中介蝮蛇毒纤溶酶基因真核表达载体的构建及其功能的研究

中国生物工程杂志

2012, Vol. 32

Issue (10): 1-6

研究报告

中介蝮蛇毒纤溶酶基因真核表达载体的构建及其功能的研究

刘玉芬, 冬黎黎, 孙玉刚, 刘鹏, 陈辉, 赵文阁

哈尔滨师范大学生命科学与技术学院哈尔滨 150025

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

全文: PDF(686 KB) HTML

摘要： 目的:蛇毒纤维蛋白溶解酶能直接溶解纤维蛋白或纤维蛋白原,在心脑血管疾病的治疗方面具有潜在的应用价值。方法:采用双酶切技术从pMD18T-FLE I克隆载体上获得中介蝮蛇毒纤溶酶基因编码区,再将其亚克隆到真核表达载体pFASTBACHTa上,经转化、筛选和鉴定,获得重组真核表达质粒pFASTBACHTa-FLE。重组质粒经小鼠尾静脉快速注入小鼠体内,进行瞬时表达。结果:经SDS-PAGE和Western blot检测,表明在小鼠肝脏组织中有重组FLE蛋白表达。免疫组织化学检测证明该蛋白在小鼠肝脏组织中大量表达。纤维蛋白平板法鉴定该重组蛋白具有较高的纤溶活性,其活性呈现出剂量相关性和时间依赖性。因此为进一步对中介蝮蛇毒纤溶酶的应用奠定了坚实的基础。

关键词： 中介蝮蛇; 纤溶酶; 真核表达; 纤溶活性

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.

Key words: Gloydius intermedius Fibrinolytic enzyme Eukaryotic expression Fibrinolytic activity

收稿日期: 2012-07-09 出版日期: 2012-10-25

ZTFLH:

Q782

基金资助: 黑龙江省自然科学基金面上项目(C200922)资助项目

通讯作者: 赵文阁,电子信箱:zhaowenge311@126.com E-mail: zhaowenge311@126.com

	服务
	把本文推荐给朋友
	加入引用管理器
	E-mail Alert
	RSS
	作者相关文章
	刘玉芬
	冬黎黎
	孙玉刚
	刘鹏
	陈辉
	赵文阁

引用本文:

刘玉芬, 冬黎黎, 孙玉刚, 刘鹏, 陈辉, 赵文阁. 中介蝮蛇毒纤溶酶基因真核表达载体的构建及其功能的研究[J]. 中国生物工程杂志, 2012, 32(10): 1-6.

LIU Yu-fen, DONG Li-li, SUN Yu-gang, LIU Peng, CHEN Hui, ZHAO Wen-ge. Expression and Function of Fibrinolytic Enzyme Gene from Gloydius intermediu Venom Gland. China Biotechnology, 2012, 32(10): 1-6.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/ 或 https://manu60.magtech.com.cn/biotech/CN/Y2012/V32/I10/1

[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.

[1]	程旭,杨雨睛,吴赛男,侯勤龙,李咏梅,韩慧明. 金黄色葡萄球菌SarA、IcaA及其融合基因的DNA疫苗构建及在小鼠免疫应答中的初步研究 ^*[J]. 中国生物工程杂志, 2020, 40(7): 41-50.
[2]	郎巧利,余琳,何麒麟,葛良鹏,杨希. 高效构建卵清白蛋白scFv噬菌体文库及其筛选 ^*[J]. 中国生物工程杂志, 2018, 38(11): 25-31.
[3]	王瑜,刘治,白文成,许丽萍,韩润林. 重组不可分型流感嗜血杆菌E蛋白与血浆脂蛋白(a)的相互作用[J]. 中国生物工程杂志, 2017, 37(12): 14-20.
[4]	王青, 徐彦召, 魏晓晓, 王秋霞, 杭柏林, 孙亚伟, 王飞飞, 胡建和. 猪繁殖与呼吸综合征病毒GP5a多克隆抗血清的制备[J]. 中国生物工程杂志, 2015, 35(8): 38-43.
[5]	王金胜, 姜浩武, 张晋霞, 潘磊, 赵凤芝, 于云飞, 蔡亚雄, 邓宁. 抗FGF2人鼠嵌合抗体在HEK293T细胞中的表达优化[J]. 中国生物工程杂志, 2014, 34(5): 14-22.
[6]	钱辉, 张充, 陆兆新, 别小妹, 赵海珍, 吕凤霞. 内生多粘芽孢杆菌EJS-3纤溶酶基因在毕赤氏酵母中的表达[J]. 中国生物工程杂志, 2014, 34(12): 45-50.
[7]	韩慧明, 李咏梅. 纳豆激酶基因在大肠杆菌中的表达及活性分析[J]. 中国生物工程杂志, 2014, 34(10): 49-54.
[8]	庞敏, 王海龙, 郭民, 郭睿. 人ANKRD49基因真核表达载体的构建及其功能的初步研究和RNA干扰靶点的鉴定[J]. 中国生物工程杂志, 2014, 34(10): 15-21.
[9]	徐文琦, 柴晓杰, 张婷, 代靖宇, 张晓琳. 胰蛋白酶抑制剂KSTI3基因新型真核表达载体的构建及其在盐藻中的表达[J]. 中国生物工程杂志, 2011, 31(8): 29-34.
[10]	黄明星, 叶韵, 韩雅莉. 纤溶活性蛋白检测方法研究进展[J]. 中国生物工程杂志, 2011, 31(10): 113-120.
[11]	方静, 周颖, 李智慧, 达飞, 胡要磊, 毛建平. 小鼠淋巴细胞抗原CTLA-4的胞外肽段表达及纯化[J]. 中国生物工程杂志, 2010, 30(11): 61-64.
[12]	王佃亮姜合作王瑞玲刘万顺张艳梅孙晋伟. 一种新型海洋纤溶酶的分离纯化与性质鉴定[J]. 中国生物工程杂志, 2010, 30(08): 47-51.
[13]	王正茂李琳管文燕李越希. 真核细胞表达单纯疱疹病毒I型包膜糖蛋白B及其抗原性与免疫原性分析[J]. 中国生物工程杂志, 2010, 30(06): 8-13.
[14]	陈武莫炜张艳玲宋钢宋后燕. 重组人纤溶酶原基因的酵母表达、产物纯化及鉴定[J]. 中国生物工程杂志, 2009, 29(10): 18-22.
[15]	刘莉,周政,采克俊,张易祥,何湃,曹访. EGFP-LacZ双报告基因真核表达载体的构建及体外表达[J]. 中国生物工程杂志, 2009, 29(01): 65-69.

Viewed

Full text

Abstract

Cited

Shared

Discussed