口蹄疫病毒3C基因与增强型绿色荧光蛋白基因的真核共表达  

Co-expression of 3C gene of foot-and-mouth disease virus and enhanced green fluorescent protein gene in Eukaryote

作　　者：魏衍全[1] 田宏[1] 吴锦艳[1] 陈妍[1] 尚佑军[1] 窦永喜[1] 刘湘涛[1] 才学鹏[1] 

机构地区：[1]中国农业科学院兰州兽医研究所家畜疫病病原生物学国家重点实验室国家口蹄疫参考实验室农业部畜禽病毒学重点开放实验室,甘肃兰州730046

出　　处：《中国兽医科学》2011年第5期479-483,共5页Chinese Veterinary Science

基　　金：国家"十一五"科技支撑计划项目(2006BAD06A03);转基因生物新品种培育重大专项(2009ZX08008-010B)

摘　　要：为验证siRNAs对口蹄疫病毒(FMDV)复制的抑制效果,用反转录聚合酶链反应(RT-PCR)扩增Asia 1型口蹄疫病毒Jiangsu/China/2005株的3C基因,克隆入增强型绿色荧光蛋白(EGFP)表达载体pEGFP-N1中,并进行双酶切、PCR及测序鉴定。将阳性重组质粒转染PK-15细胞,检测EGFP的表达和3C基因转录水平。结果显示,经PCR及双酶切鉴定,目的基因片段大小与预期相符,测序结果与Jiangsu/China/2005株相应序列一致。荧光显微镜和流式细胞仪均检测到细胞内有EGFP表达,实时荧光定量PCR检测到细胞内有3C基因的转录。证实,成功构建了FMDV 3C基因与EGFP共表达质粒并在PK-15细胞中获得了表达。To provide an experimental basis for verifying siRNAs effect on inhibition of foot-and-mouth disease virus（FMDV） replication,3C gene was amplified by reverse transcription-polymerase chain reaction （RT-PCR） from Jiangsu/China/2005 strain of Asia 1 serotype of FMDV and was cloned into pEGFP-N1 expression vector with enhanced green fluorescent protein（EGFP） gene and the constructed recombinant was confirmed by PCR,restriction analysis and sequencing. PK-15 cells were transfected with positive re combinant plasmid,the expression of EGFP and transcription level of 3C gene were determined. Both PCR and restriction analysis demonstrated that the target gene fragment was corresponding to that expected in size. The sequence of the target gene was consistent with that of corresponding gene fragment in Jiangsu/ China/2005 strain. The expression of EGFP was observed by both fluorescent microscopy and flow cytome- try. The transcription of 3C gene could be detected by real-time quantitative PCR in the transfected PK-15 cells. The recombinant plasmid for co-expression of FMDV 3C gene with EGFP gene was successfully con structed and expressed in PK 15 cells.

关 键 词：口蹄疫病毒 3C基因 增强型绿色荧光蛋白 真核共表达 

分 类 号：S852.659.6[农业科学—基础兽医学] Q503[农业科学—兽医学]