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目的构建人工miR-CCR5,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-1 CCR5基因序列设计并合成4对miRNA寡聚单链DNA,构建4个人工miR-CCR5。采用qPCR技术检测其对CCR5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;抗HIV-1_(BaL)感染实验检测其对HIV-1感染的抑制作用。结果 qPCR结果显示,4个人工miR-CCR5对CCR5基因都具有一定的沉默效果,其中miR-CCR5-4的沉默效率最高,达72%;且不会对细胞干扰素的表达产生影响。MTT实验证实miR-CCR5-4不会影响被转染细胞的活性,此外,病毒抑制实验显示,miR-CCR5-4对HIV-1_(BaL)p24蛋白的抑制作用达70.48%,效果明显。结论所获得的miR-CCR5-4可为进一步的抗HIV-1_(BaL)感染研究提供基础。
Objective To construct artificial miR-CCR5 and study its effect on HIV-1 infected host cells. Methods According to the CCR5 gene sequence of HIV-1, four miRNA oligo single-stranded DNAs were designed and synthesized based on miR-155. Four artificial miR-CCR5 plasmids were constructed. QRT-PCR was used to detect the silencing efficiency of CCR5 gene and its effect on interferon expression. The cytotoxicity of CCR5 gene to transfected cells was detected by MTT assay. The inhibitory effect on HIV-1 infection was tested by anti-HIV-1 BaL infection . Results The results of qPCR showed that four artificial miR-CCR5significantly silence CCR5 genes, of which miR-CCR5-4 had the highest silencing efficiency of 72%, and did not affect the expression of interferon. MTT assay confirmed that miR-CCR5-4 did not affect the activity of transfected cells. In addition, virus inhibition experiments showed that the inhibitory effect of miR-CCR5-4 on HIV-1 BaL p24 protein was 70.48%. Conclusions The obtained miR-CCR5-4 can provide a basis for further research on anti-HIV-1 (BaL) infection.