广西HIV-1 B亚型假病毒的构建

摘要

目的 针对广西HIV-1 B亚型毒株构建一种优化的假病毒耐药检测模型,为表型耐药检测提供成本较低、简便易行的研究工具.方法 从质粒pSV-EGFP中扩增出EGFP基因,采用双酶切法将EGFP基因克隆至骨架质粒pNL4-3.Luc.E-R-,以此构建假病毒重组骨架质粒;采用RT-PCR方法从HIV-1 B亚型慢性感染者RNA中获得包膜蛋白基因,采用双酶切法将env基因克隆至真核表达质粒pcDNATM3.1 (+),以此构建重组包膜质粒;单转染重组包膜质粒至293t细胞验证表达;共转染重组质粒至293t细胞获得假病毒;采用与TZM-b1细胞共培养法,通过荧光表达检测验证假病毒感染性.结果 两种质粒以质量比2:1(pcDNA3.1-env:pNL4-3.EGFP.E-R-)共转染至293t细胞,48 h后收集细胞上清获得假病毒.将假病毒加入到TZM-bl细胞共培养48 h后,可以观察到荧光表达.结论 带有EGFP基因重组假病毒的系统构建成功.%Objective To establish a pseudovirus system for phenotypic drug-resistance detection and provide a relatively cheap and easy method for drug-resistance testing. Methods EGFP gene was amplified from plasmid pSV-EGFP and then cloned to backbone plasmid pNL4-3.Luc. E-R-by double enzyme digestion;env gene was amplified from RNA isolated from HIV-1-infected persons and cloned to eukaryotic expression plasmid cells and EGFP or ENV expression. Pseudovirus was produced by co-transfection of two recombinant plasmids to 293t cells. Infection of pseudovirus was determined by co-cultured with TZM-b1 cells and immunofluorescent test. Results Two recombinant plasmids(mass ratio,pcDNA3.1-env:pNL4-3.EGFP.E-R-.=2:1)were co-transfected to 293t cells. Cultured supernatants containing pseudovirus were harvested at 48 h post-transfection. Fluorescence was observed in TZM-b1 cells after TZM-b1 cells were infected with pseudovirus at 48 h post-infection. Conclusion The recombinant pseudovirus carrying EGFP gene is constructed successfully and it could be used for phenotypic drug-resistance detection.