一种新的尖吻蝮蛇蛇毒类凝血酶的结构和功能研究
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摘要
心脑血管疾病如脑中风、冠心病以及外周血管的血栓栓塞等已成为了严重影响人类健康的主要疾病。作为自然界的野生资源之一,毒蛇毒液中含有许多生物活性蛋白质和多肽,其中就包括对血液系统有重要影响的组分。利用蛇毒开发新的治疗血栓的药物,成为国内外研究的热点之一。
本研究分离了广西尖吻蝮蛇毒腺组织,利用T7Select(?)Phage Display System首次构建了尖吻蝮蛇蛇毒毒腺噬菌体展示文库,以纤维蛋白/纤维蛋白原为底物,经过三轮淘选,获得了一个新的尖吻蝮蛇蛇毒类凝血酶基因;利用生物信息学技术,分析了类凝血酶基因结构特点,构建了尖吻蝮蛇蛇毒类凝血酶基因的原核表达载体pET-32a-TLE,诱导表达,亲和纯化和分子筛纯化后,获得了具有生物学活性的重组蛇毒类凝血酶蛋白。
本研究的主要实验结果如下:
1.最先构建了广西尖吻蝮蛇毒腺噬菌体展示文库,文库滴度为2.5×10~6 pfu/ml。
2.建立了使用纤维蛋白/纤维蛋白原淘选抗凝组分的方法:在固相介质上包被(ELISA板)100μl的10μg/ml纤维蛋白原后,加入扩增的噬菌体文库的裂解产物(100μl),洗去为非特异性结合的噬菌体,将特异性结合的噬菌体洗脱下来进行下一轮淘选,经过三轮“吸附一洗脱一扩增”,富集了特异性结合的噬菌体。
3.获得了一个新的尖吻蝮蛇蛇毒类凝血酶基因,基因长783 bp,能够编码260个氨基酸,GenBank Accession Number为AY861138.1,序列同源性分析,发现与GenBank登录号为AF159060类凝血酶的同源性为97%,运用互联网的http://www.expasy.org/cgi-bin/protscale.pl分析了此类凝血酶基团的亲疏水性,具有明显的亲水性,同源建模分析,发现其有三个结构域,信号肽,类胰岛素丝氨酸蛋白酶,内在的结构。
4.构建了原核表达载体pET-32a-TLE,在0.7 mmol/L的IPTG诱导下,25℃下低温诱导表达类凝血酶蛋白,在该条件下大大降低了类凝血酶蛋白的包涵体表达,为后面简便快捷的纯化蛋白奠定了基础。重组尖吻蝮蛇蛇毒类凝血酶的生物活性分析:血浆体外凝固实验表明,37℃下,4.4μg的重组类凝血酶使200μl血浆在65s钟内凝固;纤维蛋白原水解活性表明8μg重组类凝血酶蛋白在60分钟内可以降解纤维蛋白原的Aα链。
Cardiovascular and cerebrovascular diseases such as stroke,coronary heart disease and peripheral vascular thrombosis embolism have seriously impacted on quality of people life,cause a heavy burden for families.As one precious nature resources,Snake venoms contain lots of biologically active proteins and peptides,including on the components of interacting vascular system.Using snake venom to the develop new thrombosis-treatment drugs has emerged as the important researches.
In this study,we separated the tissue of Agkistrodon acutus venom gland,and constructed a phage display library of Agkistrodon acutus venom gland by use of Novagen's T7 Select(?) Phage Display System.After three rounds of panning,we obtained a new Agkistrodon acutus snake venom thrombin-like enzyme gene.Whereafter,we analyzed the gene structure of thrombin-like enzyme using bioinformatics.In order to express the thrombin-like enzyme,we constructed a prokaryon vector of pET-32a-TLE, then expressing induced by IPTG.After affinity purification and molecular sieve purification,we obtained the recombinant snake venom thrombin-like enzyme protein which keeped a goog biological activity.
The main results of our study are as follws:
1.Firstly constructed phage display library of Guangxi Agkistrodon acutus venom,after identifying,the titer of library reaching 2.5×10~6 pfu/ml.
2.Set up approach to pan anticoagulant components using the ways of using fibrin/fibrinogen:Coat 100μl 10μg/m fibrin/fibrinogen on the solid-phase medium such as ELISA plate,add amplified phage library into coated plate,wash away the non-specific phage which is binded to the plate,after three rounds of biopanning "Adsorption -Elution-Amplificationion",enrich the specific phage which is binded to the plate.
3.Obtaining a new thrombin-like enzyme gene from Agkistrodon acutus snake venom. Length is 783bp,it encodes 260 amino acids.Acquaired the GenBank Accession Number AY861138.1 after submission.It has 97%homology with the thrombin-like enzyme of Gen Bank accession nuber AF159060 through the Blast.we analysised the Hydrophilicity and hydrophobicity by the web of http://www.expasy.org/cgi-bin/protscale.pl,and found it obvious Hydrophilicity.Homology modeling analysis indicted finding that there are three structural domains,signal peptide,Tryp_Spc(Trypsin-like serine protease),and intrinsic disorder.
4.Constructed the prokaryotic expression vector pET-32a-TLE,optimized expression conditions at 0.7 mmol/L of IPTG and 25℃.Protein inclusions expression was cut down under this condition,which laid the foundation for the simple and efficient purification of the thrombin-like enzyme.
5.The biological activity of recombination Agkistrodon acutus snake venom thrombin-like was analyzed:plasma coagulation experiments in vitro showed that 4.4μg of recombinant thrombin-like enzyme could make 200μl plasma coagulation in 65 seconds at 37℃;Fibrinogen hydrolysis activity assay showed that 8μg thrombin-like enzyme could degrade the Aαchain of fibrinogen in 60 minutes throughly.
本研究分离了广西尖吻蝮蛇毒腺组织,利用T7Select(?)Phage Display System首次构建了尖吻蝮蛇蛇毒毒腺噬菌体展示文库,以纤维蛋白/纤维蛋白原为底物,经过三轮淘选,获得了一个新的尖吻蝮蛇蛇毒类凝血酶基因;利用生物信息学技术,分析了类凝血酶基因结构特点,构建了尖吻蝮蛇蛇毒类凝血酶基因的原核表达载体pET-32a-TLE,诱导表达,亲和纯化和分子筛纯化后,获得了具有生物学活性的重组蛇毒类凝血酶蛋白。
本研究的主要实验结果如下:
1.最先构建了广西尖吻蝮蛇毒腺噬菌体展示文库,文库滴度为2.5×10~6 pfu/ml。
2.建立了使用纤维蛋白/纤维蛋白原淘选抗凝组分的方法:在固相介质上包被(ELISA板)100μl的10μg/ml纤维蛋白原后,加入扩增的噬菌体文库的裂解产物(100μl),洗去为非特异性结合的噬菌体,将特异性结合的噬菌体洗脱下来进行下一轮淘选,经过三轮“吸附一洗脱一扩增”,富集了特异性结合的噬菌体。
3.获得了一个新的尖吻蝮蛇蛇毒类凝血酶基因,基因长783 bp,能够编码260个氨基酸,GenBank Accession Number为AY861138.1,序列同源性分析,发现与GenBank登录号为AF159060类凝血酶的同源性为97%,运用互联网的http://www.expasy.org/cgi-bin/protscale.pl分析了此类凝血酶基团的亲疏水性,具有明显的亲水性,同源建模分析,发现其有三个结构域,信号肽,类胰岛素丝氨酸蛋白酶,内在的结构。
4.构建了原核表达载体pET-32a-TLE,在0.7 mmol/L的IPTG诱导下,25℃下低温诱导表达类凝血酶蛋白,在该条件下大大降低了类凝血酶蛋白的包涵体表达,为后面简便快捷的纯化蛋白奠定了基础。重组尖吻蝮蛇蛇毒类凝血酶的生物活性分析:血浆体外凝固实验表明,37℃下,4.4μg的重组类凝血酶使200μl血浆在65s钟内凝固;纤维蛋白原水解活性表明8μg重组类凝血酶蛋白在60分钟内可以降解纤维蛋白原的Aα链。
Cardiovascular and cerebrovascular diseases such as stroke,coronary heart disease and peripheral vascular thrombosis embolism have seriously impacted on quality of people life,cause a heavy burden for families.As one precious nature resources,Snake venoms contain lots of biologically active proteins and peptides,including on the components of interacting vascular system.Using snake venom to the develop new thrombosis-treatment drugs has emerged as the important researches.
In this study,we separated the tissue of Agkistrodon acutus venom gland,and constructed a phage display library of Agkistrodon acutus venom gland by use of Novagen's T7 Select(?) Phage Display System.After three rounds of panning,we obtained a new Agkistrodon acutus snake venom thrombin-like enzyme gene.Whereafter,we analyzed the gene structure of thrombin-like enzyme using bioinformatics.In order to express the thrombin-like enzyme,we constructed a prokaryon vector of pET-32a-TLE, then expressing induced by IPTG.After affinity purification and molecular sieve purification,we obtained the recombinant snake venom thrombin-like enzyme protein which keeped a goog biological activity.
The main results of our study are as follws:
1.Firstly constructed phage display library of Guangxi Agkistrodon acutus venom,after identifying,the titer of library reaching 2.5×10~6 pfu/ml.
2.Set up approach to pan anticoagulant components using the ways of using fibrin/fibrinogen:Coat 100μl 10μg/m fibrin/fibrinogen on the solid-phase medium such as ELISA plate,add amplified phage library into coated plate,wash away the non-specific phage which is binded to the plate,after three rounds of biopanning "Adsorption -Elution-Amplificationion",enrich the specific phage which is binded to the plate.
3.Obtaining a new thrombin-like enzyme gene from Agkistrodon acutus snake venom. Length is 783bp,it encodes 260 amino acids.Acquaired the GenBank Accession Number AY861138.1 after submission.It has 97%homology with the thrombin-like enzyme of Gen Bank accession nuber AF159060 through the Blast.we analysised the Hydrophilicity and hydrophobicity by the web of http://www.expasy.org/cgi-bin/protscale.pl,and found it obvious Hydrophilicity.Homology modeling analysis indicted finding that there are three structural domains,signal peptide,Tryp_Spc(Trypsin-like serine protease),and intrinsic disorder.
4.Constructed the prokaryotic expression vector pET-32a-TLE,optimized expression conditions at 0.7 mmol/L of IPTG and 25℃.Protein inclusions expression was cut down under this condition,which laid the foundation for the simple and efficient purification of the thrombin-like enzyme.
5.The biological activity of recombination Agkistrodon acutus snake venom thrombin-like was analyzed:plasma coagulation experiments in vitro showed that 4.4μg of recombinant thrombin-like enzyme could make 200μl plasma coagulation in 65 seconds at 37℃;Fibrinogen hydrolysis activity assay showed that 8μg thrombin-like enzyme could degrade the Aαchain of fibrinogen in 60 minutes throughly.
引文
1.Murayama N,Hayashi MA,Ohi H,et al.:Cloning and sequence analysis of a Bothrops jararaca cDNA encoding a precursor of seven bradykinin-potentiating peptides and a C-type natriuretic peptide.Proc Natl Acad Sci U S A 1997;94(4):1189-93.
2.Doolittle RF:The structure and evolution of vertebrate fibrinogen.Ann N Y Acad Sci 1983;408:13-27.
3.Tsai IH,Wang YM,Au LC,Ko TP,Chen YH,Chu YF:Phospholipases A2 from Callosellasma rhodostoma venom gland cloning and sequencing of 10 of the cDNAs,three-dimensional modelling and chemical modification of the major isozyme.Eur J Biochem 2000;267(22):6684-91.
4.Siigur J,Samel M,Tonismagi K,Subbi J,Siigur E,Tu AT:Biochemical characterization of lebetase,a direct-acting fibrinolytic enzyme from Vipera lebetina snake venom.Thromb Res 1998;90(1):39-49.
5.Ouyang C,Hong JS,Teng CM:Purification and properties of the thrombin-like principle of Agkistrodon acutus venom and its comparison with bovine thrombin.Thromb Diath Haemorrh 1971;26(2):224-34.
6.Selistre-de-Araujo HS,de Souza EL,Beltramini LM,Ownby CL,Souza DH:Expression,refolding,and activity of a recombinant nonhemorrhagic snake venom metalloprotease.Protein Expr Purif 2000;19(1):41-7.
7.Tu AT,Baker B,Wongvibulsin S,Willis T:Biochemical characterization of atroxase and nucleotide sequence encoding the fibrinolytic enzyme.Toxicon 1996;34(11-12):1295-300.
8.Lee JW,Park W:cDNA cloning of brevinase,a heterogeneous two-chain fibrinolytic enzyme from Agkistrodon blomhoffii brevicaudus snake venom,by serial hybridization-polymerase chain reaction.Arch Biochem Biophys 2000;377(2):234-40.
9.Smith GP:Filamentous fusion phage:novel expression vectors that display cloned antigens on the virion surface.Science 1985;228(4705):1315-7.
10.Scott JK,Smith GP:Searching for peptide ligands with an epitope library.Science 1990;249(4967):386-90.
11.Wang X,Yu J,Sreekumar A,et al.:Autoantibody signatures in prostate cancer.N Engl J Med 2005;353(12):1224-35.
12.Hansen MH,Ostenstad B,Sioud M:Identification of immunogenic antigens using a phage-displayed cDNA library from an invasive ductal breast carcinoma tumour.Int J Oncol 2001;19(6):1303-9.
1.Bardwell JC,McGovern K,Beckwith J:Identification of a protein required for disulfide bond formation in vivo.Cell 1991;67(3):581-9.
2.Sanger F,Nicklen S,Coulson AR:DNA sequencing with chain-terminating inhibitors.Proc Natl Acad Sci U S A 1977;74(12):5463-7.
3.Laemmli UK:Cleavage of structural proteins during the assembly of the head of bacteriophage T4.Nature 1970;227(5259):680-5.
4.Hung CC,Chiou SH:Fibrinogenolytic proteases isolated from the snake venom of Taiwan habu:serine proteases with kallikrein-like and angiotensin-degrading activities.Biochem Biophys Res Commun 2001;281(4):1012-8.
5.Braga MD,Martins AM,de Menezes DB,et al.:Purification and biological activity of the thrombin-like substance isolated from Bothrops insularis venom.Toxicon 2007;49(3):329-38.
6.Towbin H,Staehelin T,Gordon J:Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets:procedure and some applications.Proc Natl Acad Sci U S A 1979;76(9):4350-4.
7.Ambroz KL,Zhang Y,Schutz-Geschwender A,Olive DM:Blocking and detection chemistries affect antibody performance on reverse phase protein arrays.Proteomics 2008;8(12):2379-83.
8.Naha PM,Sorrentino R:Conditional expression of the transformed phenotype in an "epithelial-like" cell line.Cell Biol Int Rep 1979;3(5):421-30.
9.Perez AV,Rucavado A,Sanz L,Calvete JJ,Gutierrez JM:Isolation and characterization of a serine proteinase with thrombin-like activity from the venom of the snake Bothrops asper.Braz J Med Biol Res 2008;41(1):12-7.
1.Gao R,Zhang Y,Meng QX,et al.:Characterization of three fibrinogenolytic enzymes from Chinese green tree viper(Trimeresurus stejnegeri) venom.Toxicon 1998;36(3):457-67.
2.Castro HC,Zingali RB,Albuquerque MG,Pujol-Luz M,Rodrigues CR:Snake venom thrombin-like enzymes:from reptilase to now.Cell Mol Life Sci 2004;61(7-8):843-56.
3.Blomback B,Blomback M,Nilsson IM:Coagulation studies on reptilase,an extract of the venom from Bothrops jararaca.Thromb Diath Haemorrh 1958;1(1):76-86.
4.Matsui T,Fujimura Y,Titani K:Snake venom proteases affecting hemostasis and thrombosis.Biochim Biophys Acta 2000;1477(1-2):146-56.
5.Braud S,Bon C,Wisner A:Snake venom proteins acting on hemostasis.Biochimie 2000;82(9-10):851-9.
6.Santos BF,Serrano SM,Kuliopulos A,Niewiarowski S:Interaction of viper venom serine peptidases with thrombin receptors on human platelets.FEBS Lett 2000;477(3):199-202.
7.Lathan LO,Staggers SL:Ancrod:the use of snake venom in the treatment of patients with heparin-induced thrombocytopenia and thrombosis undergoing coronary artery bypass grafting:nursing management.Heart Lung 1996;25(6):451-60;quiz 461-2.
8.Esnouf MP,Tunnah GW:The isolation and properties of the thrombin-like activity from Ancistrodon rhodostoma venom.Br J Haematol 1967;13(4):581-90.
9.Maeda M,Satoh S,Suzuki S,Niwa M,Itoh N,Yamashina I:Expression of cDNA for batroxobin,a thrombin-like snake venom enzyme.J Biochem 1991;109(4):632-7.
10.Pirkle H:Thrombin-like enzymes from snake venoms:an updated inventory.Scientific and Standardization Committee's Registry of Exogenous Hemostatic Factors.Thromb Haemost 1998;79(3):675-83.
11.Serrano SM,Sampaio CA,Mentele R,Camargo AC,Fink E:A novel fibrinogen-clotting enzyme,TL-BJ,from the venom of the snake Bothrops Jararaca: purification and characterization.Thromb Haemost 2000;83(3):438-44.
12.Dyr JE,Hessel B,Suttnar J,Kornalik F,Blomback B:Fibrinopeptide-releasing enzymes in the venom from the southern copperhead snake (Agkistrodon contortrix contortrix).Toxicon 1989;27(3):359-73.
13.Matsui T,Sakurai Y,Fujimura Y,et al.:Purification and amino acid sequence of halystase from snake venom of Agkistrodon halys blomhoffii,a serine protease that cleaves specifically fibrinogen and kininogen.Eur J Biochem 1998;252(3):569-75.
14.Bell WR,Jr.:Defibrinogenating enzymes.Drugs 1997;54 Suppl 3:18-30;discussion 30-1.
15.Itoh N,Tanaka N,Mihashi S,Yamashina I:Molecular cloning and sequence analysis of cDNA for batroxobin,a thrombin-like snake venom enzyme.J Biol Chem 1987;262(7):3132-5.
16.Au LC,Lin SB,Chou JS,Teh GW,Chang KJ,Shih CM:Molecular cloning and sequence analysis of the cDNA for ancrod,a thrombin-like enzyme from the venom of Calloselasma rhodostoma.Biochem J 1993;294 (Pt 2):387-90.
17.Amiconi Q Amoresano A,Boumis G,et al.:A novel venombin B from agkistrodon contortrix contortrix:evidence for recognition properties in the surface around the primary specificity pocket different from thrombin.Biochemistry 2000;39(33):10294-308.
18.Castro HC,Silva DM,Craik C,Zingali RB:Structural features of a snake venom thrombin-like enzyme:thrombin and trypsin on a single catalytic platform?Biochim Biophys Acta 2001;1547(2):183-95.
19.Suzuki T,Takahashi H:Purification of two thrombin-like enzymes from the venom of Agkistrodon caliginosus (kankoku-mamushi).Toxicon 1984;22(1):29-38.
20.Fan CY,Qian YC,Yang SL,Gong Y:Cloning,sequence analysis and expression in E.coli of the cDNA of the thrombin-like enzyme (pallabin) from the venom of Agkistrodon halys pallas.Biochem Mol Biol Int 1999;47(2):217-25.
21.Hahn BS,Yang KY,Park EM,Chang IM,Kim YS:Purification and molecular cloning of calobin,a thrombin-like enzyme from Agkistrodon caliginosus (Korean viper).J Biochem 1996;119(5):835-43.
22.Nolan C,Hall LS,Barlow GH:Ancrod,the coagulating enzyme from Malayan pit viper (Agkistrodon rhodostoma) venom.Methods Enzymol 1976;45:205-13.
23.Pirkle H,Theodor I,Miyada D,Simmons G:Thrombin-like enzyme from the venom of Bitis gaboniea.Purification,properties,and coagulant actions.J Biol Chem 1986;261(19): 8830-5.
24.Shieh TC,Tanaka S,Kihara H,Ohno M,Makisumi S:Purification and characterization of a coagulant enzyme from Trimeresurus flavoviridis venom.J Biochem 1985;98(3):713-21.
24.Oyama E,Takahashi H:Purification and characterization of a thrombin-like enzyme,elegaxobin,from the venom of Trimeresurus elegans (Sakishima-habu).Toxicon 2000;38(8):1087-100.
26.Nose T,Shimohigashi Y,Hattori S,Kihara H,Ohno M:Purification and characterization of a coagulant enzyme,okinaxobin II,from Trimeresurus okinavensis (himehabu snake) venom which release fibrinopeptides A and B.Toxicon 1994;32(12):1509-20.
27.Watanabe L,Vieira DF,Bortoleto RK,Ami RK:Crystallization of bothrombin,a fibrinogen-converting serine protease isolated from the venom of Bothrops jararaca.Acta Crystallogr D Biol Crystallogr 2002;58(Pt 6 Pt 2):1036-8.
28.Oyama E,Takahashi H:Substrate specificity of two thrombin like enzymes (elegaxobin,elegaxobin II) from the venom of Trimeresurus elegans (Sakishima-habu),using neutralizing antibody.Toxicon 2006;48(6):601-10.
29.Markland FS:Snake venoms and the hemostatic system.Toxicon 1998;36(12):1749-800.
30.Maroun RC:Molecular basis for the partition of the essential functions of thrombin among snake venom serine proteinases:the case of thrombin-like enzymes.Haemostasis 2001;31(3-6):247-56.
31.Meier J,Stocker K:Effects of snake venoms on hemostasis.Crit Rev Toxicol 1991;21(3):171-82.
2.Doolittle RF:The structure and evolution of vertebrate fibrinogen.Ann N Y Acad Sci 1983;408:13-27.
3.Tsai IH,Wang YM,Au LC,Ko TP,Chen YH,Chu YF:Phospholipases A2 from Callosellasma rhodostoma venom gland cloning and sequencing of 10 of the cDNAs,three-dimensional modelling and chemical modification of the major isozyme.Eur J Biochem 2000;267(22):6684-91.
4.Siigur J,Samel M,Tonismagi K,Subbi J,Siigur E,Tu AT:Biochemical characterization of lebetase,a direct-acting fibrinolytic enzyme from Vipera lebetina snake venom.Thromb Res 1998;90(1):39-49.
5.Ouyang C,Hong JS,Teng CM:Purification and properties of the thrombin-like principle of Agkistrodon acutus venom and its comparison with bovine thrombin.Thromb Diath Haemorrh 1971;26(2):224-34.
6.Selistre-de-Araujo HS,de Souza EL,Beltramini LM,Ownby CL,Souza DH:Expression,refolding,and activity of a recombinant nonhemorrhagic snake venom metalloprotease.Protein Expr Purif 2000;19(1):41-7.
7.Tu AT,Baker B,Wongvibulsin S,Willis T:Biochemical characterization of atroxase and nucleotide sequence encoding the fibrinolytic enzyme.Toxicon 1996;34(11-12):1295-300.
8.Lee JW,Park W:cDNA cloning of brevinase,a heterogeneous two-chain fibrinolytic enzyme from Agkistrodon blomhoffii brevicaudus snake venom,by serial hybridization-polymerase chain reaction.Arch Biochem Biophys 2000;377(2):234-40.
9.Smith GP:Filamentous fusion phage:novel expression vectors that display cloned antigens on the virion surface.Science 1985;228(4705):1315-7.
10.Scott JK,Smith GP:Searching for peptide ligands with an epitope library.Science 1990;249(4967):386-90.
11.Wang X,Yu J,Sreekumar A,et al.:Autoantibody signatures in prostate cancer.N Engl J Med 2005;353(12):1224-35.
12.Hansen MH,Ostenstad B,Sioud M:Identification of immunogenic antigens using a phage-displayed cDNA library from an invasive ductal breast carcinoma tumour.Int J Oncol 2001;19(6):1303-9.
1.Bardwell JC,McGovern K,Beckwith J:Identification of a protein required for disulfide bond formation in vivo.Cell 1991;67(3):581-9.
2.Sanger F,Nicklen S,Coulson AR:DNA sequencing with chain-terminating inhibitors.Proc Natl Acad Sci U S A 1977;74(12):5463-7.
3.Laemmli UK:Cleavage of structural proteins during the assembly of the head of bacteriophage T4.Nature 1970;227(5259):680-5.
4.Hung CC,Chiou SH:Fibrinogenolytic proteases isolated from the snake venom of Taiwan habu:serine proteases with kallikrein-like and angiotensin-degrading activities.Biochem Biophys Res Commun 2001;281(4):1012-8.
5.Braga MD,Martins AM,de Menezes DB,et al.:Purification and biological activity of the thrombin-like substance isolated from Bothrops insularis venom.Toxicon 2007;49(3):329-38.
6.Towbin H,Staehelin T,Gordon J:Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets:procedure and some applications.Proc Natl Acad Sci U S A 1979;76(9):4350-4.
7.Ambroz KL,Zhang Y,Schutz-Geschwender A,Olive DM:Blocking and detection chemistries affect antibody performance on reverse phase protein arrays.Proteomics 2008;8(12):2379-83.
8.Naha PM,Sorrentino R:Conditional expression of the transformed phenotype in an "epithelial-like" cell line.Cell Biol Int Rep 1979;3(5):421-30.
9.Perez AV,Rucavado A,Sanz L,Calvete JJ,Gutierrez JM:Isolation and characterization of a serine proteinase with thrombin-like activity from the venom of the snake Bothrops asper.Braz J Med Biol Res 2008;41(1):12-7.
1.Gao R,Zhang Y,Meng QX,et al.:Characterization of three fibrinogenolytic enzymes from Chinese green tree viper(Trimeresurus stejnegeri) venom.Toxicon 1998;36(3):457-67.
2.Castro HC,Zingali RB,Albuquerque MG,Pujol-Luz M,Rodrigues CR:Snake venom thrombin-like enzymes:from reptilase to now.Cell Mol Life Sci 2004;61(7-8):843-56.
3.Blomback B,Blomback M,Nilsson IM:Coagulation studies on reptilase,an extract of the venom from Bothrops jararaca.Thromb Diath Haemorrh 1958;1(1):76-86.
4.Matsui T,Fujimura Y,Titani K:Snake venom proteases affecting hemostasis and thrombosis.Biochim Biophys Acta 2000;1477(1-2):146-56.
5.Braud S,Bon C,Wisner A:Snake venom proteins acting on hemostasis.Biochimie 2000;82(9-10):851-9.
6.Santos BF,Serrano SM,Kuliopulos A,Niewiarowski S:Interaction of viper venom serine peptidases with thrombin receptors on human platelets.FEBS Lett 2000;477(3):199-202.
7.Lathan LO,Staggers SL:Ancrod:the use of snake venom in the treatment of patients with heparin-induced thrombocytopenia and thrombosis undergoing coronary artery bypass grafting:nursing management.Heart Lung 1996;25(6):451-60;quiz 461-2.
8.Esnouf MP,Tunnah GW:The isolation and properties of the thrombin-like activity from Ancistrodon rhodostoma venom.Br J Haematol 1967;13(4):581-90.
9.Maeda M,Satoh S,Suzuki S,Niwa M,Itoh N,Yamashina I:Expression of cDNA for batroxobin,a thrombin-like snake venom enzyme.J Biochem 1991;109(4):632-7.
10.Pirkle H:Thrombin-like enzymes from snake venoms:an updated inventory.Scientific and Standardization Committee's Registry of Exogenous Hemostatic Factors.Thromb Haemost 1998;79(3):675-83.
11.Serrano SM,Sampaio CA,Mentele R,Camargo AC,Fink E:A novel fibrinogen-clotting enzyme,TL-BJ,from the venom of the snake Bothrops Jararaca: purification and characterization.Thromb Haemost 2000;83(3):438-44.
12.Dyr JE,Hessel B,Suttnar J,Kornalik F,Blomback B:Fibrinopeptide-releasing enzymes in the venom from the southern copperhead snake (Agkistrodon contortrix contortrix).Toxicon 1989;27(3):359-73.
13.Matsui T,Sakurai Y,Fujimura Y,et al.:Purification and amino acid sequence of halystase from snake venom of Agkistrodon halys blomhoffii,a serine protease that cleaves specifically fibrinogen and kininogen.Eur J Biochem 1998;252(3):569-75.
14.Bell WR,Jr.:Defibrinogenating enzymes.Drugs 1997;54 Suppl 3:18-30;discussion 30-1.
15.Itoh N,Tanaka N,Mihashi S,Yamashina I:Molecular cloning and sequence analysis of cDNA for batroxobin,a thrombin-like snake venom enzyme.J Biol Chem 1987;262(7):3132-5.
16.Au LC,Lin SB,Chou JS,Teh GW,Chang KJ,Shih CM:Molecular cloning and sequence analysis of the cDNA for ancrod,a thrombin-like enzyme from the venom of Calloselasma rhodostoma.Biochem J 1993;294 (Pt 2):387-90.
17.Amiconi Q Amoresano A,Boumis G,et al.:A novel venombin B from agkistrodon contortrix contortrix:evidence for recognition properties in the surface around the primary specificity pocket different from thrombin.Biochemistry 2000;39(33):10294-308.
18.Castro HC,Silva DM,Craik C,Zingali RB:Structural features of a snake venom thrombin-like enzyme:thrombin and trypsin on a single catalytic platform?Biochim Biophys Acta 2001;1547(2):183-95.
19.Suzuki T,Takahashi H:Purification of two thrombin-like enzymes from the venom of Agkistrodon caliginosus (kankoku-mamushi).Toxicon 1984;22(1):29-38.
20.Fan CY,Qian YC,Yang SL,Gong Y:Cloning,sequence analysis and expression in E.coli of the cDNA of the thrombin-like enzyme (pallabin) from the venom of Agkistrodon halys pallas.Biochem Mol Biol Int 1999;47(2):217-25.
21.Hahn BS,Yang KY,Park EM,Chang IM,Kim YS:Purification and molecular cloning of calobin,a thrombin-like enzyme from Agkistrodon caliginosus (Korean viper).J Biochem 1996;119(5):835-43.
22.Nolan C,Hall LS,Barlow GH:Ancrod,the coagulating enzyme from Malayan pit viper (Agkistrodon rhodostoma) venom.Methods Enzymol 1976;45:205-13.
23.Pirkle H,Theodor I,Miyada D,Simmons G:Thrombin-like enzyme from the venom of Bitis gaboniea.Purification,properties,and coagulant actions.J Biol Chem 1986;261(19): 8830-5.
24.Shieh TC,Tanaka S,Kihara H,Ohno M,Makisumi S:Purification and characterization of a coagulant enzyme from Trimeresurus flavoviridis venom.J Biochem 1985;98(3):713-21.
24.Oyama E,Takahashi H:Purification and characterization of a thrombin-like enzyme,elegaxobin,from the venom of Trimeresurus elegans (Sakishima-habu).Toxicon 2000;38(8):1087-100.
26.Nose T,Shimohigashi Y,Hattori S,Kihara H,Ohno M:Purification and characterization of a coagulant enzyme,okinaxobin II,from Trimeresurus okinavensis (himehabu snake) venom which release fibrinopeptides A and B.Toxicon 1994;32(12):1509-20.
27.Watanabe L,Vieira DF,Bortoleto RK,Ami RK:Crystallization of bothrombin,a fibrinogen-converting serine protease isolated from the venom of Bothrops jararaca.Acta Crystallogr D Biol Crystallogr 2002;58(Pt 6 Pt 2):1036-8.
28.Oyama E,Takahashi H:Substrate specificity of two thrombin like enzymes (elegaxobin,elegaxobin II) from the venom of Trimeresurus elegans (Sakishima-habu),using neutralizing antibody.Toxicon 2006;48(6):601-10.
29.Markland FS:Snake venoms and the hemostatic system.Toxicon 1998;36(12):1749-800.
30.Maroun RC:Molecular basis for the partition of the essential functions of thrombin among snake venom serine proteinases:the case of thrombin-like enzymes.Haemostasis 2001;31(3-6):247-56.
31.Meier J,Stocker K:Effects of snake venoms on hemostasis.Crit Rev Toxicol 1991;21(3):171-82.