?acatcgcacc?tcttagtttg 420

ccttccaacc?ctcccagtat?gggctcagtt?tgctgtgtta?tgggctgggg?cacaatcaca 480

tctcctgaag?tgacttatcc?tgaagtccct?cattgtgttg?acattaacat?actccatatt 540

ccggtgtgtc?aagcagctta?cccaacaatg?tcagggaaga?acatattgtg?tgcaggtatc 600

ctggaaggag?gcaaagattc?atgtaagggc?gattctgggg?gacccctcat?ctgtaatgga 660

caaatccagg?gcattgtatc?ttgggggcgc?tttccttgtg?cccaatttct?tgaacctggc 720

atctacacca?aggtcttcga?ttataaggac?tggattgagg?gtattattgc?aggaaattca 780

aatgtgatct?gcccctagtg?acaatttttg?aaaaagataa?aaaaaaa 827

<210>3

<211>232

<212>PRT

＜213〉Lapemis (Lapemis curtus)

<400>3

Ile?Ile?Gly?Gly?Phe?Glu?Cys?Asn?Pro?Ser?Glu?Hi?s?Arg?Ser?Leu?Val

1 5 10 15

Tyr?Leu?Tyr?Asn?Ser?Ala?Gly?Phe?Phe?Cys?Ser?Gly?Thr?Leu?Leu?Asn

20 25 30

His?Glu?Trp?Val?Leu?Thr?Ala?Ala?His?Cys?Asn?Arg?Glu?Asp?Ile?Gln

35 40 45

Ile?Arg?Leu?Gly?Val?His?Asn?Val?His?Val?His?Tyr?Glu?Asp?Glu?Gln

50 55 60

Ile?Arg?Val?Pro?Lys?Glu?Lys?Leu?Cys?Cys?Leu?Ser?Thr?Asn?Asn?Cys

65 70 75 80

Thr?Gln?Phe?Ser?Gln?Asp?Ile?Met?Leu?Ile?Arg?Leu?Asn?Ser?Pro?Val

85 90 95

Asn?Tyr?Ser?Glu?His?Ile?Ala?Pro?Leu?Ser?Leu?Pro?Ser?Asn?Pro?Pro

100 105 110

Ser?Met?Gly?Ser?Val?Cys?Cys?Val?Met?Gly?Trp?Gly?Thr?Ile?Thr?Ser

115 120 125

Pro?Glu?Val?Thr?Tyr?Pro?Glu?Val?Pro?His?Cys?Val?Asp?Ile?Asn?Ile

130 135 140

Leu?HisIle?Pro?Val?Cys?Gln?Ala?Ala?Tyr?Pro?Thr?Met?Ser?Gly?Lys

145 150 155 160

Asn?Ile?Leu?Cys?Ala?Gly?Ile?Leu?Glu?Gly?Gly?Lys?Asp?Ser?Cys?Lys

165 170 175

Gly?Asp?Ser?Gly?Gly?Pro?Leu?Ile?Cys?Asn?Gly?Gln?Ile?Gln?Gly?Ile

180 185 190

Val?Ser?Trp?Gly?Arg?Phe?Pro?Cys?Ala?Gln?Phe?Leu?Glu?Pro?Gly?Ile

195 200 205

Tyr?Thr?Lys?Val?Phe?Asp?Tyr?Lys?Asp?Trp?Ile?Glu?Gly?Ile?Ile?Ala

210 215 220

Gly?Asn?Ser?Asn?Val?Ile?Cys?Pro

225 230

<210>4

<211>728

<212>DNA

＜213〉Lapemis (Lapemis curtus)

<400>4

atcattggag?gttttgaatg?taacccaagt?gaacatcgtt?cccttgtata?cttgtataac 60

tctgcagggt?ttttctgttc?agggaccttg?ctcaaccatg?aatgggtgct?caccgctgca 120

cactgcaaca?gggaagatat?ccagataagg?cttggtgtgc?ataacgtaca?tgtacactat 180

gaggatgagc?agataagggt?cccgaaggag?aagttgtgtt?gtctcagtac?caataactgt 240

acccaattta?gccaagatat?catgttgatc?aggctgaaca?gtcctgttaa?ctatagtgaa 300

cacatcgcac?ctcttagttt?gccttccaac?cctcccagta?tgggctcagt?ttgctgtgtt 360

atgggctggg?gcacaatcac?atctcctgaa?gtgacttatc?ctgaagtccc?tcattgtgtt 420

gacattaaca?tactccatat?tccggtgtgt?caagcagctt?acccaacaat?gtcagggaag 480

aacatattgt?gtgcaggtat?cctggaagga?ggcaaagatt?catgtaaggg?cgattctggg 540

ggacccctca?tctgtaatgg?acaaatccag?ggcattgtat?cttgggggcg?ctttccttgt 600

gcccaatttc?ttgaacctgg?catctacacc?aaggtcttcg?attataagga?ctggattgag 660

ggtattattg?caggaaattc?aaatgtgatc?tgcccctagt?gacaattttt?gaaaaagata 720

aaaaaaaa 728

Claims

1, a kind of snake venom thrombin-like enzyme, its amino acid residue sequence is shown in SEQ ID NO:1.

2, snake venom thrombin-like enzyme maturation protein, its amino acid residue sequence is shown in SEQ ID NO:3.

3, the encoding gene of the described snake venom thrombin-like enzyme of claim 1.

4, gene according to claim 3 is characterized in that: the base sequence of the encoding gene of described snake venom thrombin-like enzyme is shown in SEQ ID NO:2.

5, the encoding gene of the described snake venom thrombin-like enzyme maturation protein of claim 2.

6, gene according to claim 5 is characterized in that: the base sequence of described snake venom thrombin-like enzyme maturation protein encoding gene is shown in SEQ ID NO:4.

7, the expression vector that contains claim 3 or 4 described snake venom thrombin-like enzyme encoding genes.

8, the clone that contains claim 3 or 4 described snake venom thrombin-like enzyme encoding genes.

9, the engineering bacteria that contains claim 3 or 4 described snake venom thrombin-like enzyme encoding genes.

10, the expression vector that contains claim 5 or 6 described snake venom thrombin-like enzyme maturation protein encoding genes.

11, the clone that contains claim 5 or 6 described snake venom thrombin-like enzyme maturation protein encoding genes.

12, the engineering bacteria that contains claim 5 or 6 described snake venom thrombin-like enzyme maturation protein encoding genes.

13, a kind of method of expressing the described snake venom thrombin-like enzyme of claim 1 is that the recombinant expression vector that will contain claim 3 or 4 described snake venom thrombin-like enzyme encoding genes imports host cell, expresses obtaining snake venom thrombin-like enzyme.

14, method according to claim 13 is characterized in that: described host is yeast, intestinal bacteria, mammalian cell, insect cell or Bacillus subtilus; The carrier that sets out that is used to make up described recombinant expression vector is pPIC9K, pPIC9, pPIC3, pHIL-D1, pA0804, pA0815 or pPSC3K.

15, method according to claim 14 is characterized in that: described host is a yeast.

16, method according to claim 15 is characterized in that: described host is a pichia pastoris.

17, method according to claim 16 is characterized in that: described host is pichia pastoris GS115, KM7 or SMD1168.

18, a kind of method of expressing the described snake venom thrombin-like enzyme maturation protein of claim 2, be that the recombinant expression vector that will contain claim 5 or 6 described snake venom thrombin-like enzyme maturation protein encoding genes imports host cell, express obtaining the snake venom thrombin-like enzyme maturation protein.

19, method according to claim 18 is characterized in that: described host is yeast, intestinal bacteria, mammalian cell, insect cell or Bacillus subtilus; The carrier that sets out that is used to make up described recombinant expression vector is pPIC9K, pPIC9, pPIC3, pHIL-D1, pA0804, pA0815 or pPSC3K.

20, method according to claim 19 is characterized in that: described host is a yeast.

21, method according to claim 20 is characterized in that: described host is a pichia pastoris.

22, method according to claim 21 is characterized in that: described host is pichia pastoris GS115, KM7 or SMD1168.

23, a kind of medicine that has thrombolysis and prevent the thrombosis effect, its activeconstituents are described snake venom thrombin-like enzyme of claim 1 and/or the described snake venom thrombin-like enzyme maturation protein of claim 2.