蓝舌病毒NS4基因真核表达对IFN-β信号通路基因mRNA表达的影响  

Effect of bluetongue virus NS4 gene eukaryotic expression on mRNA expression of IFN-βassociated genes

作　　者：林俊泓 赵瑶 陈玉娟 王让 马鲜平 易华山[1,2] LIN Junhong;ZHAO Yao;CHEN Yujuan;WANG Rang;MA Xianping;YI Huashan(College of Veterinary Medicine,Southwest University,Rongchang,Chongqing 402460,China;Immunology Research Center,Institute of Medicine,Southwest University,Rongchang,Chongqing 402460,China)

机构地区：[1]西南大学动物医学院,重庆荣昌402460 [2]西南大学医学研究院免疫学研究中心,重庆荣昌402460

出　　处：《中国兽医学报》2022年第2期199-207,共9页Chinese Journal of Veterinary Science

基　　金：重庆市基础研究与前沿探索专项基金资助项目(cstc2018jcyjAX0615);中央高校基本科研业务专项基金资助项目(XDJK2018C060,XDJK2018C059);国家重点研发计划资助项目(2018YFD0501705)。

摘　　要：通过构建蓝舌病毒(BTV)NS4基因真核表达载体pcDNA3.1-NS4-eGFP,转染HEK-293T细胞,利用Western blot及荧光显微镜分析NS4蛋白的表达与亚细胞定位特征;pcDNA3.1-NS4-eGFP转染的HEK-293T细胞添加20 HAU/mL仙台病毒(SeV)刺激后,qRT-PCR法分析NS4基因表达对SeV诱导的上游识别基因RIG-Ⅰ、MDA5、VISA、TBK1、IKKε、IRF3、TRAF3、TRAF6、IRF9、干扰素基因(IFN-α、IFN-β)以及干扰素刺激基因ISG15和USP18的mRNA表达水平的影响。在HEK-293T细胞内转染pcDNA3.1-NS4-eGFP质粒24 h后,分别添加20 HAU/mL SeV刺激24,48 h,qRT-PCR结果表明,细胞内表达NS4-EGFP后,RIG-Ⅰ、MDA5、TRAF6、IRF9、ISG15及IFN-β基因mRNA表达极显著下降,随着SeV诱导时间的延长,VISA、TBK1、IKKε、USP18基因mRNA表达差异呈不显著趋势。本研究成功构建BTV NS4基因真核表达载体pcDNA3.1-NS4-eGFP,NS4-EGFP融合蛋白在HEK-293T细胞中主要分布于细胞核周围及细胞核内。BTV NS4基因在HEK-293T细胞内的表达显著下调SeV诱导的IFN信号通路相关基因RIG-Ⅰ、MDA5、TRAF6、IRF9、ISG15和IFN-β的表达,为进一步探究NS4基因在BTV拮抗宿主细胞免疫应答中的机制奠定基础。The eukaryotic expression vector pcDNA3.1-NS4-eGFP was constructed and transfected into HEK-293 T cells,the expression and subcellular localization of bluetongue virus NS4 protein were analyzed by Western blot and fluorescence microscope.Twenty HAU/mL Sendai virus(SeV)was added to HEK-293 T cells transfected with pcDNA3.1-NS4-eGFP to analyzed the effect of NS4 gene expression on the mRNA expression of SeV induced upstream genes RIG-Ⅰ,MDA5,VISA,TBK1,IKKε,IRF3,TRAF3,TRAF6,IRF9,interferon genes(IFN-α,IFN-β)and interferon stimulated genes ISG15,USP18 by qRT-PCR.After HEK-293 cells were transfected with pcDNA3.1-NS4-eGFP plasmid for 24 h,cells were treated with 20 HAU/mL SeV for 24,48 h respectively.The results of qRT-PCR denoted that the expression of NS4-EGFP in cells significantly lowered the mRNA expression of RIG-Ⅰ,MDA5,TRAF6,IRF9,ISG15 and IFN-β,but there was no significant difference in the mRNA expression of VISA、TBK1、IKKε、USP18 with the extension of SeV induction time.The eukaryotic expression vector pcDNA3.1-NS4-eGFP was successfully constructed,NS4-EGFP fusion protein in HEK-293 Tcells mainly distributed around and inside the nucleus.The expression of bluetongue virus NS4 gene in cells significantly down-regulated the mRNA expression of the SeV-induced interferon signaling pathway related genes RIG-Ⅰ,MDA5,TRAF6,IRF9,ISG15,IFN-β.These result lay the foundation for further investigation of the mechanism of NS4 gene in bluetongue virus infection antagonizing host cell immune response.

关 键 词：蓝舌病毒 NS4基因 IFN-Β 仙台病毒 

分 类 号：S852.65[农业科学—基础兽医学]