本文選題：RNA干擾　切入點：I_1PP2A　出處：《華中科技大學》2009年碩士論文　論文類型：學位論文

【摘要】：RNA干涉(RNAi)技術(shù)在實驗室中是一種強大的實驗工具,它是利用具有同源性的雙鏈RNA(dsRNA)誘導特異性的目標基因沉默,迅速降低基因表達水平。siRNA在RNA沉默通道中起中心作用,是對特定信使RNA(mRNA)進行降解的指導要素。本研究通過Nucleotide BLAST在線設(shè)計含有小發(fā)夾機構(gòu)的2條I1PP2A、P53和CREB對應模板DNA序列,經(jīng)過褪火、磷酸化、連接處理后克隆至psuppress質(zhì)粒,構(gòu)建重組質(zhì)粒psuppress-siI1PP2A、psuppress-siP53、psuppress-siCREB。通過酶切和測序鑒定重組產(chǎn)物的正確性。然后使用lipofectAMINE 2000轉(zhuǎn)染試劑將上述構(gòu)建好的質(zhì)粒分別轉(zhuǎn)染HEK293細胞。培養(yǎng)一定時間之后提取蛋白,利用western blot檢測I1PP2A、P53和CREB蛋白水平的變化。結(jié)果:經(jīng)酶切及測序鑒定,成功構(gòu)建了psuppress-siI1、psuppress-siP53、psuppress-siCREB的表達質(zhì)粒。它們分別可顯著抑制I1PP2A、P53和CREB的蛋白表達,與未轉(zhuǎn)染組和陰性組對照細胞相比分別降低了75.1%、90.5%、72.1%。結(jié)論:psuppress-siI1、psuppress-siP53、psuppress-siCREB重組質(zhì)粒分別能夠抑制HEK293細胞中其相應基因的蛋白表達。為進一步研究它們的蛋白功能和在AD發(fā)病機制研究提供了實驗工具。
[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.
【學位授予單位】：華中科技大學
【學位級別】：碩士
【學位授予年份】：2009
【分類號】：R346

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