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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 its effect on HIV-1 infected host cells. Methods Based on miR-155, four pairs of miRNA oligonucleotides were designed and synthesized based on HIV-1vif gene sequence. Four artificial miR-vifs were constructed. The effect of silencing vif gene on the expression of interferon was detected by qPCR. The toxicity of vif gene to transfected cells was determined by MTT assay. The inhibitory effect of HIV-1 pseudovirion on the infection was tested by the HIV-1 pseudovirion assay. Results qPCR results showed that all four artificial miR-vifs had a certain silencing effect on vif gene, of which miR-vif-1 had the highest silencing efficiency of 68%. And does not affect the expression of cellular interferons. The results of MTT assay showed that miR-vif-1 did not affect the activity of transfected cells. In addition, HIV-1 pseudovirus showed that miR-vif-1 had an inhibitory effect on HIV-1p24 by 66.5%. Conclusions The obtained miR-vif-1 has a good inhibitory effect on the replication of HIV-1, which may provide a basis for further study on anti-HIV-1 infection.