人类免疫缺陷病毒假病毒检测系统的构建

Establishment and characterization of a pseudotyped HIV assay system carrying luciferase gene

目的构建人类免疫缺陷病毒(HIV)感染性克隆并在此基础上建立携带荧光素酶基因(Luc+)的HIV假病毒感染系统。方法利用长片段PCR技术分别扩增福建省HIV分离株LWJ前后半长基因序列,以pCR-XL-TOPO为载体构建HIV感染性克隆pLWJ。将Luc+表达基因片段替换感染性克隆pLWJ中部分env区序列,从而构建出携带Luc+基因的报告基因病毒载体pLWJ-SV40-Luc+。将报告基因病毒载体和膜蛋白表达质粒VSV-G共转染293T细胞,获得携带Luc+基因的HIV假病毒。将HIV假病毒感染293T细胞,利用化学发光法检测感染细胞中Luc+相对荧光单位(RLU)。结果以HIV感染性克隆pLWJ为基础,构建出携带荧光素酶基因的VSV-G/HIV假病毒感染系统,其所产生的VSV-G/HIV假病毒仅有单轮感染活性,被感染细胞中报告基因的表达水平与加入病毒量呈剂量依赖性关系。结论成功建立具有单轮感染活性的VSV-G/HIV假病毒检测系统,为开展HIV相关基础研究提供一个技术平台。

Objective To construct and characterize a pseudotyped HIV assay system containing transient HIV infectious clone and luciferase（Luc＋） gene.Methods The two overlapping subgenomic fragments of 5′ and 3′ ends were generated by the Expand Long Template PCR System.We constructed the HIV-1 full-length infectious clone of pLWJ with pCR-XL-TOPO vector.The reporter gene vector of pLWJ-SV40-Luc＋ was reconstructed by inserting Luc＋ gene into env open reading frame of the plasmid pLWJ.Pseudoviruses were generated by co-transfection of 293T cells with the plasmid pLWJ-SV40-Luc＋ and the HIV envelope expressing plasmid of VSV-G.The supernatant of co-transfected 293T cell cultures was collected and used to infect new 293T cells.The expression of the reporter gene Luc＋ was determined by Luciferase Assay System in the infected cells.Results The pseudovirus assay system of Luc＋ reporter VSV-G/HIV was successfully established with the infectious clone of pLWJ.The generated pseudovirus had a single-round,recombinant-based viral infectivity.The expression of Luc＋ reporter gene was linearly correlated with inoculated pseudoviral dosage in 293T cells.Conclusions The new established system of pseudotyped HIV assay with a single-round,recombinant-based viral infectivity can facilitate further basic studies on HIV.