双启动子shRNA真核表达载体构建.pdfVIP

840 栽蚤葬灶躁蚤灶 Med 允熏 Oct 圆园10熏 灾燥造 38 晕燥 10 双启动子shRNA 真核表达载体的构建* 娜 仁 郭 刚 张 瑞 赵 郁 汪蓓蕾 梁东春 杨宇 吟 摘要 目的：构建含有双启动子的shRNA 真核表达载体。方法：通过PCR 分别扩增带有 BamH 玉、EcoR 玉酶切 位点的人U6 启动子序列（U6-B/E ）和带有 Bgl 域、Hind 芋酶切位点的人U6 启动子序列(U6-B/H)，用 BamH 玉与 EcoR 玉双酶切pcDN粤猿援员（垣）与PCR 产物U6-B/E 后进行连接，重组质粒命名为psPRO。再用Bgl 域与Hind 芋双酶切psPRO 与PCR 产物U6-B/H 后进行连接，构建含有双启动子的shRNA 真核表达载体。结果：酶切鉴定以及 DNA 测序结果 显示载体构建成功。 构建双启动子的shRNA 真核表达载体，使其在细胞中表达2 个不同的siRNA ，不仅筛选方 结论： 便，而且可以通过建立这 模式化工具载体，为今后RNA 干扰实验研究的开展和深入提供手段。 关键词 启动区（遗传学） 遗传载体 RNA，小分子干扰 聚合酶链反应 重组，遗传 质粒 Construction of a shRNA Eukaryotic Expression Vector of Dual Promoters NA Ren, GUO Gang, ZHANG Rui, ZHAO Yu, WANG Beilei, LIANG Dongchun, YANG Yuhong Institute of Endoc rinology, Me tabolic Diseases Hospital of Tianj in Medical Univ ers ity, Tianj in 300070, China To construct shRNA eukaryotic expression vector of dual promoters. Human U6 Abstract Obj ective ： Methods ： promoter sequences, one with BamH 玉and EcoR 玉enzyme sites（U6-B/E), another with Bgl 域and Hind 芋enzyme sites (U6- B/H), were amplified by PCR respectively. The pcDNA3.1(+) plasmid and U6-B/E were double digested by BamH 玉and EcoR 玉. The digestion product of U6 -B/E was inserted into the lined plasmid and the reconstructed plasmid was named psPRO. The psPRO plasmid and U6-B/H were then double digested by Bgl 域and Hind 芋 and the digestion product of U6-B/H was inserted into the lined plasmid. Results ：The recombinant vector was obtained with the correct sequence in the insert. Successful construction of the eukaryotic expression vector may provide the practical