具有中和活性的鸭肠炎病毒单克隆抗体的制备与鉴定  

Preparation and identification of neutralizing monoclonal antibody against duck enteritis virus

作　　者：王怡平 张忠伟 蒲路莎 赵丽丽[1] 陈洪岩[1] WANG Yi-ping;ZHANG Zhong-wei;PU Lu-sha;ZHAO Li-li;CHEN Hong-yan(State Key Laboratory of Veterinary Biotechnology/Heilongjiang Provincial Key Laboratory of Laboratory Animal and Comparative Medicine/Harbin Veterinary Research Institute,Chinese Academy of Agricultural Sciences,Harbin 150069,China;Animal Husbandry Administration of Ningjiang District of Songyuan Prefecture Jilin Province,138001China)

机构地区：[1]中国农业科学院哈尔滨兽医研究所/兽医生物技术国家重点实验室黑龙江省实验动物与比较医学重点实验室,黑龙江哈尔滨150069 [2]吉林省松原市宁江区畜牧业管理局,吉林松原138001

出　　处：《中国兽医科学》2019年第8期977-982,共6页Chinese Veterinary Science

基　　金：黑龙江省青年科学基金项目(QC2018033);黑龙江省自然科学基金重点项目(ZD2016006);中央级公益性科研院所基本科研业务费专项(1610302018013、1610302019013)

摘　　要：为制备抗鸭肠炎病毒(DEV)的单克隆抗体(MAb)并对其生物学活性进行鉴定,本研究采用蔗糖密度梯度离心法提纯DEV鸭胚成纤维细胞适应毒,以此作为抗原免疫BALB/c小鼠,随后取小鼠脾细胞与SP2/0骨髓瘤细胞融合,有限稀释法克隆杂交瘤细胞株,经间接ELISA筛选,获得了1株稳定分泌抗DEV MAb的杂交瘤细胞株2E3。鉴定结果显示,该MAb为Ig M亚类,ELISA效价可达到1∶10~5以上;间接免疫荧光试验(IFA)中MAb可与细胞中增殖的DEV结合,Western-blot试验中MAb不与变性病毒蛋白作用,表明该MAb可能是构象表位的单抗;病毒中和试验表明,该MAb具有较强的病毒中和活性。上述结果表明,本试验获得的一株具有DEV中和活性的MAb杂交瘤细胞株为建立DEV的检测方法和DEV的相关研究提供了材料支持。For preparing the duck enteritis virus(DEV) monoclonal antibody(MAb) and identifying its biological activities,in this study, we purified DEV from duck embryo fibroblast cells by sucrose density gradient centrifugation.Then used the purified DEV as antigen to immune BALB/c mice,which spleen cells were fused with SP2/0 myeloma cells.After that,an limited dilution method for cloning the hybridoma cells were performed.After screening the MAb by indirect ELISA,a hybridoma cell strain 2E3 was obtained,and the MAb was showed to be capable of neutralizing the DEV.The results showed that the MAb was Ig M subclass and the ELISA titer of the MAb could reach over 1 ∶ 10~5;In the indirect immunofluorescence(IFA)test,the MAb could bind to DEV in the cultured duck embryo fibroblast cells,while in the Western Blot test,the MAb could not react with degenerated virus proteins,it indicates that MAb should be aimed at a conformational epitope;The results of neutralization test indicated that the MAb could protect the cultured duck embryo fibroblast cells from the DEV infection.The acquired one neutralizing monoclonal antibody provided the basic material for further development of detection methods and other related researches about DEV.

关 键 词：鸭肠炎病毒 单克隆抗体 中和活性 

分 类 号：S852.659.1[农业科学—基础兽医学]