猪伪狂犬病病毒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

作　　者：王瑞宁[1,2] 王勋[3,4] 李鸽 李青梅 李存法[1] 杨苏珍[2] 郭军庆[2] WANG Ruining;WANG Xun;LI Ge;LI Qingmei;LI Cunfa;YANG Suzhen;GUO Junqing(College of Veterinary Medicine,Henan Institute of Animal Husbandry and Economics,Zhengzhou 450046,China;Institute for Animal Health,Henan Academy of Agricultural Sciences,Zhengzhou 450002,China;College of Food and Drugs,Luoyang Polytechnic,Luoyang 471023,China;Animal Diseases and Public Health Engineering Research Center of Henan Province,Luoyang 471023,China;College of Veterinary Medicine,Northwest Agriculture and Forestry University,Yangling 712100,China)

机构地区：[1]河南牧业经济学院动物医药学院,河南郑州450046 [2]河南省农业科学院动物疫病防控研究所,河南郑州450002 [3]洛阳职业技术学院食品与药品学院,河南洛阳471023 [4]河南省动物疫病与公共卫生工程研究中心,河南洛阳471023 [5]西北农林科技大学动物医学院,陕西杨凌712100

出　　处：《河南农业科学》2024年第12期167-173,共7页Journal of Henan Agricultural Sciences

基　　金：河南省重大科技专项(221100110600-03);河南省科技攻关项目(232102520012,242102111031);河南省农业科学院科技创新团队项目(2023TD03)。

摘　　要：为了开发检测猪伪狂犬病病毒(PRV)的免疫诊断试剂,使用HEK293F细胞表达的gE重组蛋白免疫BALB/c小鼠。将免疫小鼠的脾细胞与SP2/0细胞融合,应用酶联免疫吸附试验(ELISA)和免疫过氧化物酶单层细胞试验(IPMA)方法筛选阳性杂交瘤细胞株,以期获得对PRV反应性良好的抗gE重组蛋白单克隆抗体。结果显示,获得2株(2B5和8F7)稳定分泌抗PRV gE蛋白单克隆抗体的杂交瘤细胞株,其腹水单克隆抗体的ELISA效价均为1∶1 000000;单克隆抗体亚型鉴定结果表明,2株单克隆抗体的重链均为IgG1亚型,轻链均为Kappa链。单克隆抗体特异性测定结果显示,2株单克隆抗体仅与PRV反应,不与其他常见病毒发生反应;SDS-PAGE结果显示,纯化后的单克隆抗体均在约50 ku和25 ku处有纯度较高的特异性条带;间接免疫荧光试验(IFA)结果表明,制备的2株单克隆抗体与转染gE质粒的293T细胞发生特异性反应。根据Western blot结果,筛选获得的2株单克隆抗体均在120 ku附近出现特异条带,表明这2株单克隆抗体均能特异性识别PRV。综上,成功制备了2株特异性强、效价高的抗gE蛋白单克隆抗体,为后期PRV诊断试剂盒的制备提供重要的生物材料。In order to develop an immunodiagnostic reagent for porcine pseudorabies virus(PRV),BALB/c mice were immunized with the recombinant gE protein expressed in HEK293F cells.The spleen cells of immunized mice and SP2/0 myeloma cells were fused to generate hybridoma cells.Indirect ELISA and immunoperoxidase monolayer assay(IPMA)were used to screen positive hybridoma cells so as to prepare and identify monoclonal antibodies against the gE protein of PRV.The outcomes demonstrated that two hybridoma cell strains,named 2B5 and 8F7,respectively,which steadily secreted monoclonal antibody against the gE protein were developed.The ELISA titers of ascites were all 1∶1000000.IgG1 was the heavy chain and Kappa was the light chain,according to the monoclonal antibody isotype assay.The specificity assay revealed that the two monoclonal antibodies only reacted with PRV and not with other viruses.SDS-PAGE results showed that the purified monoclonal antibodies had high-purity specific bands at about 50 ku and 25 ku.The two monoclonal antibody strains could react specifically with 293T cells transfected with gE plasmid according to indirect immunofluorescence assay(IFA)detection.Western blot showed that the specific protein band appearing in the culture supernatant of two hybridoma cells was about 120 ku,indicating that the two monoclonal antibodies could specifically recognize PRV.To summarize,this study successfully prepared two strains of monoclonal antibodies against gE protein,exhibiting excellent specificity and high titer,which provided crucial biological materials required for the subsequent development of diagnostic kits.

关 键 词：猪伪狂犬病病毒 gE蛋白 单克隆抗体 间接免疫荧光试验 

分 类 号：S816[农业科学—饲料科学]