肠道病毒71型的全长cDNA感染性克隆的构建与鉴定

摘要

Objective To constructan infectious full length cDNA clone of enterovirus 71.Methods A cDNA fragment with a T7 promoter at the 5' end and a poly (A) tail at the 3' end was amplified by RT-PCR from the genome of the EV71/87-2008 Xi'an strain directly.This fragment was then ligated to pBR322 via Sal Ⅰ and BspE Ⅰ site.Then the in vitro synthesized RNA transcripts were transfected into RD cells to produce the rescued virus.The negative-strand viral RNA was detected from the RD cells lysates by RT-PCR.The one step growth curve of recovered EV71 was measured by plaque assay.Results The full length cDNA clone of EV71/87-2008 Xi'an was constructed and the infectious of this clone was validated successfully.The EV71 recovered virus was proved functionally identical to its template.Conclusions The infectious clone of EV71/87-2008 Xi'an strain was successfully constructed.%目的 构建一株肠道病毒71型的全长cDNA克隆并验证感染性.方法 用长片段高保真RT-PCR方法扩增肠道病毒71型87-2008 Xi'an株的cDNA,装入pBR322载体.将该克隆线性化,体外转录后转染RD细胞后,通过观察CPE及RT-PCR验证其感染性.随后利用空斑法测定拯救病毒的一步生长曲线.结果 成功构建了肠道病毒71型87-2008 Xi'an株的全长cDNA克隆；体外转录及转染RD细胞60 h后可观察到明显的CPE现象；RT-PCR在转染RD细胞中检测到病毒的存在；成功利用空斑法测定了拯救病毒的一步生长曲线,最大滴度达到2×107 pfu/mL.结论 成功构建了肠道病毒71型87-2008 Xi'an株的全长cDNA克隆并验证了其感染性,同时测定了拯救病毒的一步生长曲线,与野生型病毒相近.