摘要
为研究犬瘟热病毒(Canine distemper virus, CDV)不同毒株N、H蛋白调控宿主β-干扰素(IFN-β)表达的作用,将CDV野毒株LN(10)1、SD(14)7和疫苗株CDV3感染提前转染了pGL3-IFN-β报告基因质粒和pRL-TK内参质粒的MDCK细胞,分别在感染0 h、8 h、12 h和24 h后通过双荧光素酶报告系统检测试剂盒测定IFN-β启动子的活性。将CDV野毒株LN(10)1、SD(14)7和疫苗株CDV3 N和H基因克隆到pCI真核表达载体。重组表达质粒分别转染HEK293T细胞,应用Western blot检测蛋白能否正确表达。将N和H蛋白重组表达质粒连同pGL3-IFN-β报告基因质粒和pRL-TK内参质粒共转染至HEK293T细胞后,经Poly(I:C)刺激细胞,利用双荧光素酶报告系统检测系统测定IFN-β启动子活性。结果显示3种不同毒力的CDV在感染MDCK细胞后,只有CDV 3疫苗株感染后能观察到IFN-β启动子短暂激活。Western blot实验证实,3种毒株N和H蛋白在HEK293T细胞中均能正确表达。双荧光素酶报告系统对IFN-β启动子检测活性结果显示,Poly(I:C)成功激活了IFN-β信号通路,而这种激活作用在表达CDV不同毒株的N或H蛋白时均受到显著抑制(P <0.01),但不同毒株N或H蛋白对IFN-β信号通路的调控与CDV毒力无关。本研究证实了CDV野毒株和疫苗株N、H蛋白可抑制宿主IFN-β信号通路,为进一步研究CDV与宿主分子互作奠定基础。
In order to study the effects of N and H proteins of canine distemper virus(CDV)in different strains on the expression of hostβ-interferon(IFN-β).The pGL3-IFN-βreporter plasmid and the internal reference plasmid pRL-TKwere co-transfected intoMDCKcells.At least 18 h post transfection,cells were infected with wild CDV strains LN(10)1,SD(14)7 and vaccine strain CDV3,respectively.At 0 h,8 h,12 h and 24 h after infection,IFN-βpromoter activity was determined by dual luciferase reporting systemdetection kit.The NandH genes of CDV wild strains LN(10)1,SD(14)7 and vaccine strain CDV3 were cloned into pCI eukaryotic expression vector.The recombinant expression plasmid was transfected into HEK293T cells and the correct expression was confirmed byWestern blot.The target plasmid was cotransfected into HEK293Tcells with pGL3-IFN-βreporter gene plasmid and pRL-TKinternal reference plasmid.Then,the cells were stimulated with Poly(I:C)and the IFN-βpromoter activity was detected by the dual luciferase reporter system assay kit.With virus infection,a small and transient activation of the IFN-β promoter in CDV3 group,not in other groups was observed.The result of IFN-βpromoter activity detected by the dual luciferase reporter systemshowthat Poly(I:C)successfully activated the IFN signaling pathway.However,this activation was significantly inhibited by overexpression ofNorHproteins of different CDVstrains(P<0.01).However,the level of IFN-βinhibition was unrelated to whether the protein was derived from an attenuated or wild-type strain of CDV.This study confirmed that CDV N and H proteins can inhibit the host IFN-β signaling pathway,whichwould lay a foundation for the further study of CDV and hostmolecular interaction.
作者
龚成燕
李连峰
陈杰
胡博
赵建军
GONG Cheng-yan;LI Lian-feng;CHEN Jie;HU Bo;ZHAO Jian-jun(Key Laboratory of Special Animal Epidemic Disease of Ministry of Agricultural,Institute of Special Animal and Plant Sciences of Chinese Academy of Agricultural Sciences,Changchun 130112,China;State Key Laboratory of V eterinary Biotechnology,Harbin Veterinary Research Institute,Chinese Academy of Agricultural Sciences,Harbin 150069,China;College of Veterinary Medicine,Northwest Agriculture and Forestry University,Yangling 712100,China;College of Animal Science and Veterinary,Heilongjiang Bayi Agricultural University,Daqing 163319,China)
出处
《特产研究》
2021年第2期1-7,共7页
Special Wild Economic Animal and Plant Research
基金
兽医生物技术国家重点实验室开放课题(SKLVBF202004)
国家重点研发计划项目(2017YFD0501600)
国家自然科学基金(31972714)。
