猪伪狂犬病病毒gB蛋白单克隆抗体的制备及鉴定
Preparation and identification of a monoclonal antibody against pseudorabies virus gB protein
马国祥 1曹丽艳 2张娟 1张宇 2万颖 2孔祥雨 2李想通 2王娅婷 2段月月 2袁聪 2施磊 2郭庆勇 3翟少华 3郑海学 4王琦2
作者信息
- 1. 新疆农业大学动物医学学院,新疆乌鲁木齐 830052;中国农业科学院都市农业研究所,四川成都 610213;中国农业科学院兰州兽医研究所,甘肃兰州 730046
- 2. 中国农业科学院都市农业研究所,四川成都 610213;中国农业科学院兰州兽医研究所,甘肃兰州 730046
- 3. 新疆农业大学动物医学学院,新疆乌鲁木齐 830052
- 4. 中国农业科学院兰州兽医研究所,甘肃兰州 730046
- 折叠
摘要
为了制备猪伪狂犬病病毒(PRV)gB蛋白单克隆抗体,本研究利用原核表达系统表达gB蛋白的主要抗原区(540～734 aa),获得重组蛋白免疫BALB/c小鼠.利用杂交瘤技术,经过细胞筛选和亚克隆,获得1株能稳定分泌抗gB蛋白的单克隆抗体,将其命名为1C10.经间接免疫荧光试验(IFA)和免疫印迹试验(Western-blot)验证,1C10具有良好的特异性,能识别PRV感染细胞时表达的gB蛋白.亚类鉴定结果表明,1C10重链为IgG2a型,轻链为kappa型.ELISA结果表明,纯化后的腹水效价高于1∶128000.综上,本研究成功表达了 PRV gB蛋白,并制备了抗PRV gB蛋白单克隆抗体,为进一步探究PRVgB蛋白的结构和功能以及后期建立PRV诊断方法及致病机制的研究奠定了基础.
Abstract
In order to prepare monoclonal antibodies(MAbs)against the gB protein of porcine pseu-dorabies virus(PRV),the main antigen region(540-734aa)of gB protein was expressed by a prokaryotic expression system,and the recombinant protein was used to immunize BALB/c mice.Using hybridoma tech-nology,a MAb against the gB protein was obtained through the cell screening and subcloning,and named as 1C10.Indirect immunofluorescence assay(IFA)and Western-blot assay verified that 1C10 has a good specificity and could recognize the gB protein expressed in cells infected with PRV.The identification of isotyping revealed that the 1C10 was of IgG2a type for heavy chain and kappa type for light chain.The ELISA data showed that the purified ascites was higher than 1:128 000.In summary,PRV gB protein was successfully expressed and a MAb against PRV gB protein were prepared in this study,laying the founda-tion for further study on the structure and function of PRV gB protein,as well as the establishment of diagnostic methods and pathogenic mechanisms in the later stage.
关键词
猪伪狂犬病病毒/gB蛋白/原核表达/单克隆抗体Key words
porcine pseudorabies virus/gB protein/prokaryotic expression/monoclonal antibodies引用本文复制引用
基金项目
四川省科技计划重点研发项目(2022YFN0008)
四川省青年学者基金项目(21JCQN0175)
中国农业科学院创新工程项目(ASTIP2022-34-IUA-07)
中国农业科学院青年英才项目(21ZD3NA001)
出版年
2024
国家科技期刊平台
NSTL
万方数据