Construction of HIV-1 B subtype pseudovirus system in Guangxi / 实用医学杂志

ABSTRACT

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-envpNL4-3.EGFP.E-R-.=21)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.