TAT-Apoptin融合蛋白分泌表达载体的构建及其活性检测

 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。

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

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.coli. Methods Chemically synthesized TAT sequence and the apoptin gene sequence were ligated together. The TAT-Apoptin gene was sub-cloned into the multiple clone sites of plasmid pET22b+ to construct prokaryotic secretory expression vector of pET22b+-TAT-Apoptin, was and then transformed into E.coliBL21(DE3). Expression of E.coliBL21(DE3) was induced by IPTG, the recombinant protein was secreted into periplasmic space of E.coli. Protein was detected by SDS-PAGE. TAT-Apoptin fusion protein in periplasmic space was incubated with gastric cancer 823 cells, and the anti-tumor effect on 823 cells was evaluated through methyl thiazolyl terazolium (MTT) assay. Results TAT-Apoptin fusion protein can be secreted into periplasmic space of E.coliin soluble state. The maximal anti-tumor effect on 823 cells was 83.95%. Conclusion The recombinant TAT-Apoptin fusion protein vector was successfully established, and the secreted fusion protein has biological activities.