Apoptin原核表达载体的构建及在大肠杆菌中的表达

目的构建Apoptin的原核表达载体,并制备抗原物质Apoptin融合蛋白。方法在获得Apoptin融合基因的基础上,成功构建了Apoptin的高效原核表达载体pET-28a( )-Apoptin,将该质粒转化至大肠杆菌E.coliBL21(DE3)受体菌中,以IPTG对其进行诱导表达,聚丙烯酰胺凝胶电泳分析目的蛋白。结果转化有Apoptin的原核表达载体pET-28a( )-Apoptin的大肠杆菌E.coliBL21(DE3)经IPTG诱导后,经SDS-PAGE分析,在相对分子质量约17 000的位置出现目的蛋白条带,大小与Apoptin融合蛋白一致。结论Apoptin原核表达载体pET-28a( )-Apoptin能够表达出Apoptin融合蛋白,为进一步的Apoptin研究和制备Apoptin抗体奠定了基础。

Construction of a prokaryotic expression vector for Apoptin and expression in E.coli

Objective To construct an Apoptin prokaryotic vector,aiming to produce antigenic fusion protein Apoptin. Methods The Apoptin gene was amplified from the template of plasmid pSSCHG/NT4-Apoptin-HA2-TAT by PCR.The Apoptin was sub-cloned into the multiple clone sites of plasmid pET-28a( ) to get the prokaryotic vector of pET-28a( )-Apoptin,which was transformed into E.coli BL21(DE3).Expression of E.coli BL21(DE3) was induced by IPTG.The specific protein expression was detected by SDS-PAGE. Results The fusion protein was expressed with high efficiency in E.coli BL21(DE3) transformed by pET-28a( )-Apoptin after induction with IPTG.The specific fusion protein had an apparent related molecular weight of about 17 000 ku as indicated by SDA-PAGE analysis. Conclusion The Apoptin prokaryotic expression vector with pET-28a( )-Apoptin can effectively express Apoptin fusion protein,laying a foundation for further study of Apoptin and preparation of antibodies against Apoptin.