北京生物醫(yī)學工程

BACE1基因RNA干擾質(zhì)粒的構(gòu)建及干擾效果的鑒定

Construction and identification of RNA interference plasmid targeting BACE1 gene

作者：張璇胡海梅

單位：深圳市精神衛(wèi)生中心(廣東深圳518020)

關(guān)鍵詞： BACE1；基因；RNA干擾；neuro-2a細胞

分類號：

出版年·卷·期（頁碼）：2013·32·6（579-582）

摘要：

目的構(gòu)建BACE1基因干擾質(zhì)粒，并研究其在neuro-2a細胞中的表達，為以其為靶點的基因治療提供穩(wěn)定轉(zhuǎn)染的質(zhì)粒。方法選擇人、小鼠和大鼠的BACE1基因共有序列為干擾靶點，設(shè)計3組連接有GFP的干擾質(zhì)粒，將構(gòu)建好的質(zhì)粒轉(zhuǎn)染neuro-2a細胞，通過Real time RT-PCR及Western-blotting的方法分別在RNA及蛋白質(zhì)水平上檢測BACE1的表達，以分析其干擾的效率。結(jié)果經(jīng)酶切及測序證實，插入的DNA片段序列與設(shè)計序列完全一致。與空質(zhì)粒對照組相比，3個干擾靶點對BACE1基因的表達均有不同程度的抑制作用。其中pYr-1.1-siBACE1在mRNA水平及蛋白質(zhì)水平干擾效果均最好。結(jié)論成功構(gòu)建了BACE1基因的干擾質(zhì)粒pYr-1.1-siBACE1，并能有效抑制neuro-2a細胞內(nèi)源性BACE1基因表達，為靶向BACE1基因的治療提供了有力的工具。
關(guān)鍵詞

Objective To construction RNA interference plasmid targeting BACE1 gene，and detect the expression in neuro-2a cells in order to provide stable plasmid for BACE1-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 BACE1-targeting gene therapy.

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