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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。
RNA interference can effectively inhibit the expression of specific genes and become an important method in HIV gene therapy. In order to prevent the emergence of virus escape strains, we constructed shRNA recombinant lentiviral vectors targeting vpr and tat of HIV-1 regulatory genes and double-expressing recombinant lentiviral vectors containing these two shRNAs respectively. The vectors were composed of U6 and H1 The two promoters were started and cotransfected with the target gene tat and vpr expression plasmids in 293T cells, and the expression abundance of the target genes in the cells was measured by the fluorescence quantitative PCR. The results showed that the recombinant lentivirus The inhibition rate of vector on vpr and tat expression can reach 89.20% and 62.00% respectively. P24 ELISA showed that the inhibitory rate of plvx-vpr-tat shRNA on virus packaging yield could reach 99.05%, which was higher than that of single shRNA. HIV-1 NL4-3 in vitro MT4 cell challenge Toxicity experiments show that double shRNA recombinant lentiviral vector has a strong ability to inhibit viral replication.