广西HIV-1 B亚型假病毒的构建

Construction of HIV-1 B subtype pseudovirus system in Guangxi

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p SV-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p NL4-3.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pc DNATM3.1(+),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(pc DNA3.1-env:p NL4-3.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 andprovide a relatively cheap and easy method for drug-resistance testing. Methods EGFP gene was amplified fromplasmid p SV-EGFP and then cloned to backbone plasmid p NL4-3.Luc. E-R-by double enzyme digestion;env genewas amplified from RNA isolated from HIV-1-infected persons and cloned to eukaryotic expression plasmid cellsand EGFP or ENV expression. Pseudovirus was produced by co-transfection of two recombinant plasmids to 293 tcells. Infection of pseudovirus was determined by co-cultured with TZM-b1 cells and immunofluorescent test.Results Two recombinant plasmids（mass ratio,pc DNA3.1-env：p NL4-3.EGFP.E-R-. = 2：1）were co-transfectedto 293 t cells. Cultured supernatants containing pseudovirus were harvested at 48 h post-transfection. Fluorescencewas 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 phenotypicdrug-resistance detection.