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.
出处
《畜牧兽医学报》
CAS
CSCD
北大核心
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)
