通过设计发夹RNA抑制HIV病毒NEF基因表达的实验研究

目的 实验研究shRNA能否抑制宿主细胞Hele细胞中NEF基因的表达.方法 给HIV病毒宿主细胞Hele细胞加入预先设计好的4对shRNA,同时设置对照组,提取细胞总RNA和细胞总蛋白,设计NEF和GAPDH的引物,利用SYBR Green实时荧光定量PCR检测NEF的基因水平,利用western blotting检测NEF的蛋白水平.结果 定量PCR和Western-blot方法检测显示,NEF-miRNA-1、NEF-miRNA-2、NEF-miRNA-3、NEF-miRNA-4干扰质粒对靶基因mRNA表达都有抑制作用,其中NEF-miRNA-3干扰质粒抑制作用最显,抑制率达87％.结论 可以通过设计shRNA去抑制HIV病毒的复制.

The inhibition effect of shRNA of NEF of HIV

OBJECTIVE To detect the effect of shRNA inhibiting the expression of NEF in hele cells.METHODS Four pairs of shRNA were transfect four recombinant plasmids into Hele cells.The controls were established,and the RNA and protein were extracted.The primers of NEF and GAPDH were designed.The gene expression of NEF was detected by qPCR,and the protein expression was detected by western blotting.RESULTS The result of qPCR and western blotting indicated the NEF-shRNA-1,NEF-shRNA-2,NEF-shRNA-3 and NEF-shRNA-4 interfering veetors could inhibit NEFmRNA expression.The inhibition rate ofNEF-shRNA-3 was 87%.The inhibition rate of NEF protein of NEF-shRNA-3 was 87% detected by western blotting.CONCLUSION shRNA could inhibit the replication of HIV.