TAT-Apoptin融合蛋白分泌表达载体的构建及其活性检测  

Construction of Prokaryotic Secretory Expression Vector of TAT-Apoptin and Expression in E. coli

作　　者：刘雪梅[1] 崔剑[1] 侯伟健[2] 李其久[1] 贲松彬[1] 

机构地区：[1]辽宁大学生命科学院生化与分子生物学教研室,沈阳110036 [2]中国医科大学基础医学院组织工程教研室,沈阳110001

出　　处：《中国医科大学学报》2013年第1期45-48,共4页Journal of China Medical University

基　　金：辽宁省教育厅高校科研计划(2009A310);辽宁省科学技术计划项目(2010225036);沈阳市科技计划项目(2009-15)

摘　　要：目的构建具有自主跨膜能力的Apoptin融合蛋白表达载体,使其分泌表达,避开包涵体复性,简化制备工艺。方法人工合成反式激活蛋白(TAT)序列,与Apoptin序列融合,连接到pET22b+载体上,构建原核分泌表达载体pET22b+-TAT-Apoptin,IPTG诱导目的蛋白分泌表达到大肠杆菌周质空间,提取蛋白行SDS-PAGE分析,MTT法检测分泌表达的融合蛋白对胃癌823细胞的抑制作用。结果重组TAT-Apoptin蛋白可分泌至大肠杆菌周质空间中,以可溶状态存在,对胃癌823细胞的最大抑制率为83.95%。结论成功构建重组TAT-Apoptin蛋白表达载体,分泌表达的可溶性融合蛋白具有生物活性。Objective To simplify the preparation process of Apoptin by constructing a TAT-Apoptin fusion protein expression vector with independent ability to cross membrane in E. coil Methods Chemically synthesized TAT sequence and the Apoptin gene sequence were hgated together. The TAT-Apoptin gene was sub-cloned into the rnuhiple clone sites of plasroid pET22b＋ to construct prokaryofic secretory expression vector of pET22b＋-TAT-Apoptin, was and then transformed into E. coli BI21 （DE3）. Expression of E. coli BL21 （ DE3 ） was in- duced by IPTG, the recombinant protein was secreted into pefiplasmic space of E. coil Protein was detected by SDS-PAGE. TAT-Apoptin fusion protein in pefiplasmie space was incubated with gastric cancer 823 cells, and the anti-tumor effect on 823 cells was evaluated through methyl thiazolyl terazolium （MTF） assay. Results TAT-Apeptin fusion protein can be secreted into periplasmic space of E. coli in soluble state. The maximal anti-tumor effect on 823 cells was 83.95%. Conclusion The recombinant TAT-Apoptin fusion protein vector was suc- cessfully established, and the secreted fusion protein has biological activities.

关 键 词：反式激活蛋白 凋亡素 分泌表达 凋亡 

分 类 号：Q786[生物学—分子生物学]