發(fā)布時(shí)間：2018-05-14 11:22

  本文選題：Argonaute2/Ago2 + DFFA��； 參考：《天津醫(yī)科大學(xué)》2008年碩士論文

【摘要】：目的： RNA干擾是一種由siRNA或dsRNA介導(dǎo)的基因沉默表達(dá)的方式。通過(guò)與靶mRNAde特異性結(jié)合,這些小RNA與Argonaute (AGO)蛋白及相關(guān)因子形成RNA誘導(dǎo)的沉默復(fù)合體(RISC),后者完成對(duì)靶mRNA的剪切或者翻譯抑制。前面通過(guò)酵母雙雜交篩選出與人類(lèi)Argonaute2 (Ago2)相關(guān)蛋白——DFFA和PSMC3,本實(shí)驗(yàn)在此基礎(chǔ)上,進(jìn)一步通過(guò)免疫共沉淀驗(yàn)證它們與Ago2相互作用的特異性,通過(guò)熒光實(shí)驗(yàn),我們推測(cè)這兩種蛋白在體內(nèi)miRNA或者siRNA介導(dǎo)的mRNA剪切中發(fā)揮著重要功能。 方法： 利用免疫共沉淀的方法在細(xì)胞內(nèi)驗(yàn)證DFFA、PSMC3同人類(lèi)Ago2蛋白之間相互作用的特異性,通過(guò)GST pull-down驗(yàn)證DFFA同Argonaute2 PIWI結(jié)構(gòu)域(hAgo2 PIWI)體外結(jié)合作用。構(gòu)建內(nèi)源性miR-21介導(dǎo)的GFP陽(yáng)性Read-Out報(bào)告系統(tǒng),通過(guò)該系統(tǒng)驗(yàn)證在分別敲除DFFA和PSMC3的情況下,被抑制的GFP表達(dá)效果是否得以增強(qiáng)；利用siRNA-GFP和表達(dá)GFP的質(zhì)粒作為研究siRNA介導(dǎo)的GFP表達(dá)抑制,觀察在分別敲除DFFA和PSMC3的情況下GFP的表達(dá)強(qiáng)度變化,間接推測(cè)兩者在由小RNA介導(dǎo)的RNA干擾過(guò)程中的功能。 結(jié)果： 1.免疫共沉淀證實(shí)外源性DFFA和PSMC3同轉(zhuǎn)染的hAgo2 PIWI存在著特異的相互作用。進(jìn)一步的實(shí)驗(yàn)證明,DFFA和PSMC3 Flag融合蛋白的N端結(jié)構(gòu)域可能與全長(zhǎng)的hAgo2蛋白相互結(jié)合。 2.通過(guò)GST-pull down體外結(jié)合實(shí)驗(yàn)證實(shí)DFFA同hAgo2 PIWI的相互作用。 3.敲除了DFFA、PSMC3對(duì)miR-21介導(dǎo)的內(nèi)源封閉報(bào)告系統(tǒng)產(chǎn)生的積極地作用。 4.敲除了DFFA、PSMC3對(duì)si-GFP介導(dǎo)的GFP抑制產(chǎn)生了逆轉(zhuǎn)的效果。 結(jié)論： DFFA在體內(nèi)、體外均同hAgo2 PIWI存在著相互作用,PSMC3在體內(nèi)同hAgo2 PIWI存在著相互作用；DFFA和PSMC3 Flag融合蛋白的N端結(jié)構(gòu)域可能與全長(zhǎng)的hAgo2蛋白相互結(jié)合。敲除實(shí)驗(yàn)證實(shí)DFFA、PSMC3均參與了由miRNA或者siRNA介導(dǎo)的mRNA剪切過(guò)程,推測(cè)兩者可能是新的RISC組件。
[Abstract]:Objective: RNA interference is a gene silencing expression mediated by siRNA or dsRNA. By binding specifically to the target mRNAde, these small RNA proteins and related factors form RNA induced silencing complex RISC, which inhibits the splicing or translation of the target mRNA. The proteins associated with human Argonaute2 Ago2 (DFFA and PSMC3) were screened by yeast two-hybrid. On this basis, the specificity of their interaction with Ago2 was further verified by immunoprecipitation, and fluorescence assay was used. We speculate that these two proteins play an important role in miRNA or siRNA mediated mRNA cleavage in vivo. Methods: The specificity of the interaction between DFFA-PSMC3 and human Ago2 protein was verified by immunoprecipitation in vitro, and the binding of DFFA to Argonaute2 PIWI domain hAgo2 PIWI was confirmed by GST pull-down in vitro. An endogenous miR-21 mediated GFP positive Read-Out reporting system was constructed to verify whether the inhibition of GFP expression was enhanced when DFFA and PSMC3 were knocked out respectively. SiRNA-GFP and GFP expression plasmids were used to study the inhibition of GFP expression mediated by siRNA. The changes of GFP expression intensity were observed when DFFA and PSMC3 were knocked out, respectively, and the function of GFP in RNA interference mediated by small RNA was inferred indirectly. Results: 1. Immunoprecipitation confirmed that exogenous DFFA and PSMC3 had specific interactions with transfected hAgo2 PIWI. Further experiments showed that the N-terminal domain of DFFA and PSMC3 Flag fusion protein may interact with the full-length hAgo2 protein. 2. The interaction between DFFA and hAgo2 PIWI was confirmed by GST-pull down binding assay in vitro. 3. Knock out the positive role of DFFAP PSMC3 in miR-21-mediated closed reporting systems. 4. Knockout of DFFAA PSMC3 reversed the inhibition of GFP mediated by si-GFP. Conclusion: DFFA has interaction with hAgo2 PIWI in vivo and in vitro. PSMC3 interacts with hAgo2 PIWI in vivo and the N-terminal domain of PSMC3 Flag fusion protein may bind to the full-length hAgo2 protein. Knockout experiments confirmed that both DFFA-PSMC3 were involved in the mRNA shearing process mediated by miRNA or siRNA, which suggested that both of them might be new RISC components.
【學(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2008
【分類(lèi)號(hào)】：R346

【共引文獻(xiàn)】

相關(guān)博士學(xué)位論文 前1條

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相關(guān)碩士學(xué)位論文 前3條

1 劉寧;對(duì)蝦血細(xì)胞T7噬菌體展示文庫(kù)構(gòu)建及功能基因淘選[D];山東大學(xué);2006年

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3 孔鵬洲;AGO2相互作用蛋白PSMC3、DFFA對(duì)RNAi功能的影響[D];天津醫(yī)科大學(xué);2007年

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本文編號(hào)：1887657