猪传染性胃肠炎病毒重组N蛋白单克隆抗体的制备及鉴定  

Development and identification of monoclonal antibodies against recombinant nucleoprotein of transmissible gastroenteritis virus

作　　者：黄小波[1] 李娜[1,2] 文心田[1] 曹三杰[1] 余慧[1] 杨恒[1] 秦学远[1,3] 尹杰[1] 

机构地区：[1]四川农业大学动物医学院动物传染病与基因芯片实验室四川省动物疫病与人类健康重点实验室,四川雅安625014 [2]西南交通大学机械工程学院,四川成都610000 [3]四川省动物疫病预防控制中心,四川成都610000

出　　处：《中国兽医科学》2009年第11期994-998,共5页Chinese Veterinary Science

基　　金：教育部"长江学者和创新团队发展计划"创新团队项目(IRT0848);四川省教育厅资助项目(08zb075)

摘　　要：以纯化的猪传染性胃肠炎病毒(TGEV)重组N蛋白免疫BALB/c小鼠,取免疫小鼠的脾细胞与SP2/0骨髓瘤细胞进行融合,通过间接ELISA法对融合细胞进行筛选,采用有限稀释法对阳性细胞进行4次亚克隆,获得了3株能稳定分泌N蛋白单克隆抗体的杂交瘤细胞,分别命名为JY(4)-1、JY(4)-2、JY(4)-3。分别采用体外培养法、诱导腹水法和实体瘤法制备单克隆抗体,并用间接ELISA法检测其效价,结果显示,这3株杂交瘤细胞的细胞培养上清、小鼠腹水和血清的单抗效价均分别为1∶512、1∶105、1∶105,其中以诱导腹水法制备的单抗产量和效价最佳。间接免疫荧光试验结果显示,这3株单抗均能与TGEV感染的ST细胞产生特异性免疫荧光,而不与猪细小病毒、猪生殖与呼吸综合征病毒、猪轮状病毒等发生交叉反应;经鉴定,3株单抗的亚类均为IgG2a,这3株杂交瘤细胞具有良好的遗传稳定性。The monoclonal antibodies（McAbs） against transmissible gastroenteritis virus（TGEV） N protein were developed by fusing SP2/0 cells with splenocytes of BALB/c mice immunized with the purified recombinant N protein of TGEV.Three hybridoma cells,JY（4）-1,JY（4）-2 and JY（4）-3,were exa-mined by an indirect ELISA and subcloned 4 times by the limited dilution.The titers of 3 hybridoma cells＇culture supernatants,murine ascites and sera were 1∶512,1∶105 or 1∶105 in the indirect ELISA,respectively.The inducing ascites method was the best for producing monoclonal antibodies.The 3 McAbs were in strong reactivity with the ST cells infected with TGEV and no cross-reaction was observed between the McAbs and porcine parvovirus,or porcine reproductive and respiratory syndrome virus or porcine rotavirus in the indirect immunofluorescence assay.The three McAbs,being classified into IgG2a,were highly stable.

关 键 词：猪传染性胃肠炎病毒 重组N蛋白 单克隆抗体 杂交瘤 

分 类 号：S852.659.6[农业科学—基础兽医学]