本文選題：干擾素調(diào)節(jié)因子3 + 啟動子��； 參考：《南京醫(yī)科大學(xué)》2011年博士論文

【摘要】：干擾素調(diào)節(jié)因子3 (interferon regulatory factor 3, IRF-3)是調(diào)節(jié)Ⅰ型干擾素(IFN-α/β)基因表達的關(guān)鍵轉(zhuǎn)錄因子,在固有免疫反應(yīng)和獲得性免疫反應(yīng)中的起重要性作用。目前,國內(nèi)外關(guān)于IRF-3的研究幾乎全部集中在IRF-3參與的抗病毒自身免疫信號通路中的作用,而關(guān)于IRF-3的自身轉(zhuǎn)錄調(diào)控機制尚不十分清楚。生物信息學(xué)分析結(jié)果表明IRF-3第一內(nèi)含子和第二內(nèi)含子內(nèi)有新的轉(zhuǎn)錄起始位點(transcription start site, TSS),表明該基因有多個啟動子和多個剪接異構(gòu)體,但現(xiàn)有文獻未見該分子多個啟動子鑒定和詳細的剪接異構(gòu)體分析的報道。 本研究中通過5'末端快速cDNA末端擴增法(rapid amplification of cDNA ends, RACE),發(fā)現(xiàn)IRF-3第二內(nèi)含子內(nèi)存在兩個新的轉(zhuǎn)錄起始位點,分別位于第三外顯子前718bp和162bp,與之對應(yīng)的新的剪接異構(gòu)體命名為intron 2 variant 1(Int2V1)和intron 2 variant 2(Int2V2)。RT-PCR發(fā)現(xiàn)二者在多數(shù)組織細胞中表達,并存在表達差異。為探索新的剪接異構(gòu)體Int2V1的轉(zhuǎn)錄調(diào)控機制,分別克隆不同長度的5'側(cè)翼啟動子區(qū)域進行熒光素酶功能分析,發(fā)現(xiàn)Int2V1的核心啟動子位于轉(zhuǎn)錄起始位點上游159~100bp之間,結(jié)合點突變、過表達和RNA干擾實驗以及凝膠遷移實驗(electrophoretic mobility shift assay, EMSA)和染色質(zhì)免疫沉淀法(chromatin immunoprecitation, ChIP)證實轉(zhuǎn)錄因子特異性蛋白1(specificity protein, Sp1)是調(diào)節(jié)其啟動子活性及mRNA水平的關(guān)鍵轉(zhuǎn)錄因子。EB病毒(Epstein-Barr varius)感染患兒外周血中Int2V1 mRNA水平較健康患兒低。病毒雙鏈RNA(double stranded RNA, dsRNA)類似物Poly(I:C)和雙鏈DNA(double stranded DNA, dsDNA)類似物poly(dA:dT)轉(zhuǎn)染293T細胞發(fā)現(xiàn)隨著轉(zhuǎn)染時間和劑量的增加Int2V1啟動子活性隨之增加。Poly(I:C)刺激組隨時間延長Int2V1 mRNA表達量升高。poly(dA:dT)處理組,Int2V1在6-8小時表達量到達高峰,隨后下降。 本研究拓展了IRF-3啟動子和剪接異構(gòu)體的探索,完善了IRF-3轉(zhuǎn)錄調(diào)控的關(guān)鍵信息。
[Abstract]:Interferon regulatory factor 3 (IRF-3) is a key transcription factor in the regulation of IFN- 偽 / 尾 gene expression and plays an important role in the innate and acquired immune response. At present, almost all the studies on IRF-3 at home and abroad focus on the role of IRF-3 in the anti-virus autoimmune signaling pathway, but the mechanism of autotranscription regulation of IRF-3 is not very clear. Bioinformatics analysis showed that there were new transcription start sites in the first and second introns of IRF-3, indicating that the gene had multiple promoters and splicing isomers. However, the identification of multiple promoters and the detailed analysis of splicing isomers have not been reported in the literature. In this study, we found that there are two new transcriptional initiation sites in the second intron of IRF-3 by rapid cDNA terminal amplification method. The new splicing isomers were named intron 2 variant 1 (Int2V1) and intron 2 variant 2(Int2V2).RT-PCR, respectively, and they were found to be expressed differently in most tissue cells. In order to explore the transcriptional regulation mechanism of new splicing isomer Int2V1, luciferase function analysis was performed by cloning 5 'flanking promoter regions with different lengths. It was found that the core promoter of Int2V1 was located between the upstream 159~100bp of transcription initiation site, and the binding point was mutated. Overexpression and RNA interference assay, gel migration assay, electrophoretic mobility shift assay, EMSA) and chromatin immunoprecipitation (Chip-immunoprecipitation) confirmed that transcription factor-specific protein 1(specificity (Sp1) is the key transcription factor to regulate its promoter activity and mRNA level. EB disease The level of Int2V1 mRNA in peripheral blood of children with Epstein-Barr varius infection was lower than that of healthy children. 293T cells were transfected with virus double-stranded RNA(double stranded RNA, dsRNA) analogues PolyRNA(double stranded RNA, dsRNA): C) and double-stranded DNA(double stranded DNA, dsDNA) analogues polyDNA: DT). It was found that the activity of Int2V1 promoter increased with the increase of transfection time and dose, and the activity of Int2V1 promoter increased with the increase of transfection time and dose. The expression of Int2V1 reached its peak at 6-8 hours. Then it went down. This study expanded the exploration of IRF-3 promoter and splicing isomer and improved the key information of IRF-3 transcription regulation.
【學(xué)位授予單位】：南京醫(yī)科大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2011
【分類號】：R392.1

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本文編號：1899685