中文摘要：

根据口蹄疫病毒的感染特征及其在临床发病过程中呈现的症状,筛选抗口蹄疫病毒单链抗体基因。通过EcoRⅠ/NotⅠ双酶切中间质粒pGEX-ScFv获得单链抗体基因片段,并克隆至反转录病毒载体质粒pFB-neo,构建反转录病毒穿梭载体质粒pFB-ScFv。将pFB-ScFv与辅助质粒pVPack-GP、pVPack-10A1共转染HEK293T细胞,包装重组反转录病毒MMLV-ScFv。同时,构建含增强型绿色荧光蛋白基因的重组反转录病毒MMLV-EGFP作为对照。利用所包装重组反转录病毒感染猪成纤维细胞,通过G418筛选获得携带目的基因的单克隆细胞集落。试验结果表明,本试验在成功构建反转录病毒穿梭载体pFB-ScFv和pFB-EGFP的基础上,获得了分别携带抗口蹄疫病毒单链抗体基因和增强型绿色荧光蛋白基因的反转录病毒,并最终筛选出分别携带上述基因的猪成纤维细胞克隆,为抗口蹄疫病毒转基因动物的研究奠定了基础。

英文摘要：

According to characteristics of the foot and mouth disease virus infection in the pathogenesis of the clinical presentation of symptoms,screening single-chain antibody against foot and mouth disease virus gene.By EcoRⅠ/NotⅠ double digestion to obtain intermediate plasmid pGEX-ScFv single chain antibody gene fragment and cloned into retroviral vector plasmid pFB-neo,to build anti-retroviral shuttle vector plasmid pFB-ScFv.PFB-ScFv with the helper plasmid pVPack-GP,pVPack-10A1 were cotransfected into HEK293T cells,packaging recombinant retroviruses MMLV-ScFv.Meanwhile,building enhanced green fluorescent protein recombinant retroviruses MMLV-EGFP as a control.Useing recombinant retrovirus infect swine fibroblasts,obtained the target gene carrying monoclonal colony by G418 selection.Restriction endonuclease detection,fluorescence detection and reverse transcription polymerase chain reaction and other experimental results show that,In this study,based on the successful construction of anti-retroviral shuttle vector pFB-ScFv and pFB-EGFP,obtained anti-FMD virus were carrying single-chain antibody gene and enhanced green fluorescent protein retrovirus.And eventually were selected to carry the gene cloned pig fibroblasts for the anti-FMD virus transgenic animal research laid the foundation.