HIV-1 vif基因的RNA干扰体外研究  

In vitro study of RNA interference on vif gene of HIV-1

作　　者：江雪艳[1] 卢洪洲[1,2,3] 陈秋丽[4] 潘卫[4] 张云智[1] 沈银忠[1] 

机构地区：[1]上海市复旦大学附属公共卫生临床中心感染科,上海201508 [2]复旦大学附属华山医院传染科,上海200040 [3]复旦大学上海医学院内科学系,上海200032 [4]第二军医大学微生物教研室,上海200433

出　　处：《复旦学报（医学版）》2009年第1期32-36,共5页Fudan University Journal of Medical Sciences

基　　金：上海市科委自然科学基金项目(06ZR1401);国家自然科学基金项目(30771983)

摘　　要：目的利用RNAi技术对HIV-1 vif基因的转录后水平进行下调,达到抑制vif蛋白表达的目的,为新的抗HIV治疗和预防研究提供理论基础。方法通过体外转录法合成siRNA,与转接有vif基因的表达质粒共同转染HEK293T细胞,荧光显微镜下观察干扰效果,并利用Real-time PCR和Western blot对HIV-1 vif分别从转录和表达水平进行验证。结果将pEGFP-N1-vif重组载体转染HEK 293T细胞后,结果显示vif蛋白可以在细胞中高水平表达;针对vif设计的3段特异的siRNA可以有效且特异地降低vif蛋白的表达。结论RNAi技术可以下调HIV-1蛋白的表达,此技术可以作为一项新的治疗和预防HIV-1的方法用于进一步的研究。Objective In order to explore the potential effects of gene therapy for HIV-1 treatment and precaution, we studied the suppression of RNA interference on HIV-1 vif protein by directing vif to post-transcriptional level. Methods siRNAs, synthesized by transcription in vitro, and the plasmid expressing vif gene and EGFP were cotransfeeted into HEK 293T cell, to express the vif protein and investigate the suppression effect of siRNA on HIV-1 vif under fluorescent microscope. This effect was confirmed by real time PCR at transcription level and Western blot at expression level. Results HIV-1 vif protein exhibited high expression in HEK 293T cells after transfecting pEGFP- N1-HIV-1/vif plamid into HEK 293T cell alone; the three siRNAs designed in this study targeted to vif suppressed HIV-1 vif protein expression specifically. Conclusions The use of RNAi, a new gene therapy tool, was proved to be useful in inhibiting HIV-1 vif expression. This technique deserves further study as a potential therapeutic tool for HIV-1.

关 键 词：人类免疫缺陷病毒(HIV) vif基因 vif蛋白 RNAI siRNA 人胚肾293T细胞 pEGFP—N1质粒 

分 类 号：Q754[生物学—分子生物学]