口蹄疫病毒Asia1型猪源中和抗体的筛选与抗原表位鉴定  

Screening and Identification of the Antigenic Epitope of Pig Neutralizing Antibodies against Foot-and-mouth Disease Virus Serotype Asia 1

作　　者：董开恒 黄书伦 李凤娟 李坤[2,3] 刘果 张强 包慧芳[2,3] 李洪炫 卢曾军[2,3] 张小丽 DONG Kaiheng;HUANG Shulun;LI Fengjuan;LI Kun;LIU Guo;ZHANG Qiang;BAO Huifang;LI Hongxuan;LU Zengjun;ZHANG Xiaoli(College of Veterinary Medicine,Gansu Agricultural University,Lanzhou 730070,China;State Key Laboratory for Animal Disease Control and Prevention,National Foot-and-Mouth Diseases Reference Laboratory,College of Veterinary Medicine,Lanzhou University,Lanzhou Veterinary Research Institute,Chinese Academy of Agricultural Sciences,Lanzhou 730000,China;Gansu Province Research Center for Basic Disciplines of Pathogen Biology,Lanzhou 730046,China)

机构地区：[1]甘肃农业大学动物医学院,兰州730070 [2]中国农业科学院兰州兽医研究所,动物疫病防控全国重点实验室,兰州大学动物医学与生物安全学院,国家口蹄疫参考实验室,兰州730000 [3]甘肃省病原生物学基础学科研究中心,兰州730046

出　　处：《畜牧兽医学报》2024年第11期5211-5221,共11页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：“十四五”国家重点研发项目(2021YFD1800300)。

摘　　要：Asia1型口蹄疫病毒(FMDV)仍然在我国周边国家存在,对我国畜牧业造成长期威胁。本研究旨在利用单个B细胞抗体技术研制Asia1型FMDV的中和性单克隆抗体,以此为工具,鉴定Asia1型FMDV的保护性抗原位点。以FMDV Asia1/JS/05为诱饵抗原,通过流式细胞术分选免疫猪PBMCs中的抗原特异性B细胞,通过巢式PCR扩增单个B细胞IgG抗体重链与轻链可变区基因序列,分别构建IgG抗体重链与轻链表达质粒,将其共转染CHO-S细胞进行抗体表达;通过间接ELISA、间接免疫荧光试验(IFA)、病毒中和试验(VNT)验证抗体反应性和中和活性,利用免疫印迹、中和抗体逃逸突变株筛选鉴定中和抗体所识别的抗原表位类型和抗原表位关键氨基酸。结果显示:获得了5株反应性良好的Asia1型FMDV特异性抗体,其中PD3和PD7为中和抗体,两株中和抗体识别相同表位,关键氨基酸为VP2蛋白72位残基(D)。本研究首次获得Asia1型FMDV特异性猪源中和抗体,鉴定VP272D是中和表位的关键氨基酸,进一步丰富了Asia1型FMDV抗原位点信息,为FMDV Asia1型分子疫苗和新诊断检测技术研究的奠定了基础。Foot-and-mouth disease virus(FMDV)serotype Asia 1 is still circulating in Asia countries around China,which will be a long-term threat to animal husbandry.The aim of this study was to screen porcine monoclonal neutralizing antibodies against Asia1 FMDV using single B-cell antibody technology,and to identify the antigenic sites recognized by neutralizing antibodies.FMDV Asia1/JS/05 inactivated whole virus particles were used as the bait antigen to sort antigen-specific B cells in PBMCs of immunized porcine by flow cytometry.The sequences of variable regions of the heavy and light chain of the IgG were amplified by nested PCR.The expression plasmids of the IgG heavy and light chain were constructed respectively and co-transfected into CHO-S cells to express the whole IgG antibodies.The reactivity and neutralization ability of expressed antibodies were verified by indirect ELISA,indirect immunofluorescent assay(IFA)and viral neutralizing test(VNT).The epitopes as well as the critical amino acids recognized by the neutralized antibody were identified using Western blot and neutralizing antibody escape mutants screening.Five Asia 1 FMDV-specific antibodies were obtained,of which PD3 and PD7 showed neutralizing ability.These two neutralizing antibodies recognized the same epitope and the key amino acids residue was D 72 of the VP2 protein.In this study,neutralizing antibody against Asia 1 FMDV from pig were obtained for the first time,which provides further information of the protective antigen sites of Asia1 FMDV,and make a basis for the design of molecular vaccine and the development of new tools for diagnosis of FMD Asia 1.

关 键 词：口蹄疫病毒 Asia1型 猪源单克隆抗体 抗原表位 

分 类 号：S852.659.6[农业科学—基础兽医学]