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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mR-NA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。
Objective To investigate whether shRNA can inhibit NEF gene expression in Hele cells. Methods Four pre-designed shRNAs were added to Hele cells of HIV virus. At the same time, the control group was set up to extract total cellular RNA and total cellular protein. NEF and GAPDH primers were designed. The gene level of NEF was detected by SYBR Green real-time PCR The protein level of NEF was detected by western blotting. Results The results of quantitative PCR and Western-blot showed that NEF-miRNA-1, NEF-miRNA-2, NEF-miRNA-3 and NEF-miRNA-4 interfere with the expression of mR- -miRNA-3 interference plasmid showed the most significant inhibitory rate of 87%. Conclusion shRNA can be designed to inhibit HIV replication.