靶向HIV-1 vif的高效人工miRNA的构建及慢病毒介导的体外抗病毒研究  

Construction of Highly Effective Artifical miRNA Targeted to HIV-1 vif and the Lentiviral-mediated Antiviral Research in vitro

作　　者：张涛[1] 程通[1] 魏丽华[1] 张雅丽[1] 王颖彬[1] 蔡毅君[1] 张军[1] 夏宁邵[1] 

机构地区：[1]厦门大学国家传染病诊断试剂与疫苗工程技术研究中心,厦门361005

出　　处：《病毒学报》2010年第1期8-15,共8页Chinese Journal of Virology

基　　金：福建省自然科学基金计划资助项目(基金项目编号:C0710041)

摘　　要：RNAi在HIV-1的治疗研究中得到了广泛的应用,构建高效并且安全的RNAi抗病毒元件是开展相关研究的基础。vif37是以前的研究中筛选获得的靶向HIV-1 vif的高抑制效率兼具保守性的RNAi靶点。miRNA在抑制效率和启动子的选择上比常用的shRNA具有优势。本研究探索以人工miRNA结构引发vif37靶点对应的RNAi。本研究以天然的miR-155前体为骨架,采用步移的方式设计了3个靶向vif37的人工miRNA,以RNA聚合酶Ⅱ类启动子指导表达。miRNA表达质粒和HIV-1的感染性克隆pNL4-3共转染的结果显示,3个人工miR-NA中只有miR-vif37H具有抑制效果,效率与shRNA-vif37相当。与携带靶序列的荧光素酶报告质粒共转染实验证明miR-vif37H具有良好的抑制特异性。用表达miR-vif37H的重组慢病毒转导HIV-1的敏感细胞MT-4,克隆化后获得稳定表达miR-vif37H的细胞株MT-4-miR37H,该细胞可以有效抑制HIV-1的体外复制。实时RT-PCR检测结果显示,与shRNA-vif37相比,miR-vif37H的表达水平明显下降,安全性更好。进一步的实时RT-PCR检测结果还显示,miR-vif37H在细胞内的稳定表达不会影响内源代表性miRNA(miR-181和miR-16)的加工以及干扰素应答相关基因Stat1的mRNA水平。miR-vif37H是一个特异的高效RNAi元件,为vif37靶点的进一步应用研究奠定了基础。Effective and specific RNA interference （RNAi） elements are essential for the RNAi-based antHIV-1 research which has achieved extensive application, vif37 targeted to HIV-1 vif is a highly effective and conserved RNAi target obtained from the previous study on screening. In this study, we explored the construction of artificial miRNAs to induce RNAi targeted to vif37, which had advantages on inhibition efficiency and flexibility of promoter selection. Three artificial miRNA targeted to vif37 were constructed by walking method using native miR-155 as a backbone and expressed by RNA polymerase Ⅱ promoter. Then, expression vectors of artificial miRNA were co-transfected with HIV-1 infectious clone pNL4-3 to score its inhibition ability and showed that only miR-vif37 had the significant inhibition efficiency similar to shRNA-vif37. Subsequently, co-transfections with luciferase reporter plasmids into which different target sequences were inserted proved the specificity of miR-vif37H. The replication of HIV-1 was inhibited in MT-4-miR37H cells which could express miR-vif37H stably and were cloned from MT-4 cells transducted with recombinant lentiviral vectors containing the miR-vif37H expression element. Real-time RT-PCR revealed that miR-vif37H had much lower expression level than shRNA-vif37. Results also showed that intracellular miR-181 and miR-16 expression levels and statl mRNA levels were not effected by the expression of miR-vif37H in MT-4-miR37H cells. We conclude miR-vif37 is a specific and highly effective artificial miRNA which will promote the further application of vif37 target.

关 键 词：RNA干扰 HIV-1 VIF 人工miRNA 

分 类 号：R373.9[医药卫生—病原生物学]