禽传染性支气管炎病毒nsp4蛋白多克隆抗体制备及其生物学功能研究  

Preparation and biological function research on polyclonal anti⁃body against avian infectious bronchitis virus nsp4 protein

作　　者：卢彦澎 林丽婷 任丽娜 晋英浩 张桃妮 张愉 韦兰萍 吕迪 磨美兰[1,2,3,4] LU Yan-peng;LIN Li-ting;REN Li-na;JIN Ying-hao;ZHANG Tao-ni;ZHANG Yu;WEI Lan-ping;LYU Di;MO Mei-lan(Animal Science and Technology College,Guangxi University,Nanning,Guangxi 530004,China;Guangxi Engineering Research Center of Veterinary Biologics,Nanning,Guangxi 530004,China;Guangxi Key Laboratory of Livestock and Poultry Breeding Disease Prevention and Control,Nanning,Guangxi 530004,China;Guangxi Colleges and Universities Key Laboratory of Prevention and Control for Animal Disease,Nanning,Guangxi 530004,China)

机构地区：[1]广西大学动物科学技术学院,广西南宁530004 [2]广西兽用生物制品工程研究中心,广西南宁530004 [3]广西畜禽繁育与疾病防控重点实验室,广西南宁530004 [4]广西高校动物疫病预防与控制重点实验室,广西南宁530004

出　　处：《南方农业学报》2023年第8期2423-2435,共13页Journal of Southern Agriculture

基　　金：广西自然科学基金项目(2019GXNSFAA245009,2020GXNSFDA297004)。

摘　　要：【目的】筛选出禽传染性支气管炎病毒(IBV)非结构蛋白4(nsp4)抗原性较好的氨基酸区域并制备多克隆抗体,为进一步研究nsp4蛋白在IBV复制过程中的功能提供物质基础,也为研发IBV新型诊断试剂盒与疫苗打下基础。【方法】对IBV Beaudette株nsp4蛋白进行原核表达,以融合蛋白nsp4作为免疫原免疫健康日本大耳白兔和健康阴性鸡,制备相应的兔多克隆抗体和鸡多克隆抗体,并通过间接ELISA检测、Western blotting检测及IFA验证等对制备获得的多克隆抗体进行生物学功能研究。【结果】选取nsp4蛋白第408~514位氨基酸作为原核表达序列,成功构建了nsp4蛋白原核表达载体pCZN-1-HIS-nsp4和nsp4蛋白真核表达载体pVAX1-nsp4-HA。原核表达的融合蛋白nsp4为13.6 kD,与预期结果相符,能与IBV GX-YL5株和M41株全病毒阳性血清反应。制备的兔、鸡多克隆抗体血清效价分别为1∶128000和1∶6400,均能与融合蛋白nsp4和IBV全病毒反应,且与IBV GX-YL5株、Beaudette株和M41株感染鸡胚肾细胞(CEK)表达的nsp4蛋白反应,还能与转染状态及感染IBV Beaudette株的非洲绿猴肾细胞(Vero)表达nsp4蛋白反应。实时荧光定量PCR检测结果表明,经nsp4蛋白真核表达载体pVAX1-nsp4-HA转染后,Vero细胞的病毒载量呈逐渐上升趋势,即体外过表达nsp4蛋白能促进IBV的复制。【结论】制备获得的2种IBV nsp4多克隆抗体效价高、反应性良好、特异性强,可作为细胞定位及表达时相分析等蛋白特性研究的工具,用于解析nsp4蛋白在IBV增殖过程中的作用机制,也为其他冠状病毒nsp4蛋白的研究提供参考。【Objective】This paper screened out the amino acid region with good antigenicity of non-structural protein 4(nsp4)of avian infectious bronchitis virus(IBV)and prepared polyclonal antibody,so as to provide a material basis for further research on the function of nsp4 protein in the process of IBV replication and lay a foundation for the research and development of new diagnostic kits and vaccines for IBV.【Method】In this study,nsp4 protein of IBV Beaudette strain was prokaryotically expressed.Rabbit and chicken polyclonal antibodies were prepared by immunizing Japanese white rabbit and healthy negative chicken with the fusion protein nsp4 as immunogen.And the biological function of the prepared polyclonal antibodies were investigated by indirect enzyme linked immunosorbent assay(ELISA),Western blotting and indirect immunofluorescence assay(IFA).【Result】The nsp4 protein prokaryotic expression vector pCZN-1-HISnsp4 and nsp4 protein eukaryotic expression vector pVAX1-nsp4-HA were successfully constructed by selecting amino acids at positions 408-514 of nsp4 protein as prokaryotic expression sequences.The prokaryotic expression of fusion protein nsp4 was 13.6 kD,which was consistent with its predicted size,and the fusion protein nsp4 was able to react with positive whole virus serum against IBV GX-YL5 and M41 strains.The serum titers of prepared rabbit and chicken polyclonal antibodies were 1∶128000 and 1∶6400 respectively.Additionally,both polyclonal antibodies could react with the fusion protein nsp4 and IBV whole virus.Moreover,they could react with nsp4 protein expressed in chicken embryonic kidney(CEK)cells infected with IBV GX-YL5,Beaudette and M41 strains,and could react with nsp4 protein expressed in the transfected or IBV infected African green monkey kidney cells(Vero)Beaudette strain.The results of real-time fluorescence quantitative PCR showed that the viral loads of Vero cells increased gradually after transfection of nsp4 protein eukaryotic expression vector pVAX1-nsp4-HA.In other words,in

关 键 词：禽传染性支气管炎病毒(IBV) nsp4蛋白 多克隆抗体 反应性 特异性 

分 类 号：S852.659.6[农业科学—基础兽医学]