单链抗体2F3表达条件的优化及其提纯和性质研究  

Studies on the Optimal Expression Condition,Purification and Its Characterization of ScFv-2F3

作　　者：罗元明[1] 牟颖[1] 魏景艳[1] 阎岗林[1] 罗贵民[1] 

机构地区：[1]吉林大学分子酶学工程教育部重点实验室,长春130023

出　　处：《生物工程学报》2002年第1期74-78,共5页Chinese Journal of Biotechnology

基　　金：国家 8 6 3高技术研究发展计划项目 (No .10 3 13 0 1 0 5 );国家自然科学基金项目 (No .2 0 0 72 0 10 );吉林大学博士后人员科研启动基金资助~~

摘　　要：将构建好的单链抗体 2F3表达载体pTMF2F3ScFv转化到大肠杆菌BL2 1(DE3)。先挑选出表达量高的单克隆 ,然后让其在 37℃进行表达 ,并将表达时的培养条件进行优化。实验结果表明 :最佳诱导条件为开始诱导时的菌体密度OD590nm =1.0～ 1.8,所加异丙基 β D 硫代半乳糖苷 (IPTG)的浓度为 0 3～ 0 5mmol L ,诱导时间 7h ,优化后目的蛋白表达量占菌体总蛋白的 2 0 % ,并用发酵罐成功地进行了扩大培养 ,筛选了洗涤包涵体的最佳条件。采用两步法对包涵体复性进行了研究 ,用Westernblotting及ELISA法检测了所表达的单链抗体及其生物活性 。The expression vectors of the gene encoding ScFv-2F3 were transformed into \%E.coli BL21\%(DE3).Clones of higher expression were first selected,then were grown in the presence of IPTG at 37℃ to induce its expression.The culture conditions were carefully optimized.It was found that optimal conditions were as follows:the induction was started as OD 590 reached to 1.0～1.8;the concentration of IPTG was 0.3～0.5mmol/L and induction time is 7h.The yield of ScFv-2F3 expressed in the selected clones is about 20% of the total proteins.The optimal culture conditions were successfully applied to fermenter of 50 L.The conditions of washing the inclusion bodies were also optimized.A two-step method was used to renature the inclusion body.The expression product of interest and its biological activities were characterized with Western blotting and ELISA.A novel selenium-containing single-chain abzyme with GPX activity was prepared.

关 键 词：单链抗体2F3 表达条件 优化 提纯 性质 包涵体 ELISA 抗体酶 

分 类 号：Q813.2[生物学—生物工程]