表达萤光素酶的HIV药物筛选细胞模型的建立  

Construction of an Anti-HIV Drug-Screening Cell Model Using Luciferase as a Reporter

作　　者：张昕[1] 安小平[1] 陈斌[1] 张宝中[1] 王盛[1] 刘大斌[1] 童贻刚[1] 

机构地区：[1]军事医学科学院微生物流行病研究所病原微生物生物安全国家重点实验室,北京100071

出　　处：《生物技术通讯》2010年第5期666-669,共4页Letters in Biotechnology

基　　金：国家科技重大专项(2008ZX10001-013)

摘　　要：目的:构建基于萤光素酶的单次复制人免疫缺陷病毒(HIV)细胞模型,用于抗HIV药物的筛选。方法:构建含萤光素酶报告基因的假型慢病毒质粒,将疱疹性口炎病毒外膜糖蛋白(VSV-G)的表达质粒、HIV-1 Rev蛋白表达质粒、HIV Gag-Pol蛋白表达质粒和含萤光素酶报告基因的重组慢病毒质粒共转染HEK 293FT细胞,制备假型慢病毒;在假型慢病毒生产和再感染新鲜HEK 293FT细胞的过程中加入逆转录酶和蛋白酶抑制剂(如AZT),检测再感染的细胞中萤光素酶的表达水平,从而判断药物对HIV的抑制作用。结果:构建了含萤光素酶报告基因的重组慢病毒质粒pLenti-Luc;利用已知抗HIV药物AZT进行测试,发现HIV药物处理组细胞中萤光素酶活性远低于对照组。结论:建立了基于萤光素酶的HIV药物筛选细胞模型,该系统使用单次复制的报告病毒,具有良好的安全性,而使用萤光素酶基因作为报告基因使该系统具备极高的敏感性,该系统适合于进行高通量药物筛选。Objective： To construct single cycle replication HIV cell model for anti-HIV drug screening.Methods： Lentivirus vector expressing luciferase reporter gene was constructed and was co-transfected with plasmids expressing vesicular stomatitis virus glycoprotein VSV-G and HIV-1 Rev protein and HIV Gag-Pol polyprotein,into HEK 293FT cells.VSV-G pseudo-typed lentivirus harboring luciferase gene was collected from the cell culture su-pernatant and was used to infect naive HEK 293FT cells.The infection and subsequent cell culture were in the presence of anti-HIV drugs（reverse transcriptase inhibitors or protease inhibitors,like AZT）.Relative luciferase activities were examined to evaluate the inhibition effect of the anti-HIV drugs.Results： We have generated a HIV cell model with a recombinant lentivirus vector which expressed luciferase as a reporter.When the anti-HIV drug AZT was used to inhibit the VSV-G pseudo-typed lentivirus,drastic decrease in luciferase activity was observed compared with the control.Conclusion： An anti-HIV drug screening cell model was established with VSV-G pseu-do-typed lentivirus expressing luciferase as reporter.The single cycle replication feature of this model provides very good safety and the luciferase system produces great sensitivity.This cell model is also suitable for high-throughput anti-HIV drug screening.

关 键 词：人免疫缺陷病毒 假型慢病毒 单次复制 药物筛选 

分 类 号：Q78[生物学—分子生物学] Q24