草鱼NF-κB信号通路激活及其调控的分子机制

发布时间：2018-01-11 06:12

  本文关键词：草鱼NF-κB信号通路激活及其调控的分子机制　出处：《南昌大学》2017年博士论文　论文类型：学位论文

  更多相关文章： 草鱼 核转录因子-κB 蛋白激酶R IKK激酶复合物β亚基 NF-κB抑制蛋白α 负反馈调节

【摘要】：核转录因子-κB(NF-κB)是一种广泛存在于无脊椎和脊椎动物的重要的转录调节因子。NF-κB通过调控多种靶基因的表达在炎症、免疫、氧化应激、细胞增殖、细胞凋亡等生理病理过程中起到重要作用。快速反应的先天性免疫在鱼类的免疫应答反应中起着重要的作用,NF-κB被激活后可以启动机体的先天免疫从而抵抗病原的入侵。草鱼是中国的主要淡水经济鱼类之一,在养殖过程中容易被病原感染造成经济损失。对草鱼NF-κB的研究有助于了解草鱼的免疫应答机制,为进一步阐明鱼类的先天性免疫机制奠定理论基础。本研究通过同源克隆和RACE方法克隆获得了草鱼的IKK激酶复合物的β亚基(CiIKKβ),以及草鱼NF-κB的三个亚基p65基因(Cip65),p105基因(Cip105)和c-Rel基因(Cic-Rel)。CiIKKβ全长cDNA为3428 bp,序列分析表明CiIKKβ具有典型的IKKβ蛋白结构特征,系统进化树结果表明Ci IKKβ在进化过程中与斑马鱼的亲缘关系最近。CiIKKβ在草鱼不同的器官组织中都有表达。Poly I:C刺激草鱼肾细胞(CIK)后,CiIKKβ表达量增加,表明CiIKKβ与细胞的抗病毒活动有关。Cip65的全长cDNA为2481 bp、Cip105的全长cDNA为3760 bp,Cic-Rel的全长cDNA为2618bp。序列分析结果表明草鱼的这三个NF-κB亚基在进化中保守。系统进化树的结果表明草鱼的这三个NF-κB亚基均与鱼类的亲缘性高。Poly I:C和LPS的刺激下,草鱼各组织中的Cip65,Cip105和Cic-Rel的表达水平均呈现上调,说明它们与草鱼的抗病毒或抗菌的生理过程有关。草鱼PKR(Ci PKR)通过介导细胞信号转导在抗病毒的免疫应答中发挥作用。为了分析CiPKR介导NF-κB信号通路的机制,利用免疫共沉淀(co-IP)和GST pull-down方法证明了CiPKR能与CiIKKβ直接结合,CiIKKβ能与草鱼NF-κB抑制蛋白α(CiIκBα)相互作用。这初步表明了CiPKR能够通过与CiIKKβ的结合来介导NF-κB信号通路,当然这还需要进一步研究。为了进一步研究草鱼的NF-κB经典信号通路,本研究设计构建了一系列带FLAG或HA标签的质粒载体进行co-IP和GST pull-down实验。结果表明Cip65能与Cip105或其剪切后形成的草鱼p50(Cip50)形成二聚体,Cip65和Cic-Rel均能与CiIκBα发生蛋白结合。以上结果表明了草鱼的NF-κB经典信号通路是存在的并且高度保守。为了分析草鱼的NF-κB经典信号通路的负反馈机制,利用5′-Full RACE方法确定了CiIκBα和Cip105的转录起始位点(TSS),通过锚定PCR的方法分别克隆得到CiIκBα和Cip105的启动子。其中在本研究中成功得到了距离转录起始位点414 bp的CiIκBα启动子序列,启动子序列分析结果表明该序列含有1个非典型TATA盒,2个RelA结合位点和2个NF-κB结合位点。本研究中得到距离转录起始位点1469bp的Cip105启动子序列,启动子序列分析结果显示该序列含有1个RelA结合位点,1个NF-κB结合位点和1个Sp1结合位点。同时为研究Cip65对CiIκBα和Cip105基因转录调控的机制,原核表达并纯化获得Cip65的ORF全长和截断蛋白。通过体外凝胶阻滞实验表明Cip65全长蛋白对CiIΚBα和Cip105启动子有亲和性。将pGL3-CiIκBα和pGL3-Cip105启动子荧光素酶报告基因载体质粒和pCDNA3.1-Cip65-ORF,pCDNA3.1-Cip65-ΔC,pCDNA3.1-Cip65-ΔN真核表达载体质粒分别共转染CIK细胞,检测荧光素酶的活性。结果表明在CIK细胞中Cip65-ORF能够增强CiIκBα和Cip105的转录活性。
[Abstract]:Nuclear factor kappa B (NF- K B) is a kind of widely exist in important invertebrate and vertebrate transcription regulatory factor.NF- kappa B by regulating the expression of multiple target genes in immune, inflammation, oxidative stress, cell proliferation, apoptosis and other physiological and pathological processes play an important role in innate immunity. The rapid reaction plays an important role in the immune response of fish in the reaction of NF- kappa B was activated after can start the body's innate immune intrusion to resist pathogens. Grass carp is one of the main Chinese freshwater fishes, in the breeding process to pathogen infection caused economic losses. Based on the grass carp NF- kappa B a help to understand the mechanism of immune response of grass carp, which lays the theoretical foundation for further elucidation of the mechanisms of innate immunity of fish. This research has obtained the IKK kinase complex grass carp Yaji by homologous cloning and RACE cloning method (CiIK K and grass carp NF-), Beta Kappa B three subunit p65 gene (Cip65), P105 gene (Cip105) and c-Rel (Cic-Rel).CiIKK beta gene full-length cDNA was 3428 BP, the sequence analysis showed that CiIKK beta IKK protein has typical structure, the result of phylogenetic tree showed that the genetic relationship and in zebrafish the evolutionary process of Ci IKK.CiIKK in grass carp beta beta recently in different organs and tissues have expression of.Poly stimulated I:C grass carp (CIK) after renal cell, the expression of CiIKK increased, cDNA showed that the total length of about.Cip65 CiIKK and beta cell antiviral activity was 2481 BP, full length cDNA of Cip105 was 3760 BP, length cDNA Cic-Rel 2618bp. sequence analysis showed that the three NF- kappa B subunit of grass carp's conservative in evolution. The phylogenetic tree showed that the three NF- kappa B subunit of grass carp and fish relative high.Poly I:C and LPS under the stimulation of the organization of grass carp Cip65, Cip105 The expression level and Cic-Rel showed up-regulated, indicating that they associated with grass carp antivirus or antibacterial physiological process. Grass carp PKR (Ci PKR) play a role in the immune response through antiviral mediated signal transduction. In order to analyze the mechanism of CiPKR mediated by NF- B pathway, by CO immunoprecipitation (co-IP) and the GST pull-down method to prove that CiPKR can be directly combined with the CiIKK beta, CiIKK and grass carp NF- Beta Kappa B inhibitory protein (CiI alpha kappa B alpha) interactions. These preliminary results suggest that CiPKR can be mediated by NF- B pathway by binding to CiIKK beta, which further research is needed to further study. Grass carp NF- classic kappa B signaling pathway, this study designed and constructed by co-IP and GST pull-down, a series of experiments with FLAG or HA tag plasmid. The results showed that Cip65 and Cip105 can be formed or cut after the grass carp P50 (Cip50) two Dimers, Cip65 and Cic-Rel can bind CiI kappa B alpha protein. The above results indicated that the grass carp NF- kappa B classical signaling pathway exists and is highly conservative. In order to negative feedback mechanism analysis of grass carp NF- classic kappa B signaling pathway, the transcription initiation site of CiI kappa B alpha and Cip105 use 5 '-Full RACE (TSS), CiI kappa B alpha and Cip105 promoter were cloned by anchored PCR. In this study successfully get the distance of transcription start site 414 BP CiI kappa B alpha promoter sequence, promoter sequence analysis showed that the sequence contains 1 atypical TATA box, 2 RelA and 2 NF- binding sites B binding site. From the transcription initiation site of 1469bp Cip105 promoter sequence in this study, promoter sequence analysis showed that this sequence contains 1 RelA binding sites, 1 NF- and 1 B binding site Sp1 binding position At the same time. To investigate the mechanism of Cip65 CiI kappa B alpha and Cip105 gene transcription, prokaryotic expression and purification of ORF full-length and truncated Cip65 protein. Cip65 showed that the full-length protein affinity of CiI kappa B alpha and Cip105 promoter in vitro by EMSA. The pGL3-CiI kappa B alpha and pGL3-Cip105 promoter luciferase reporter plasmid and the pCDNA3.1-Cip65-ORF pCDNA3.1-Cip65- pCDNA3.1-Cip65-, Delta C, Delta N eukaryotic expression plasmid were co transfected into CIK cells, the activity of luciferase was detected. The results show that Cip65-ORF can enhance the transcriptional activity of CiI kappa B alpha and Cip105 in CIK cells.

【学位授予单位】：南昌大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：S943;Q78

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