羊口疮病毒ORF128锚蛋白对HEK293T细胞NF-κB信号通路的调节作用及机制  

Regulating effect of ORF128 on NF-κB signaling pathway of HEK293T cells and its mechanism

作　　者：安雪珂 贾怀杰[2] 冯园 陈国华[2] 何小兵[2] 景志忠[2] 王晓霞 AN Xueke;JIA Huaijie;FENG Yuan;CHEN Guohua;HE Xiaobing;JING Zhizhong;WANG Xiaoxia(Institute of Nutrition and Food Hygiene, School of Public Health, Lanzhou University, Lanzhou 730000;2State Key Laboratory of Veterinary of Etiological Biology, Key Laboratory of Veterinary Public Health of Agricultural Ministry, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou 730046;Xiangtan Central Hospital, Xiangtan 411100, China)

机构地区：[1]兰州大学公共卫生学院营养与食品卫生学研究所,甘肃兰州730000 [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农业部兽医公共卫生重点实验室,甘肃兰州730046 [3]湘潭市中心医院,湖南湘潭411100

出　　处：《细胞与分子免疫学杂志》2019年第3期243-249,共7页Chinese Journal of Cellular and Molecular Immunology

基　　金："十三五"国家重点研发计划项目(2017YFD0500903);国家自然科学基金青年项目(81501734);甘肃省农业生物技术专项(GNSW-2012-16)

摘　　要：目的研究羊口疮病毒(ORFV)感染HEK293T宿主细胞后,其编码的锚蛋白ORF128蛋白对宿主核因子κB(NF-κB)信号通路的影响及其机制。方法将来自ORFV/QH02/2010株的ORF128 DNA序列分别构建至真核表达载体pCMV-tag2B和pEGFP-N1;在病毒感染细胞期间,利用反转录PCR检测ORF128 mRNA水平、激光共聚焦显微镜检测ORF128蛋白的亚细胞定位;利用双荧光素酶报告基因检测系统分析ORF128对NF-κB信号通路的调节作用, Western blot法检测NF-κBp65的核移位以及NF-κB抑制蛋白α(IκBα)的蛋白磷酸化水平。结果 ORF128蛋白在病毒感染早期表达且定位于宿主细胞核, ORF128蛋白可抑制NF-κB转录因子报告载体荧光素酶活性; ORF128的表达抑制NF-κBp65的核移位和磷酸化的IκBα(p-IκBα)的降解。结论 ORF128蛋白通过抑制p-IκBα蛋白的降解过程,阻止NF-κBp65蛋白核移位,进而抑制宿主NF-κB信号通路的活化。Objective To elucidate the regulating effect of orf virus(ORFV) encoded ORF128 on NF-κB signaling pathway during the infection of HEK293T cells with ORFV and the underlying mechanism. Methods The ORF128 DNA sequences from ORFV/QH02/2010 strain were constructed into eukaryotic expression vectors pCMV-tag2B and pEGFP-N1. During viral infection of cells, the level of ORF128 mRNA was detected by reverse transcription PCR and the subcellular localization of ORF128 protein by laser confocal microscopy. A dual luciferase reporter assay system was used to analyze the regulating effect of ORF128 on NF-κB signaling pathway, and Western blot analysis to detect the nuclear translocation of NF-κBp65 and the phosphorylation of IκBα protein(p-IκBα). Results ORF128 protein was expressed at the early stage and localized in the cell nuclear during ORFV infection, and it inhibited the expression of NF-κB reporter luciferase activity. The expression of the protein blocked the nuclear translocation of NF-κBp65 and the degradation of p-IκBα. Conclusion ORF128 inhibits the activation of NF-κB signaling pathway by blocking the nuclear translocation of NF-κBp65 and the degradation of p-IκBα.

关 键 词：羊口疮病毒(ORFV) 锚蛋白 核因子κB( NF-κB) 羊口疮病毒蛋白128(ORF128) 

分 类 号：R392-33[医药卫生—免疫学] S718.85[医药卫生—基础医学]