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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∶90~1∶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 construct gp160 DNA of CD4 + T cells as a template and construct HIV-1 proviral virus, which is proved to be infectious by neutralization test. Methods Three patients with HAART were enrolled in this study. Peripheral blood mononuclear cells (PBMCs) were isolated and CD4 + T cells were purified. The DNA was extracted and used as a template to amplify the full-length gp160 gene. The cloned To pcDNA3.1 (+) expression vector, confirmed by digestion positive clones. This positive clone was co-transfected with pNL4-3 plasmid to obtain HIV-1 pseudovirion. Immune rabbit serum was used to verify the infection activity of pseudoviruses. Results A pseudovirus was successfully obtained. The half-inhibitory concentration (IC50) titer was determined to be between 1:90 and 1: 270 with the rabbit serum after immunization. There was a good linear relationship between the addition of virus and cell infection rate, indicating that the pseudovirus infection system can reflect the content of infectious virus well through cell infection rate. Conclusion The HIV-1B / C pseudotyped virus with infection activity was obtained.