猪细小病毒4型单克隆抗体制备及抗原表位鉴定  

Preparation and epitope identification of monoclonal antibodies against porcine parvovirus type 4

作　　者：柴书军[1] 杨苏珍[1] 刘运超[1] 冯华[1] 魏蔷[1] 王方雨[1] 金前跃[1] 张改平[1,2] CHAI Shujun;YANG Suzhen;LIU Yunchao;FENG Hua;WEI Qiang;WANG Fangyu;JIN Qianyue;ZHANG Gaiping(Henan Province Key Laboratory of Animal Immunology,Henan Academy of Agricultural Sciences,Zhengzhou 450002,China;Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses,Yangzhou University,Yangzhou,225009,China)

机构地区：[1]河南省农业科学院动物免疫学重点实验室,河南郑州450002 [2]江苏高校动物重要疫病与人兽共患病防控协同创新中心,扬州大学,江苏扬州225009

出　　处：《畜牧与兽医》2024年第8期77-84,共8页Animal Husbandry & Veterinary Medicine

基　　金：国家重点研发计划项目(2017YDF0501103);河南省农业科学院自主创新项目(2023ZC085,2023ZC087,2024ZC099)。

摘　　要：为了制备猪细小病毒(PPV)4型Cap蛋白单克隆抗体(mAb)并鉴定其抗原表位,试验采用原核表达的Cap重组蛋白免疫BALB/c小鼠,将其脾细胞与SP2/0细胞进行融合,通过酶联免疫吸附测定(ELISA)、血凝抑制(HI)和免疫过氧化物酶单层细胞试验(IPMA)筛选阳性克隆,最终获得2E7、4D6和5C9这3株特异性杂交瘤细胞株;然后用mAb对Cap蛋白多肽进行ELISA检测,鉴定分析其抗原表位。结果显示:4D6和5C9 mAb识别表位为线性表位,分别为387RRQDN^(391)和578QRKE^(581),而2E7 mAb识别的表位为构象表位;通过对Cap蛋白3D结构定位分析,387RRQDN^(391)和578QRKE^(581)抗原表位位于蛋白表面,且在PPV4亚型中高度保守,而与猪瘟病毒(CSFV)、猪伪狂犬病病毒(PRV)、猪圆环病毒2型(PCV-2)、猪繁殖与呼吸综合征病毒(PRRSV)、猪乙型脑炎病毒(JEV)和猪博卡病毒(PBoV)基因序列同源性低。本研究为PPV4诊断试剂的开发和新型疫苗的研制提供了参考。In order to develop monoclonal antibodies(mAb)against the Cap protein of porcine parvovirus(PPV)type 4 and to identify its antigenic epitopes,a series of experiments were conducted.Firstly,the Cap recombinant protein was expressed in prokaryotic cells and used to immunize BALB/c mice.The spleen cells from the immunized mice were then fused with SP2/0 myeloma cells.Next,positive clones were screened using ELISA,hemagglutination inhibition assay(HI),and immunoperoxidase monolayer assay(IPMA),resulting in the generation of three specific hybridoma cell lines,namely 2E7,4D6,and 5C9.Finally,the Cap protein peptides were subjected to ELISA using the mAb,and the antigenic epitopes were characterized and analyzed.The results revealed that the 4D6 and 5C9 mAb recognized linear epitopes,specifically at residues 387RRQDN^(391) and 578QRKE^(581),respectively.On the other hand,the epitope recognized by the 2E7 mAb was of a conformational nature.Based on the 3D structural analysis of the Cap protein,it was determined that the 387RRQDN391 and 578QRKE^(581) epitopes were located on the protein surface,highly conserved among PPV4 subtypes,and they displayed low homology with the gene se⁃quences of CSFV,PRV,PCV-2,PRRSV,JEV,and PBoV.The findings of this study laid a foundation for the development of diagnostic reagents for PPV4 and the design of novel vaccines.

关 键 词：猪细小病毒4型 单克隆抗体 抗原表位 

分 类 号：S852.6[农业科学—基础兽医学]