逆转录病毒载体介导的口蹄疫病毒衣壳前体基因和绿色荧光蛋白基因在BHK-21细胞中的表达  

Expression of the Capsid Precursor Protein Gene of Foot-and-mouth Disease Virus and Green Fluorescent Protein Gene in BHK-21 Cells Mediated by Retroviral Vector

作　　者：李炯[1] 刘艳红[1] 安芳兰[1] 刘俊林[1] 刘湘涛[1] 尚佑军[1] 殷宏[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/农业部畜禽病毒学重点开放实验室/农业部草食动物疫病重点开放实验室,兰州730046

出　　处：《畜牧兽医学报》2009年第3期376-382,共7页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：国家"863计划"(2006AA10A204);甘肃省科技攻关项目(ZGS-052-A41-0006-03);中国农业科学院兰州兽医研究所所长基金项目

摘　　要：为建立逆转录病毒载体介导的口蹄疫病毒衣壳前体蛋白哺乳动物细胞表达体系,将口蹄疫病毒(FMDV)衣壳前体基因(P1)和绿色荧光蛋白基因(EGFP)依次插入逆转录病毒载体pBABEpuro构建重组逆转录病毒载体pBABEpuro-P1-2A-EGFP。将重组逆转录病毒载体和pVSV-G质粒载体共转染GP2-293包装细胞,用产生的重组逆转录病毒感染幼仓鼠肾细胞(BHK-21)。荧光显微镜下观察绿色荧光蛋白的表达,用嘌呤霉素筛选抗性细胞,间接免疫荧光方法检测细胞中FMDV衣壳前体蛋白的表达。结果表明,重组逆转录病毒载体pBABEpuro-P1-2A-EGFP构建正确,绿色荧光蛋白和FMDV衣壳前体蛋白在细胞中能稳定表达。FMDV衣壳前体蛋白基因细胞表达体系的成功建立为进一步开展口蹄疫亚单位疫苗的研制奠定了基础。Here we constructed a retroviral vector mediated mammalian cell expression system of the capsid precursor protein of foot-and-mouth disease virus （FMDV）. The recombinant retroviral vector pBABEpuro-P1-2A-EGFP was constructed by inserting capsid precursor protein gene （P1） of FMDV and enhance green fluorescent protein gene（EGFP） into pBABEpuro by turns. Both the recombinant retroviral vector and the pVSV-G plasmid were co-transfected into packa- ging cell GP2-293 by liposome mediated transduction. And then, the pseudovirus were produced. The pseudovirus infected BHK-21 cells and resistance cells were screened by puromycin-resistant. The green fluorescent protein were observed under fluorescence microscope. The expression of capsid precursor protein gene of FMDV was detected by indirect immunofluorescence assay. The recombinant retroviral vector pBABEpuro-P1-2A-EGFP was constructed successfully. The capsid precursor protein of FMDV and green fluorescent protein could express in BHK-21 cells. The mammalian cell expression system of the capsid precursor protein of FMDV had been constructed successfully, which laid a foundation of development of FMDV subunit vaccine.

关 键 词：逆转录病毒载体 口蹄疫病毒 衣壳前体基因 绿色荧光蛋白基因 BHK-21细胞 

分 类 号：S852.659.61[农业科学—基础兽医学] Q78[农业科学—兽医学]