鸭呼肠孤病毒p18重组蛋白表达、纯化及其单克隆抗体制备  

Expression,Purification and Monoclonal Antibodies Preparation of Recombinat p18 Protein for Duck Reovirus

作　　者：黄聪 刘志艺 刘忠媛 黄远玲 李嘉 刘静宜 刘英楠 陈鸿军[1] 陈宗艳[1] HUANG Cong;LIU Zhiyi;LIU Zhongyuan;HUANG Yuaning;LI Jia;LIU Jingyi;LIU Yingnan;CHEN Hongjun;CHEN Zongyan(Shanghai Veterinary Research Institute,Chinese Academy of Agricultural Sciences,Shanghai 200241)

机构地区：[1]中国农业科学院上海兽医研究所,上海200241

出　　处：《中国家禽》2023年第12期53-58,共6页China Poultry

基　　金：“十四五”国家重点研发计划项目(2022YFD1801000)。

摘　　要：研究旨在制备DRV p18蛋白单克隆抗体。试验通过原核表达并纯化DRV p18重组蛋白,经BCA蛋白浓度测定后免疫BALB/c小鼠;通过细胞融合技术制备杂交瘤细胞,获得的p18单克隆抗体通过ELISA、IFA和Western blot试验进行鉴定,并将杂交瘤细胞腹腔注射小鼠获得单抗腹水,通过Western blot试验验证其特异性。结果显示:纯化的重组p18蛋白浓度为1.37 mg/mL,经过三轮亚克隆获得4株分泌单克隆抗体的杂交瘤细胞株1E3、2C4、4A2和4D4,亚型鉴定1E3和4D4重链为IgG1型、2C4重链为IgM型、4A2为IgG2b型,轻链均为κ型;4株单克隆抗体均能使感染DRV的BHK-21细胞发出特异性荧光,并在18 ku处存在特异性条带;4D4腹水ELISA效价为1∶409600,Western blot效价达1∶102400,IFA效价为1∶51200;4D4与DRV反应,而不与其他禽源常见病毒反应。研究成功制备了抗DRV p18蛋白的单克隆抗体,为进一步研探究p18蛋白的功能奠定基础。To prepare monoclonal antibody against duck reovirus(DRV)p18 protein,DRV p18 recombinant protein was pre-pared and the concentration was determined by BCA,which was used as immunogen to immunize BALB/c mice.Hybridoma cells were prepared by cell fusion technique to obtain monoclonal antibodies and p18 monoclonal antibodies were identified by ELISA,IFA and Western blot.Hybridoma cells were injected intraperitoneally into mice to obtain monoclonal antibody ascites,and the specificity was verified by Western blot test.The results showed that the concentration of purified recombinant p18 protein was 1.37 mg/mL.Four hybridoma cell lines(1E3,2C4,4A2 and 4D4)secreting monoclonal antibody were successfully obtained by three subclones.The subtypes were identified as IgG1 with 1E3 and 4D4 heavy chain,IgM with 2C4 heavy chain,IgG2b with 4A2,andκwith light chain.The four strains of monoclonal antibody could make BHK-21 cells infected with DRV emit specific fluores-cence and showed specific bands at 18 ku.The titer of 4D4 ascites was 1∶409600 by ELISA,1∶102400 by Western blot and 1∶51200 by IFA.4D4 could react with DRV,but not with other common avian viruses.It was suggested that monoclonal antibodies against DRV p18 protein were successfully prepared,which laid a foundation for further research on the function of p18 protein.

关 键 词：鸭呼肠孤病毒 p18蛋白 单克隆抗体 

分 类 号：S855.3[农业科学—临床兽医学]