靶向核仁素RNA干扰载体的构建与筛选

Construction and Screening of RNAi Vector Targeting Nucleolin

目的:设计靶向大鼠核仁素的RNAi序列,构建6个RNAi真核表达载体,采用转染工具细胞筛选有效靶点。方法:根据GenBank中的核仁素序列,设计特异性siRNA序列,将模板序列克隆至pGCsi-lencerTMU6/Neo/GFP质粒中,通过测序鉴定后,将重组质粒用脂质体转染技术导入H9C2细胞,荧光显微镜检测GFP表达以判断转染效率,Western-blot检测靶细胞中核仁素蛋白水平的表达,筛选有效干扰序列。结果:经测序鉴定,克隆的RNAi打靶序列完全正确,无碱基突变。转染H9C2细胞36 h后,荧光显微镜下可检测到GFP标记的核仁素蛋白的表达,转染效率达75%以上。Western-blot结果显示3号核仁素干扰质粒(Ncl-3)能明显下调H9C2细胞的核仁素表达。结论:成功构建靶向核仁素RNA干扰载体,并筛选出效能最优序列,为进一步相关研究奠定基础。

Objective：To design RNAi sequences targeting nucleolin and to construct six RNAi eukaryotic expression vectors. To screen the most efficient knock - down sequence by transfecting above to H9C2 cells. Methods： Six specific siRNA sequences were designed according to the nucleolin sequence in GenBank. The sequences were cloned into pGCsilencerTM U6/Neo/GFP and the sequencing was performed. The recombinant plasmids were transfected into H9C2 cells. The transfection efficiency was detected with GFP expression by fluorescent microcope. Western - blot method was used to detect the nucleolin proteins and chose the most potent plasmid. Results：The correct results showed that the recombinant plasmids have special fragments, as demonstrated by DNA Sequencing. After 36 hours, GFP could express with the efficiency over 75 % in H9C2 cells transfected with the recombinant plas- mids. Western- blot results showed Ncl- 3 has the most potent compared with the others, as specifically inhibit the expression of nucleolin in H9C2 cells. Conclusion ： The RNAi vectors targeting nucleolin are constructed successfully, and from them Ncl - 3 showed the most potent. The successful construction of RNAi expressing plasmid targeting nucleolin makes it possible for the further studies.