伪狂犬病病毒VHS蛋白单克隆抗体的制备及其抗原表位鉴定  

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

作　　者：赵桐 潘梦娇 刘克森 白娟[1] 马梓承 刘星[1] ZHAO Tong;PAN Mengjiao;LIU Kesen;BAI Juan;MA Zicheng;LIU Xing(College of Veterinary Medicine,Nanjing Agriculture University/Key Laboratory of Animal Bacteriology,Ministry of Agriculture and Rural Affairs,Nanjing 210095,China)

机构地区：[1]南京农业大学动物医学院/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》2025年第5期99-106,共8页Animal Husbandry & Veterinary Medicine

基　　金：江苏省杰出青年基金项目(BK20230039,BK20241567);江苏省农业科技自主创新资金项目(CX(23)3073);国家自然基金面上项目(32272985,32402870)。

摘　　要：为制备伪狂犬病病毒(PRV)VHS蛋白单克隆抗体,通过原核表达系统将目的基因克隆至原核表达载体pET-32a(+),获得pET-32a-VHS重组质粒,成功制备了PRV VHS重组蛋白,将纯化后的蛋白作为免疫原接种BALB/c雌鼠,经过细胞融合、间接ELISA筛选和3轮亚克隆获得了2株PRV VHS蛋白的单克隆抗体,分别命名为1H8和4C3。使用间接ELISA方法检测,2株VHS单抗细胞均能稳定分泌抗体,且腹水抗体效价均达到1∶409 600。1H8和4C3的轻链均属于Kappa型,重链皆为IgG1亚类。Western blot和间接免疫荧光试验(IFA)显示,2株杂交瘤细胞株分泌的单抗均能与PRV病毒蛋白发生特异性反应。通过VHS蛋白的截短表达和Western blot鉴定出1H8单抗可以识别抗原表位^(241)VAPEDVKLKY^(250),单抗4C3可以识别抗原表位^(103)RADGDGAADAPP^(114)。本研究制备出2株VHS的单克隆抗体,鉴定出2个新的抗原表位,为后续研究PRV VHS蛋白功能及其在病毒感染和免疫应答中的作用提供了重要依据。This study was to prepare monoclonal antibody against Pseudorabies virus(PRV) VHS protein.The target gene was cloned into the prokaryotic expression vector pET-32a(+) through the prokaryotic expression system,pET-32a-VHS recombinant plasmids were obtained,and PRV VHS recombinant proteins were successfully prepared.The proteins were purified and inoculated into BALB/c female mice as immunogens.After cell fusion,indirect ELISA screening and 3 rounds of subcloning,two monoclonal antibodies to the PRV VHS proteins were obtained,and were named 1H8 and 4C3,respectively.The indirect ELISA assay showed that both VHS monoclonal antibody strains could secrete antibody steadily,and the titer of the ascites antibody reached 1∶409 600.The light chains of 1H8 and 4C3 belonged to the Kappa type,and the heavy chains belonged to the IgG1 subclass.Western blot and the indirect immunofluorescence assay(IFA) showed that the monoclonal antibodies secreted by the two hybridoma cell lines could react specifically with the PRV virus protein.According to the truncated expression of the VHS protein and Western blot analysis,the 1H8 monoclonal antibody could recognize epitope ^(241)VAPEDVKLKY^(250),and the 4C3 monoclonal antibody could recognize epitope ^(103)RADGDGAADAPP^(114).In summary,two monoclonal antibodies to VHS were prepared and two new cell epitopes were identified here.This provided important material support for further research on the function of PRV VHS proteins and their role in viral infection and immune response.

关 键 词：伪狂犬病病毒 VHS蛋白 单克隆抗体 抗原表位 

分 类 号：S852[农业科学—基础兽医学]