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| Expression and Function of Fibrinolytic Enzyme Gene from Gloydius intermediu Venom Gland |
| LIU Yu-fen, DONG Li-li, SUN Yu-gang, LIU Peng, CHEN Hui, ZHAO Wen-ge |
| College Life Science and Technology, Harbin Normal University, Harbin 150025, China |
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Abstract Objective Fibrinolytic enzymes (FLE) which wildly exist in many kinds of snake venoms as a kind of novel powerful thrombotic agent can directly lysis fibrin or fibrinogen.It has the highest value in anti-thrombotic diseases. Methods:The FLE sequence, cloned from Gloydius intermedius venom gland, which was cutted by double enzyme digestion from pMD18T-FLE plasmid was subcloned to pFASTBACHTa eukaryotic expressive vector. Recombinant eukaryotic expressive plasmid, pFASTBACHTa-FLE, was constructed after transformation, selection and identification. The recombinant plasmid was injected into mice in large quantity and large volume in a short time through tail vein. Conclusion: Recombinant FLE was examined in liver tissue by SDS-PAGE and Western blot. Immunohistochemistry showed that there were a great of recombinant FLE proteins could be found in liver tissue. The fibrinolytic activity of recombinant protein revealed by the modified fibrin plate method demonstrated that it had efficiently fibrinolytic activity to be dose-dependent and time-relative. Therefore, a solid foundation for using snake venom FLE from Gloydius intermedius was built.
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Received: 09 July 2012
Published: 25 October 2012
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[1] 赵文阁. 黑龙江省两栖爬行动物志. 北京:科学出版社,2008.208-212. Zhao W G. Fauna of amphibians and reptiles in Heilongjiang province. Beijing:Science Press, 2008.208-212.
[2] 赵文阁,刘晓龙,郭玉民.中介蝮在黑龙江省的发现.四川动物,2000,19(3):157-158. Zhao W G, Liu X L, Guo Y M. Discovery of Gloydius intermedius snake in Heilongjiang province. Sichuan Journal of Zoology, 2000,19(3):157-158.
[3] 赵尔宓,黄美华,宗愉.中国动物志爬行纲有鳞目蛇亚目. 北京:科学出版社,1998.402-407. Zhao E M, Huang M H, Zong Y. Ophidia, Squamata, Reptilia of China Fauna. Beijing: Science Press, 1998.402-407.
[4] Doley R, Kini R M. Protein complexes in snake venom. Cellular and Molecular Life Sciences, 2009, 66: 2851-2871.
[5] Koh D C, Armugam A, Jeyaseelan K. Snake venom components and their applications in biomedicine. Cellular and Molecular Life Sciences, 2006, 63: 3030-3041.
[6] Margie P. From viper’s venom to drug design: treating hypertention. The Faseb Journal, 2004, 18(4):421-424.
[7] 王春玲, 曹小红, 闫仲丽. 江浙蝮蛇蛇毒中抗肿瘤蛋白的分离纯化及活性研究. 中国生物工程杂志, 2005,27(4): 278-281. Wang C L, Cao X H, Yan Z L. Study on purification and anti-tumor activity of Agkistrodom halys Pallas. China Biotechnology, 2005,27(4): 278-281.
[8] 孟秀君,王洪新,王轶晶,等. 白眉蝮蛇毒凝血酶应用于消化道出血的临床观察.医学理论与实践,2006,19(10):1147-1148. Meng X J, Wang H X, Wang Y J, et al. Haemostatic effect of hemocoagulase for injection in bleeding of the gastro intestinal tract. The Journal of Medical Theory and Practice, 2006,19(10):1147-1148.
[9] Furtado M F, Maruyama M. Comparative study of nine bothrops snake venoms from adult snakes and their offspring.Toxicom,1991,29:219-229.
[10] Markland F S. Snake venom fibrinogenolytic and fibrinolytic enzymes: an Updated inventory. Thromb Haemost,1998, 79: 668-674.
[11] Ouyang C, Huang T F. Purification and characterization of the fibrinolytic principle of agkistrodon acutus venom. Biochimicaet Biophysica Acta, 1976, 439(1): 146-153.
[12] 于德涵, 刘玉芬, 赵文阁. 中介蝮蛇毒纤溶酶基因克隆及生物信息学分析. 中国农学通报, 2009, 25(6): 59-63. Yu D H, Liu Y F,Zhao W G. Cloning and bioinformatic analysis of fibrinolytic enzyme from Gloydius intermedius.Chinese Agricultural Science Bulletin, 2009, 25(6): 59-63.
[13] Bjarnason J B, Fox J W. Hemorrhagic metalloproteinases from snake venom. Pharmacology and Therapeutics, 1994, 62: 325-372.
[14] 张箴波, 张学荣, 舒雨雁. 蛇毒纤溶酶的性质及应用研究. 蛇志, 2005, 17(3): 174-176. Zhang Z B, Zhang X R, Shu Y Y. The study of application and characterization of fibrinolytic enzyme from snake venom. Journal of Snake, 2005, 17(3): 174-176.
[15] Swenson S, Markland F S. Snake venom fibrin(ogen)olytic enzyms. Toxicon, 2005, 45(8): 1021-1039.
[16] Toombs C F. Alfimeprase: Pharmacology of a novel fibrinolytic metalloproteinase for thrombosis. Haemostasis, 2001, 31: 141-147.
[17] 殷志扬, 张陆勇, 季慧芳,等. 蛇毒纤溶酶的一般药理作用研究. 中国药科大学学报, 1998, 29(2): 132-134. Yin Z Y, Zhang L Y, Ji H F, et al. Experimental studies of the general pharmacology of nevin fibrinolytic enzyme.Journal of China Pharmaceutical University, 1998, 29(2): 132-134.
[18] 于德涵. 中介蝮蛇毒纤溶酶基因克隆及原核表达. 哈尔滨:哈尔滨师范大学硕士论文, 2009. Yu D H. Cloning and prokaryotic expression of fibrinolytic enzyme gene from Gloydius intermedius. Harbin:Harbin Normal University,2009.
[19] 张守涛, 周雁胜, 赖学华,等. 蛇毒纤溶酶Alfimeprase在大肠杆菌中的可溶表达和纯化. 中国生物工程杂志, 2006, 26(3): 31-36. Zhang S T, Zhou Y S, Lai X H, et al. Soluble expression and purification of snake venoms fibrino(geno)lytic emzyme alfimeprase in E.coli.China Biotechnology, 2006, 26(3): 31-36.
[20] Shi J, Zhang S T, Zhang X J, et al. Expression, purification, and activity identification of alfimeprase in Pichia pastoris. Protein Expression and Purification, 2007, 54(2): 240-246.
[21] Zhang G, Budker V, Wolff J A. High levels of foreign gene expression in Hepatocytes after tail vein injections of naked plasmid DNA. Human Gene Therapy, 1999, 10: 1735-1737.
[22] Liu F, Song Y K, Liu D. Hydrodynamics-based transfection in animals by systemic administration of plasmid DNA. Gene Therapy, 1999, 6(7): 1258-1266.
[23] 朱传龙, 宁琴, 严伟明,等. 尾静脉高压注射质粒DNA后体内表达的规律. 中国比较医学杂志, 2006, 16(4): 226-229. Zhu C L, Ning Q, Yan W M, et al. Plasmid DNA distribution and gene expression generated via tail vein hydrodynamic injection.Chinese Journal of Comparative Medicine, 2006, 16(4): 226-229. |
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