基于Semliki森林病毒复制子的新型RNAi质粒载体的构建

Construction of a Novel RNAi Vector Based on Replicon Derived from Semliki Forest Virus

目的:以semliki森林病毒复制子为基础,构建一类可迅速高效表达shRNA的新型RNAi载体。方法:以Semliki森林病毒衍生的复制子载体pSFV1为骨架,用CMV IE启动子替换SP6启动子并在3′-UTR下游插入SV40 polyA转录终止子,在原26S亚基因组启动子后插入带有相应改良多克隆位点的shRNA表达元件,同时加入抗新霉素选择复合体,并去掉3′-UTR的重复序列。所获载体用于沉默EGFP基因,通过体外细胞转染、病毒颗粒制备、荧光显微镜观察、RT-PCR分析等初步验证、评估其效果。结果:构建了基于Semliki森林病毒复制子的新型RNAi质粒载体pSFV-RNAi Ready。经体外实验初步证实,该载体直接转染细胞,或与辅助载体共转染,制备成具有感染能力的重组病毒颗粒后使用,均可高水平表达shRNA,沉默目的基因。其中使用病毒颗粒抑抑效率可高达90%以上。结论:该载体的成功构建,可望显著拓宽SFV载体的应用范围,丰富RNAi实施手段,并用于相关科学研究及基因药物技术开发。

Objoetive：To construct a novel RNAi vector bassed on replicon derived from Semliki Forest Virus.Methods：Use plamid pSFV1 as the template,and modify it as follow：A,Replace SP6 promotor by CMV IE promoter;B,add new MCS and shRNA expression system;C,add Neomycin resistence gene;D,deleted reapting sequence in 3＇-UTR of SFV.Then the novel plasmid was used in silencing of EGFP gene in two methods,plasmid dierectly or recombinant virus particles,which was prepared by cotranfected the ex- pression vectors with the helper vectors.The expression of EGFP were observed under inverted fluorescence microscope and investigated by RT-PCR.Results：The novel pSFV-RNAi Ready vector was constructed successfully.It can be used both in highly efficient expression of foreign shRNA and preparation of RVP ex vivo.Results of microscopic observation and RT-PCR indicated that pSFV-RNAi Ready could silence the aim gene with a very high inhibition ratio （〉90%） and much more longer time than siRNA （〉10 d）.Conclusion：The novel pSFV-RNAi Ready vector system might not only widen the use range of SFV vector obviously,but also add new methods to prac- tise RNAi.It might have a brilliant future in scientific research and gene therapautic medicine development.