發(fā)布時(shí)間：2018-03-04 15:10

  本文選題：STING　切入點(diǎn)：啟動(dòng)子　出處：《南京醫(yī)科大學(xué)》2017年碩士論文　論文類(lèi)型：學(xué)位論文

【摘要】：背景:固有免疫反應(yīng)是機(jī)體抵抗病毒感染的第一道防線(xiàn),在抗病毒免疫過(guò)程中具有關(guān)鍵作用。干擾素基因刺激因子(stimulator of interferon genes,STING)是抗DNA病毒信號(hào)通路中重要的接頭蛋白,還可作為模式識(shí)別受體(pattern recognition receptor,PRR)直接識(shí)別cAMP、cGMP等參與抗胞內(nèi)菌感染,此外STING還參與抗RNA病毒的免疫,并在抗腫瘤免疫及自身免疫性疾病中均具有重要的作用。目前對(duì)STING的研究主要集中在其活化下游激酶TANK結(jié)合激酶 1(TANK binding kinase 1,TBK1)和干擾素刺激因子 3(Interferon Regulatory Factor 3,IRF3)的分子機(jī)制及其在不同病原體誘導(dǎo)宿主產(chǎn)生固有免疫應(yīng)答過(guò)程中的功能。而STING在進(jìn)化上高度保守,人源性的STING和鼠源性的STING具有很高的同源性,因此小鼠常被作為STING功能研究中重要的動(dòng)物模型,但兩者仍因種屬的特異性在某些方面仍存在差異。本課題組前期對(duì)人源性STING的啟動(dòng)子進(jìn)行了相關(guān)研究,目前尚無(wú)對(duì)小鼠STING基因啟動(dòng)子方面的研究。因此,本課題主要針對(duì)小鼠STING基因啟動(dòng)子的特征和調(diào)控機(jī)制進(jìn)行研究。目的:尋找并克隆鑒定小鼠干擾素基因刺激因子(STING)的啟動(dòng)子區(qū)及其啟動(dòng)子核心功能區(qū)域,并進(jìn)一步鑒定調(diào)控小鼠STING基因啟動(dòng)子基本轉(zhuǎn)錄活性的關(guān)鍵轉(zhuǎn)錄因子,為揭示小鼠STING基因的表達(dá)調(diào)控機(jī)制及種屬特異性奠定基礎(chǔ)。方法:通過(guò)生物信息數(shù)據(jù)庫(kù)預(yù)測(cè)啟動(dòng)子位置,以NIH3T3細(xì)胞抽提的基因組DNA為模板,利用PCR技術(shù)擴(kuò)增獲取小鼠STING 5'上游的啟動(dòng)子區(qū)序列(1005nt,-828～+177),將此核酸序列亞克隆至無(wú)啟動(dòng)子活性的PGL3-Basic載體的多克隆位點(diǎn),構(gòu)建熒光素酶重組報(bào)告質(zhì)粒。將構(gòu)建的重組報(bào)告質(zhì)粒瞬時(shí)轉(zhuǎn)染NIH3T3細(xì)胞及HEK293細(xì)胞,使用熒光素酶報(bào)告基因檢測(cè)系統(tǒng)檢測(cè)其熒光素酶活性,鑒定其是否具有啟動(dòng)子活性。利用步移缺失突變分析法對(duì)STING基因啟動(dòng)子5'端進(jìn)行缺失突變,分別亞克隆到pGL3-Basic載體上,瞬時(shí)轉(zhuǎn)染細(xì)胞后檢測(cè)各截短片段的熒光素酶活性,比較相對(duì)熒光素酶活性(RLU)后定位STING基因啟動(dòng)子的核心功能區(qū)域。利用生物信息學(xué)軟件分析核心啟動(dòng)子區(qū)并預(yù)測(cè)潛在的轉(zhuǎn)錄因子結(jié)合位點(diǎn),進(jìn)一步利用定點(diǎn)突變技術(shù)、si-RNA干擾及基因過(guò)表達(dá)技術(shù)、染色質(zhì)免疫沉淀(chromatin immunoprecitation,ChIP)等實(shí)驗(yàn)方法,分析轉(zhuǎn)錄因子對(duì)小鼠STING啟動(dòng)子活性的影響及其作用機(jī)制。結(jié)果:經(jīng)雙酶切、基因測(cè)序鑒定,成功克隆并構(gòu)建了含有小鼠STING啟動(dòng)子區(qū)序列的熒光素酶重組報(bào)告質(zhì)粒pSTING-1005。熒光素酶活性實(shí)驗(yàn)結(jié)果顯示,與載體pGL3-Basic比較,小鼠STING啟動(dòng)子重組報(bào)告質(zhì)粒在NIH3T3細(xì)胞及HEK 293細(xì)胞中均表現(xiàn)出明顯的啟動(dòng)子活性。進(jìn)一步分析STING基因5'端缺失的截短片段的熒光素酶活性后發(fā)現(xiàn),STING基因的核心啟動(dòng)子區(qū)域位于-77～+177nt之間。利用生物信息學(xué)軟件對(duì)小鼠STING基因核心啟動(dòng)子區(qū)域進(jìn)行預(yù)測(cè),該區(qū)域內(nèi)包含了 GATA1、Sp1/Sp3、STAT和IK2等轉(zhuǎn)錄因子結(jié)合位點(diǎn)。將潛在的轉(zhuǎn)錄因子結(jié)合位點(diǎn)的關(guān)鍵性堿基進(jìn)行定點(diǎn)突變,實(shí)驗(yàn)結(jié)果表明轉(zhuǎn)錄因子GATA1和Sp1/Sp3結(jié)合位點(diǎn)對(duì)維持小鼠STING基因的基本轉(zhuǎn)錄活性具有重要作用。同時(shí)利用si-RNA干擾及基因過(guò)表達(dá)實(shí)驗(yàn)進(jìn)一步證實(shí)了 GATA1和Sp3可以調(diào)控STING基因啟動(dòng)子活性,從而影響其翻譯和表達(dá)。ChIP實(shí)驗(yàn)結(jié)果進(jìn)一步證明了轉(zhuǎn)錄因子GATA1和Sp3可以與小鼠STING基因啟動(dòng)子直接結(jié)合。結(jié)論:克隆并成功構(gòu)建了小鼠STING基因啟動(dòng)子的熒光素酶重組報(bào)告質(zhì)粒,通過(guò)截短突變確定其核心啟動(dòng)子區(qū)域位于轉(zhuǎn)錄起始位點(diǎn)-77至+177 nt內(nèi),轉(zhuǎn)錄因子GATA1和Sp3可正向調(diào)控小鼠STING基因啟動(dòng)子的活性。ChIP實(shí)驗(yàn)結(jié)果進(jìn)一步證明了轉(zhuǎn)錄因子GATA1和Sp3對(duì)STING基因啟動(dòng)子的作用是通過(guò)與STING的啟動(dòng)子區(qū)特定的核酸序列直接結(jié)合而實(shí)現(xiàn)。
[Abstract]:Background: the innate immune response is the first line of the body against infection, plays a key role in antiviral immune process. Interferon gene stimulating factor (stimulator of interferon genes, STING) is an important signaling adaptor protein against DNA virus, also can be used as pattern recognition receptors (pattern, recognition receptor, PRR) direct recognition cAMP, cGMP participate in anti intracellular bacteria infection, STING also participate in immunity against RNA virus, and plays an important role in anti-tumor immunity and autoimmune diseases. The current research on STING mainly focus on the activation of downstream kinase TANK binding kinase 1 (TANK binding 1 kinase, TBK1) and interferon stimulating factor 3 (Interferon Regulatory 3 Factor, IRF3) and its molecular mechanism in different pathogens induce the process of innate immune response function. STING in the evolution of high Conservative, human STING and rat STING with high homology, so the mouse is often regarded as an important animal model for functional studies of STING, but they are still due to species specificity in some aspects still exist differences. The human STING promoter of ourprevious the related research, there is no study on promoter of mouse STING gene aspects. Therefore, this paper focuses on the mouse STING gene promoter characteristics and regulation mechanism were studied. Objective: to search and clone identification of mouse interferon stimulating factor gene (STING) promoter region and its promoter core functional areas, and further identification key transcriptional regulation of the mouse STING gene promoter transcriptional activity, lay the foundation for revealing the mechanism of expression regulation of mouse STING gene and species specificity. Methods: the biological information database to predict. Rotor position, using the genomic DNA of NIH3T3 cells was extracted as template, amplified the sequence of mouse STING promoter region upstream of the 5'by using the technology of PCR (1005nt, -828 ~ +177), the multiple cloning sites of the nucleic acid sequence was subcloned into plasmid PGL3-Basic promoter activity, luciferase reporter plasmid to construct recombinant constructs. The recombinant reporter plasmids were transiently transfected into NIH3T3 cells and HEK293 cells using luciferase reporter the luciferase activity, identification of its promoter activity. Using step mutation analysis of STING gene promoter 5' terminal deletion mutant shift deletion, were subcloned into pGL3-Basic vector and detect the truncated luciferase activity instantaneous after the transfection, relative luciferase activity (RLU) core functional areas after the localization of STING gene promoter by bioinformatics software analysis. The core promoter region and prediction of potential transcription factor binding sites, further using site directed mutagenesis technique, si-RNA interference and gene expression technology, chromatin immunoprecipitation (chromatin immunoprecitation, ChIP) and other experimental methods, analysis of transcription factor promoter activity of STING mice and its mechanism. Results: after double enzyme digestion, gene sequencing was successfully cloned and constructed with mouse STING promoter sequence of the recombinant luciferase reporter plasmid pSTING-1005. luciferase activity assay showed that, compared with the vector pGL3-Basic, the recombinant mouse STING promoter reporter plasmids in NIH3T3 cells and HEK 293 cells showed obvious promoter activity. Further analysis of the truncated fragments of STING gene 5'terminal the lack of luciferase activity after the discovery, the core promoter region of STING was located at -77 ~ +177nt. The use of biological Bioinformatics software to predict the mouse STING gene core promoter region, the region contains GATA1, Sp1/Sp3, STAT and IK2 transcription factor binding sites. The potential transcription factor binding sites are the key bases of site directed mutagenesis, the experimental results show that the transcription factor GATA1 and Sp1/Sp3 binding sites plays an important role in maintaining basic transcription the activity of mouse STING gene. At the same time by si-RNA interference and overexpression experiments further confirmed that GATA1 and Sp3 can regulate STING promoter activity, thus affecting the translation and expression of.ChIP experimental results further proved that the transcription factor GATA1 and Sp3 can be directly combined with mouse STING gene. Conclusion: cloned and constructed successfully luciferase reporter plasmid of recombinant mouse STING gene promoter, the truncated mutation determines its core promoter region in transcription initiation Site of -77 to +177 in NT, the activity of.ChIP experimental results of transcription factor GATA1 and Sp3 can positively regulate the mouse STING gene promoter further proved that the transcription factor GATA1 and Sp3 promoter of STING gene is achieved through direct binding with STING promoter region specific nucleic acid sequences.

【學(xué)位授予單位】：南京醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：R392

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