小反刍兽疫病毒分离株China/Tib/07辅助质粒及微型基因组的构建和鉴定

摘要

本研究旨在构建小反刍兽疫病毒(PPRV)的辅助质粒和微型基因组并验证其功能,为建立PPRV反向遗传操作平台奠定基础.首先构建表达PPRV分离株China/Tib/07的N、P和L蛋白的辅助质粒pCI-N、pCI-P和pCI-L.通过间接免疫荧光试验验证3个辅助质粒转染细胞后的蛋白表达情况,并从mRNA水平上验证蛋白表达的正确性；扩增PPRV基因组两末端序列和eGFP报告基因,三者通过overlap-PCR连接并克隆至转录载体pOL-TV5中构建微型基因组.将构建的微型基因组分别与辅助病毒和3个辅助质粒进行共转染.经鉴定各辅助质粒完全正确,构建的微型基因组不论与辅助病毒还是与3个辅助质粒共转染,报告基因均表达.本试验成功构建了PPRV分离株的表达N、P和L蛋白的3个辅助质粒以及微型基因组,并用微型基因组验证了3个辅助质粒可以作为PPRV反向遗传操作的辅助质粒,为该病毒感染性克隆的构建奠定了基础.%The research was conducted to construct and identify the support plasmids and minigenome of Peste des petits ruminants virus (PPRV) in order to lay a foundation for the establishment of a reverse genetic system for rescue of PPRV. Support plasmids of pCI-N, pCI-P and pCI-L according to the PPRV isolate, China/Tib/07, were constructed and functions were identified by IFA and RT-PCR after transfection. At the same time, the two end sequences of the genome of the PPRV isolate, along with the reporter gene of eGFP were amplified, then the 3 fragments were connected by overlap-PCR and cloned into the transcription vector, pOLTV5, the minigenome was constructed and then co-transfected with the support virus and the 3 support plasmids respectively. As a result, the 3 support plasmids were identified to be correct and the reporter gene of eGFP expressed when the minigenome transfected with either the support virus or the 3 support plasmids. In conclusion, support plasmids of the N, P and L genes and minigenome of the PPRV were constructed successfully. Furthermore, the 3 plasmids worked well with the minigenome, which indicated that the 3 plasmids can work as the support plasmids for the study of reverse genetic operations of China/Tib/07.