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

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

摘要:

摘要:【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。

Abstract:

Abstract:［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 singleround replicative pseudovirus in the future．