β分泌酶RNA干扰质粒的构建与鉴定  

Construction and Identification of Small Interfering RNA Plasmid Specific for BACE in vitro

作　　者：潘岩[1] 朱文斌[1] 陈雪梅[1] 徐运[1] 

机构地区：[1]南京大学医学院附属鼓楼医院神经内科

出　　处：《生命科学研究》2007年第3期200-205,共6页Life Science Research

基　　金：江苏省自然科学基金资助项目(BK2005002)

摘　　要：选择人、小鼠和大鼠的BACE1基因的共有序列为干扰的靶标,设计4对干扰片段连接至带有GFP和U6 RNA聚合酶启动子的PRNATU6.1载体.将构建好的质粒转染HEK293T细胞,通过RT-PCR和Westernblotting的方法分别在RNA和蛋白水平上来检测BACE1和Aβ,以鉴定其干扰效率,并通过夹心ELISA的方法进一步在过表达蛋白APP和BACE1的HEK293T细胞体系中验证干扰质粒对Aβ1-42生成的影响.RT-PCR和Western blotting结果提示β分泌酶的siRNA能干扰BACE1和AβRNA和蛋白水平表达,效率分别达到90%和80%.Western blotting和ELISA结果提示,β分泌酶的siRNA也降低Aβ1-42生成.The consensus BACE1 gene sequence of human, mouse and rat were used as target, and four designed dsRNA molecules were produced through the constructed GFP plasmids that contain DNA templates for the synthesis of siRNA under the control of U6 promoter. HEK293T cell was transfeeted with those plasmids, and the interference efficiency was assayed through RT-PCR and Western blotting. Finally effective plasmids could inhibit BACE1 RNA and protein expression, which the inhibition ratios were about 80% and 90% respectively. These plasmids were proved to be useful to decrease the Aβ1-42 generation by sandwich ELISA in HEK293T cell system which expressed protein APP and BACE1 at high levels.

关 键 词：Alzheimer’s病 RNAI Β分泌酶 卢淀粉样肽 

分 类 号：Q189[生物学—神经生物学]