论文部分内容阅读
目的克隆东方马脑炎病毒(EEEV)衣壳蛋白(Capsid)基因,构建其真核表达载体,检测该基因在293细胞中的表达,并初步研究其对干扰素(IFN)应答基因表达的影响。方法构建包含EEEV capsid基因的真核表达载体pcDNA3-Flag-capsid,以威格拉斯脂质体转染293细胞进行瞬时表达,Western印迹检测其表达情况;应用定量PCR比较IFN效应基因Gbp1,Gbp2和Isg15在pcDNA3-Flag-capsid和空载体转染细胞中mRNA表达差异。结果构建了EEEV Capsid真核表达质粒,并且该质粒能够在293细胞中有效表达,同时定量PCR结果表明,与空载体转染对照组相比,外源导入EEEV Capsid抑制IFN应答基因Gbp1,Gbp2和Isg15的转录。结论真核表达EEEVCapsid抑制细胞内IFN应答基因的表达,为研究EEEV致病机制奠定基础。
Objective To clone the capsid protein of eastern equine encephalitis virus (EEEV) and construct its eukaryotic expression vector to detect the expression of this gene in 293 cells and to study its effect on the expression of interferon (IFN) . Methods The eukaryotic expression vector pcDNA3-Flag-capsid containing EEEV capsid gene was constructed and transfected into 293 cells by Wieglas liposome for transient expression. The expression of Gbp1, Gbp2 and IFN- Isg15 in pcDNA3-Flag-capsid and empty vector transfected cells mRNA expression differences. Results The EEEV Capsid eukaryotic expression plasmid was constructed and expressed efficiently in 293 cells. The results of quantitative PCR showed that EEEV Capsid inhibited the expression of Gbp1, Gbp2 and Gbp2 in 293 cells Isg15 transcription. Conclusion Eukaryotic expression of EEEVCapsid inhibits the expression of intracellular IFN-responsive genes and lay the foundation for the study of the pathogenesis of EEEV.