伪狂犬病病毒VP22蛋白单克隆抗体制备及其抗原表位鉴定  

Monoclonal antibodies preparation and antigenic epitope identification of VP22 protein of pseudorabies virus

作　　者：潘梦娇 刘德鹏 郭磊[1] 赵桐 白娟[1] 姜平[1] 刘星[1] PAN Mengjiao;LIU Depeng;GUO Lei;ZHAO Tong;BAI Juan;JIANG Ping;LIU Xing(Nanjing Agriculture University/Key Laboratory of Animal Bacteriology,Ministry of Agriculture and Rural Affairs,Nanjing 210095,China)

机构地区：[1]南京农业大学/农业农村部动物细菌学重点实验室,江苏南京210095

出　　处：《畜牧与兽医》2024年第5期82-89,共8页Animal Husbandry & Veterinary Medicine

基　　金：国家自然科学基金项目(32272985);财政部和农业农村部国家现代农业产业技术体系专项(CARS-35)。

摘　　要：为制备伪狂犬病病毒(PRV)VP22蛋白单克隆抗体,将目的基因克隆至原核表达载体pET-28a(+),构建pET-28a-VP22原核表达质粒,并利用大肠杆菌表达系统,获得重组VP22蛋白。将纯化后的蛋白免疫6~8周龄BALB/c雌鼠,通过杂交瘤细胞融合技术及间接ELISA方法筛选,经3次亚克隆后获得4株PRV VP22蛋白的单克隆抗体1D6、1D9、1B12和2C10。间接ELISA检测4株杂交瘤细胞传至15代均能稳定分泌抗体,且细胞上清液抗体效价均为1∶6400。经亚型鉴定,4株单克隆抗体的重链均是IgG1亚类,轻链皆属于κ型。Western blot和间接免疫荧光试验(IFA)结果表明,制备的单克隆抗体均可与PRV发生特异性反应,并鉴定出2个抗原表位;Western blot和共聚焦试验结果表明,VP22蛋白是病毒早期蛋白,且早期存在于细胞质,晚期移位至细胞核并在核中积累。本研究制备的单克隆抗体将为后续探索PRV VP22蛋白的功能提供材料支撑。In order to prepare monoclonal antibodies against VP22 protein of pseudorabies virus(PRV),the target gene was cloned into the prokaryotic expression vector pET-28a(+),and the pET-28a-VP22 prokaryotic expression plasmid was constructed.The recombinant VP22 protein was obtained by using the E.coli expression system.Then,6-8 week-old female BALB/c mice were immunized with the puri-fied protein,and four monoclonal antibodies 1D6,1D9,1B12 and 2C10 to the PRV VP22 protein were obtained after three rounds of sub-cloning by means of hybridoma cell fusion and indirect ELISA.Indirect ELISA detected that 4 hybridoma cells were able to stably secrete an-tibodies till the 15th generation,and the antibody titer of the cell supernatant was 1:6400 According to the subtype identification,the heavy chains of the four monoclonal antibodies belonged to IgG1,and the light chains were theκtype.The results of Western blot and indirect im-munofluorescence assay(IFA)showed that all the monoclonal antibodies prepared in this study could react specifically with PRV and identify two epitopes.Meanwhile,the results of Western blot and confocal microscopy showed that the VP22 protein was a viral early protein,early presenting in the cytoplasm and late infection translocating to and accumulating in the nucleus.In this study,the monoclonal antibodies against the VP22 protein of PRV provided technical support for further exploration of the biological function and virulence mechanism of the protein.

关 键 词：伪狂犬病病毒 VP22蛋白 单克隆抗体 抗原表位 

分 类 号：S852[农业科学—基础兽医学]