CN1060806C - Gene sequence of fibrin ferment of Agkistrodon acutus snakes - Google Patents
Gene sequence of fibrin ferment of Agkistrodon acutus snakes Download PDFInfo
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- CN1060806C CN1060806C CN97106706A CN97106706A CN1060806C CN 1060806 C CN1060806 C CN 1060806C CN 97106706 A CN97106706 A CN 97106706A CN 97106706 A CN97106706 A CN 97106706A CN 1060806 C CN1060806 C CN 1060806C
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Abstract
Batroxobin is obtained from Agkistrodon acutus snakes by separation and purification, the sequence of 15 amino acids at the N-terminal of the batroxobin is measured, and an upstream primer is designed on the basis of the sequence, wherein the upstream primer contains a translation initiation codon ATG, which is favorable for the expression of the gene. Subsequently, according to a degenerate downstream primer designed for the region which has high homology with a non-coding region of batroxobin 3' of venom from other Ankistrodon acutus snakes, total RNA is extracted from venom gland, and the gene is amplified by PCR. Full-sequence measurement after clone shows that the cDNA of the mature protein can code 699 nucleotides, namely 233 amino acids. The successful clone of the gene and the gene sequence measurement create favorable conditions for the development of gene engineering products.
Description
The invention belongs to the echidnotoxin field, particularly about the gene order of sTLE.
Agkistrodon acutus (Agkistrodon acutus, being commonly called as Agkistrodon) snake venom thrombin-like enzyme (Thrombin-like enzyme) has and the similar effect of blood plasma zymoplasm, it can directly make the Fibrinogen in the blood change scleroproein into, and generation is condensed, Fibrinogen is that molecular weight is 340,000 protein, be made up of three polypeptide chains that are referred to as A α, B β and γ respectively, these three polypeptide chains are connected with disulfide linkage again and have formed dimeric form in fibrinogen molecule.Fibrinogen is under the effect of zymoplasm, respectively scleroproein A peptide on two A α chains and the scleroproein B peptide on the B β chain are scaled off, remaining α, β and γ chain is referred to as fibrin monomer after having excised A peptide and B peptide, and they can automatically be met end to end by hydrogen bond and form the fibrin polymerization body.In addition, because zymoplasm can also activate Hageman factor I, be that plasma fibrin connects enzyme, the Hageman factor I (XIIIa) that has activated can make the acid amides of the glutamine on the scleroproein molecule and ∈ on the Methionin-amino constitute peptide bond, from and make the crosslinked gathering of scleroproein molecule side direction, form insoluble crosslinked fibrin polymer.
Snake venom thrombin-like enzyme can make equally that A peptide and B peptide scale off on the fibrinogen molecule, formed fibrin monomer also can be met end to end, but because Thrombin-like enzyme can not activate Hageman factor I, therefore can not make the crosslinked gathering of scleroproein molecule side direction, formed like this fibrin polymerization body is insecure, not only broken up by blood flow easily, and very responsive to the plasmin in the blood plasma (Plasmin), be easy under the effect of plasmin, be decomposed.People utilize snake venom thrombin-like enzyme to be different from this characteristic of blood plasma zymoplasm just, be applied to the clinical treatment thrombotic diseases, its mechanism of action mainly shows two aspects: at first be that Fibrinogen in the patient blood that is under the hypercoagulative state is degraded under the snake venom thrombin-like enzyme effect, fibrinogenic content reduces greatly, be optimum defibrination raw state, and formed thus fibrin polymerization body is easy to be degraded by plasmin in the body, and broken up by blood flow, make patient be not easy to form again thrombus, thereby have the effect of anti-freezing.Just owing to fibrinogen content in the blood significantly reduces, influenced by biological regulation and control and feedback ground impels vascular endothelial cell release tPA, it is tissue plasminogen activator, the tPA Profibrinolysin (Plasminogen) in the deexcitation blood plasma again becomes plasmin, the latter can make fibrin degradation or make thrombolysis, thereby plays the effect of thrombolysis.So Thrombin-like enzyme direct effect clinically is anti-freezing, its supervention effect is a thrombolysis.
Abroad snake venom thrombin-like enzyme has been carried out the deep research of system, and Thrombin-like enzyme is applied to the clinical treatment thrombotic diseases.Ancrod is the trade name of red mouthful of pallas pit viper (Agkistrodon rhodostoma) snake venom thrombin-like enzyme, at first be by Ewart (Biochem.J.1970,118,585-593) wait people's purifying to obtain, (Thromb.Haemostasis 1985 for Latallo, 50,604-609) then big quantity research has been made in its clinical application.Batroxobin is the Thrombin-like enzyme in South America spearhead Pallas pit viper (Bothrops atrox) snake venom, people such as Hollemand have carried out scrutiny (J.Biol.Chem.1976 to its zymetology and physiological property, 251,1663-1669), be developed to clinical application and widespread use by Japan and Sweden.Their gene order is published, but gene engineering product is not also developed.
At present domestic employed clinically Thrombin-like enzyme is and extracts from the snake venom of agkistrodon acutus, Jiangsu and Zhejiang Provinces pallas pit viper and agkistrodon halys ussuriensis, through clinical practice for many years, finds that sTLE has best curative effect.Yet the snake venom resource is very limited, if obtain product by genetically engineered, is a up-and-coming approach.For the exploitation of gene engineering product from now on, it is a significant research topic that sTLE is carried out gene sequencing.
, the present invention seeks to by measuring one section aminoacid sequence of sTLE N end, the synthetic upstream and downstream primer of design is used amplification of PCR method and clone, the thrombase-like gene of being cloned is measured obtained gene order for this reason.
The present invention's purifying from the agkistrodon acutus snake venom obtains Thrombin-like enzyme, measures its one section aminoacid sequence of N end, according to this section sequence and other snake kind Thrombin-like enzyme homology structurally, designs and synthesizes the upstream and downstream Oligonucleolide primers.From agkistrodon acutus poison gland, separate relevant mRNA then as template, obtain cDNA, increase and cloned the full gene of Thrombin-like enzyme, the complete sequence of assay determination gene with the PCR method through reverse transcription.
1, total RNA extracts
Downcut snakehead, take out poison gland immediately, quick-frozen in dry ice, place-70 ℃ standby.Adopt acid guanidine thiocyanate-phenol-chloroform single stage method (to consult Chomczynski P.et al.AnalyticalBiochemistry.1987,162:156) the total RNA of extracting poison gland.
2, design of primers foundation, primer synthetic method
Aminoacid sequence according to the Thrombin-like enzyme maturation protein N end of measuring, structure is: VIGGVECDINEHRFL, designed the upstream oligonucleotide primer of degeneracy, i.e. 5 ' CTTCTAGAATGGTCATTGGAGGTGA (T) TGAG (A) TG 3 ', and add atg start codon---ATG and XbaI site at the N of maturation protein end, be beneficial to this expression of gene research and operate; According to the structural homology of other snake kind Thrombin-like enzyme (Itoh N., et al 1987, The Journalof Biology Chemistry 262:3132; Au L.C., et al 1993, Biochem.J. 294:387), has designed the downstream Oligonucleolide primers of degeneracy, i.e. 5 ' TTTCTCCTCTTG (A) AG (C) TA (T) TTTCAAA3 ' at the conservative section of 3 ' non-coding region.Primer is synthetic on the Bechman oligo 1000 primer synthesizers that Bechman company produces, and primer is seen Fig. 2.
3, reverse transcription, pcr amplification and gene clone
With the total RNA of agkistrodon acutus poison gland is template, and downstream primer is that primer is done reverse transcription.The reverse transcription test kit is available from Gibco BRL company, and operation is undertaken by its specification sheets.Go on foot gained cDNA is template in the past, carries out pcr amplification with synthetic upstream and downstream primer.Pcr amplification goes out the dna fragmentation of a 700bp, sees Fig. 3 agarose gel electrophoretogram.The dna fragmentation that amplifies is inserted between the XbaI and EcoRV restriction enzyme site of phagemid carrier pBlueScript-SK (plasmid vector is available from Stratagene company) by the abundant enzymolysis of XbaI, cuts evaluation through blue hickie screening and enzyme, obtains 10 positive colonies.
4, sTLE gene identification
The present invention is to two positive colony T
3Gene and T
2Gene mutation body carries out complete sequence determination, and most of sequence wherein is that two-way order-checking is determined.T
3And T
2The Nucleotide of mutant and amino acid complete sequence are seen Fig. 1.T
3699 Nucleotide of cDNA reading frame coding, i.e. 233 amino acid, this albumen and the Thrombin-like enzyme homology that derives from copperhead Pallas pit viper (Agkistrodon contortrix), huge Pallas pit viper snake kinds such as (Lahcesis mutus) are seen Fig. 4 between 70-80%.And, compare with 15 amino acid of Agkistrodon snake venom thrombin-like enzyme N end of having measured, identical substantially with it by the corresponding aminoacid sequence that cDNA derives, this has illustrated T
3The cDNA new thrombase-like gene that does not appear in the newspapers of encoding.T
2And T
3Marked difference is that 656 bit bases are at T
2In become A, form a terminator codon TAG, thereby T
2Reading frame so far stop.So T
2CDNA encode 651 Nucleotide, i.e. 217 amino acid.T
2And T
3CDNA encoded protein matter sequence is almost completely identical, only T
2The C end of coded protein has lacked 16 amino acid, in addition, and T
2The 5th of encoded protein matter is Val and T
3Be Asp, be a mutant of Thrombin-like enzyme.
Utilize above-mentioned synthetic upstream and downstream primer, through pcr amplification and clone, obtain 10 positive colonies, wherein two positive colonies are carried out gene sequencing, obtained a kind of gene order and a kind of sTLE gene mutation body sequence that is encoded to sTLE.For those other 8 positive colonies of determined sequence not, new sTLE gene mutation body wherein also may appear, and two positive colonies measuring sequence are carried out genetic modification, also can obtain artificial directed mutants of sTLE and the interfertile gene of sTLE.
Advantage of the present invention: agkistrodon acutus is the distinctive snake kind of China, the vigor of its Thrombin-like enzyme and curative effect are much better than the Thrombin-like enzyme in Jiangsu and Zhejiang Provinces pallas pit viper and the agkistrodon halys ussuriensis, yet snake venom resource-constrained, the present invention clone obtains thrombase-like gene and measures its gene order, has created good condition for developing gene engineering product from now on.
The present invention is further elaborated by the following drawings and embodiment:
Description of drawings:
The Nucleotide of Fig. 1 sTLE and aminoacid sequence.A:T
3The nucleotide sequence of Thrombin-like enzyme; B:T
3The aminoacid sequence of Thrombin-like enzyme; C:T
2The nucleotide sequence of thrombase-like gene mutant; D:T
2The aminoacid sequence of thrombase-like gene mutant.
Fig. 2 synthetic oligonucleotide upstream primer and downstream primer
The agarose gel electrophoresis figure of Fig. 3 PCR product
The comparison of several venin-derived Thrombin-like enzyme aminoacid sequences of Fig. 4
Embodiment 1:
1, total RNA extracts
Downcut snakehead, take out poison gland immediately, quick-frozen in dry ice, place-70 ℃ standby.Take out 100mg poison gland tissue, add the 1ml solution D and (contain the 4M guanidinium isothiocyanate, 25mM Trisodium Citrate, pH7.0; 5% sarcosyl (Sarcosyl), 0.1M beta-mercaptoethanol), in tissue homogenizer fully after the homogenate, homogenate is changed in the 10ml centrifuge tube, the sodium acetate (pH4) that adds 0.1ml 2M again, the water-saturated phenol of 1ml and the chloroform of 0.2ml: primary isoamyl alcohol (49: 1) mixture, behind the mixing that fully vibrates, leave standstill 15min on ice.Sample at Hitachi's refrigerated centrifuge (Hitach Centrifuge20PR-52D) with 10000g in 4 ℃ of centrifugal 20min, draw the upper strata water, with equal-volume Virahol mixing, put-20 ℃ freezing at least 1 hour.With the centrifugal 20min collecting precipitation of 10000g and be dissolved in the 0.3ml solution D, add 0.03ml 2M sodium acetate and 0.3ml Virahol, mixing, place-20 ℃ freezing 1 hour.Be dissolved in the H that 0.3ml coke diethyl phthalate (DEPC) was handled again with the centrifugal 20min collecting precipitation of 10000g
2O adds 0.03ml sodium acetate and 0.75ml ethanol, mixing, place-20 ℃ freezing 1 hour, with the centrifugal 20min collecting precipitation of 10000g, precipitate with 75% washing RNA, after the seasoning RNA is dissolved in the DEPC treated water, packing, place-70 ℃ standby.
2, design of primers foundation, primer synthetic method
The present invention is according to the aminoacid sequence of the Thrombin-like enzyme maturation protein N end of measuring, structure is VIGGVECDINEHRFL, the upstream oligonucleotide primer of design degeneracy is: 5 ' CTTCTAGAATGGTCATTGGAGGTGA (T) TGAG (A) TG 3 ', and add atg start codon---ATG and XbaI site at the N of maturation protein end, be beneficial to this expression of gene research and operate.According to the structural homology of other snake kind Thrombin-like enzyme (Itoh N., et al 1987, The Journal ofBiology Chemistry 262:3132; Au L.C., et al 1993, Biochem.J. 294:387), designs downstream primer 5 ' TTTCTCCTCTTG (A) AG (C) TA (T) TTTCAAA3 ' of degeneracy at the conservative section of 3 ' non-coding region.Primer is synthetic on the Bechman Oligo 1000 primer synthesizers that Bechman company produces, and the upstream of design and downstream Oligonucleolide primers are seen Fig. 2.
3, reverse transcription, pcr amplification and gene clone
With the total RNA of agkistrodon acutus poison gland is template, and above-mentioned downstream primer is that primer is done reverse transcription.The reverse transcription test kit is available from Gibco BRL company, and operation is undertaken by its specification sheets.
Go on foot gained cDNA is template in the past, carries out pcr amplification with above-mentioned upstream and downstream primer.The cumulative volume of reaction is 50ul, wherein MgCl
2Final concentration be 1.5mM, the final concentration of dNTP is 0.15mM, adds the Taq archaeal dna polymerase (available from auspicious imperial company) of 5ul 10 * PCR damping fluid and 1-2 unit..Reaction conditions is: 93 ℃ of sex change 10min, and 48 ℃ of annealing 1min, 72 ℃ are extended 2min, carry out 5 circulations, 93 ℃ of sex change 1min then, 55 ℃ of annealing 1min, 72 ℃ are extended 2min, carry out 30 circulations again, last 72 ℃ of insulation 10min.Get the 5u1 reaction product through 1% agarose electrophoresis inspection.Pcr amplification goes out the dna fragmentation of a 700bp, and the agarose gel electrophoresis figure of PCR product sees Fig. 3.Product chloroform extracting, ethanol sedimentation is dissolved in the 15ul water standby.
The dna fragmentation that amplifies is inserted between the XbaI and EcoRV restriction enzyme site of phagemid carrier pBlueScript-SK (available from Stratagene company) by the abundant enzymolysis of XbaI, cuts evaluation through blue hickie screening and enzyme, obtains 10 positive colonies.
Embodiment 2: the sTLE gene identification
The present invention is to two positive colony T
3And T
2Carry out complete sequence determination, most of sequence wherein is two-way mensuration.[d-
35S] dATP is available from Amersham company, and sequencing kit is available from U.S.B. company, and concrete operations are with reference to specification sheets, T
3Gene and T
2The Nucleotide of mutant and amino acid complete sequence are seen Fig. 1.T
3699 Nucleotide of cDNA reading frame coding, i.e. 233 amino acid, this albumen and the Thrombin-like enzyme homology that derives from copperhead Pallas pit viper (Agkistrodon contortrix), huge Pallas pit viper snake kinds such as (Lahcesis mutus) are between 70-80%, see Fig. 4, catalytic residue His41, Asp86 and Ser179 and Cys the (the 7th, 26,42,74,118,139,150,164,175,185,200 and 231) the position very conservative; And, compare with 15 amino acid of Agkistrodon snake venom thrombin-like enzyme N end of having measured, identical substantially with it by the corresponding aminoacid sequence that cDNA derives, this has all illustrated T
3The cDNA new thrombase-like gene that does not appear in the newspapers of encoding.
T
2And T
3Marked difference is that 656 bit bases are at T
2In become A, form a terminator codon TAG, thereby T
2Reading frame so far stop.So T
2CDNA encode 651 Nucleotide, i.e. 217 amino acid.T
2And T
3CDNA encoded protein matter sequence is almost completely identical, only T
2The C end of coded protein has lacked 16 amino acid, in addition, and T
2The 5th of encoded protein matter is Val and T
3Be Asp, be a mutant of Thrombin-like enzyme.
Claims (2)
1、,DNA：ATGGTCATTGGAGGTGATGAGTGTGACATAAATGAACATCGTTTCCTTGTAGCCTTCTTT 60 M V I G G D E C D I N E H R F L V A F FAACACTACTGGATTTTTCTGTGGTGGGACTTTGATCAACCCGGAATGGGTGGTCACTGCT 120 N T T G F F C G G T L I N P E W V V T AGCACACTGCGACAGTACAAATTTCCAGATGCAGCTTGGTGTGCATAGCAAAAAGGTACTA 180 A H C D S T N F Q M Q L G V H S K K V LAATGAGGATGAGCAGACAAGAAACCCAAAGGAGAAGTTCATTTGTCCCAATAAGAACAAC 240 N E D E Q T R N P K E K F I C P N K N NAATGAAGTACTGGACAAGGACATCATGTTGATCAAGCTGGACAAACCTATTAGCAACAGT 300 N E V L D K D I M L I K L D K P I S N SAAACACATCGCGCCTCTCAGCTTGCCTTCCAGCCCTCCCAGTGTGGGCTCAGTTTGCCGT 360 K H I A P L S L P S S P P S V G S V C RATTATGGGATGGGGATCAATCACACCTGTrAAAGAGACTTTTCCCGATGTCCCTTATTGT 420 I M G W G S I T P V K E T F P D V P Y CGCTAACATTAACCTACTTGATCATGCAGTGTGTCAAACAGGTTATCCAAGTTGCTGGCGG 480 A N I N L L D H A V C Q T G Y P S C W RAATACAACATTGTGTGCAGGTTTCCTGGAAGGAGGCAAAGATACATGTGGGGGTGACTCT 540 N T T L C A G F L E G G K D T C G G D SGGGGGGCCCCTCATCTGTAATGGACAATTCCAGGGCATTGTATCTTATGGGGCGCATTCT 600 G G P L I C N G Q F Q G I V S Y G A H STGTGGCCAAGGTCCTAAGCCTGGTATCTACACCAATGTCTTCGATTATACTGACTGGATC 660 C G Q G P K P G I Y T N V F D Y T D W ICAGAGAAATATTGCAGGAAATACAGATGCAACTTGCCCCCCGTGA 705 Q R N I A G N T D A T C P P---
2, a kind of mutant of sTLE gene is characterized in that having following DNA Nucleotide and amino acid sequence corresponding:
ATGGTCATTGGAGGTGTTGAATGTGACATAAATGAACATCGTTTCCTTGTAGCCTTCTTT 60
M V I G G V E C D I N E H R F L V A F F
AACACTACTGGATTTTTCTGTGGTGGGACTTTGATCAACCCGGAATGGGTGGTCACTGCT 120
N T T G F F C G G T L I N P E W V V T A
GCACACTGCGACAGTACAAATTTCCAGATGCAGCTTGGTGTGCATAGCAAAAAGGTACTA 180
A H C D S T N F Q M Q L G V H S K K V L
AATGAGGATGAGCAGACAAGAAACCCAAAGGAGAAGTTCATTTGTCCCAATAAGAACAAC 240
N E D E Q T R N P K E K F I C P N K N N
AATGAAGTACTGGACAAGGACATCATGTTGATCAAGCTGGACAAACCTATTAGCAACAGT 300
N E V L D K D I M L I K L D K P I S N S
AAACACATCGCGCCTCTCAGCTTGCCTTCCAGCCCTCCCAGTGTGGGCTCAGTTTGCCGT 360
K H I A P L S L P S S P P S V G S V C R
ATTATGGGATGGGGATCAATCACACCTGTTAAAGAGACTTTTCCCGATGTCCCTTATTGT 420
I M G W G S I T P V K E T F P D V P Y C
GCTAACATTAACCTACTTGATCATGCAGTGTGTCAAACAGGTTATCCAAGTTGCTGGCGG 480
A N I N L L D H A V C Q T G Y P S C W R
AATACAACATTGTGTGCAGGTTTCCTGGAAGGAGGCAAAGATACATGTGGGGGTGACTCT 540
N T T L C A G F L E G G K D T C G G D S
GGGGGGCCCCTCATCTGTAATGGACAATTCCAGGGCATTGTATCTTATGGGGCGCATTCT 600
G G P L I C N G Q F Q G I V S Y G A H S
TGTGGCCAAGGTCCTAAGCCTGGTATCTACACCAATGTCTTCGATTATACTGACTAGATC 660
C G Q G P K P G I Y T N V F D Y T D?---
CAGAGAAATATTGCAGGAAATACAGATGCAACTTGCCCCCCGTGA
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| CN97106706A CN1060806C (en) | 1997-11-12 | 1997-11-12 | Gene sequence of fibrin ferment of Agkistrodon acutus snakes |
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| CN1060806C true CN1060806C (en) | 2001-01-17 |
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Families Citing this family (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1218747C (en) * | 2003-08-14 | 2005-09-14 | 吴忠 | Medicinal use of agkistrodon acutus thrombase for treating hemorrhagic disease |
| CN1324132C (en) * | 2004-10-19 | 2007-07-04 | 北京大学 | Snake venom thrombin-like enzyme and its encoding gene and application |
| JP2008522633A (en) * | 2004-12-15 | 2008-07-03 | 中山大学 | Five-step snake venom fibrinolytic enzyme and its use |
| WO2006066441A1 (en) * | 2004-12-20 | 2006-06-29 | Sun Yat-Sen University | A fibrinolysin of agkistrodon acutus venom and its usage |
| CN100351381C (en) * | 2005-09-02 | 2007-11-28 | 中山大学 | Novel thrombase-like gene and its use |
| CN101684151B (en) * | 2008-09-26 | 2012-06-20 | 江苏正大天晴药业股份有限公司 | Protein matter with coagulation activity |
| CN102559721B (en) * | 2012-01-05 | 2013-03-20 | 哈尔滨师范大学 | Gloydius ussuriensis venom FLE (fibrin olytic enzyme) gene and encoding protein thereof as well as preparation method of eukaryon expression product of Gloydius Ussuriensis venom FLE gene |
| CN102719461A (en) * | 2012-05-16 | 2012-10-10 | 中国农业大学 | Gene sequence and amino acid sequence of snake venom plasmin of agkistrodon blomhoffii ussurensis |
| CN102851303A (en) * | 2012-09-24 | 2013-01-02 | 福州大学 | Thrombin-like enzyme gene and application thereof |
Citations (3)
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|---|---|---|---|---|
| CN86104422A (en) * | 1986-06-24 | 1988-01-06 | 中国医科大学 | The preparation method of snake venom antithrombotic enzyme |
| CN1077390A (en) * | 1993-02-27 | 1993-10-20 | 军事医学科学院放射医学研究所 | A kind of novel thrombolytic drug and preparation thereof |
| CN1120070A (en) * | 1994-12-26 | 1996-04-10 | 合肥兆峰科大药业有限公司 | Point pallas pit viper poisonous fibre-dissolving enzyme and its purifying method |
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1997
- 1997-11-12 CN CN97106706A patent/CN1060806C/en not_active Expired - Fee Related
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN86104422A (en) * | 1986-06-24 | 1988-01-06 | 中国医科大学 | The preparation method of snake venom antithrombotic enzyme |
| CN1077390A (en) * | 1993-02-27 | 1993-10-20 | 军事医学科学院放射医学研究所 | A kind of novel thrombolytic drug and preparation thereof |
| CN1120070A (en) * | 1994-12-26 | 1996-04-10 | 合肥兆峰科大药业有限公司 | Point pallas pit viper poisonous fibre-dissolving enzyme and its purifying method |
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