鸡源EphrinA2基因的克隆表达及其单克隆抗体研制  

Cloning and Expression of Chicken EphrinA2 Gene and Generation of Its Monoclonal Antibody

作　　者：相汝苹 姚晓慧 谢泉[1,2,3,4] 万志敏 邵红霞 秦爱建[1,2,3,4] 叶建强 李拓凡[1,2,3,4] XIANG Ruping;YAO Xiaohui;XIE Quan;WAN Zhimin;SHAO Hongxia;QIN Aijian;YE Jianqiang;LI Tuofan(Ministry of Education Key Laboratory for Avian Preventive Medicine,Key Laboratory of Jiangsu Preventive Veterinary Medicine,Yangzhou University,Yangzhou,Jiangsu 225009;Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses,Yangzhou,Jiangsu 225009;Institutes of Agricultural Science and Technology Development,Yangzhou University,Yangzhou,Jiangsu 225009;Joint International Research Laboratory of Agriculture and Agri-Product Safety,Ministry of Education,Yangzhou University,Yangzhou,Jiangsu 225009)

机构地区：[1]扬州大学兽医学院,禽类预防医学教育部重点实验室,江苏省动物预防医学重点实验室,江苏扬州225009 [2]江苏高校动物重要疫病与人兽共患病防控协同创新中心,江苏扬州225009 [3]扬州大学农业科技发展研究院(国际联合实验室),江苏扬州225009 [4]教育部农业与农产品安全国际合作联合实验室,江苏扬州225009

出　　处：《中国家禽》2023年第9期25-30,共6页China Poultry

基　　金：江苏省自然科学基金青年项目(BK20220579);中国博士后科学基金面上资助项目(2020M681747);国家自然科学面上基金项目(31972661)。

摘　　要：研究旨在研制抗鸡EphrinA2蛋白的特异性单克隆抗体。试验用PCR和同源重组技术构建了鸡EphrinA2真核及原核表达载体,以纯化的EphrinA2原核表达产物免疫小鼠,将小鼠脾细胞与骨髓瘤细胞SP2/0融合,用间接ELISA方法筛选出阳性细胞克隆;用SDS-PAGE、间接免疫荧光试验和Western blot验证表达产物及单抗的特性。结果显示:原核表达的重组蛋白His-EphrinA2-RBD主要位于细菌包涵体中;获得2株能稳定分泌抗EphrinA2蛋白特异性抗体的杂交瘤细胞株,分别命名为4H6和1G9;单克隆抗体4H6不仅能特异性识别真核表达载体pcDNA3.1-EphrinA2在DF-1细胞中表达的EphrinA2蛋白,而且能有效识别LMH和DF-1细胞中内源性的EphrinA2蛋白。结果表明,单克隆抗体4H6与鸡EphrinA2蛋白反应性和特异性均良好,能够应用于鸡EphrinA2相关基础研究。The purpose of this study was to develop specific monoclonal antibodies against chicken EphrinA2 protein.The eukaryotic and prokaryotic expression vectors of chicken EphrinA2 gene were constructed by PCR and homologous recombination techniques.Mice were immunized with purified EphrinA2 prokaryotic expression product.The mouse spleen cells were fused with SP2/0 myeloma cells,and the positive cell clones were screened by indirect ELISA.The characteristics of the expressed product and monoclonal antibody were verified by SDS-PAGE,indirect immunofluorescence assay and Western blot.The results showed that the recombinant prokaryotic protein His-EphrinA2-RBD was mainly expressed in pellet.Two hybridoma cell lines(designated as,4H6 and 1G9,respectively)which could stably secrete specific antibodies against chicken EphrinA2 were screened by ELISA.Indirect immunofluorescence assay and Western blot showed that monoclonal antibody 4H6 could not only specifically recognize the overexpressed chEphrinA2 protein in DF-1 cells transfected with eukaryotic expression vector pcDNA3.1-EphrinA2,but also effectively identify the endogenous EphrinA2 protein in LMH and DF-1 cells.The results showed that monoclonal antibody 4H6 possesses good reactivity and specificity with chicken EphrinA2 protein,and could be applied in chicken EphrinA2 related basic researches.

关 键 词：鸡源EphrinA2 蛋白表达 单克隆抗体 间接免疫荧光试验 Western blot 

分 类 号：S855.3[农业科学—临床兽医学]