几种AD相关基因的siRNA质粒的构建及其鉴定

发布时间：2018-03-07 16:29

本文选题：RNA干扰　切入点：I_1PP2A　出处：《华中科技大学》2009年硕士论文　论文类型：学位论文

【摘要】：RNA干涉(RNAi)技术在实验室中是一种强大的实验工具,它是利用具有同源性的双链RNA(dsRNA)诱导特异性的目标基因沉默,迅速降低基因表达水平。siRNA在RNA沉默通道中起中心作用,是对特定信使RNA(mRNA)进行降解的指导要素。本研究通过Nucleotide BLAST在线设计含有小发夹机构的2条I1PP2A、P53和CREB对应模板DNA序列,经过褪火、磷酸化、连接处理后克隆至psuppress质粒,构建重组质粒psuppress-siI1PP2A、psuppress-siP53、psuppress-siCREB。通过酶切和测序鉴定重组产物的正确性。然后使用lipofectAMINE 2000转染试剂将上述构建好的质粒分别转染HEK293细胞。培养一定时间之后提取蛋白,利用western blot检测I1PP2A、P53和CREB蛋白水平的变化。结果:经酶切及测序鉴定,成功构建了psuppress-siI1、psuppress-siP53、psuppress-siCREB的表达质粒。它们分别可显著抑制I1PP2A、P53和CREB的蛋白表达,与未转染组和阴性组对照细胞相比分别降低了75.1%、90.5%、72.1%。结论:psuppress-siI1、psuppress-siP53、psuppress-siCREB重组质粒分别能够抑制HEK293细胞中其相应基因的蛋白表达。为进一步研究它们的蛋白功能和在AD发病机制研究提供了实验工具。
[Abstract]:RNA interference RNAi (RNAi) is a powerful experimental tool in the laboratory. It is used to induce specific target gene silencing by using homologous double-stranded RNAs RNAs, thus rapidly reducing the level of gene expression. SiRNAs play a central role in the RNA silencing pathway. In this study, Nucleotide BLAST was used to design two I1PP2ANP53 and CREB corresponding template DNA sequences by Nucleotide BLAST, and then cloned into psuppress plasmids after mellowing, phosphorylation and ligation. The recombinant plasmid psuppress-siIpP2An psuppress-siP53 psuppress-siCREBwas constructed. The recombinant product was confirmed by restriction endonuclease digestion and sequencing. Then the constructed plasmid was transfected into HEK293 cells using lipofectAMINE 2000 transfection reagent. The protein was extracted after a certain time of culture. Results: the expression plasmids of psuppress-siIpsuppress-siP53 and CREB were successfully constructed by restriction endonuclease digestion and sequencing. The expression of psuppress-siP53 and CREB proteins were significantly inhibited by western blot. Compared with the untransfected cells and the negative control cells, 75.1% and 72.1% psuppress-siI1psuppress-siI-1 psuppress-p53 psuppress-siCREB recombinant plasmids could inhibit the protein expression of their corresponding genes in HEK293 cells respectively. In order to further study their protein function and study the pathogenesis of AD, the recombinant plasmid psuppress-siCREB can inhibit the expression of its corresponding genes in HEK293 cells. Experimental tools are provided.
【学位授予单位】：华中科技大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R346

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