摘要
目的构建携带绿色荧光蛋白基因的HIV假病毒载体,并研究该载体包装的HIV假病毒的感染活性,为进一步进行HIV生物学研究与中和抗体实验室评价搭建安全的技术平台。方法将增强型绿色荧光蛋白(enhancedgreenfluorescentprotein,EGFP)基因插入到骨架质粒pNL43LucR-E-的nef基因读码框,获得携带EGFP基因的质粒pNL43EGFPR-E-;通过将pNL43EGFPR-E-与编码HIV膜蛋白的质粒共转染293T细胞,收集上清,获得携带EGFP基因的HIV假病毒。该假病毒感染CD4+CCR5+的HOS细胞后可表达绿色荧光,这样通过流式细胞仪可测定表达绿色荧光的细胞数与细胞感染率。结果携带绿色荧光蛋白基因的HIV假病毒感染CD4+CCR5+的HOS细胞后,被感染的细胞可以表达绿色荧光蛋白,细胞的感染率与病毒加入量呈线性关系。结论获得了携带绿色荧光蛋白基因的HIV假病毒载体,并建立了具有单轮感染活性的HIV假病毒感染的检测方法。
Objective To construct and characterize a pseudotyped HIV containing enhanced green fluorescent protein（EGFP） gene, which will serve as a useful tool for HIV virological study and neutralization assays. Methods The plasmid pNL43 EGFP R-E- carrying EGFP gene was obtained by inserting EGFP gene into nef open reading frame of the plasmid pNL43 Luc R-E-. Then EGFP reporter pseudoviruses were generated by cotransfection of 293T cells with the plasmid pNL43 EGFP R-E- and HIV envelope expressing plasmid. Supematant of co-transfected 293T cells were collected and used to infect CD4^＋ CCR5^＋ and CD4^＋ CXCR4^＋ HOS cells that were engineered to co-express CD4^＋ and CCR5 or CXCR4 receptors. Forty-eight hours after infection, GFP positive HOS cells were determined by cell flow cytometry analysis. Results HIV pseudoviruses containing EGFP gene were capable of infecting CD4 ^＋ CCR5 ^＋ HOS cells and the infected HOS cells could express EGFP protein. EGFP positive cells linearly correlated with the pseudoviral dosage inoculated into HOS cells. In addition, the pseudoviruses only had single-muM infectivity and were unable to replicate after entering HOS cells. Conclusion A pseudotyped HIV backbone vector containing EGFP gene has been successfully constructed and a platform to use a single-round replicative pseudovims has been established.
出处
《中华微生物学和免疫学杂志》
CAS
CSCD
北大核心
2006年第5期394-398,共5页
Chinese Journal of Microbiology and Immunology
关键词
HIV
绿色荧光蛋白
假病毒
中和抗体
HIV
Green fluorescent protein
Pseudovims
Neutralization antibody
