多聚精氨酸蛋白转导域-凋亡素融合蛋白的原核表达及其生物活性

Prokaryotic Expression and Biological Activity of Poly-Arg Protein Transduction Domain-Apoptin Fusion Protein

目的原核表达多聚精氨酸蛋白转导域-凋亡素融合蛋白,并检测其生物活性。方法应用PCR法扩增Arg9-VP3序列,与载体pET-43.1a连接后,转化E.coliBL21(DE3),IPTG诱导表达。表达产物经Ni2+-NTA纯化后,进行肠激酶裂解、超滤浓缩,并检测其生物活性。结果重组表达质粒pET-43.1a-Arg9-VP3经酶切鉴定和序列分析,证明构建正确。转化E.coliBL21(DE3)后,重组蛋白获得可溶性表达。纯化的融合蛋白纯度达90%以上,可抑制HeLa细胞增殖。结论已成功地在大肠杆菌中表达了多聚精氨酸蛋白转导域-凋亡素融合蛋白,纯化的融合蛋白具有诱导HeLa细胞凋亡的能力。

Objective To express poly-Arg protein transduction domain （PTD）-apoptin fusion protein in prokaryotic cells and determine its biological activity. Methods Arg9-VP3 sequence was amplified by PCR and inserted into vector pET-43.1a, and the constructed recombinant plasmid pET43.1a-Arg9-VP3 was transformed to E. coli BL21 （DE3） for expression under induction of IPTG. The expressed product was purified by Ni2＋-NTA chromatography, lysed with enterokinase, concentrated by ultrafiltration and determined for biological activity. Results Both restriction analysis and sequencing proved that recombinant plasmid pET43.1a-Arg9-VP3 was constructed correctly. Recombinant fusion protein was expressed in a soluble form, reached a purity of more than 90% after purification and showed inhibitory effect on the proliferation of HeLa cells. Conclusion Poly-Arg PTD-apoptin fusion protein was successfully expressed in E. coli and purified , which induced the apoptosis of HeLa cells.