人抗肌萎缩蛋白Dp71 shRNA载体构建与检测  

Construction and evaluation of human Dp71 shRNA vector

作　　者：谭思创[1] 陈志康[2] 曹小霞[3] 文俏程[2] 张蔚林[2] 曾庆仁[4] 谭斯品 

机构地区：[1]中南大学湘雅二医院心胸外科,长沙410011 [2]中南大学湘雅医院胃肠外科,长沙410008 [3]中南大学基础医学院病理生理学系,长沙410078 [4]中南大学细胞与分子生物学实验教学中心,长沙410013

出　　处：《中南大学学报（医学版）》2014年第4期338-343,共6页Journal of Central South University ：Medical Science

基　　金：国家自然科学基金(30800550;81371839);湖南省自然科学基金(10JJ4016)~~

摘　　要：目的:构建有效针对人Dystrophin Dp71基因的短发夹RNA(short hairpin RNA,shRNA)真核表达载体,并验证其干扰效果。方法:设计合成3对针对人Dystrophin Dp71基因的和1对无同源性的siRNA片段,在两端和中间加上酶切位点和Loop环。将合成的DNA片段插入到干扰载体pRNAT-U6.1/Neo中,经过酶切和测序验证,成功构建人Dp71基因的shRNA和空白对照载体。将各干扰载体和空白载体转染人正常胃黏膜上皮细胞(gastric epithelial cells,GES-1)和人支气管上皮细胞(human bronchial epithelium,HBE),Western印迹检测Dp71 shRNA真核表达载体的干扰效率。结果:酶切和测序验证均表明各Dp71-shRNA载体构建成功。将空载体及各Dp71 shRNA载体分别转染GES-1和HBEC,和空白细胞对照及shRNA空白载体组相比,3组Dp71-shRNA能够明显抑制Dp71蛋白表达,但是3组质粒的抑制效率有一定的差异,以Dp71 shRNA2对Dp71表达的干扰效率最强。结论:成功构建了3个有效针对人Dystrophin Dp71基因的shRNA干扰载体,3组质粒都能有效地抑制Dp71在GES-1和HBEC中的表达,其中以Dp71-shRNA2的抑制效率最高。Objective: To construct effective short hairpin RNA (shRNA) recombinant plasmids targeting humanDystrophin Dp71 gene, and evaluate their interference effciency. Methods: hTree pairs of siRNA sequences targeting human Dp71 gene and one pair of control siRNA sequence were designed, synthesized, and then inserted into the pRNAT-U6.1/Neo vector. hTe shRNA recombinant vectors were evaluated by enzyme digestion and sequencing. Dp71-shRNA and control shRNA plasmids were transfected into human normal gastric epithelial cells (GES-1) and humanbronchial epithelium (HBE). Western blot was used to evaluate its interfering effciency. Results: Restriction enzyme digestion and sequencing showed that the Dp71-shRNA vectors were successfully constructed. Western blot displayed that Dp71 protein expression was reduced to a signiifcant degree atfer transfection with the 3 Dp71-shRNA plasmids, and Dp71-shRNA2 plasmid inhibit the Dp71 expression most effciently. Conclusion: Dp71-shRNA vectors have been successfully constructed. The 3 Dp71-shRNA plasmids can inhibit Dp71 expression in GES-1 and HBEC, with Dp71-shRNA2 plasmid displaying the highest inhibition effciency.

关 键 词：DYSTROPHIN Dp71 shRNA载体 干扰效率 

分 类 号：R746.2[医药卫生—神经病学与精神病学]