鸭坦布苏病毒E蛋白结构域Ⅰ-Ⅱ与Ⅲ的反应原性和免疫原性比较  

Comparison of reactogenicity and immunogenicity of duck Tembusu virus E protein domainsⅠ-ⅡandⅢ

作　　者：曲哲会[1,2,3] 鲁绍芳 郭晓秋 陈敏[1] 焦凤超[1,2] 王梦瑶[1] 魏星 马君钊 张茹 黄立 张喜文 QU Zhehui;LU Shaofang;GUO Xiaoqiu;CHEN Min;JIAO Fengchao;WANG Mengyao;WEI Xing;MA Junzhao;ZHANG Ru;HUANG Li;ZHANG Xiwen(College of Animal Science and Veterinary Medicine,Xinyang Agriculture and Forestry University,Xinyang 464000,China;Henan Province Engineering Technology Research Center for Waterfowl Resources Development and Utilization and Epidemic Disease Prevention and Control,Xinyang 464000,China;Xinyang City Key Laboratory of Integrated Technology Research for Prevention and Control of Major Livestock and Poultry Diseases,Xinyang 464000,China)

机构地区：[1]信阳农林学院动物科技学院,河南信阳464000 [2]河南省水禽资源开发利用与疫病防控工程技术研究中心,河南信阳464000 [3]信阳市畜禽重大疫病防控综合技术研究重点实验室,河南信阳464000

出　　处：《黑龙江畜牧兽医》2023年第20期89-95,100,共8页Heilongjiang Animal Science And veterinary Medicine

基　　金：河南省科技攻关项目(222102110188);信阳农林学院高水平科研孵化器建设项目(FCL202004);信阳农林学院青年科研基金项目(20200113);信阳农林学院科技创新团队建设项目(XNKJTD-014);信阳市创新应用专项(20200016)。

摘　　要：为了比较鸭坦布苏病毒(duck Tembusu virus,DTMUV)E蛋白结构域Ⅰ-Ⅱ(EDⅠ-Ⅱ)与结构域Ⅲ(EDⅢ)的反应原性和免疫原性,试验将重组质粒pET-EDⅠ-Ⅱ[EDⅠ-Ⅱ基因插入表达载体pET-30a(+)构建的重组质粒]和pET-EDⅢ[EDⅢ基因插入表达载体pET-30a(+)构建的重组质粒]分别转化至大肠杆菌(E.coli)Rosetta(DE3)感受态细胞,经IPTG诱导后,对表达产物进行SDS-PAGE分析和Western-blot鉴定,采用亲和层析法纯化重组蛋白EDⅠ-Ⅱ和EDⅢ,通过Dot-blot试验和间接ELISA试验比较二者的反应原性;将重组蛋白EDⅠ-Ⅱ和EDⅢ免疫小鼠,通过测定免疫小鼠血清中特异性抗体、病毒中和(VN)抗体和细胞因子(IL-6、IFN-γ)水平比较两者的免疫原性;将DTMUV分离株感染重组蛋白EDⅠ-Ⅱ和EDⅢ免疫小鼠,采用实时荧光定量PCR方法检测小鼠肺脏、肝脏和脑组织中DTMUV RNA相对表达水平,比较重组蛋白EDⅠ-Ⅱ和EDⅢ对免疫小鼠的保护力。结果表明:含重组质粒pET-EDⅠ-Ⅱ和pET-EDⅢ诱导4 h的菌体蛋白经SDS-PAGE分析和Western-blot鉴定分别在分子量约33 ku和17 ku位置出现明显条带,可与鸡抗DTMUV多克隆抗体发生特异性免疫反应;经Dot-blot试验和间接ELISA试验分析发现,重组蛋白EDⅠ-Ⅱ比重组蛋白EDⅢ具有更好的反应原性;重组蛋白EDⅠ-Ⅱ免疫小鼠后第42天血清中特异性抗体水平、IFN-γ和IL-6水平均极显著高于重组蛋白EDⅢ(P<0.01或P<0.001),但VN抗体水平极显著低于重组蛋白EDⅢ(P<0.01);免疫小鼠感染DTMUV后,重组蛋白EDⅢ免疫小鼠肺脏、肝脏和脑组织中DTMUV mRNA相对表达水平均显著或极显著低于重组蛋白EDⅠ-Ⅱ免疫小鼠(P<0.05或P<0.01),即EDⅢ蛋白诱导小鼠产生的免疫应答较EDⅠ-Ⅱ蛋白对于抑制DTMUV复制的效果更好。说明EDⅠ-Ⅱ蛋白更适合作为以E蛋白及相关特异性抗体为基础的免疫学检测方法研究的目标蛋白,而EDⅢ蛋白更适合作为DTMUV新型疫苗研究的目标蛋白。In order to compare the reactogenicity and immunogenicity of duck Tembusu virus(DTMUV)E protein domainsⅠ-Ⅱ(EDⅠ-Ⅱ)andⅢ(EDⅢ),in this experiment,the recombinant plasmids pET-EDI-Ⅱ(recombinant plasmid constructed by EDⅠ-Ⅱgene insertion expression vector pET-30a[+])and pET-EDⅢ(recombinant plasmid constructed by EDⅢgene insertion expression vector pET-30a[+])were transformed respectively into E.coli Rosetta(DE3)competent cells;after IPTG induction,SDS-PAGE analysis and Western-blot identification of expression products were performed.The recombinant proteins EDI-Ⅱand EDⅢwere purified by affinity chromatography,and the reactogenicity of them was compared by Dot-blot test and indirect ELISA test.The recombinant proteins EDI-Ⅱand EDⅢwere immunized into mice;the immunogenicity of them was compared by determining the levels of specific antibodies,virus-neutralizing(VN)antibodies and cytokine(IL-6,IFN-γ)levels in the sera of immunized mice.The DTMUV isolate was infected in recombinant proteins EDI-Ⅱand EDⅢ-immunized mice;real-time PCR was used to detect the relative expression level of DTMUV RNA in mouse lung,liver and brain tissues.The protective effect of recombinant proteins EDI-Ⅱand EDⅢon immunized mice was compared.The results showed that the proteins from the bacteria containing recombinant plasmids pET-EDI-Ⅱand pET-EDⅢafter 4 h induction showed that the obvious bands at the molecular weight of about 33 ku and 17 ku respectively,by SDS-PAGE analysis and Western⁃blot identification,which could have specific immune reaction with chicken anti-DTMUV polyclonal antibodies.The analysis of Dot-blot test and indirect ELISA test showed that the recombinant protein EDI-Ⅱhad better reactogenicity than the recombinant protein EDⅢ.The levels of specific antibodies,IFN-γand IL-6 in sera on the 42nd day after immunization of recombinant protein EDI-Ⅱwere significantly higher than those of EDⅢprotein(P<0.01 or P<0.001),but the level of VN antibody was significantly lower than that

关 键 词：鸭坦布苏病毒 E蛋白 结构域 反应原性 免疫原性 

分 类 号：S852.651.1[农业科学—基础兽医学]