【摘要】 目的构建表达小鼠动力蛋白激活蛋白1(dynactin-1)特异性shRNA的慢病毒载体,并检测其对小鼠足细胞dynactin-1的敲低效果。方法针对小鼠dynactin-1mRNA序列,设计合成3种shRNA,克隆到入门质粒pENTR/pTER中,再利用LR反应重组到pLenti X2Puro慢病毒目的质粒,经过酶切测序鉴定后,将慢病毒质粒和包装质粒共转染293FT细胞,包装得到病毒颗粒。各组shRNA病毒载体转染小鼠足细胞后,用嘌呤霉素抗性筛选细胞,利用蛋白质印迹法检测各组细胞中dynactin-1蛋白的表达水平。结果构建的各组shRNA入门质粒和慢病毒载体经酶切及测序鉴定正确;慢病毒载体与慢病毒包装质粒共转染293FT细胞,制备病毒颗粒,转染小鼠足细胞,经嘌呤霉素筛选获得稳定表达shRNA的足细胞系;蛋白质印迹检测结果表明转染dynactin-1-shRNA组的dynactin-1蛋白表达降低。结论构建的小鼠dynactin-1-shRNA慢病毒载体能有效降低小鼠足细胞中dynactin-1蛋白表达,为进一步研究dynactin-1在足细胞中的功能奠定了基础。更多还原

【Abstract】 Objective To construct the lentiviral vector carrying shRNA of mouse dynactin-1 and to identify its knockdown efficiency of dynactin-1 by infecting mouse podocytes. Methods Three pairs of shRNA targeting mouse dynactin-1 were synthesized and subcloned into BglⅡ-HindⅢ digested pENTR/pTER entry vectors.Then the entry vectors were transferred into pLenti X2 Puro destination vector by LR Clonase reaction.All the constructs were verified by restriction enzyme digestion and sequencing.The pLentiX2 Puro/dynactin-1-shRNA vectors and the packaging vectors were co-transfected into 293FT cells to produce dynactin-1-shRNA lentiviruses,which were used to transfect podocytes and the cells were screened by puromycin resistance.The expression of dynactin-1 protein in podcytes was analyzed by Western blotting analysis. Results Restriction enzyme digestion and sequencing analysis confirmed that the dynactin-1-shRNA lentivirus vector was successfully constructed.The podocyte cell lines stably expressing dynactin-1-shRNA were obtained by puromycin selection.Western blotting analysis indicated that dynactin-1 protein expression was down-regulated by dynactin-1-shRNA lentiviral vector in podocytes. Conclusion We have successfully constructed lentiviral vector of dynactin-1-shRNA,which can effectively down-regulate dynactin-1 protein expression,providing a basis for further studying the role of dynactin-1 in podocytes.更多还原