【摘要】 家蚕核型多角体病毒(Bombyx mori nucleopolyhedrovirus,Bm NPV)基因组中的orf90基因(Bm90)是一个未知功能的保守基因。利用λRed重组系统和Bac-to-Bac系统构建野生型病毒Wt Bacmid-polh-egfp、Bm90基因缺失型病毒Bm90ko-polh-egfpBacmid及Bm90基因补回型病毒Bm90re-polh-egfp-Bacmid,并制备Bm90蛋白的多克隆抗体,研究Bm90基因在病毒感染周期中的表达以及对病毒增殖和组装的影响。将3种重组病毒分别转染Bm N细胞,Western blot检测显示Bm90蛋白在病毒转染Bm N细胞后72 h有表达;但用荧光显微镜观察Bm90基因缺失型病毒转染的Bm N细胞中没有出现绿色荧光,且检测其病毒滴度为0;用透射电子显微镜观察在Bm90基因缺失型病毒转染的Bm N细胞中没有子代病毒粒子出现。研究结果表明,Bm90基因缺失会抑制子代病毒粒子的组装和产生,使病毒失去侵染性,Bm90基因是病毒增殖及组装的必需基因。更多还原

【Abstract】 orf90 (Bm90) gene is one of the conserved genes with unknown function in Bombyx mori nucleopolyhedrovirus( Bm NPV) genome. The wild type virus Wt Bacmid-polh-egfp,Bm90 knockout virus Bm90ko-polh-egfp-Bacmid and Bm90 repair virus Bm90re-polh-egfp-Bacmid were constructed by λRed recombination system and Bac-to-Bac system,and a polyclonal antibody against Bm90 protein was prepared to study the expression of Bm90 and its effect on the proliferation and assembly of Bm NPV in the whole virus infection cycle. The three types of virus DNA were respectively used to transfect Bm N cells. Bm90 protein was detected in Bm N cells at 72 h post transfection by Western blotting. No fluorescence was observed in Bm N cells transfected with Bm90ko-polh-egfp-Bacmid DNA under fluorescence microscope and the viral titer was 0. Observation under transmission electron microscope showed that no nucleocapsid appeared in Bm N cells transfected with Bm90ko-polh-egfp-Bacmid DNA. In summary,knockout of Bm90 could inhibit production and assembly of infectious budded virus and eliminate the infectivity of Bm NPV,demonstrating that Bm90 gene is required for the production and assembly of nucleocapsids.更多还原