RNA聚合酶Ⅱ启动子驱动的长双链RNA干扰载体的构建与验证

Construction and verification of long double - stranded RNA interference vector driven by RNA polymerase Ⅱ promoter

为了探索构建由RNA聚合酶Ⅱ型启动子驱动、能有效避免干扰素反应的表达长双链RNA的RNA干扰载体,试验在转录单元两端各添加了一个自剪切核酶序列,在转录后切成RNA出核所必要的5'帽子和3'polyA尾,以绿色荧光蛋白(EGFP)基因为报告基因插入2个核酶之间作为试验载体,以表达红色荧光蛋白基因的载体作转染效率校准,通过瞬间转染对荧光蛋白的表达情况进行观察和RT-PCR分析。结果表明:在转染效率基本一致的情况下,试验组未观察到绿色荧光;2个核酶能够完全自剪切,试验组细胞质中有EGFP mRNA的存在,但与正常对照组相比下降了46.1%。说明研究设计的载体达到了延迟表达RNA出核的目的,为用该载体表达长双链RNA时在其出核之前被Dicer酶有效切割成小双链RNA从而避免其出核引起干扰素反应提供了时间保障。

To explore and construct a long double - stranded RNA interference expression vector driven by RNA polymerase I] promoter, which can effectively avoid interferon response, an eukaryotic expression vector was constructed, in which each end of its transcription unit was added a self cutting ribozyme sequence; the 5＇ Cap and 3＇ polyA tail were cut off, which was the necessary structure for a transcription product to ex- port from nucleus, and the green fluorescent protein （EGFP） gene as reporter gene was inserted between the two self cutting ribozyme se- quence. At the same time, a red fluorescent protein gene expression vector was used as a reporter structure to normalize the different transfection experiment. The expression of the fluorescent protein was observed and analyzed by RT - PCRT through transient transfection experiment. The results showed that there was no green fluorescent being observed for the experiment group at basically same transfection efficiency. RT - PCR results showed that the two self cutting ribozyme sequence could work efficiently, there were EGFP mRNAs in the cytoplasm, but the concentra- tion of the EGFP mRNAs in the cytoplasm of the test group cell dropped 46.1% compare with the control. The results indicate that the expres- sion of EGFP mRNA has been delayed to be exported from the nucleus. This will provide enough time for Dicer enzyme to cut the long double strand RNA into small double strand RNA before it being export from nucleus, and will efficiently avoid the interferon response to provide time protection when the long double strand RNA being expressed in the nucleus.