小鼠干扰素基因刺激因子启动子的克隆鉴定与转录调控机制的初探

发布时间：2018-03-04 15:10

本文选题：STING　切入点：启动子　出处：《南京医科大学》2017年硕士论文　论文类型：学位论文

【摘要】：背景:固有免疫反应是机体抵抗病毒感染的第一道防线,在抗病毒免疫过程中具有关键作用。干扰素基因刺激因子(stimulator of interferon genes,STING)是抗DNA病毒信号通路中重要的接头蛋白,还可作为模式识别受体(pattern recognition receptor,PRR)直接识别cAMP、cGMP等参与抗胞内菌感染,此外STING还参与抗RNA病毒的免疫,并在抗肿瘤免疫及自身免疫性疾病中均具有重要的作用。目前对STING的研究主要集中在其活化下游激酶TANK结合激酶 1(TANK binding kinase 1,TBK1)和干扰素刺激因子 3(Interferon Regulatory Factor 3,IRF3)的分子机制及其在不同病原体诱导宿主产生固有免疫应答过程中的功能。而STING在进化上高度保守,人源性的STING和鼠源性的STING具有很高的同源性,因此小鼠常被作为STING功能研究中重要的动物模型,但两者仍因种属的特异性在某些方面仍存在差异。本课题组前期对人源性STING的启动子进行了相关研究,目前尚无对小鼠STING基因启动子方面的研究。因此,本课题主要针对小鼠STING基因启动子的特征和调控机制进行研究。目的:寻找并克隆鉴定小鼠干扰素基因刺激因子(STING)的启动子区及其启动子核心功能区域,并进一步鉴定调控小鼠STING基因启动子基本转录活性的关键转录因子,为揭示小鼠STING基因的表达调控机制及种属特异性奠定基础。方法:通过生物信息数据库预测启动子位置,以NIH3T3细胞抽提的基因组DNA为模板,利用PCR技术扩增获取小鼠STING 5'上游的启动子区序列(1005nt,-828～+177),将此核酸序列亚克隆至无启动子活性的PGL3-Basic载体的多克隆位点,构建荧光素酶重组报告质粒。将构建的重组报告质粒瞬时转染NIH3T3细胞及HEK293细胞,使用荧光素酶报告基因检测系统检测其荧光素酶活性,鉴定其是否具有启动子活性。利用步移缺失突变分析法对STING基因启动子5'端进行缺失突变,分别亚克隆到pGL3-Basic载体上,瞬时转染细胞后检测各截短片段的荧光素酶活性,比较相对荧光素酶活性(RLU)后定位STING基因启动子的核心功能区域。利用生物信息学软件分析核心启动子区并预测潜在的转录因子结合位点,进一步利用定点突变技术、si-RNA干扰及基因过表达技术、染色质免疫沉淀(chromatin immunoprecitation,ChIP)等实验方法,分析转录因子对小鼠STING启动子活性的影响及其作用机制。结果:经双酶切、基因测序鉴定,成功克隆并构建了含有小鼠STING启动子区序列的荧光素酶重组报告质粒pSTING-1005。荧光素酶活性实验结果显示,与载体pGL3-Basic比较,小鼠STING启动子重组报告质粒在NIH3T3细胞及HEK 293细胞中均表现出明显的启动子活性。进一步分析STING基因5'端缺失的截短片段的荧光素酶活性后发现,STING基因的核心启动子区域位于-77～+177nt之间。利用生物信息学软件对小鼠STING基因核心启动子区域进行预测,该区域内包含了 GATA1、Sp1/Sp3、STAT和IK2等转录因子结合位点。将潜在的转录因子结合位点的关键性碱基进行定点突变,实验结果表明转录因子GATA1和Sp1/Sp3结合位点对维持小鼠STING基因的基本转录活性具有重要作用。同时利用si-RNA干扰及基因过表达实验进一步证实了 GATA1和Sp3可以调控STING基因启动子活性,从而影响其翻译和表达。ChIP实验结果进一步证明了转录因子GATA1和Sp3可以与小鼠STING基因启动子直接结合。结论:克隆并成功构建了小鼠STING基因启动子的荧光素酶重组报告质粒,通过截短突变确定其核心启动子区域位于转录起始位点-77至+177 nt内,转录因子GATA1和Sp3可正向调控小鼠STING基因启动子的活性。ChIP实验结果进一步证明了转录因子GATA1和Sp3对STING基因启动子的作用是通过与STING的启动子区特定的核酸序列直接结合而实现。
[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.

【学位授予单位】：南京医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R392

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