猪繁殖与呼吸综合征病毒感染激活IL-10的分子机制研究

项目名称： 猪繁殖与呼吸综合征病毒感染激活IL-10的分子机制研究

项目编号： No.30871871

项目类型： 面上项目

立项/批准年度： 2009

项目学科： 轻工业、手工业

项目作者： 肖少波

作者单位： 华中农业大学

项目金额： 35万元

中文摘要： 猪繁殖与呼吸综合征病毒（PRRSV）具有超强的免疫抑制能力，白细胞介素10(IL-10)是一种免疫抑制因子。为了探讨PRRSV感染激活IL-10的分子细节，本课题构建了猪IL-10启动子及其转录因子结合位点突变体的荧光素酶报告质粒，克隆了20多个相关的信号分子，分析了PRRSV感染猪肺泡巨噬细胞后IL-10表达的动力学，发现PRRSV激活IL-10依赖于MAPK p38、转录因子NF-κ#21644;TLR通路；筛选了可能参与PRRSV激活IL-10的PRRSV编码蛋白，发现PRRSV结构蛋白GP4、GP5和N蛋白具有激活IL-10的功能，进一步通过缺失或点突变证实GP4的74-84aa是激活IL-10的关键区域。同时，还发现不同PRRSV分离株激活IL-10的能力存在差异，而且miRNA-106a、miRNA-365、miR-125b等参与PRRSV调控IL-10、IL-6、NF-κ#31561;细胞因子或转录因子，从一个新的角度揭示了PRRSV免疫调节机制的复杂性。课题执行期内分别在EID、JBC、Mol Immunol、Dev & Comp Immunol等期刊发表研究论文10篇，培养研究生5名。

中文关键词： 猪繁殖与呼吸综合征病毒；IL-10；转录因子；信号通路；免疫抑制

英文摘要： Porcine reproductive and respiratory syndrome virus (PRRSV) possesses the stronger ability to inhibit host's innate immunity. Interleukin 10 (IL-10) is a known immunosuppressive factor. In order to investigate the underlying mechanism(s) that PRRSV activates IL-10, a series of luciferase reporters with mutation in the different transcription factor binding sites of IL-10 promoter were constructed, and nearly 20 signaling molecules associated with IL-10 pathway were cloned. By analyzing the expression dynamics of IL-10 in PRRSV-infected porcine alveolar macrophages (PAMs), we found that PRRSV infection induced IL-10 mRNA and protein expression in a time- and dose-dependent manner. Treatment with specific inhibitors or siRNA demonstrated that NF-κand MAPK p38 are requested for PRRSV-induced IL-10. Furthermore, the Toll like receptor (TLR) signaling pathway but not RIG-I signaling pathway is involved in PRRSV-induced IL-10 activation. We also screened the PRRSV-encoded structural proteins and found that overexpression of GP4, GP5, or N protein alone could activate IL-10. Analyses of a series of mutation or deletion constructs demonstrated that the domain of 78-84 amino acid resides in GP4 was the key region responsible for IL-10 activation. In addition, we also showed that different PRRSV isolates exhibited different ability to activate IL-10. And we found that some microRNAs, including miR-106a, miR-365, and miR-125b, regulated the expression of IL-10, IL-6, and NF-κ respectively. Collectively, these results indicated that PRRSV possesses a very complex immuno-regulation mechanism. During the period of execution of this project, 10 research articles have been published in professional journals, such as EID, JBC, Mol Immunol, and Dev & Comp Immunol. In addition, this project trained 2 post-graduate master students and 3 doctoral students.

英文关键词： Porcine reproductive and respiratory syndrome virus;IL-10;transcription factor;signaling pathway;immune suppression