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| 人抗肌萎缩蛋白Dp71 shRNA载体构建与检测 | |
| 引用本文： | 谭思创,陈志康,曹小霞,文俏程,张蔚林,曾庆仁,谭斯品.人抗肌萎缩蛋白Dp71 shRNA载体构建与检测[J].中南大学学报(医学版),2014,39(4):338-343. |
| 作者姓名： | 谭思创 陈志康 曹小霞 文俏程 张蔚林 曾庆仁 谭斯品 |
| 作者单位： | 中南大学 1. 湘雅二医院心胸外科，长沙 410011；2. 湘雅医院胃肠外科，长沙 410008； 3. 基础医学院病理生理学系，长沙 410078；4. 细胞与分子生物学实验教学中心，长沙 410013 |
| 基金项目： | 国家自然科学基金(30800550，81371839)；湖南省自然科学基金(10JJ4016)。This work was supported by National Natural Science Fund of China (30800550, 81371839) and the Natural Science Fund of Hunan Province, P. R. China (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。 |
| 关 键 词： | Dystrophin Dp71 shRNA 载体 干扰效率 |
Construction and evaluation of human Dp71 shRNA vector |
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| TAN Sichuang,CHEN Zhikang,CAO Xiaoxia,WEN Qiaocheng,ZHANG Weilin,ZENG Qingr.Construction and evaluation of human Dp71 shRNA vector[J].Journal of Central South University (Medical Sciences)Journal of Central South University (Medical Sciences),2014,39(4):338-343. | |
| Authors: | TAN Sichuang CHEN Zhikang CAO Xiaoxia WEN Qiaocheng ZHANG Weilin ZENG Qingr |
| Institution: | 1. Department of Cardiovascular and Thoracic Surgery, Second Xiangya Hospital, Central South University, Changsha 410011; 2. Department of Gastrointestinal Surgery, Xiangya Hospital, Central South University, Changsha 410008; 3. Department of Pathophysiology, School of Basic Medicine, Central South University, Changsha 410078; 4. Cell and Molecular Biology Experimental Teaching Center, Central South University, Changsha 410013, China |
| Abstract: | Objective: To construct effective short hairpin RNA (shRNA) recombinant plasmids targeting human Dystrophin Dp71 gene, and evaluate their interference efficiency. Methods: Three 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. The 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 human bronchial epithelium (HBE). Western blot was used to evaluate its interfering efficiency. 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 significant degree after transfection with the 3 Dp71-shRNA plasmids, and Dp71-shRNA2 plasmid inhibit the Dp71 expression most efficiently. 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 efficiency. |
| Keywords: | Dystrophin Dp71 shRNA plasmid inhibition efficiency |
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