猪伪狂犬病病毒变异株HN1201 gE蛋白单克隆抗体的制备与鉴定  

Preparation and identification of monoclonal antibody against gE protein of porcine pseudorabies virus variant strain HN1201

作　　者：牛欣蕊 付朋飞 鲁维飞[1,2,3] 张超[1,2,3] 褚贝贝[1,2,3] 王江[1,2,3] 曾磊[1,2,3] NIU Xinrui;FU Pengfei;LU Weifei;ZHANG Chao;CHU Beibei;WANG Jiang;ZENG Lei(College of Veterinary Medicine,Henan Agricultural University,Zhengzhou 450046,China;Key Laboratory of Animal Biochemistry and Nutrition,Ministry of Agriculture and Rural Affairs,Henan Agricultural University,Zhengzhou 450046,China;Key Laboratory of Animal Growth and Development of Henan,Henan Agricultural University,Zhengzhou 450046,China;College of Life Science and Engineering,Henan University of Urban Construction,Pingdingshan,Henan 467036,China)

机构地区：[1]河南农业大学动物医学院,河南郑州450046 [2]河南农业大学农业农村部动物生化与营养重点实验室,河南郑州450046 [3]河南农业大学河南省动物生长发育调控重点实验室,河南郑州450046 [4]河南城建学院生命科学与工程学院,河南平顶山467036

出　　处：《中国兽医学报》2023年第8期1604-1611,1617,共9页Chinese Journal of Veterinary Science

基　　金：河南省自然科学基金资助项目(202300410213);河南省教学改革资助项目(2021SJGLX351)。

摘　　要：旨在获得用于伪狂犬病病毒(pseudorabies virus,PRV)实验室检测和临床诊断所需的PRV gE单克隆抗体,用灭活的PRV变异株PRV HN1201免疫BALB/c小鼠,采用细胞融合技术将免疫小鼠脾细胞与SP2/0细胞融合,经免疫过氧化物酶单层细胞试验(immune peroxidase monolayer assay,IPMA)和免疫印迹试验(Western blot)筛选,获得2株分泌PRV gE抗体的杂交瘤细胞(1-F11-3、3-E11-3),抗体亚类分别为IgG2a和IgM,轻链均为Kappa链。间接免疫荧光试验(IFA)检测结果显示单克隆抗体可与不同PRV毒株反应,而不与PRV Bartha K61反应;Western blot、IPMA、IFA试验结果显示2株单克隆抗体均能特异性识别PRV gE蛋白,与其他病毒无交叉反应。Western blot试验鉴定2株抗体的抗原识别序列分别为^(64)GDDRRAGFGSALASLR^(79)和^(156)PPEVPRLRRGPPIVTPERWS^(175)。腹水纯化后的1-F11-3、3-E11-3抗体用于Western blot检测的最高稀释比分别为1∶14000和1∶12000,1-F11-3的IFA效价为2^(-14),3-E11-3不可用于IFA检测。结果表明,本研究制备的PRV gE抗体特异性强、灵敏度高,为建立快速、高效的PRV免疫学检测方法奠定了基础。The aim of this research is to obtain monoclonal antibodies against PRV gE for laboratory detection and clinical diagnosis of pseudorabies virus(PRV),BALB/c mice were immunized with inactivated pseudorabies virus variant PRV HN1201,splenic cells of immunized mice were fused with SP2/0 cells by cell fusion technique,two hybridoma cells that could secrete PRV gE monoclonal antibody:(1-F11-3,3-E11-3)d were screened by immune peroxidase monolayer assay(IPMA)and Western blot.The subclasses of antibodies were IgG2a and IgM respectively,and the light chains were Kappa chains.Indirect immunofluorescence assay(IFA)results showed that the monoclonal antibodies could react with different strains of PRV,but it did not react with PRV Bartha K61.Western blot,IPMA and IFA tests results showed that the two monoclonal antibodies could specifically recognize PRV gE protein,and they had no cross-reaction with other viruses.The antigen recognition sequences were^(64)GDDRRAGFGSALASLR^(79)and^(156)PPEVPRLRRGPPIVTPERWS^(175) respectively.The highest dilution ratios of 1-F11-3 and 3-E11-3 antibodies purified from ascites for Western blot detection were 1∶14000and 1∶12000,respectively,the IFA titer of 1-F11-3 was 2^(-14),3-E11-3cannot be used in IFA tests.PRV gE antibody prepared in this study has strong specificity and high sensitivity,laying a foundation for the establishment of rapid and efficient PRV immunological detection technology.

关 键 词：PRV HN1201 gE蛋白 单克隆抗体 IGG2A IGM 

分 类 号：S852.65[农业科学—基础兽医学]