携带GFP发光基因人猴嵌合免疫缺陷病毒SHIV的构建及活性检测

目的获得正常感染宿主细胞并稳定表达绿色荧光的SHIV毒株,为后期建立发光SHIV/恒河猴感染模型奠定基础。方法通过分子克隆手段,将绿色荧光蛋白基因克隆到携带HIV-1包膜蛋白的SHIV病毒全基因组中,并在细胞水平检测各毒株的感染活性及荧光蛋白表达能力。结果得到一株可表达绿色荧光蛋白的病毒株SHIV-KB9nefGFP,并具有感染TZM-bl细胞系及猴PBMC的能力。结论该毒株在宿主细胞恒河猴PBMC中具有一定复制能力,希望通过后续的猴体内传代实验获得毒力更强的发光病毒。

Construction and Characterization of a New SHIV Clone Carrying GFP Gene

Objective To engineer recombinant strains of simian-human immunodeficiency virus（SHIV） stably expressing green fluorescent protein（EGFP）.A replication-competent SHIV construct containing the green fluorescent gene with the ability to infect rhesus monkeys would serve as an important tool in AIDS research.Methods A SHIV strain was constructed by inserting the EGFP genes into the nef gene of SHIV KB9.The infection activity and bright fluorescence expression of the SHIV clone was determined in vitro in TZM-bl cells and macaque PBMCs.Results Replication-competent virus and bright fluorescence of infected cells were obtained with one construct,in which EGFP was inserted into the SHIV nef locus.This strain was infectious to rhesus PBMC and TZM-bl cells.Green fluorescing cells were detected by direct microscopic visualization.Conclusions A recombinant and replication-competent SHIV strain expressing EGFP is engineered.It is suggested that the SHIV-KB9nefGFP could be used as a tool to directly detect infected cells and aid in the immunophenotypic characterization of these cells.