口蹄疫病毒O型OHM/02株全长cDNA感染性克隆的构建

摘要

为构建O型口蹄疫病毒(FMDV)的感染性克隆,采用 RT-PCR将 O型 FMDV OHM/02株全长基因组分为5个重叠的片段进行扩增,克隆至载体 pUC57中,获得全长 cDNA克隆 pPO-1.将线性化的pPO-1与含有编码 T7 RNA 聚合酶的真核表达重组质粒共转染 BHK-21细胞,出现致细胞病变效应(CPE).对拯救的病毒进行RT-PCR扩增、酶切、测序,表明人工设计的分子标记B am HⅠ酶切位点消失,排除了亲本毒株污染的可能性.间接免疫荧光试验可以检测到绿色荧光,电镜观察可观察到病毒粒子,表明构建了具有感染性的 OHM/02毒株全长 cDNA克隆.病毒生长曲线表明,拯救病毒与亲本病毒的复制能力和增殖特性相似.OHM/02株全长 cDNA感染性克隆的构建及拯救可为FMDV致病机理的研究提供一种重要工具,可促进疫苗的研发.%To establish the infectious cDNA clone of foot-and-mouth disease virus(FMDV),the complete ge-nome of a strain belonging to OHM/02 was amplified by RT-PCR and cloned into vector pUC57 to con-struct FMDV recombinant plasmid.The recombinant plasmid was transfected into BHK-21 cells with the plasmid that expresses T7 RNA polymerase.The typical CPE caused by rescued FMDV was observed.The rescued FMDV,which has a deleted B am HⅠ site to differentiate contamination from parental FMDV,was successfully obtained.Fluorescent light was observed by the indirect immunofluorescence assay and FMDV particles were observed by the immune electron microscope compared its parental virus.This stable infec-tious clone is an important tool for study molecular pathogenesis of FMDV and development of novel vac-cine against FMDV.