鹅星状病毒2型单克隆抗体制备及其抗原表位鉴定  

Preparation of monoclonal antibodies against goose astrovirus type 2 and identification of antigen epitopes

作　　者：王雯 佟贺 杨兴淼 曹瑞兵[1] WANG Wen;TONG He;YANG Xingmiao;CAO Ruibing(College of Veterinary Medicine,Nanjing Agricultural University,Nanjing 210095,China)

机构地区：[1]南京农业大学动物医学院,江苏南京210095

出　　处：《南京农业大学学报》2025年第2期401-409,共9页Journal of Nanjing Agricultural University

基　　金：江苏现代农业(水禽)产业技术体系疾病防控创新团队项目(JATS[2023]324)。

摘　　要：[目的]本研究旨在制备抗鹅星状病毒(goose astrovirus,GAstV)单克隆抗体并鉴定其识别的抗原表位,为快速检测GAstV感染和研究P2蛋白的结构与功能奠定基础。[方法]通过原核表达系统表达并纯化GAstV-2 P2蛋白,用其免疫6周龄BALB/c雌鼠,利用杂交瘤技术,经过细胞筛选和亚克隆,获得3株能稳定分泌抗P2蛋白的单克隆抗体,利用Western blot(WB)和间接免疫荧光(IFA)鉴定单抗特异性,用获得的单抗对GAstV感染阴、阳性肾脏切片进行免疫组化检测,用间接ELISA测定单抗效价和亚型,同时构建P2蛋白的不同截短体来鉴定单抗识别抗原表位。[结果]本研究共获得3株能稳定分泌抗P2蛋白抗体的杂交瘤细胞,命名为1B10、3C6、5B1。单抗效价分别为1∶2048000、1∶512000、1∶256000,亚型鉴定结果显示,3株单抗的重链均为IgG2b,轻链均为Kappa型。WB和IFA结果显示,3株单抗均可以特异性结合鹅星状病毒。免疫组化结果显示,5B1和3C6可以应用于免疫组化的检测。抗原表位鉴定结果显示,1B10识别_(109)TSTTSTGGLITELRN_(123),3C6识别_(206)LTDPEEDDDPLS_(217),5B1识别_(96)LNPELETAVLRV_(107)。[结论]本研究成功制备能识别不同抗原表位的3株单克隆抗体,为快速检测GAstV感染和研究P2蛋白的结构与功能奠定物质基础。[Objectives]This study aimed to prepare monoclonal antibodies against goose astrovirus(GAstV)and identify the antigenic epitopes recognized by them,laying the foundation for rapid detection of GAstV infection and studying the structure and function of P2 protein.[Methods]GAstV-2 P2 protein was expressed and purified by prokaryotic expression system,and then used as an immunogen to immunize six-week-old BALB/c female mice.Three strains of monoclonal antibodies against P2 protein were obtained by hybridoma technology,cell screening and subcloning.Western blot and indirect immunofluorescence(IFA)were used to identify the specificity of monoclonal antibodies,immunohistochemical detection of GAstV infected positive and negative kidney sections was performed using the obtained monoclonal antibody,and the titer and subtype of monoclonal antibodies were determined by indirect ELISA.Simultaneously constructing different truncated versions of P2 protein to identify antigen epitopes recognized by monoclonal antibodies.[Results]This study obtained three hybridoma cells that could stably secrete anti P2 protein antibodies,named 1B10,3C6,and 5B1.The titers of monoclonal antibodies were 1∶2048000,1∶512000,and 1∶256000,respectively.The subtype identification results showed that the heavy chain of the three monoclonal antibodies was IgG2b,and the light chain was Kappa type.Western blot and IFA results showed that all three monoclonal antibodies could specifically bind to goose astrovirus.Immunohistochemical results showed that 5B1 and 3C6 could be used for immunohistochemical detection.The results of antigen epitope identification showed that 1B10 recognized _(109)TSTTSTGGLITELRN_(123),3C6 recognized _(206)LTDPEEDDDPLS_(217),and 5B1 recognized _(96)LNPELETAVLRV 107.[Conclusions]This study successfully prepared three monoclonal antibodies that recognize different antigenic epitopes,laying the foundation for rapid detection of GAstV infection and studying the structure and function of P2 protein.

关 键 词：鹅星状病毒 P2蛋白 原核表达 单克隆抗体 抗原表位 

分 类 号：S852.6[农业科学—基础兽医学]