重组腺病毒HIV-1 vpr基因的构建和表达

目的 构建携带HIV-1 vpr基因的重组腺病毒,使CD4 T淋巴细胞C8166内源性的高表达Vpr蛋白.方法 利用AdEasy-1系统, 通过将含有目的 基因片段的穿梭载体pAdTrack-CMV-vpr和骨架质粒pAdEasy-1在BJ5183细菌内同源重组的方法构建重组腺病毒质粒Ad-vpr, 用脂质体法将重组质粒转染至HEK293A细胞包装, 获得重组腺病毒Ad-vpr, 荧光显微镜观察Ad-vpr感染C8166细胞GFP的表达 , Western blotting鉴定Vpr在C8166细胞内的特异性表达,流式细胞术检测Ad-vpr感染 C8166细胞的效率.结果 成功构建携带HIV-1 vpr基因的重组腺病毒 ,Western blotting结果表明重组腺病毒Ad-vpr感染的C8166细胞内源性的高表达Vpr 蛋白,流式细胞术检测结果表明Ad-vpr感染C8166细胞效率高(44.07±3.62)%.结论 成功构建出携带HIV-1 vpr基因的重组腺病毒,使C8166细胞内源性的高表达Vpr蛋白.

Construction and expression of recombinant adenovirus carrying HIV-1 vpr gene

Objective To construct the recombinant adenoviral vector expressing HIV-1 vpr gene and to induce viral protein R(Vpr)highly expressed in C8166 cells.Methods The HIV-1 vpr gene was cloned to the shuttle plasmid pAdTrack-CMV.The linearized resultant plasmid and adenoviral backbone plasmid pAdEasy-1 were co-transfected into E.coli BJ5183.Recombinant adenoviral plasmids were screened and identified,the linearized recombinant plasmid was transfected into HEK293A for adenovirus package through lipofectamine 2000.Viral productions were monitored by GFP expression.Results The results shown that the recombinant adenovirus carrying HIV-1 vpr gene was constructed successfully.High expression of HIV-1 vpr gene in C8166 was detected by Western blotting and the high infection rate was detected by FACS.Conclusion Recombinant adenovirus Ad-vpr is successfully constructed and the Vpr protein is specially expressed in C8166 cells.