猪繁殖与呼吸综合征病毒（PRRSV）诱导白介素-12（IL-12）产生的分子机制研究

发布时间：2018-01-09 03:18

本文关键词：猪繁殖与呼吸综合征病毒（PRRSV）诱导白介素-12（IL-12）产生的分子机制研究　出处：《中国农业大学》2016年博士论文　论文类型：学位论文

【摘要】：猪繁殖与呼吸综合征(Porcine Reproductive and Respiratory Syndrome, PRRS)是由猪繁殖与呼吸综合征病毒(Porcine Reproductive and Respiratory Syndrome Virus, PRRSV)感染引起的传染性疾病,给全球的养猪业造成了重大的经济损失。PRRS的主要症状是妊娠母猪的繁殖障碍以及仔猪的严重呼吸道疾病。自1987年首次在北美被报道以来,PRRS已经广泛传播至全球几乎所有的养猪国家。2006年,我国爆发了由高致病性PRRSV (highly pathogenic PRRSV, HP-PRRSV)感染引起的非典型性猪繁殖与呼吸综合征,其特点为高热、高发病率和高死亡率。PRRSV感染的重要特征之一是细胞因子异常表达,从而引起免疫抑制。白细胞介素-12(IL-12)是一种由IL-12p40和IL-12p35亚基组成的异二聚体细胞因子,也被称作自然杀伤细胞刺激因子(NKSF)或胞毒淋巴细胞成熟因子(CLMF)。IL-12的主要功能之一是刺激Thl细胞的分化,从而促进细胞介导的免疫反应的发生,对于病毒的清除具有重要意义。已有的研究表明IL-12在预防和控制PRRSV中发挥着重要作用。本论文对PRRSV调控IL-12产生的机制进行了研究,对于进一步明晰PRRSV的致病机理具有重要的意义。本课题首先在体外条件下用PRRSV高致病毒株(HP-PRRSV)和普通毒株CH-la感染猪肺泡巨噬细胞(PAM),发现两种毒株均能够明显诱导IL-12p40和IL-12p35的表达。动物感染实验表明,HP-PRRSV可以显著上调猪肺中IL-12p40以及IL-12p35的表达,但在其他组织中未能检测到IL-12亚基表达水平的升高。与此相一致,在感染PRRSV的猪肺中同样检测到了IFN-γ的表达。为了进一步研究PRRSV诱导IL-12的分子机制,本文中克隆了猪IL-12p40的5’端基因序列,然后通过染色体步移技术获得猪IL-12p40的启动子序列,并对其调控元件进行分析。分析结果显示,猪IL-12p40基因启动子与人和小鼠的IL-12p40启动子在核心区域上高度保守,猪IL-12p40启动子中含有ISRE、NF-κB、C/EBPβ、AP-1和TATA box等转录调控元件。信号通路抑制处理实验表明PRRSV可以通过激活p38 MAPK、JNK和NF-κB信号通路诱导IL-12p40产生,而PRRSV感染对IL-12p35表达的诱导则主要依赖于p38 MAPK和PKC信号通路。AP-1是位于JNK信号途径下游的转录因子,AP-1抑制剂同样可以显著抑制P-PRRSV感染诱导的IL-12p40表达,从而进一步证明PRRSV感染对IL-12p40表达的诱导依赖于JNK-AP-1信号通路。此外,我们还通过比较PRRSV和PRV、SeV感染后细胞中IL-12的表达水平发现,PRRSV虽然可以诱导IL-12亚基的表达,但是其诱导能力明显低于其他病毒。综上,本论文发现PRRSV感染可以诱导IL-12p40和IL-12p35的表达。PRRSV对IL-12p40的诱导依赖于JNK-AP-1和NF-κB信号途径,而对IL-12p35的诱导则依赖于p38 MAPK和PKC信号通路。本论文的研究结果对于进一步阐明PRRSV诱导IL-12产生的分子机制具有重要的参考价值。
[Abstract]:Porcine Reproductive and Respiratory Syndrome. PRRSs are produced by porcine reproductive and respiratory syndrome virus (PRRSs). Porcine Reproductive and Respiratory Syndrome Virus. Infectious diseases caused by infection of PRRSVs. The major symptoms of PRRS are reproductive disorders in pregnant sows and severe respiratory diseases in piglets. Since 1987, it has been reported for the first time in North America. PRRS has been widely spread to almost all pig-raising countries in the world. In 2006, the outbreak of highly pathogenic PRRSV pathogenic PRRSV in China. Atypical porcine reproductive and respiratory syndrome caused by HP-PRRSV.The abnormal expression of cytokines is one of the important characteristics of high fever, high morbidity and high mortality. Interleukin-12 (IL-12) is a heterodimer cytokine composed of IL-12p40 and IL-12p35 subunits. Also known as natural killer cell stimulating factor (NKSF) or cytotoxic lymphocyte maturation factor (CLMFF), IL-12 is one of the main functions of stimulating the differentiation of Thl cells. Thus promote the occurrence of cell-mediated immune response. IL-12 plays an important role in the prevention and control of PRRSV. The mechanism of IL-12 production by PRRSV has been studied in this paper. Research. It is of great significance to further understand the pathogenesis of PRRSV. Firstly, we used HP-PRRSVV, a highly virulent strain of PRRSV, in vitro. And common virus strain CH-la infected porcine alveolar macrophages (PAM). It was found that both strains could induce the expression of IL-12p40 and IL-12p35. HP-PRRSV could significantly up-regulate the expression of IL-12p40 and IL-12p35 in pig lung. However, no increase in the expression of IL-12 subunits was detected in other tissues. The expression of IFN- 纬 was also detected in porcine lungs infected with PRRSV. In order to further study the molecular mechanism of IL-12 induced by PRRSV. The 5'terminal gene sequence of porcine IL-12p40 was cloned and the promoter sequence of porcine IL-12p40 was obtained by chromosome step technique. The results showed that porcine IL-12p40 gene promoter and human and mouse IL-12p40 promoter were highly conserved in the core region. The porcine IL-12p40 promoter contained ISREN- 魏 B C- / EBP 尾. Transcriptional regulatory elements such as AP-1 and TATA box. Signal pathway inhibition experiments showed that PRRSV could activate p38 MAPK. JNK and NF- 魏 B signaling pathway induce IL-12p40 production. However, the induction of IL-12p35 expression by PRRSV infection mainly depends on p38 MAPK and PKC signaling pathway. AP-1 is a transcription factor downstream of JNK signaling pathway. AP-1 inhibitor also significantly inhibited the expression of IL-12p40 induced by P-PRRSv infection. It is further demonstrated that the induction of IL-12p40 expression by PRRSV infection depends on the JNK-AP-1 signaling pathway. In addition, we compare PRRSV with PRV. The expression level of IL-12 in SeV infected cells showed that although it could induce the expression of IL-12 subunit, its induction ability was significantly lower than that of other viruses. It is found that PRRSV infection can induce the expression of IL-12p40 and IL-12p35. The induction of IL-12p40 by PRRSv depends on JNK-AP-1 and NF- 魏. B signaling pathway. The induction of IL-12p35 depends on p38. MAPK and PKC signaling pathways. The results of this paper have important reference value for further elucidating the molecular mechanism of IL-12 production induced by PRRSV.
【学位授予单位】：中国农业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S852.651

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