齐帕特罗人工抗原合成及其多克隆抗体制备  

Synthesis of Zilpaterol Complete Antigen and Preparation of Polyclonal Antibody

作　　者：郭东光[1] 陈明艳 冯春花 王丰 卞爽丽 李文明 王凯露 吕薇薇 郑文珺 陈子怡 孙国鹏[1] 李雪华 田金河 李鹏[1] 朱艳平[1] 岳锋[1] 王选年[1] GUO Dong-guang;CHEN Ming-yan;FENG Chun-hua;WANG Feng;BIAN Shuang-li;LI Wen-ming;WANG Kai-lu;LV Wei-wei;ZHENG Wen-jun;CHEN Zi-yi;SUN Guo-peng;LI Xue-hua;TIAN Jin-he;LI Peng;ZHU Yan-ping;YUE Feng;WANG Xuan-nian(School of Life Science and Basic Medicine/Henan Engineering Laboratory for Molecular Diagnosis of Animal Diseases,Xinxiang University,Xinxiang 453000,China;School of Life Science,Zhengzhou University,Zhengzhou 450000,China;Xinxiang City Agricultural Comprehensive Administrative Law Enforcement Detachment,Xinxiang 453000,China)

机构地区：[1]新乡学院生命科学与基础医学学院/动物疫病分子诊断河南省工程实验室,河南新乡453000 [2]郑州大学生命科学技术学院,河南郑州450000 [3]新乡市农业综合行政执法支队,河南新乡453000

出　　处：《动物医学进展》2022年第5期6-12,共7页Progress In Veterinary Medicine

基　　金：河南省科技攻关项目(192102110066);国家重点研发计划子课题(2018YFC1602900);新乡市重大科技专项(ZD2020007);新乡市科技攻关项目(GG2019019);国家自然科学基金项目(32102632);新乡学院博士科研启动经费项目(1366020120)。

摘　　要：为制备齐帕特罗(ZIL)完全抗原,获得抗ZIL多克隆抗体(pAb),将ZIL与4-溴丁酸乙酯反应后,采用EDC/NHS方法使ZIL-丁酸盐中间体与牛血清白蛋白(BSA)和鸡卵清蛋白(OVA)进行偶联,分别用紫外全波长扫描(UV)、聚丙烯酰胺凝胶电泳(SDS-PAGE)进行鉴定后,通过免疫新西兰大白兔获得抗ZIL多抗血清,并对抗体效价、半数抑制浓度(IC_(50))和特异性进行鉴定。HPLC-MS检测结果显示,改造后的主产物分子质量为348.21 u,约占整个反应体系产物的92.23%,与ZIL-丁酸盐分子质量完全相符;SDS-PAGE电泳结果显示,BSA/OVA在电泳中的迁移速率明显大于偶联产物;UV结果显示,相比载体蛋白,偶联产物ZIL-BSA/OVA的特征吸收峰均发生了明显变化;ELISA效价检测结果表明,ZIL多抗血清效价在1∶6400以上,通过建立标准曲线测得IC_(50)分别为0.38 ng/mL和1.13 ng/mL,并且与沙丁胺醇(SAL)、特布他林(TBL)、溴布特罗(BRO)、西马特罗(CIM)、克仑特罗(CLB)、盐酸多巴胺(DH)、莱克多巴胺(RAC)、达氟沙星(DAN)、泰乐菌素(TYL)均不存在任何交叉反应。成功制备了ZIL完全抗原ZIL-BSA/OVA,获得了一种高灵敏度和高特异性的抗ZIL pAb,为后续建立ZIL免疫学快速检测方法奠定了基础。In order to prepare zilpaterol(ZIL)complete antigen and obtain anti-ZIL polyclonal antibody(pAb),ZIL was reacted with ethyl 4-bromobutyrate,and the intermediate of ZIL butyrate was coupled with bovine serum albumin(BSA)and chicken ovalbumin(OVA)by EDC/NHS method.Ultraviolet full-wavelength scanning(UV)and polyacrylamide gel electrophoresis(SDS-PAGE)were used to identify the complete antigen.Then the anti-ZIL pAb was obtained after immunization of New Zealand white rabbits.The antibody titer,half inhibitory concentration(IC_(50))and specificity of the antibody were identified.The results of HPLC-MS showed that the molecular weight of the modified product was 348.21,which was consistent with that of ZIL butyrate,it accounts for about 92.23%of the reaction system products.SDS-PAGE results showed that the migration rate of BSA/OVA was significantly higher than that of coupling product.UV results showed that,the characteristic absorption peaks of ZIL-BSA/OVA were obviously changed as compared with the carrier protein.The results of ELISA showed that the titer of ZIL pAb was above 1∶6400.The IC_(50) of ZIL pAb were 0.38 ng/mL and 1.13 ng/mL,respectively.There was no cross reaction with salbutamol(SAL),terbutaline(TBL),brobuterol(BRO),cimaterol(CIM),clenbuterol hydrochloride(CLB),dopamine hydrochloride(DH),ractopamine hydrochloride(RAC),dafloxacin(DAN)and tylomectin(TYL).In this study,the ZIL complete antigen ZIL-BSA/OVA was successfully prepared,and an anti-ZIL pAb with high sensitivity and specificity was obtained,which laid a research foundation for the subsequent establishment of a rapid immunological detection method for ZIL.

关 键 词：齐帕特罗 新型瘦肉精 半抗原 完全抗原 多克隆抗体 

分 类 号：S87[农业科学—畜牧兽医] S852.4