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为研究结核分枝杆菌(Mycobacterium tuberculosis,M.tb)与丙型肝炎病毒(Hepatitis C virus,HCV)感染之间的相关关系,本文研究毒性毒株M.tb H37Rv的RD13区中的Rv2645基因对免疫细胞中干扰素刺激基因(Interferonstimulated gene 15,ISG15)表达的影响,进而探讨ISG15的表达与HCV感染之间的相关性。利用携带Rv2645基因的无毒牛结核分枝杆菌卡介苗(Bacille calmette guerin,BCG)(即rBCG::Rv2645,rBCG)与亲本BCG分别感染小鼠或刺激小鼠巨噬细胞系RAW264.7,运用RT-qPCR以及蛋白印迹的方法检测,比较rBCG与BCG感染对小鼠CD4+T细胞及RAW264.7细胞中ISG15的mRNA及蛋白质表达的影响。同时克隆ISG15真核表达质粒和ISG15的沉默表达质粒-ISG15-shRNA,运用RT-qPCR及蛋白印迹分别检测ISG15过表达及沉默后,对HCV感染的人肝癌细胞系Huh7.5.1中HCV的mRNA、HCV非结构蛋白NS3及核心蛋白Core的表达。我们发现相对于BCG,携带Rv2645的BCG感染之后脾脏CD4+T细胞及体外RAW264.7中ISG15的mRNA及蛋白质的水平明显升高。此外,ISG15过表达导致Huh7.5.1中HCV的mRNA、NS3蛋白及Core蛋白的水平明显升高,而ISG15沉默后ISG15和HCV的mRNA的水平明显下调。本研究首次发现M.tb Rv2645能上调小鼠CD4+T细胞及巨噬细胞中ISG15的表达;而ISG15能促进Huh7.5.1中HCV增殖。本研究对阐明M.tb毒性菌株H37Rv的Rv2645基因的功能,以及为研究MTB感染与HCV感染的分子机制提供参考依据。
In order to study the correlation between Mycobacterium tuberculosis (M.tb) and Hepatitis C virus (HCV) infection, we studied the Rv2645 gene pair in RD13 region of virulent strain M.tb H37Rv Immune cells interferon stimulating gene (Interferonstimulated gene15, ISG15) expression, and then explore the expression of ISG15 and HCV infection between the correlation. Mice were infected with BCG or non-toxic Bacillus calmette guerin (BCG) carrying Rv2645 gene (ie, rBCG :: Rv2645, rBCG) -qPCR and Western blotting were used to detect the effect of rBCG and BCG infection on mRNA and protein expression of ISG15 in mouse CD4 + T cells and RAW264.7 cells. The ISG15 eukaryotic expression plasmid and the ISG15 silencing expression plasmid-ISG15-shRNA were cloned. The expression of HCV mRNA and protein in HCV-infected human hepatocellular carcinoma cell line Huh7.5.1 were detected by RT-qPCR and Western blotting respectively after overexpression and silencing of ISG15. Expression of HCV nonstructural protein NS3 and core protein Core. We found significantly increased mRNA and protein levels of spleen CD4 + T cells and ISG15 in RAW264.7 after BCG infection with Rv2645 relative to BCG. In addition, ISG15 overexpression resulted in significantly increased levels of HCV mRNA, NS3 protein and Core protein in Huh7.5.1, whereas ISG15 and HCV mRNA levels were significantly down-regulated after ISG15 silencing. This study, for the first time, found that M.tb Rv2645 up-regulated the expression of ISG15 in mouse CD4 + T cells and macrophages, while ISG15 promoted the proliferation of HCV in Huh7.5.1. This study elucidated the function of Rv2645 gene of M.tb virulent strain H37Rv and provided a reference for the study of the molecular mechanism of MTB infection and HCV infection.