BACE1基因RNA干扰质粒的构建及干扰效果的鉴定

目的构建BACE1基因干扰质粒，并研究其在neuro．2a细胞中的表达，为以其为靶点的基因治疗提供稳定转染的质粒。方法选择人、小鼠和大鼠的BACE1基因共有序列为干扰靶点，设计3组连接有GFP的干扰质粒，将构建好的质粒转染nellro-2a细胞，通过RealtimeRT—PCR及Western—blotting的方法分别在RNA及蛋白质水平上检测BACE1的表达，以分析其干扰的效率。结果经酶切及测序证实，插入的DNA片段序列与设计序列完全一致。与空质粒对照组相比，3个干扰靶点对BACE1基因的表达均有不同程度的抑制作用。其中pYr-1．1-siBACE1在mRNA水平及蛋白质水平干扰效果均最好。结论成功构建了BACE1基因的干扰质粒pYr-1．1-siBACE1，并能有效抑制neuro．2a细胞内源性BACE1基因表达，为靶向BACE1基因的治疗提供了有力的工具。

Construction and identification of RNA interference plasmid targeting BACE1 gene

Objective To construction RNA interference plasmid targeting BACE1 gene, and detect the expression in neuro-2a cells in order to provide stable plasmid for BACEl-targeting gene therapy. Methods The consensus BACE1 gene sequence of human, mouse and rat were used as target, and three RNA interference plasmid connected with GFP were designed. Neuro-2a cells were transfected with those plasmids, and the expression of RNA and protein were detected through Real time RT-PCR and Western-blotting so as to assay the interference efficiency. Results RNA interference plasmids were correct confirmed by digestion and DNA sequencing. Three sites of siBACE1 had distinct effect for inhibition of BACE1 gene compared to control group. And pYr-1. 1-siBACE1 was the most effective one in the suppression of BACE1 gene with significant reduction of mRNA and protein level. Conclusions RNA interference plasmid pYr-1.1- siBACE1 is successfully constructed and the expression of endogenous BACE1 gene in neuro-2a cells is effectively inhibited. This offers a powerful tool for BACEl-targeting gene therapy.