诺如病毒衣壳蛋白在Tn5细胞中的表达及纯化

Expression of noro virus capsid protein in Tn5 cells

将人诺如病毒VA387株ORF2基因插入载体pFastBac1中,转化DH10Bac感受态细胞获得Bacmid-NoV-ORF2;脂质体介导转染sf9昆虫细胞,获得表达NoV-ORF2的重组杆状病毒Ac-NoV-ORF2.冻融破碎经Ac-NoV-ORF2感染的Tn5细胞,离心收集上清进行分子筛纯化.10%SDS-PAGE分析结果显示,重组病毒感染的Tn5细胞可见特异的蛋白条带;目的蛋白条带为相对分子质量59kD和56kD的两个条带;电镜观察发现表达的诺如病毒衣壳蛋白成功装配成了大小约为40~50nm的VLPs.结果表明在Tn5细胞中实现了诺如病毒衣壳蛋白的表达和VLPs的装配.

The ORF2 gene sequence of NoVs VA387was synthesized and inserted into vector pFastBacl. The DH10Bac competent cells were transformed by pFastBacl-NoV-ORF2 to obtain Bacmid-NoV- ORF2. The sf9 cells transfected by Bacmid-NoV-ORF2 were harvested as recombinant baculovirus Ac- NoV-ORF2 virus. Tn5 cells were infected by Ac-NoV-ORF2 virus and clarified by centrifugation. Then the supernatant was extracted and purified by molecular sieve purified. Capid protein were collected, an- alyzed for purity by 10% SDS-PAGE, and observed by electron microscopy. The Tn5 cells transfected with Ac-NoV-ORF2 virus showed specific protein by 10% SDS-PAGE profile. The result of 10% SDS- PAGE profile showed that the capsid protein mainly located in two bands with relative molecular masses of 59 kD and 56 kD. Electron microscopy showed that the expressed capid protein was assembled to VLPs at sizes of about 40--50 nm. So the capsid protein of NoVs was successfully expressed in Tn5 cells and assembled into VLPs.