大黄鱼TLR5与NF-κB家族部分基因的克隆与功能研究
发布时间：2018-08-01 10:41
【摘要】：本研究在实验室前期获得大黄鱼(Larimichthys crocea)两种类型TLR5(LcTLR5M,LcTLR5S)cDNA序列的基础上,分别克隆了它们的启动子序列,通过片段缺失,定点突变等技术,筛选并验证了其启动子结构及转录结合位点;构建了LcTLR5M和LcTLR5S的亚细胞定位质粒,研究了其在细胞内的表达特征;构建了它们的过表达质粒,将其分别以及共同与NF-κB家族p65基因启动子共转染细胞系,检测了LcTLR5M和LcTLR5S过表达对NF-κB-p65启动活性的影响。克隆了大黄鱼NF-κB家族RelB基因的全长cDNA序列,分析了它的分子结构及组织表达谱,检测了病原相关分子模式(PAMP)刺激后,其在细胞系内的时空表达特征;研究了RelB亚细胞定位并分析了其过表达后对TNF-α等部分细胞因子的影响及IκB家族IκB-α对其调控机制。扩增了NF-κB家族成员p65基因的启动子序列,检测了其活性,以及病原刺激后对其启动活性的影响;研究了p65基因亚细胞定位及分析了其过表达对下游细胞因子IL-1β、TNF-α和IFN-I基因的启动子活性的影响,及IκB-α对其调控机制。结果如下:(1)大黄鱼TLR5M启动子区域在-1829bp至+189bp之间,无CpG岛区域,转录结合位点含有CEBP、Oct-1、GATA-1、AP1等元件,系列片段缺失分析基本启动子活性所需的顺式作用元件可能位于-417至+189之间,负调控元件可能位于-799至-417之间。大黄鱼TLR5S启动子区域在-1932bp至+106bp之间,无CpG岛区域,转录结合位点含有CEBP、SP1、Oct-1、AP1、NF-B等元件,片段缺失表明TLR5S基因顺式作用元件可能位于-808至+106之间,Oct-1和NF-κB结合位点对TLR5S启动子活性有正调控作用且大黄鱼NF-κB家族亚基p65过表达可显著上调TLR5S启动子活性,表明预测正确。亚细胞定位分析表明,大黄鱼TLR5M蛋白主要定位在细胞膜上,而TLR5S蛋白主要定位在细胞浆中。TLR5M过表达对NF-κB-p65启动子的活性无显著影响,而TLR5S过表达可以显著上调NF-κB-p65启动活性。(2)大黄鱼RelB(LcRelB)基因全长为1946bp,ORF为1788bp,编码595个氨基酸,包含RHD-DNA-bind结构域和IPT结构域,等电点p I=5.6,分子量为66.5KDa。进化分析表明,大黄鱼RelB基因与红鳍东方渶亲缘关系最近;LcRelB广泛分布于多种组织,其中在血细胞中含量最高,其次是脾脏和头肾,皮肤中含量最低;LPS和poly I:C刺激后,大黄鱼LCK细胞系中LcRelB上调表达显著,而PGN免疫刺激后表达下调(p0.05)。LcRelB蛋白定位于细胞核中。LcRelB过表达后,其下游细胞因子IL-1β启动子活性显著下调,而TNF-α和IFN-I启动子活性显著上调(p0.05),而当NF-κB家族抑制因子Lc IκB-α与其共转染后,可以显著抑制TNF-α启动子活性(p0.01)。(3)Lcp65启动子区域为-1695bp至+111bp,预测其转录结合位点含有USF、AP1、SRF、IRF-1、NF-κB等元件,含有CpG岛以及(GT)n重复区域;LPS,PGN,Flagellin刺激后均能够显著诱导p65启动活性上调,其中Flagellin诱导能力最为显著(p0.01);亚细胞定位表明,Lcp65蛋白主要定位于细胞核中;Lcp65过表达后能显著上调其下游细胞因子IL-1β、TNF-α和IFN-I启动子的活性,其中,TNF-α的启动活性最强。抑制因子Lc IκB-α与其共转染后,可以显著抑制其对TNF-α启动子活性的诱导(p0.01)。
[Abstract]:In this study, on the basis of two types of TLR5 (LcTLR5M, LcTLR5S) cDNA sequence of Larimichthys crocea in the laboratory, the promoter sequences were cloned respectively. The promoter structure and transcriptional binding sites were screened and verified by the technique of fragment deletion and fixed-point mutation, and the subcellular binding of LcTLR5M and LcTLR5S was constructed. The expression of the plasmid was studied in the cell, and their overexpressed plasmids were constructed to co transfect the cell lines with the NF- kappa B family p65 promoter, respectively. The effect of LcTLR5M and LcTLR5S overexpression on the activation of NF- kappa B-p65 was detected. The full-length cDNA sequence of the NF- kappa B family RelB gene of the rhubarb fish was cloned, and the analysis was made. Its molecular structure and tissue expression profiles were used to detect the spatio-temporal expression characteristics of the pathogen associated molecular model (PAMP), and to study the location of RelB subcellular localization and to analyze the effect of its overexpression on TNF- alpha and the regulation mechanism of I kappa B- alpha of the I kappa B family. The p65 gene of NF- kappa B family members was amplified. The promoter sequence was used to detect its activity and the effect of pathogen stimulation on its activity. The effect of over expression of p65 gene on the promoter activity of the downstream cytokine IL-1 beta, TNF- A and IFN-I genes and the regulation mechanism of I kappa B- a were studied. The results were as follows: (1) the TLR5M promoter region of the yellow croaker was in -1829 Between BP and +189bp, there is no CpG island region, and the transcription binding site contains CEBP, Oct-1, GATA-1, AP1 and other components. The sequence of sequence deletion analysis of the basic promoter activity may be located between -417 to +189, and the negative regulatory element may be between -799 to -417. The transcriptional binding site contains CEBP, SP1, Oct-1, AP1, NF-B and other components. The deletion of fragment indicates that the cis acting element of the TLR5S gene may be located between -808 and +106. Oct-1 and NF- kappa B binding sites have positive regulation on the activity of the TLR5S promoter. The subcellular localization analysis showed that the TLR5M protein was mainly located on the cell membrane, while the TLR5S protein was mainly located in the cytoplasm, and the.TLR5M overexpression had no significant effect on the activity of NF- kappa B-p65 promoter, while TLR5S overexpression could significantly increase the activation activity of NF- kappa B-p65. (2) the RelB (LcRelB) gene of large yellow croaker was 1946bp, ORF was 1788bp, which encodes 595 amino acids, contains the RHD-DNA-bind domain and IPT domain, isoelectric point P I=5.6. The molecular weight of 66.5KDa. evolution analysis shows that the RelB gene is closest to the red fin Orient; LcRelB is widely distributed in a variety of tissues, with the highest content in the blood cells, followed by the spleen and the head kidney, and the lowest in the skin. After the stimulation of LPS and poly I:C, the expression of LcRelB in the LCK cell line of the large yellow croaker was significantly up-regulated, while the expression of P0.05.LcRelB protein was down regulated after.LcRelB overexpression in the nucleus after PGN immunization, and the activity of the downstream cytokine IL-1 beta promoter was down significantly down, while TNF- alpha and IFN-I promoter activity were significantly up-regulated. Factor Lc I kappa B- alpha and co transfection can significantly inhibit the activity of TNF- alpha promoter (P0.01). (3) Lcp65 promoter region is -1695bp to +111bp, and predicts its transcriptional binding sites contain USF, AP1, SRF, IRF-1, and repeating regions. The Flagellin induction ability was most significant (P0.01); subcellular localization showed that Lcp65 protein was mainly located in the nucleus; Lcp65 overexpression could significantly increase the activity of IL-1 beta, TNF- A and IFN-I promoter, among which TNF- a was the strongest. The inhibitory factor Lc I kappa B- alpha and co transfection could significantly inhibit it. Induction of TNF- alpha promoter activity (P0.01).
【学位授予单位】：集美大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S917.4;Q78
本文编号：2157308
[Abstract]:In this study, on the basis of two types of TLR5 (LcTLR5M, LcTLR5S) cDNA sequence of Larimichthys crocea in the laboratory, the promoter sequences were cloned respectively. The promoter structure and transcriptional binding sites were screened and verified by the technique of fragment deletion and fixed-point mutation, and the subcellular binding of LcTLR5M and LcTLR5S was constructed. The expression of the plasmid was studied in the cell, and their overexpressed plasmids were constructed to co transfect the cell lines with the NF- kappa B family p65 promoter, respectively. The effect of LcTLR5M and LcTLR5S overexpression on the activation of NF- kappa B-p65 was detected. The full-length cDNA sequence of the NF- kappa B family RelB gene of the rhubarb fish was cloned, and the analysis was made. Its molecular structure and tissue expression profiles were used to detect the spatio-temporal expression characteristics of the pathogen associated molecular model (PAMP), and to study the location of RelB subcellular localization and to analyze the effect of its overexpression on TNF- alpha and the regulation mechanism of I kappa B- alpha of the I kappa B family. The p65 gene of NF- kappa B family members was amplified. The promoter sequence was used to detect its activity and the effect of pathogen stimulation on its activity. The effect of over expression of p65 gene on the promoter activity of the downstream cytokine IL-1 beta, TNF- A and IFN-I genes and the regulation mechanism of I kappa B- a were studied. The results were as follows: (1) the TLR5M promoter region of the yellow croaker was in -1829 Between BP and +189bp, there is no CpG island region, and the transcription binding site contains CEBP, Oct-1, GATA-1, AP1 and other components. The sequence of sequence deletion analysis of the basic promoter activity may be located between -417 to +189, and the negative regulatory element may be between -799 to -417. The transcriptional binding site contains CEBP, SP1, Oct-1, AP1, NF-B and other components. The deletion of fragment indicates that the cis acting element of the TLR5S gene may be located between -808 and +106. Oct-1 and NF- kappa B binding sites have positive regulation on the activity of the TLR5S promoter. The subcellular localization analysis showed that the TLR5M protein was mainly located on the cell membrane, while the TLR5S protein was mainly located in the cytoplasm, and the.TLR5M overexpression had no significant effect on the activity of NF- kappa B-p65 promoter, while TLR5S overexpression could significantly increase the activation activity of NF- kappa B-p65. (2) the RelB (LcRelB) gene of large yellow croaker was 1946bp, ORF was 1788bp, which encodes 595 amino acids, contains the RHD-DNA-bind domain and IPT domain, isoelectric point P I=5.6. The molecular weight of 66.5KDa. evolution analysis shows that the RelB gene is closest to the red fin Orient; LcRelB is widely distributed in a variety of tissues, with the highest content in the blood cells, followed by the spleen and the head kidney, and the lowest in the skin. After the stimulation of LPS and poly I:C, the expression of LcRelB in the LCK cell line of the large yellow croaker was significantly up-regulated, while the expression of P0.05.LcRelB protein was down regulated after.LcRelB overexpression in the nucleus after PGN immunization, and the activity of the downstream cytokine IL-1 beta promoter was down significantly down, while TNF- alpha and IFN-I promoter activity were significantly up-regulated. Factor Lc I kappa B- alpha and co transfection can significantly inhibit the activity of TNF- alpha promoter (P0.01). (3) Lcp65 promoter region is -1695bp to +111bp, and predicts its transcriptional binding sites contain USF, AP1, SRF, IRF-1, and repeating regions. The Flagellin induction ability was most significant (P0.01); subcellular localization showed that Lcp65 protein was mainly located in the nucleus; Lcp65 overexpression could significantly increase the activity of IL-1 beta, TNF- A and IFN-I promoter, among which TNF- a was the strongest. The inhibitory factor Lc I kappa B- alpha and co transfection could significantly inhibit it. Induction of TNF- alpha promoter activity (P0.01).
【学位授予单位】：集美大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S917.4;Q78
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