丙型肝炎病毒核心蛋白和NS5A蛋白对NF-κB的激活研究

发布时间：2018-01-15 07:37

本文关键词：丙型肝炎病毒核心蛋白和NS5A蛋白对NF-κB的激活研究　出处：《武汉大学》2005年博士论文　论文类型：学位论文

【摘要】：丙型肝炎病毒(HCV)是引起丙型肝炎的病原体,HCV感染常常导致慢性持续性感染,最终导致肝硬化甚至肝癌,但关于其致病及宿主免疫防御机制还不是很清楚。核因子κB(NF-κB)是一种重要的细胞核转录因子,能调节大量基因特别是免疫应答和炎症反应相关基因的转录,在许多免疫和炎症反应的调节中起着重要作用,同时也影响着细胞的生长、分化、凋亡、癌变和个体发育等,功能涉及到许多生理、病理过程。本论文研究了HCV的Core和NS5A两种蛋白与NF-κB信号途径的关系,目的是探索HCV致病的分子机制。首先我们构建了core基因的重组真核表达质粒pcDNA-△core519、pcDNA-△core474、pcDNA-△core(76-573)、pcDNA-△core(76-474)和NS5A基因的重组表达质粒p3NS5A,将各个重组质粒单独与NF-κB报告质粒pNF-κB-Luc或是将core基因质粒和p3NS5A混合后与报告质粒共转染转染Cos-7、HeLa和Huh7三种细胞系,检测它们对NF-κB活性的影响,同时用Western-blot和间接免疫荧光实验检测了蛋白的表达定位情况。虫荧光素酶实验表明Core蛋白能够在Huh7细胞中激活NF-κB,且其N端1-25氨基酸为其NF-κB激活功能所必需的,其C端的疏水性区域对其NF-κB激活功能也有一定的影响;Core蛋白在Huh7细胞中对NF-κB的激活作用是剂量依赖性的。但Core蛋白在Cos-7和HeLa细胞中对NF-κB活性没有明显影响,表明Core蛋白对NF-κB激活是有细胞选择性的;NS5A蛋白单独在三种细胞系中对NF-κB活性都没有影响,但它能增强全长Core蛋白在Huh7细胞中对NF-κB的激活作用,但对缺失的Core蛋白的突变体没有增强作用,表明NS5A只能和全长Core蛋白作用;并且NS5A蛋白对Core蛋白对NF-κB激活的增强作用与NS5A的蛋白量在一定范围内呈相关性。同时用Westem-blot检测了Core蛋白和NS5A蛋白在Huh-7细胞系中单独或共表达时NF-κB/p65,NF-κB/p50和IκB-α三种NF-κB相关因子的表达情况。结果表明Core蛋白和NS5A蛋白的表达没有提高NF-κB蛋白表达水平,而是引起了IκB-α的降解,推测其引起的NF-κB的激活是由与促进IκB-α磷酸化从而被降解而导致的。我们的研究结果表明NS5A蛋白对Core蛋白激活NF-κB具有协同作用,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

【学位授予单位】：武汉大学
【学位级别】：博士
【学位授予年份】：2005
【分类号】：R373

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