miRNA调控p53抗传染性法氏囊病病毒免疫的分子机制

MicroRNAs (miRNAs) are one class of non-coding RNAs of lengths ranging from 18 to 23 nucleotides (nt) that play critical roles in a wide variety of biological processes including the p53-mediated antiviral immune response .Infection bursal disease virus(IBDV) causes severe immunosuppressive disease in young chickens and continues to pose a significant threat to the poultry.To control the spread of IBDV infection and reduce the pathogenesis of IBD, it is important to understand the role of miRNA in regulation of p53-mediated antiviral immune response in IBDV infected chickens. Based on the bioinformatics prediction, our current project aims to identify the antiviral immune related miRNA which involved in the regulation of p53 functions in IBDV infected chickens. The 3-untranslated region (UTR) reporter analysis and RNAi silencing technique will be used to identify putative targets of the p53 related miRNAs. In addition, over-expression or decreased expression of putative miRNAs in B cells and SPF chickens will be tried as the models to analyze the influence of antiviral immune response. The harvested bursal tissues and spleens and serum would be examined for the following:1-histopathological lesions, 2-immunohistochemical detection of virus antigen, T cells and macrophages, 3-mononuclear cell and expression of virus-induced innate, proinflammatory cytokines, chemokines by quantitative real time RT-PCR(qRT-PCR),4-T cells responses and CTL would be analyzed by flow cytometry, 5-IBDV specific IgG isotypes in serum 10 days after IBDV infection are analyzed by ELISA .This study can help us to understand the molecular pathogenesis mechanisms of miRNA in regulation of p53 gene mediated antiviral immune response in IBDV infected chicken cells and SPF chicken, which are very important for the establishment of effective novel approach for the control and prevention of IBD.

microRNA (miRNA)是存在于细胞内的非编码小RNA，在宿主细胞和病毒基因表达调控途径中发挥着关键作用。在肿瘤抑制蛋白p53抗传染性法氏囊病病毒（IBDV）复制中的作用也是如此。本实验室前期发现p53具有显著的抗IBDV复制的功能，本项目拟在此功能研究基础上，用生物信息学方法预测调控p53基因的miRNA，运用双荧光素酶报告系统和RNAi沉默技术分析miRNA对p53基因的调控作用，进一步在细胞和鸡体内进行miRNA的过表达与抑制表达的抗病毒免疫应答分析，着重检测细胞因子变化、T细胞和单核巨噬细胞反应、IBDV含量的变化、IBDV特异抗体水平，并结合鸡的临床与病理变化等指标进行综合分析，解析miRNA调控p53基因抗IBDV免疫的分子机制，并发掘调控p53基因的miRNA分子靶标，为IBD新型药物及疫苗设计提供科学依据。

microRNA (miRNA)是存在于细胞内的非编码小RNA，在宿主细胞和病毒基因表达调控途径中发挥着关键作用。在肿瘤抑制蛋白p53抗传染性法氏囊病病毒（IBDV）复制中的作用也是如此。本实验室前期发现p53具有显著的抗IBDV复制的功能，本项目拟在此功能研究基础上，用生物信息学方法预测调控p53基因的miRNA，运用双荧光素酶报告系统和RNAi沉默技术分析miRNA对p53基因的调控作用，解析miRNA调控p53基因抗IBDV免疫的分子机制，并发掘调控p53基因的miRNA分子靶标，为IBD新型药物及疫苗设计提供科学依据。 . 本研究以IBDV为模式病毒，采用体外感染模式，在IBDV感染的DF-1和DT40细胞中，p53的表达水平和活性均显著升高。p53过表达可以抑制IBDV的复制，并激活鸡体抗病毒天然免疫基因（IPS-1、IRF3、PKR、OAS、Mx）的表达水平上调；p53抑制表达则得到相反的结果；表明p53可以激活鸡体的抗病毒天然免疫反应发挥抗IBDV感染作用。用生物信息学方法和双荧光素酶报告系统分析发现gga-miR-2127可以作用于p53的3’UTR；qRT-PCR和免疫印迹分析表明gga-miR-2127可以使p53的蛋白水平表达降低，但mRNA水平没有变化；gga-miR-2127过表达可以使鸡体的抗病毒天然免疫基因表达降低，IBDV复制水平升高。本研究表明：gga-miR-2127可以作用于p53的3’UTR调控p53的表达从而影响其下游抗病毒天然基因的表达来发挥抗IBDV感染的作用。