本文關(guān)鍵詞：草魚NF-κB信號通路激活及其調(diào)控的分子機制　出處：《南昌大學(xué)》2017年博士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 草魚 核轉(zhuǎn)錄因子-κB 蛋白激酶R IKK激酶復(fù)合物β亞基 NF-κB抑制蛋白α 負反饋調(diào)節(jié)

【摘要】：核轉(zhuǎn)錄因子-κB(NF-κB)是一種廣泛存在于無脊椎和脊椎動物的重要的轉(zhuǎn)錄調(diào)節(jié)因子。NF-κB通過調(diào)控多種靶基因的表達在炎癥、免疫、氧化應(yīng)激、細胞增殖、細胞凋亡等生理病理過程中起到重要作用�？焖俜磻�(yīng)的先天性免疫在魚類的免疫應(yīng)答反應(yīng)中起著重要的作用,NF-κB被激活后可以啟動機體的先天免疫從而抵抗病原的入侵。草魚是中國的主要淡水經(jīng)濟魚類之一,在養(yǎng)殖過程中容易被病原感染造成經(jīng)濟損失。對草魚NF-κB的研究有助于了解草魚的免疫應(yīng)答機制,為進一步闡明魚類的先天性免疫機制奠定理論基礎(chǔ)。本研究通過同源克隆和RACE方法克隆獲得了草魚的IKK激酶復(fù)合物的β亞基(CiIKKβ),以及草魚NF-κB的三個亞基p65基因(Cip65),p105基因(Cip105)和c-Rel基因(Cic-Rel)。CiIKKβ全長cDNA為3428 bp,序列分析表明CiIKKβ具有典型的IKKβ蛋白結(jié)構(gòu)特征,系統(tǒng)進化樹結(jié)果表明Ci IKKβ在進化過程中與斑馬魚的親緣關(guān)系最近。CiIKKβ在草魚不同的器官組織中都有表達。Poly I:C刺激草魚腎細胞(CIK)后,CiIKKβ表達量增加,表明CiIKKβ與細胞的抗病毒活動有關(guān)。Cip65的全長cDNA為2481 bp、Cip105的全長cDNA為3760 bp,Cic-Rel的全長cDNA為2618bp。序列分析結(jié)果表明草魚的這三個NF-κB亞基在進化中保守。系統(tǒng)進化樹的結(jié)果表明草魚的這三個NF-κB亞基均與魚類的親緣性高。Poly I:C和LPS的刺激下,草魚各組織中的Cip65,Cip105和Cic-Rel的表達水平均呈現(xiàn)上調(diào),說明它們與草魚的抗病毒或抗菌的生理過程有關(guān)。草魚PKR(Ci PKR)通過介導(dǎo)細胞信號轉(zhuǎn)導(dǎo)在抗病毒的免疫應(yīng)答中發(fā)揮作用。為了分析CiPKR介導(dǎo)NF-κB信號通路的機制,利用免疫共沉淀(co-IP)和GST pull-down方法證明了CiPKR能與CiIKKβ直接結(jié)合,CiIKKβ能與草魚NF-κB抑制蛋白α(CiIκBα)相互作用。這初步表明了CiPKR能夠通過與CiIKKβ的結(jié)合來介導(dǎo)NF-κB信號通路,當(dāng)然這還需要進一步研究。為了進一步研究草魚的NF-κB經(jīng)典信號通路,本研究設(shè)計構(gòu)建了一系列帶FLAG或HA標簽的質(zhì)粒載體進行co-IP和GST pull-down實驗。結(jié)果表明Cip65能與Cip105或其剪切后形成的草魚p50(Cip50)形成二聚體,Cip65和Cic-Rel均能與CiIκBα發(fā)生蛋白結(jié)合。以上結(jié)果表明了草魚的NF-κB經(jīng)典信號通路是存在的并且高度保守。為了分析草魚的NF-κB經(jīng)典信號通路的負反饋機制,利用5′-Full RACE方法確定了CiIκBα和Cip105的轉(zhuǎn)錄起始位點(TSS),通過錨定PCR的方法分別克隆得到CiIκBα和Cip105的啟動子。其中在本研究中成功得到了距離轉(zhuǎn)錄起始位點414 bp的CiIκBα啟動子序列,啟動子序列分析結(jié)果表明該序列含有1個非典型TATA盒,2個RelA結(jié)合位點和2個NF-κB結(jié)合位點。本研究中得到距離轉(zhuǎn)錄起始位點1469bp的Cip105啟動子序列,啟動子序列分析結(jié)果顯示該序列含有1個RelA結(jié)合位點,1個NF-κB結(jié)合位點和1個Sp1結(jié)合位點。同時為研究Cip65對CiIκBα和Cip105基因轉(zhuǎn)錄調(diào)控的機制,原核表達并純化獲得Cip65的ORF全長和截斷蛋白。通過體外凝膠阻滯實驗表明Cip65全長蛋白對CiIΚBα和Cip105啟動子有親和性。將pGL3-CiIκBα和pGL3-Cip105啟動子熒光素酶報告基因載體質(zhì)粒和pCDNA3.1-Cip65-ORF,pCDNA3.1-Cip65-ΔC,pCDNA3.1-Cip65-ΔN真核表達載體質(zhì)粒分別共轉(zhuǎn)染CIK細胞,檢測熒光素酶的活性。結(jié)果表明在CIK細胞中Cip65-ORF能夠增強CiIκBα和Cip105的轉(zhuǎn)錄活性。
[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.

【學(xué)位授予單位】：南昌大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2017
【分類號】：S943;Q78

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