HIV-1B/C重组毒株gp160假病毒的构建及其活性检测

Construction of a HIV-1 B/C recombinant subtyped pseudovirus and evaluation of its activities

目的以CD4＋T细胞DNA为模板,进行gp160扩增,构建艾滋病病毒Ⅰ型（HIV-1）前病毒假病毒,通过中和试验验证其具有感染活性。方法选择高效抗反转录病毒治疗（HAART）成功的病人3例,分离外周血单核细胞（PBMC）,纯化CD4＋T细胞,提取DNA,以其为模板扩增gp160全长基因,并克隆到pcDNA3.1（＋）表达载体上,酶切验证得到阳性克隆。将此阳性克隆和pNL4-3质粒共转染,获得HIV-1假病毒。用免疫兔血清验证假病毒的感染活性。结果成功地获得了1株假病毒。用免疫后的兔血清测定半数抑制浓度（IC50）的滴度约在1∶901∶270之间。病毒的加入量与细胞感染率之间存在良好的线性关系,说明该假病毒感染系统可以通过细胞感染率较好地反映出感染性病毒的含量。结论获得了具有感染活性的HIV-1B/C重组型假病毒。

Objective To extract viral DNA from the CD＋4 T cells of the HIV-1 infected patients and to construct HIV-1 pseudotyped virus,which will serve as a useful tool for neutralization assay.Methods The blood samples were collected from HIV-1 infected patients and the peripheral blood mononuclear cells were separated.The provirus DNA was extracted and gp160 was amplified by PCR.The Env containing PCR amplicons was ligated into the pcDNA3.1（＋） expression vector after appropriate restriction enzyme digestion.And then a pseudovirus-based neutralization assay was established.Results HIV pesudovirus containing luciferase gene was capable of infecting Tzm-bl cells.Conclusion The B/C recombinant subtyped pseudovirus has been successfully constructed and a platform to use a single-round replicative pseudovirus has been established.