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

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。

Expression and Function of Fibrinolytic Enzyme Gene from Gloydius intermediu Venom Gland

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.