本文關(guān)鍵詞：丙型肝炎病毒核心蛋白和NS5A蛋白對NF-κB的激活研究　出處：《武漢大學(xué)》2005年博士論文　論文類型：學(xué)位論文

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【摘要】：丙型肝炎病毒(HCV)是引起丙型肝炎的病原體,HCV感染常常導(dǎo)致慢性持續(xù)性感染,最終導(dǎo)致肝硬化甚至肝癌,但關(guān)于其致病及宿主免疫防御機制還不是很清楚。核因子κB(NF-κB)是一種重要的細胞核轉(zhuǎn)錄因子,能調(diào)節(jié)大量基因特別是免疫應(yīng)答和炎癥反應(yīng)相關(guān)基因的轉(zhuǎn)錄,在許多免疫和炎癥反應(yīng)的調(diào)節(jié)中起著重要作用,同時也影響著細胞的生長、分化、凋亡、癌變和個體發(fā)育等,功能涉及到許多生理、病理過程。 本論文研究了HCV的Core和NS5A兩種蛋白與NF-κB信號途徑的關(guān)系,目的是探索HCV致病的分子機制。 首先我們構(gòu)建了core基因的重組真核表達質(zhì)粒pcDNA-△core519、pcDNA-△core474、pcDNA-△core(76-573)、pcDNA-△core(76-474)和NS5A基因的重組表達質(zhì)粒p3NS5A,將各個重組質(zhì)粒單獨與NF-κB報告質(zhì)粒pNF-κB-Luc或是將core基因質(zhì)粒和p3NS5A混合后與報告質(zhì)粒共轉(zhuǎn)染轉(zhuǎn)染Cos-7、HeLa和Huh7三種細胞系,檢測它們對NF-κB活性的影響,同時用Western-blot和間接免疫熒光實驗檢測了蛋白的表達定位情況。蟲熒光素酶實驗表明Core蛋白能夠在Huh7細胞中激活NF-κB,且其N端1-25氨基酸為其NF-κB激活功能所必需的,其C端的疏水性區(qū)域?qū)ζ銷F-κB激活功能也有一定的影響;Core蛋白在Huh7細胞中對NF-κB的激活作用是劑量依賴性的。但Core蛋白在Cos-7和HeLa細胞中對NF-κB活性沒有明顯影響,表明Core蛋白對NF-κB激活是有細胞選擇性的;NS5A蛋白單獨在三種細胞系中對NF-κB活性都沒有影響,但它能增強全長Core蛋白在Huh7細胞中對NF-κB的激活作用,但對缺失的Core蛋白的突變體沒有增強作用,表明NS5A只能和全長Core蛋白作用;并且NS5A蛋白對Core蛋白對NF-κB激活的增強作用與NS5A的蛋白量在一定范圍內(nèi)呈相關(guān)性。 同時用Westem-blot檢測了Core蛋白和NS5A蛋白在Huh-7細胞系中單獨或共表達時NF-κB/p65,NF-κB/p50和IκB-α三種NF-κB相關(guān)因子的表達情況。結(jié)果表明Core蛋白和NS5A蛋白的表達沒有提高NF-κB蛋白表達水平,而是引起了IκB-α的降解,推測其引起的NF-κB的激活是由與促進IκB-α磷酸化從而被降解而導(dǎo)致的。 我們的研究結(jié)果表明NS5A蛋白對Core蛋白激活NF-κB具有協(xié)同作用,HCV
[Abstract]:Hepatitis C virus (HCV) is caused by hepatitis C virus pathogens, HCV infection often leads to chronic persistent infection, eventually lead to cirrhosis and liver cancer, but about its pathogenesis and host immune defense mechanism is not very clear. The nuclear factor kappa B (NF- K B) is an important nuclear transcription factor, regulating a large number of genes in particular genes related to immune response and inflammation, plays an important role in the regulation of immune and inflammatory responses, but also affects cell growth, differentiation, apoptosis, cancer and individual development, function related to many physiological and pathological processes.
In this paper, the relationship between the Core and NS5A two proteins of HCV and the NF- kappa B signaling pathway was studied in order to explore the molecular mechanism of HCV pathogenicity.
First, we constructed a recombinant eukaryotic expression plasmid of core gene of pcDNA- core519, pcDNA- core474, pcDNA- core (76-573), pcDNA- core (76-474) and NS5A gene recombinant expression plasmid p3NS5A, the recombinant plasmid NF- B reporter plasmid alone with kappa kappa B-Luc pNF- or core gene and plasmid p3NS5A when mixed with the report plasmid was co transfected into Cos-7, HeLa and Huh7 three cell lines, detection of their effects on NF- kappa B activity, at the same time by Western-blot and indirect immunofluorescence assay to detect the expression and location of protein. The luciferase experiments showed that Core protein can activate NF- kappa B in Huh7 cells, and the N 1-25 amino acid activation required for the function of NF- kappa B, C terminal hydrophobic region has certain effect on its NF- kappa B activation; Core protein in Huh7 cell activation of NF- K B is dose dependent. But Core egg In the Cos-7 and HeLa cells of NF- kappa B activity had no significant effect, suggesting that the Core protein on the activation of NF- kappa B is a selective cell; NS5A protein alone in three cell lines of NF- kappa B activity had no effect, but it can enhance the full-length Core protein in Huh7 cells of NF- kappa B active role, but the deletion mutants of the Core protein did not enhance the effect, and can only show that the NS5A full-length Core protein; NS5A protein on Core and protein to enhance protein interaction with NS5A NF- kappa B activation in a certain range of correlation.
At the same time, Westem-blot was used to detect Core protein and NS5A protein in Huh-7 cells alone or co expression of NF- K B/p65, expression of NF- K B/p50 and I kappa B- alpha three NF- kappa B related factors. The results showed that the expression of Core protein and NS5A protein did not increase NF- kappa B protein expression level, but caused the degradation of I kappa B- alpha, presumably caused by NF- activation of kappa B is composed of I kappa B- alpha and promote the phosphorylation and degradation is caused.
Our results show that NS5A protein has a synergistic effect on the activation of NF- kappa B by Core protein, HCV

【學(xué)位授予單位】：武漢大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2005
【分類號】：R373

【參考文獻】

相關(guān)期刊論文 前1條

1 ;丙型肝炎防治指南[J];中華預(yù)防醫(yī)學(xué)雜志;2004年03期

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本文編號：1427420