【作者】 王彬；

【导师】 郑世军；

【作者基本信息】 中国农业大学 ， 预防兽医学， 2018， 博士

【摘要】 传染性法氏囊病(Infectious Bursal Disease,IBD)是由传染性法氏囊病病毒(Infectious Bursal Disease Virus,IBDV)引起的禽的一种急性、高度接触性传染病。该病以法氏囊损伤和免疫抑制为主要特征。MicroRNAs(miRNAs)是一种长度为18-25个核苷酸的非编码的小分子RNA,通过结合特定基因转录的mRNA3’UTR区的互补位点导致mRNA降解从而抑制蛋白翻译。miRNAs在宿主与病原的相互作用中发挥重要作用,尤其对病毒感染引起的先天性免疫反应产生影响。然而,miRNAs在IBDV感染所引起的宿主细胞反应中的作用与机理还不清楚,需要深入研究。本研究利用高通量测序技术,筛选IBDV感染DF-1细胞后差异表达的宿主miRNAs,发现369种miRNAs表达量显著上调,169种miRNAs显著下调。为了验证差异表达的miRNAs与IBDV感染的相关性,我们选取了 22种与免疫、病毒感染、炎症反应以及细胞凋亡相关的miRNAs作为我们的研究对象。利用半数组织细胞感染量实验(TCID50)、蛋白质免疫印迹(WesternBlot)、间接免疫荧光实验(IFA)和荧光定量PCR等实验技术,发现在DF-1细胞中过表达gga-miR-155抑制IBDV复制,而以miRNA抑制剂抑制细胞内源gga-miR-155的表达则可以促进IBDV的复制。进一步研究发现,gga-miR-155增强IBDV诱导下Ⅰ型干扰素的表达。利用TargetScan和miRanda预测软件,发现了 gga-miR-155在宿主细胞内的两个潜在的作用靶点SOCS1和TANK(SOCS1和TANK是免疫信号转导中的负调控分子)。利用荧光素酶报告系统、Western Blot、RNAi等方法,证实gga-miR-155直接靶向作用于Ⅰ型干扰素通路上的两个负调控蛋白SOCS1和TANK。上述结果表明,gga-miR-155通过靶向SOCS1和TANK蛋白,促进Ⅰ型干扰素的表达,抑制IBDV的复制。综上所述,本研究发现gga-miR-155靶向作用于免疫信号负调控蛋白SOCS1和TANK促进Ⅰ型干扰素的表达,抑制IBDV的复制。该研究结果揭示了 gga-miR-155抑制IBDV复制的分子机理;为深入解析IBDV感染的致病机理提供了参考依据。更多还原

【Abstract】 Infectious bursal disease(IBD)is an acute,highly contagious avian disease caused by IBD virus(IBDV),characterized by severe damage in the bursal of Fabricius(BF)that caused immunosuppression in young chickens.MicroRNAs(miRNAs)are a family of small,non-coding RNAs,ranging in length from 18 to 25 nucleotides,which mediate degradation of RNA and translational suppression by binding to complementary sites primarily in the 3’UTRs of mRNAs of targeted gene.miRNAs play a critical role in host-pathogen interactions,especially in innate immune response to viral infections.However the effects of miRNA in host cells on IBDV infection and the underlying mechanism have not yet been elucidated.We performed deep sequencing to examine miRNA expression profiles in DF-1 cells infected with IBDV Lx strain.Among the differentially expressed miRNAs,369 miRNAs were significantly upregulated and 169 downregulated.Twenty-two miRNAs were selected as candidates for IBDV replication analysis as they played varied roles in cancer,inflammation,and immune responses.Employing TCID50,Western Blot,IFA and real-time PCR assays,we found that transfection of DF-1 cells with gga-miR-155 markedly suppressed IBDV replication,while blockage of the endogenous gga-miR-155 by inhibitors enhanced IBDV replication.Furthermore,our data showed that gga-miR-155 enhanced the expression of type I interferon in DF-1 cells post IBDV infection.Using TargetScan and miRanda prediction softwares,we found two putative gga-miR-155 targeted genes SOCS1 and TANK(two negative regulators of type Ⅰ IFN signaling).Importantly,we confirmed that gga-miR-155 targeted SOCS1 and TANK,two negative regulators of type Ⅰ IFN signaling,exploying dual-luciferase reporter gene assay and Western Blot assays.These results indicate that gga-miR-155 enhanced type I interferon expression via targeting SOCS1 and TANK,suppressing IBDV repliaction.In summary,we found that gga-miR-155 enhanced type I IFN signaling and suppressed IBDV replication via targeting SOCS1 and TANK,two negative regulators in the immune response.These findings have revealed the molecular mechanism of inhibition of IBDV replication by gga-miR-155,and provided insights for further studies of the pathogenesis of IBDV infection.更多还原