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. coli BL21(DE3). Expression of E. coli BL21(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. coli in 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.%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.
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