呼吸道合胞病毒感染调控NLRC5表达及其意义

发布时间：2018-01-27 13:47

本文关键词： 呼吸道合胞病毒 NLRC5 MHC I 单克隆抗体　出处：《南京大学》2014年硕士论文　论文类型：学位论文

【摘要】：呼吸道合胞病毒(Respiratory syncytial virus, RSV)为负义单链RNA病毒,是导致婴幼儿下呼吸道感染的主要病原体,主要引起毛细支气管炎和肺炎。婴幼儿RSV感染与青少年期过敏性哮喘的发生发展密切相关,是诱发哮喘的高危因素。环境以及基因因素可能与这一现象有关,但是具体原因不清。细胞免疫在RSV感染清除和致病方面具有重要作用,其中CD8+T淋巴细胞是清除感染的主要效应细胞。CD8+T细胞通过与MHC I分子相互作用识别并杀伤宿主细胞,在清除感染的同时也付出了免疫损伤的代价。宿主细胞感染RSV后,MHC I分子表达明显升高,增强了CD8+T细胞介导的免疫杀伤反应,是RSV感染造成免疫损伤的主要原因。本论文主要研究了呼吸道合胞病毒感染调控NLRC5表达及其意义。NLRC5是天然免疫识别受体NLRs家族中新发现的一个蛋白,由1866个氨基酸残基组成。为了检查NLRC5在RSV感染过程中的表达,我们首先制备了NLRC5的多抗和单克隆抗体。通过抗原分析,我们选取NLRC5 C-端含258个氨基酸残基的蛋白片段作为抗原,然后在大肠杆菌表达系统制备了相应的重组蛋白,分别免疫新西兰兔和小鼠,分别制备了多抗和单克隆抗体。单克隆抗体的特异性通过ELISA筛选,免疫印迹方法验证表明该抗体具有特异识别人源NLRC5的能力。我们利用这些试剂对RSV感染过程中NLRC5表达以及MHCI分子的表达调控进行了研究,发现RSV感染通过诱导干扰素释放以及病毒双链RNA两个通路诱导了NLRC5表达。RSV感染过程中NLRC5表达与MHC I上调的关系通过两种方法验证：1)外源性过表达NLRC5可以明显促进MHC I表达；2)siRNA抑制NLRC5表达可以明显降低RSV感染过程中MHC I的表达。综上所述,本文首次证明了RSV感染可以显著诱导NLRC5表达,并且初步阐述了NLRC5在RSV感染过程中调控MHC I表达的重要作用,这可能是RSV所致免疫病理损伤以及诱发哮喘的潜在原因。
[Abstract]:Respiratory syncytial virus Respiratory syncytial virus (RSVV) is a negative sense single-stranded RNA virus. Infantile RSV infection is the main pathogen of infantile lower respiratory tract infection, which mainly causes bronchiolitis and pneumonia. Infant RSV infection is closely related to the occurrence and development of juvenile allergic asthma. Environment and genetic factors may be related to this phenomenon, but the specific reasons are unclear. Cellular immunity plays an important role in the clearance and pathogenesis of RSV infection. Among them, CD8 T lymphocytes are the main effector cells. CD8 T cells recognize and kill host cells by interacting with MHC I molecules. After the host cells infected with RSV, the expression of MHCI increased significantly, which enhanced the immune response mediated by CD8 T cells. In this paper, we studied the regulation of NLRC5 expression in respiratory syncytial virus infection and its significance. NLRC5 is a natural immune recognition receptor NLRs family. A newly discovered protein. In order to detect the expression of NLRC5 in the process of RSV infection, we first prepared the polyclonal antibody and monoclonal antibody of NLRC5. We selected the protein fragment containing 258 amino acid residues at the C-terminal of NLRC5 as antigen and then prepared the corresponding recombinant protein in Escherichia coli expression system to immunize New Zealand rabbits and mice respectively. Polyclonal antibodies and monoclonal antibodies were prepared respectively. The specificity of monoclonal antibodies was screened by ELISA. The immunoblotting assay showed that the antibody had the ability to specifically recognize human NLRC5. We used these reagents to regulate the expression of NLRC5 and MHCI molecules in the process of RSV infection. Research. It was found that RSV infection induces the expression of NLRC5 and MHC in the process of NLRC5 infection by inducing interferon release and viral double-stranded RNA. I-Up relationship: two ways to verify the relationship: 1). Exogenous overexpression of NLRC5 could significantly promote the expression of MHC I. 2inhibition of NLRC5 expression by siRNA can significantly reduce the expression of MHC I in the process of RSV infection. In conclusion, it is the first time that RSV infection can significantly induce NLRC5 expression. The important role of NLRC5 in regulating the expression of MHC I in the process of RSV infection was discussed, which may be the potential cause of RSV induced immunopathologic injury and asthma.
【学位授予单位】：南京大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R373

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