摘要
旨在探索牛结节性皮肤病病毒(lumpy skin disease virus, LSDV)ORF140在病毒感染宿主细胞中的作用。利用前期构建的LSDV ORF140缺失株,荧光定量分析缺失ORF140基因在LSDV感染宿主细胞后细胞因子转录水平的变化,同时选取LSDV保守基因ORF077建立检测LSDV ORF077的标准曲线,检测LSDV ORF140缺失株与野毒株在不同时间点的拷贝数变化,然后通过蛋白质谱技术和免疫共沉淀技术分析140蛋白与宿主细胞内存在相互作用的蛋白,最后敲低此蛋白的基因转录水平检测宿主细胞内细胞因子转录水平的变化。结果:LSDV ORF140缺失株会引起宿主细胞β-catenin基因转录水平显著下调(P<0.000 1)且缺失株拷贝数始终比野毒株高,推测LSDV ORF140基因的存在能激活宿主细胞的Wnt/β-catenin(Wnt/β-连环蛋白信号通路)信号通路,并且该通路的激活会影响LSDV的复制,宿主细胞中的连接斑珠蛋白(JUP)与LSDV 140蛋白存在相互作用,敲低宿主细胞JUP基因转录水平,发现β-catenin基因转录水平下调(P<0.05),感染LSDV后,β-catenin基因转录水平仍处于下调状态,并且检测到此时病毒拷贝数比野毒株高(P<0.05),以此推测LSDV ORF140是通过与JUP结合激活Wnt/β-catenin信号通路,从而影响LSDV的复制。综上,本研究证实LSDV 140蛋白能够与宿主细胞中的JUP蛋白结合,激活Wnt/β-catenin信号通路,从而抑制LSDV的复制,为揭示LSDV与宿主之间的相互作用及LSDV的致病机制提供参考。
The aim of this study was to explore the role of the Lumpy skin disease virus(LSDV)ORF140 in infected host cells.Using a previously constructed LSDV ORF140 deletion mutant in the laboratory,fluorescence quantitative analysis was performed to assess the changes in cytokine transcription levels after the deletion of ORF140 in LSDV-infected host cells.Simultaneously,a standard curve for detecting LSDV ORF077 was established,and the copy number changes of LSDV ORF140 deletion mutant and wild-type strain at different time points were measured.Then,protein mass spectrometry and immunoprecipitation techniques were employed to analyze proteins that interact with the 140 protein in host cells. Finally,the transcription levels of genes were knocked down to examine changes in cytokine transcription levelswithin the host cells. The results showed that the LSDV ORF140 deletion mutant significantly downregulated the transcription levels of thehost cell β-catenin gene ( P<0. 000 1) and maintained a higher copy number than the wild-type strain,suggesting that the presence of theLSDV ORF140 gene activated the host cell's Wnt /β-catenin signaling pathway,and the activation of this pathway affected LSDV replication.Junction Plakoglobin ( JUP) in the host cells interacted with the LSDV 140 protein. Knocking down the transcription levels of the host cellJUP gene resulted in downregulation of the β-catenin gene transcription level ( P<0. 05) . After LSDV infection,the β-catenin gene transcriptionlevel remained downregulated,and a higher virus copy number than the wild-type strain was detected ( P<0. 05) . It is inferred thatLSDV ORF140 activated the Wnt /β-catenin signaling pathway by binding to JUP,thereby affecting LSDV replication. This study confirmedfor the first time that LSDV 140 protein can bind to JUP in host cells,activating the Wnt /β-catenin signaling pathway to inhibit LSDV replication.This finding provided reference for the study of the interaction between LSDV and its host and the pathogenic mechanism of LSDV.
作者
王敏
温渊
曹钰莹
刘聪
李超
汤芳
戴建君
薛峰
WANG Min;WEN Yuan;CAO Yuying;LIU Cong;LI Chao;TANG Fang;DAI Jianjun;XUE Feng(Joint International Cooperation Laboratory of Animal Health and Food Safety,Nanjing Agricultural University,Nanjing 210095,China;China Animal Health and Epidemiology Center/Key Laboratory of Animal Biosafety Risk Prevention and Control(South),Ministry of Agriculture and Rural Affairs/Key Laboratory of Animal Biosafety,Qingdao 266032,China;School of Life Science and Technology,China Pharmaceutical University,Nanjing 211199,China)
出处
《畜牧与兽医》
北大核心
2025年第3期103-110,共8页
Animal Husbandry & Veterinary Medicine
基金
国家重点研发项目(2021YFD1800500)。
