利用报告基因比较悬浮细胞与贴壁细胞对HIV-1假病毒感染效率的差异

【目的】研究用人免疫缺陷病毒(Human immunodeficiency virus,HIV)-1假病毒感染带有β-半乳糖苷酶(β-galactosidase,β-gal)报告基因和HIV受体CD4+CCR5+的Tzmbl细胞,分析悬浮状态与贴壁状态对HIV-1假病毒感染Tzmbl细胞的影响,为进一步进行HIV生物学研究与中和抗体实验室评价提供实验基础。【方法】通过将pNL43 R-E-与编码HIV膜蛋白的质粒共转染293T细胞,收集上清,获得HIV假病毒。该假病毒感染悬浮的和贴壁的Tzmbl细胞后可表达β-gal报告蛋白,通过X-gal染色和仪器分析可测定表达β-gal报告基因的细胞数与细胞感染率。【结果】HIV假病毒感染悬浮细胞的效率高于其对贴壁的Tzmbl细胞感染的效率,且细胞的感染率的改变与病毒的型相关。【结论】该研究结果可为进一步利用具有单轮感染活性的HIV假病毒进行生物研究和中和抗体实验提供研究方法。

Using reporter gene to compare infection efficiency of HIV-1 pseudovirus to suspended and adherent cells

Objective] In order to compare the infection of HIV-1 pseudovirus to suspended and adherent cells,Tzmbl cells containing β-gal(β-galactosidase) reporter gene were used here to do the analysis.Methods] HIV-1 pseudoviruses were generated by co-transfection of 293T cells with the plasmid pNL43R-E-and HIV envelope expressing plasmid.Supernatant of co-transfected 293T cells was collected and used to infect Tzmbl cells with or without trypsin treatment.Forty-eight hours after infection,β-gal positive Tzmbl cells and virus infection were determined using X-gal staining and β-glo(β-galactosidase) assay.Results] The efficiency of HIV pseudoviruses infection of suspended Tzmbl cell was higher than that of adherent cells and the increase of infection correlated with the pseudoviral subtype.Conclusion] This study may provide a useful method for HIV biological study and neutralization assays using a single-round replicative pseudovirus in the future.