重组蛇毒纤溶因子特异性相关突变体的构建、表达和纯化  

Construction, Expression and Purification of Specificity-related Muta tion of Recombinant Nonhemorrhagic Fibrin(ogen)olytic Metalloprotease

作　　者：马涛[1] 邱鹏新[1] 颜光美[1] 

机构地区：[1]中山大学基础医学院药理学教研室,广州510080

出　　处：《热带医学杂志》2005年第3期257-259,共3页Journal of Tropical Medicine

基　　金：广东省科技厅计划资助项目(No.2003A10905)

摘　　要：目的通过重组蛇毒纤溶因子特异性相关突变体的构建、表达和纯化,初步考查其特异性变化。方法SOEingPCR方法构建突变体,突变体在P.pastoris酵母中诱导表达。表达蛋白经过柱纯化,纯化蛋白用SDS-PAGE和Westernblot鉴定,并用MALDI-TOF初步分析其作用于氧化胰岛素B链的特异性。结果SDS-PAGE和Westerenblot证实得到纯化的突变体。MALDI-TOF质谱分析揭示,rFⅡ及突变体对氧化胰岛素B链的早期裂解都发生在第12～13位点,但次级的裂解模式有所不同。结论删除的序列可能与rFⅡ的特异性相关。Objective To investigate the role of deleted sequence found b y alignment in specificity of onhemorrhagic recombinant fibrin(ogen)olytic metal loprotease. Methods SOEing PCR was used to generate mutated DNA fragment. The m utated fibrin(ogen)olytic metalloprotease was expressed in transformed P.pastori s cells and the purified proteins were analyzed by SDS-PAGE and Western blot.The n,MALDI-TOF was used to analyze the cleavage specificity of the enzyme on the ox idized insulin-B chain. Results Mutation was confirmed by SDS-PAGE and Western blot. MALDI-TOF analysis revealed the different mode of secondary cleavage site. Conclusion The deleted sequence in rFⅡ may be responsible for the specificity of the enzyme.

关 键 词：蛇毒 重组 溶纤 突变 序列 特异性 

分 类 号：R96[医药卫生—药理学]