靶向HIV-1细胞受体基因CCR5的人工miRNA的构建与鉴定  

Construction and Identification of Artificial miRNA Targeting to HIV-1 Cell Receptor Gene CCR5

作　　者：王颖彬[1] 程通[1] 魏丽华[1] 张涛[1] 梁露萍[1] 夏宁邵[1] 

机构地区：[1]厦门大学生命科学学院,国家传染病诊断试剂与疫苗工程技术研究中心,福建厦门361005

出　　处：《厦门大学学报（自然科学版）》2010年第3期410-416,共7页Journal of Xiamen University：Natural Science

基　　金：“艾滋病和病毒性肝炎等重大传染病防治”科技重大专项课题:“艾滋病毒诊断技术研究与产品研制”(2008ZX10001-013);教育部科学技术研究重大项目培育基金(705031);福建省自然科学基金计划资助项目(C0710041);福建省科技重大专项(2004YZ01)

摘　　要：趋化因子CCR5是HIV-1入侵机体细胞的主要辅助受体之一,通过抑制CCR5的表达可以在一定程度上阻断HIV-1的复制进程.miRNA介导的RNA干扰相比经典的siRNA在应用中具有更多的特点.本研究的目的是构建可特异有效靶向CCR5的人工miRNA元件.本研究以CCR5基因序列为模板设计了14个miRNA靶序列,以天然miR-30a为基础骨架通过靶序列区替换构建人工miRNA元件.通过将miRNA表达质粒与携带靶序列区的报告质粒的共转染抑制实验检测不同miRNA的抑制效率,结果显示miCCR-13A具有较好的抑制效率和特异性.进一步将miCCR-13A表达载体转染CCR5阳性的HIV-1受体细胞株TZM-b1,RT-PCR检测显示miCCR-13A可在细胞中获得有效表达,同时应用免疫流式细胞术检测显示在miCCR-13A转染细胞相比对照细胞的CCR5蛋白的检测活性明显降低.进一步的研究显示miCCR-13A在细胞中不会影响细胞活性和干扰素效应相关基因osa1和stat1 mRNA水平,具有良好的特异性.本研究获得的可特异靶向CCR5的人工miRNA元件可为进一步的抗HIV-1研究提供重要基础.The chemokine receptor CCR5 is one of the major co-receptors for HIV-1 cellular entry.HIV-1 replication can be blocked to some extent by inhibiting the expression of CCR5.RNA interfence（RNAi） is a sequence-specific intracellular mechanism of gene silence.Recent studies have shown that microRNAs（miRNAs） posses more characteristics of inhibition and expression mechanisms than small interfering RNAs（siRNAs）.In this study,we explored the construction of artificial miRNA element against CCR5 effectively and specifically.Fourteen miRNA sequences were selected based on the sequence of CCR5 gene and the target sequence in the stem-loop structure of the native miR-30a was replaced with the artifical miRNA sequence separately.Using various reporter plasmids containing different target sequences,the activities of the constructs against CCR5 were determined and miCCR-13A was selected from the candidates.miCCR-13A expression vector was then transfected into CCR5-positive TZM-b1 cells,miCCR-13A expression was confirmed by RT-PCR detection in transformed cells,and the CCR5 expression was effectively inhibited by miCCR-13A in transformed cells comparing to control cells.Results also showed that cell viabilities and intracellular stat1 and osa1 mRNA levels were not affected by miCCR-13A expression in transformed cells.miCCR-13A is a promising candidate for future research on HIV-1 therapy.

关 键 词：CCR5 RNA干扰 人工miRNA 

分 类 号：R373[医药卫生—病原生物学]