本文選題：豬　切入點：IFIT2　出處：《東北農(nóng)業(yè)大學》2015年碩士論文　論文類型：學位論文

【摘要】：IFITs家族是干擾素刺激基因(Interferon-stimulated genes,ISGs)家族的重要成員,大多數(shù)IFITs基因都由2個外顯子組成。在正常情況下,細胞中不表達IFITs基因,但在干擾素、病毒、微生物的病原相關分子模式等的刺激下,IFITs基因高效、快速表達。對IFIT2的研究多是在人和小鼠上進行的,目前尚未見有關豬IFIT2的研究報告。本課題組在前期的研究中,克隆了豬IFIT2基因并對其啟動子活性區(qū)進行了初步分析,發(fā)現(xiàn)-1836~-1661 bp和-389~-310bp分別存在著負調控元件,-1661~-1397 bp和-310~-126 bp分別存在著正調控元件。為了進一步確定豬IFIT2基因的轉錄調控元件,本研究在生物信息學預測的基礎上,采用真核表達和雙熒光素酶報告系統(tǒng),在體外培養(yǎng)的PK15細胞內(nèi)分析相應序列缺失對啟動子活性的影響。得到的主要研究結果如下:(1)生物信息學分析發(fā)現(xiàn),在-1838~-1661 bp中存在Delta EF1元件。在-1661~-1579 bp中存在1個ISRE(ISREIII)元件。在-389~-310 bp中存在Id3結合位點。在-310~-126 bp中存在2個ISRE元件(ISREI和ISREII)和2個NF-kappa B結合位點(NF-kappa BI和NF-kappa BII)。(2)利用重疊延伸PCR方法對豬IFIT2啟動子區(qū)不同元件進行了缺失突變,獲得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元件缺失的突變體,并以p GL3-Basic為載體骨架,成功構建了熒光報告基因質粒: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)雙熒光素酶報告系統(tǒng)分析表明,Delta EF1抑制豬IFIT2基因轉錄,NF-kappa Bs促進轉錄,ISREs是正調控轉錄的順式作用元件。并且在-310~-126 bp區(qū),2個ISIREs間、2個NF-Kappa Bs間以及NF-Kappa B與ISRE間存在著協(xié)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.
【學位授予單位】：東北農(nóng)業(yè)大學
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：S828;Q78

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