整合埃博拉病毒囊膜蛋白的伪病毒的构建

Construction of Ebola virus glycoprotein-pseudotyped lentivirus

埃博拉病毒(EBOV)是一种能够引起人类和灵长类动物的烈性出血热的病原体,致死率高达90%。对该病毒活病毒的操作仅能够在生物安全4级实验室进行,高级别的生物安全要求限制了病毒的研究。为建立一个能够表达EBOV囊膜蛋白(GP)的伪病毒,本研究利用表达GP基因的重组质粒,与囊膜蛋白缺失、表达绿色荧光蛋白(GFP)的人免疫缺陷病病毒1型(HIV-1)基因组的重组质粒共转染人胚胎肾细胞(293T),以包装整合含有囊膜蛋白的伪型EBOV。结果表明,EBOV GP蛋白能够正确组装至HIV-1病毒表面;利用组装的伪型病毒粒子感染293T、人宫颈癌细胞(Hela)、人骨肉瘤细胞(Ghost)和非洲绿猴肾细胞(Vero)几种细胞系,结果显示在不同细胞系中均可检测到GFP蛋白的表达,表明组装的伪型病毒可以模拟野生型病毒感染相关细胞。本研究构建的伪型EBOV作为活病毒的替代工具,为研究EBOV的感染机制及中和抗体的筛选提供了一个良好的平台。

Ebola virus, the pathogen of Ebola haemorrhagic fever, causes human and primates severe disease and high case-fatality rates （up to 90%）. As a highly contagious pathogen, the operation of live viruses has to be conducted in biosafety- level 4 facilities. In order to develop a surrogate for live virus, Ebola virus glycoprotein （Ebola-GP） expressing recombinant plasmid was co-transfected with human immunodeficiency syndrome virus （HIV-1） based retroviral vector into human embryonic kidney （HEK） 293T cells for the construction of the Ebola-GP pseudotyped lentivirus. Results showed that the Ebola GP successfully packaged with H1V-1 viral genome. In addition, the expression of reporter gene （GPF） could be detected in Ghost, Vero, 293T and Hela cells when the pseudotyped viruses were used to infect these cells lines, suggesting that the GP-pseudotyped lentiviruses were able to mimic the live viruses to infect target cells. This study provided an practically platform for further research on the mechanism of virus infection and Ebola virus neutralizing antibody screening.