猪IFIT2基因转录调控元件鉴定

发布时间：2018-03-17 17:49

本文选题：猪　切入点：IFIT2　出处：《东北农业大学》2015年硕士论文　论文类型：学位论文

【摘要】：IFITs家族是干扰素刺激基因(Interferon-stimulated genes,ISGs)家族的重要成员,大多数IFITs基因都由2个外显子组成。在正常情况下,细胞中不表达IFITs基因,但在干扰素、病毒、微生物的病原相关分子模式等的刺激下,IFITs基因高效、快速表达。对IFIT2的研究多是在人和小鼠上进行的,目前尚未见有关猪IFIT2的研究报告。本课题组在前期的研究中,克隆了猪IFIT2基因并对其启动子活性区进行了初步分析,发现-1836~-1661 bp和-389~-310bp分别存在着负调控元件,-1661~-1397 bp和-310~-126 bp分别存在着正调控元件。为了进一步确定猪IFIT2基因的转录调控元件,本研究在生物信息学预测的基础上,采用真核表达和双荧光素酶报告系统,在体外培养的PK15细胞内分析相应序列缺失对启动子活性的影响。得到的主要研究结果如下:(1)生物信息学分析发现,在-1838~-1661 bp中存在Delta EF1元件。在-1661~-1579 bp中存在1个ISRE(ISREIII)元件。在-389~-310 bp中存在Id3结合位点。在-310~-126 bp中存在2个ISRE元件(ISREI和ISREII)和2个NF-kappa B结合位点(NF-kappa BI和NF-kappa BII)。(2)利用重叠延伸PCR方法对猪IFIT2启动子区不同元件进行了缺失突变,获得ΔDelta EF1、ΔISREIII、ΔId3-ttg、ΔId3、ΔISREI、ΔISREII、ΔISREI-II、ΔNF-Kappa BI、ΔNF-Kappa BII、ΔNF-Kappa BI-II、ΔNF-Kappa BI+ISREI-II、ΔNF-Kappa BII+ISREI-II、ΔNF-Kappa BI-II+ISREI-II元件缺失的突变体,并以p GL3-Basic为载体骨架,成功构建了荧光报告基因质粒:p GL3-ΔDelta EF1、p GL3-ΔISREIII、p GL3-ΔId3-ttg、p GL3-ΔId3、p GL3-ΔISREI、p GL3-ΔISREII、p GL3-ΔISREI-II、p GL3-ΔNF-Kappa BI、p GL3-ΔNF-Kappa BII、p GL3-ΔNF-Kappa BI-II、p GL3-ΔNF-Kappa BI+ISREI-II、p GL3-ΔNF-Kappa BII+ISREI-II、p GL3-ΔNF-Kappa BI-II+ISREI-II。(3)双荧光素酶报告系统分析表明,Delta EF1抑制猪IFIT2基因转录,NF-kappa Bs促进转录,ISREs是正调控转录的顺式作用元件。并且在-310~-126 bp区,2个ISIREs间、2个NF-Kappa Bs间以及NF-Kappa B与ISRE间存在着协同作用。
[Abstract]:The IFITs family is an important member of the Interferon-stimulated genes (ISGs) family, and most of the IFITs genes are composed of two exons. Under normal circumstances, the IFITs gene is not expressed in the cells, but in interferon and virus. Under the stimulation of pathogen-associated molecular patterns of microbes, the IFIT2 gene is highly efficient and rapidly expressed. Most of the studies on IFIT2 have been carried out in humans and mice, but no reports on porcine IFIT2 have been reported at present. The porcine IFIT2 gene was cloned and its promoter active region was preliminarily analyzed. It was found that there were positive regulatory elements in -18361-1661bp and -389- 310bp, respectively. In order to further determine the transcriptional regulatory elements of porcine IFIT2 gene, the positive regulatory elements were found in porcine IFIT2 gene. In this study, based on bioinformatics prediction, eukaryotic expression and double luciferase reporting system were used. The effect of sequence deletion on promoter activity was analyzed in PK15 cells cultured in vitro. The main results were as follows: 1) Bioinformatics analysis showed that, There are Delta EF1 elements in -1838 ~ 1661 BP, one ISREISREIII element in -1661 ~ 1579 BP, Id3 binding site in -389 ~ 310bp, two ISRE elements in -310 ~ 126bp, and two NF-kappa B binding sites, NF-kappa BI and NF-kappa BII-II). Methods the deletion mutation of different elements in porcine IFIT2 promoter region was carried out. The mutants of 螖 Delta EF1, 螖 ISREIII, 螖 Id3-ttg, 螖 Id3, 螖 ISREI, 螖 ISREIIII, 螖 ISREI-II, 螖 NF-Kappa BI, 螖 NF-Kappa BII, 螖 NF-Kappa BI-II, 螖 NF-Kappa BI ISREI-II, 螖 NF-Kappa BII ISREI-II, 螖 NF-Kappa BI-II ISREI-II missing mutants were obtained. The fluorescent reporter gene plasmid pGL3- 螖 Delta EF1pGL3- 螖 ISREIIIpGL3- 螖 ID3-ttgtgp GL3- 螖 ID3- 螖 ID3REIpGL3- 螖 ISREI-IIp GL3- 螖 NF-Kappa BIP-GL3- 螖 NF-Kappa BIIIP-GL3- 螖 NF-Kappa BI ISREI-IIP GL3- 螖 NF-Kappa ISREI-IIp GL3- 螖 NF-Kappa BII ISREI-IIp GL3- 螖 NF-Kappa BI-II ISREI-IIp GL3- 螖 NF-Kappa BI-II REI-II.app3) has been successfully constructed. The cis-acting elements controlled transcription. There was a synergistic effect between -310 ~ 126bp, 2 ISIREs, 2 NF-Kappa Bs and NF-Kappa B and ISRE.
【学位授予单位】：东北农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S828;Q78

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