登革病毒衣壳蛋白在大肠杆菌中的表达及其自组装活性的研究

Expression and self-assembly of dengue virus capsid protein

采用高保真RT-PCR自登革2型病毒43株基因组RNA中扩增全长C基因及缺失羧基端Cv片段,分别构建可表达C及Cv的重组质粒pLEX-C和pLEX-Cv,转化E.coliGI724后用色氨酸诱导表达。经SDS-PAGE分析,表达的C及Cv蛋白相对分子质量分别约为12000和10000,分别约占菌体蛋白总量的19%和13%。Western印迹检测表明重组表达的C蛋白均可被特异识别登革病毒衣壳蛋白的单克隆抗体特异识别。表达的蛋白经过硫酸铵沉淀和蔗糖密度梯度离心后,通过琼脂糖凝胶电泳和负染电镜均未能检测到衣壳样颗粒的存在,说明登革病毒衣壳蛋白可能不具体外自组装活性。

The C and C v gene fragments were amplified by high fidelity RT-PCR from dengue virus Chinese isolate D2-43.Then the products of RT-PCR were cloned into T vector and cut with KpnI and Bam HI.The pLEX expres-sion vectors of C and C v gene were constructed and expressed in E.coli strain GI724induced by tryptophan.The expressed recombinant proteins were identified by SDS-PAGE and Western Blot.The relatively molecular weight of the expressed C and C v proteins were12kD and10kD,and the expressed output of the recombinant proteins were approximate19%and13%of the total bacterial protein.The results of Western Blot indicated the expressed C and C v proteins could react with the MAb8H8that specifically identified the capsid protein of dengue-2virus.No cap-sid-like particles were detected by both native agarose gel assay and negative-stain electron microscopy.And so the dengue capsid protein might not self-assembly in vitro.