慢病毒介导双shRNA靶向于HIV的抑制作用研究  

Inhibition of HIV-1 in Vitro by Combination of Vpr and Tat Specific Short Hairpin RNA via Lentiviral Vectors

作　　者：郝彦哲[1] 滕智平[2] 杨怡姝[1] 孙晓娜[1] 马晶[2] 金孝华[2] 曾毅[1,2] 

机构地区：[1]北京工业大学生命科学与生物工程学院,北京100124 [2]中国疾病预防控制中心病毒病预防控制所传染病预防控制国家重点实验室,北京100052

出　　处：《病毒学报》2013年第2期126-131,共6页Chinese Journal of Virology

基　　金：973计划"应用纳米技术解决新型抗艾滋病药物的若干关键科学问题"(2009CB010200;2009CB930202)

摘　　要：RNA干扰可以有效地抑制特定基因的表达,在HIV基因治疗中成为一种重要的方法。为了防止病毒逃逸株的出现,本研究分别构建了靶向于HIV-1调控蛋白基因vpr和tat的shRNA重组慢病毒载体以及含有这两种shRNA的双表达重组慢病毒载体,分别由U6和H1两种启动子启动,通过与靶向基因tat和vpr表达质粒在293T细胞中进行共转染,采用荧光定量PCR方法测定细胞内靶向基因的表达丰度,结果显示双表达shRNA的重组慢病毒载体对vpr和tat的表达抑制率分别可以达到89.20%和62.00%。与pNL4-3病毒包装质粒共转染,P24ELISA显示plvx-vpr-tat shRNA对病毒包装产量的抑制率可以达到99.05%,比单个shRNA的抑制率都要强,HIV-1NL4-3体外对MT4细胞攻毒实验表明双shRNA重组慢病毒载体具有很强的抑制病毒复制的能力。Gene therapy offers the promise of curing the HIV-infected patients. Specific, potent, and sustained short hairpin RNA （shRNA）-mediated gene silencing is crucial for the successful application of RNA interference technology to therapeutic interventions. To reduce the probability of viral escape mutants, in this study, we constructed lentiviral vector containing vpr and tat shRNA, respectively, furthermore the bispecific lentiviral vector harboring vpr and tat shRNA expression cassettes from U6 promotor and H1 promotor was cotransfected with recombinant plasmid expressing the vpr and tat gene. The result showed that the bispecific lentiviral vector plvx-vpr-tatshRNA could inhibit the vpr and tat effectively,with ratios of 89.20% and 62.00% respectively. When cotransfected with pNL4-3 in 293T cell, plvxvpr-tatshRNA showed higher efficacy in down regulating the HIV NL4-3 packaging production than the plvx-vprshRNA or plvx-tatshRNA individually. MT4 cell clones transduced with recombinant lentiviral vectors were screened and challenged with HIV NL4-3. P24 ELISA test showed that MT4 transduced with the combinational lentiviral vector could inhibit virus replication efficiently.

关 键 词：HIV 基因治疗 RNA干扰 SHRNA 

分 类 号：R512.91[医药卫生—内科学]