干扰素调节因子3第二内含子中新剪接异构体2核心启动子的功能特征

发布时间：2018-11-12 18:11

【摘要】：目的: 干扰素调节因子3(Interferon regulatory factor3,IRF-3)是IRF家族的一个成员,是调节I型干扰素(即干扰素α和β)基因表达的关键转录因子,参与抗病毒感染,包括SARS病毒和丙型肝炎病毒感染,抗细胞内细菌感染。本课题组的前期工作抽取肾上腺嗜铬细胞瘤组织RNA,通过5'RACE(rapid amplification of cDNA ends)在IRF-3基因第二内含子内、第三外显子前162位发现新的转录起始位点,与之对应的新的剪接异构体命名为Int2V2。本课题克隆IRF-3基因第二内含子中新剪接异构体2(intron2 variant2,Int2V2)启动子区,研究其转录调控机制以及不同病毒类似物对Int2V2启动子的影响,为最终阐明IRF-3多个剪接异构体的转录调控奠定基础。方法: 1.以人类基因组DNA为模板,通过PCR定向克隆策略,构建了覆盖IRF-3基因第二内含子内新转录起始位点(第三外显子前162bp)上下游800bp区域的荧光素酶报告基因重组体。2.应用TFSEARCH ver.1.3软件对Int2V2启动子区序列进行分析,对可能存在的转录因子结合位点进行预测,根据转录因子及转录起始位点,采用PCR的方法构建一系列缺失突变体,并瞬时转染AD293及Hela细胞,用Luciferase检测报告基因启动子的活性,计算相对荧光素酶活性单位(RLU)。3.利用点突变技术及过表达试验验证转录因子对Int2V2启动子活性的调节。4.利用共转染研究不同病毒类似物(polyI:C、polydA:dT)对Int2V2启动子及其mRNA水平的影响。结果: 1.酶切、测序鉴定证实成功构建含有IRF-3基因第二内含子中新转录起始位点上下游800bp的启动区的表达质粒。2.启动子活性分析表明,IRF-3第二内含子中新转录起始位点上游区域的启动子活性与正常的pGL3-Basic基本质粒比较,其启动子活性增加了10倍,提示新转录起始位点上游具启动子活性。-317～+30区域的启动子活性显著升高,而-110～+30区域的启动子活性明显下降,表明维持其基本转录活性的必需调控元件在-317 bp至-110 bp之间,采用转录因子结合位点预测分析软件分析表明,该区域内存在转录因子cAMP应答元件结合蛋白(cAMP response element binding protein,CREB)结合位点。3.将CREB结合位点进行点突变后启动子活性下降近50%,过表达后启动子活性上升近40%。4.病毒类似物刺激HEK293细胞后Int2V2启动子活性及mRNA水平均有升高。结论 1. IRF-3第二内含子内新转录起始位点上游具启动子活性。2. -317 bp至-110 bp区存在必需调控元件,转录因子CREB可能参与其转录调控。3.转录因子CREB对启动子活性具有调控作用。4.病毒类似物可影响新启动子的活性及其mRNA水平。
[Abstract]:Objective: interferon regulatory factor 3 (Interferon regulatory factor3,IRF-3) is a member of the IRF family and a key transcription factor regulating the gene expression of interferon type I (interferon 伪 and 尾), and is involved in antiviral infection. Including SARS virus and hepatitis C virus infection, anti-bacterial infection in cells. In our previous work, we extracted RNA, from adrenal pheochromocytoma and discovered new transcriptional initiation sites in the second intron of the IRF-3 gene and in the first 162 positions of the third exon through 5'RACE (rapid amplification of cDNA ends). The corresponding new splicing isomer is named Int2V2. In this study, we cloned the new splicing isomer 2 (intron2 variant2,Int2V2) promoter in the second intron of IRF-3 gene and studied its transcriptional regulation mechanism and the effect of different viral analogues on Int2V2 promoter. It lays a foundation for elucidating the transcriptional regulation of multiple splicing isomers of IRF-3. Methods: 1. Using human genomic DNA as template, the recombinant luciferase reporter gene covering the upstream and downstream 800bp region of the second intron of IRF-3 gene (pre-exon 3 162bp) was constructed by PCR directed cloning strategy. 2. The sequence of Int2V2 promoter region was analyzed by TFSEARCH ver.1.3 software, and the possible transcription factor binding sites were predicted. According to transcription factors and transcription initiation sites, a series of deletion mutants were constructed by PCR method. After transient transfection of AD293 and Hela cells, the activity of reporter gene promoter was detected by Luciferase, and the relative luciferase activity unit (RLU). 3 was calculated. Point mutation technique and overexpression test were used to verify the regulation of transcription factors on the activity of Int2V2 promoter. 4. 4. The effect of different viral analogue (polyI:C,polydA:dT) on Int2V2 promoter and its mRNA level was studied by co-transfection. Results: 1. Restriction endonuclease digestion and sequencing confirmed the successful construction of the promoter region containing the upstream and downstream 800bp in the second intron of the IRF-3 gene. Promoter activity analysis showed that the promoter activity of the upstream region of the new transcriptional initiation site in the second intron of IRF-3 was 10 times higher than that of the normal pGL3-Basic basic plasmid. These results suggested that the promoter activity in the upstream of the new transcription initiation site was significantly increased in the region of -317 ~ 30, but decreased in the region of -110 ~ 30. The essential regulatory elements for maintaining their basic transcriptional activity ranged from -317 bp to -110 bp. The analysis of transcription factor binding site predictive analysis software showed that the transcription factor cAMP responsive element binding protein (cAMP response element binding protein, existed in the region. CREB) binding site. The promoter activity decreased nearly 50% after point mutation of CREB binding site, and increased nearly 40% after overexpression. The activity of Int2V2 promoter and the level of mRNA increased after HEK293 cells stimulated by virus analogue. Conclusion 1. The promoter activity was found in the upstream of the new transcriptional initiation site in the second intron of IRF-3. 2. From -317 bp to -110 bp, there are necessary regulatory elements, and the transcription factor CREB may be involved in its transcriptional regulation. 3. 3. Transcription factor CREB can regulate promoter activity. 4. Viral analogues can affect the activity of the new promoter and its mRNA level.
【学位授予单位】：南京医科大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R346

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