趋化因子受体基因CCR5人工miRNA的构建与功能分析  

Construction and Function Analysis of Artificial miRNA Targeting to HIV-1 CCR5 Gene

作　　者：王芳宇[1,2] 周云[1,2] 何丽芳[1] 滕涛[1] 杨海[1] 谭潇 

机构地区：[1]衡阳师范学院生命科学与环境学院功能金属有机材料湖南省重点实验室,湖南衡阳421008 [2]南华大学药学与生命科学学院,湖南衡阳421001 [3]Laboratory of Molecular and Functional Enzymology,UPMC-Sorbonne University,franceparis75205

出　　处：《中国皮肤性病学杂志》2017年第4期359-364,共6页The Chinese Journal of Dermatovenereology

基　　金：湖南省自然科学基金重点项目(09JJ3034)

摘　　要：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 effects on HIV-1 infected host cells.Methods Four pairs of miRNA oligonucleotides were designed and synthesized based on HIV-1 CCR5 gene sequences to construct four artificial miR-CCR5, which derived from the native miR-CCR5 gene sequence. Then qPCR was used to investigate the gene silence efficiency and the expression of IFN-β gene; the cell toxicity was tested by MTT assays and HIV-1 infection was analyzed by detecting p24 protein content.Results The qPCR results showed that the four miR-CCR5 could downregulate CCR5 gene expression, in which the miR-CCR5-4 presented the highest gene silence efficiency that reached 72%. Further experiments confirmed that miR-CCR5-4 had neither effect on the viability of the transfected cells nor interference the expression of IFN-β gene. Furthermore, the results verified that miR-CCR5-4 has inhibition effect of 70.48% on HIV-1 replication.Conclusion The miR-CCR5-4 could effectively suppress HIV-1 replication and would be a useful candidate for further study on anti-HIV-1 infection.

关 键 词：HIV-1 CCR5基因 RNA干扰 人工miRNA 

分 类 号：Q786[生物学—分子生物学]