HIV-1 vif基因人工miRNA的构建和功能分析

Construction and Function Analysis of Artificial miRNA Targeting to HIV-1 Vif Gene

目的构建人工miR-vif,并研究其对HIV-1感染宿主细胞的影响。方法以miR-155为基础骨架,根据HIV-1vif基因序列设计并合成4对miRNA寡聚单链DNA,构建4个人工miR-vif。采用qPCR技术检测其对vif基因的沉默效率和对干扰素表达的影响;MTT法测定其对被转染细胞的毒性;HIV-1假病毒实验技术检测其对感染的抑制作用。结果 qPCR结果显示,4个人工miR-vif对vif基因都具有一定的沉默效果,其中miR-vif-1的沉默效率最高,达68%。且不会对细胞干扰素的表达产生影响。MTT实验证实miR-vif-1不会影响被转染细胞的活性,此外,HIV-1假病毒实验显示,miR-vif-1对HIV-1p24的抑制作用达66.5%,效果明显。结论所获得的miR-vif-1对HIV-1的复制具有良好的抑制作用,可为进一步的抗HIV-1感染研究提供基础。

Objective To construct artificial miR-vif and study their effects on HIV-1 infection. Methods To replace the stem sequence in the stem-loop structure of the well-characterized native miR-155 with siRNA sequences targeting to HIV-1 vif gene, and construct four artificial miR-vif. To detect the vif gene silence efficiency and the expression of IFN-~ gene by qPCR; to test the cell toxicity through MTr assays and analyze the HIV-1 infection by using HIV-1 pseudotyped virus experiment. Results The qPCR results showed that four miR- vif could downregulate vif gene expression, and miR-vif-1 presented the highest gene silence efficiency, reached 68%. The further experiments confirmed that miR-vif-1 had neither effect on the viability of the transfected ceils nor interference the expression of IFN-β gene. In addition, the results verified that miR-vif- I has inhibition effect of 66.5% on HIV-1 replication. Conclusion The miR-vif-1 could efficiently suppress HIV-1 replication and would be a useful candidate for further study on anti-HIV-1 infection.