猪RIG-I真核表达载体的构建及其抗口蹄疫病毒作用研究  

Construction of Porcine RIG-I Eukaryotic Expressing Plasmid and Its Antiviral Effects Research against Foot and mouth Disease Virus

作　　者：王国庆[1] 朱紫祥[2] 曹伟军[2] 杨帆[2] 毛箬青[2] 李丹[2] 刘磊[1] 郑海学[2] 

机构地区：[1]甘肃农业大学动物医学院,兰州730070 [2]中国农业科学院兰州兽医研究所家畜疫病病原生物学国家重点实验室国家口蹄疫参考实验室,兰州730046

出　　处：《畜牧兽医学报》2015年第4期600-607,共8页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：国家自然科学基金项目(31302118;31402179);甘肃省杰出青年基金项目(145RJDA328);甘肃省科技重大专项项目(1302NKDA027);国际原子能项目(16025/R0)

摘　　要：为研究模式识别受体分子RIG-I是否具有抑制口蹄疫病毒(FMDV)复制的作用,本研究从猪的PK15细胞中提取RNA,通过分段扩增与融合PCR的方法,扩增猪的RIG-I的完整CDS序列,进一步构建猪RIG-I的真核表达质粒。通过Western blotting和间接免疫荧光试验对构建的真核表达质粒进行表达验证,证明其成功表达,同时证明猪RIG-I蛋白定位于细胞的细胞质中。感染试验发现FMDV能够诱导细胞内RIG-I的转录上调,这表明两者间存在着重要联系。过表达试验证实RIG-I具有抑制FMDV复制的作用,而下调表达RIG-I可以促进FMDV的复制,这表明RIG-I在机体抗口蹄疫病毒感染过程中发挥着重要作用。本研究的开展,为进一步探索RIG-I抗口蹄疫病毒的分子机制提供了理论支持;为口蹄疫病毒感染过程中,天然免疫系统抗病毒机制研究奠定了基础。To investigate whether pattern recognition receptor RIG-I has antiviral role against foot-and-mouth disease virus(FMDV),we extracted porcine cellular RNA from PK15 cells,and the complete CDS region of RIG-I was obtained by using two-segmental amplification and fusion PCR methods.Subsequently,the eukaryotic expressing plasmid was constructed.The expression of the plasmid was confirmed by Western blotting and indirect fluorescence assay(IFA),which showed RIG-I was successfully expressed.The IFA result indicated porcine RIG-I protein was located in the cellular cytoplasm.The results of infection assay suggested that FMDV infection induced the upregulation of RIG-I expression,which implied the potential connection between RIGI and FMDV.Over-expression assay confirmed that RIG-I inhibited FMDV replication,and downregulation of RIG-I significantly promoted FMDV replication.These results indicated that RIG-Ihad antiviral effect against FMDV.In conclusion,this study provided references for further research on antiviral mechanism of RIG-I against FMDV.It also paves ways for the research of antiviral mechanism of innate immune system after FMDV infection.

关 键 词：口蹄疫病毒 RIG-I 天然免疫 抗病毒作用 

分 类 号：S852.65[农业科学—基础兽医学]