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Bam 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 genome of a strain belonging to OHM/02 was amplified by RT-PCR and cloned into vector pUC57 to construct 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 Bam HⅠsite 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 infectious clone is an important tool for study molecular pathogenesis of FMDV and development of novel vaccine against FMDV.
作者
苗书魁
魏玉荣
魏婕
米晓云
汪萍
马文戈
王延
薛英
易忠
黄炯
MIAO Shu-kui;WEI Yu-rong;WEI Jie;MI Xiao-yun;WANG Ping;MA Wen-ge;WANG Yan;XUE Ying;YI Zhong;HUANG Jiong(Institute of Veterinary Medicine/Research Center of Animal Clinical,Xinjiang Acadamy of Animal Sciences,Urumqi,Xinjang,830011,China;Tecon Biology Co.Ltd,Urumqi,Xinjang,830030,China)
出处
《动物医学进展》
北大核心
2018年第1期1-6,共6页
Progress In Veterinary Medicine
基金
国家科技支撑计划项目(2013BAD12B04)
新疆维吾尔自治区科研机构创新发展专项资金项目(2016D04008)
新疆维吾尔自治区公益性科研院所基本科研业务经费项目(2014
2015)
关键词
口蹄疫病毒
全长CDNA
转染
病毒拯救
Foot-and-mouth disease virus
full length cDNA
transfection
virus rescue
