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在線設計含有小發(fā)夾機構的2條I1PP2A、P53和CREB對應模板DNA序列,經過褪火、磷酸化、連接處理后克隆至psuppress質粒,構建重組質粒psuppress-siI1PP2A、psuppress-siP53、psuppress-siCREB。通過酶切和測序鑒定重組產物的正確性。然后使用lipofectAMINE 2000轉染試劑將上述構建好的質粒分別轉染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。結果:經酶切及測序鑒定,成功構建了psuppress-siI1、psuppress-siP53、psuppress-siCREB的表達質粒。它們分別可顯著抑制I1PP2A、P53和CREB的蛋白表達,與未轉染組和陰性組對照細胞相比分別降低了75.1%、90.5%、72.1%。結論:psuppress-siI1、psuppress-siP53、psuppress-siCREB重組質粒分別能夠抑制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

【參考文獻】

相關期刊論文 前6條

1 谷海剛,龍大宏;神經營養(yǎng)因子基因治療老年性癡呆的研究進展[J];廣州醫(yī)學院學報;2002年03期

2 張旺明,牛東濱,徐如祥,王曉民;中樞神經系統(tǒng)疾病的基因治療[J];基礎醫(yī)學與臨床;2002年03期

3 龍大宏,楊丹迪,羅秀梅,洪樂鵬,許孟杰,李沃棠;神經生長因子促進老年鼠大腦皮質膽堿能纖維損傷后再生[J];解剖學雜志;2001年05期

4 李承晏;老年性癡呆的治療進展[J];臨床內科雜志;2003年12期

5 龍大宏,姚志彬,李沃棠,許孟杰;神經生長因子對老年癡呆鼠學習記憶能力的影響[J];中國行為醫(yī)學科學;2001年01期

6 屈秋民,喬晉,楊劍波,韓建峰,羅國剛,張輝,武成斌,王小娟,霍東紅,楊華,李正儀,鄧美英,韓雪梅,趙松珍,于普林,張振馨;西安地區(qū)中老年人的癡呆患病率調查[J];中華老年醫(yī)學雜志;2001年04期

，

本文編號：1580057