Preparation and interaction mechanism analysis of single‑chain fragment variables against phenylethanolamine A  

作　　者：Long Li Ren Hou Huaming Li Shiyun Han Jixiang Liang Yu Si Dapeng Peng 

机构地区：[1]State Key Laboratory of Agricultural Microbiology,National Reference Laboratory of Veterinary Drug Residues(HZAU)and MOA Key Laboratory for Detection of Veterinary Drug Residues,Huazhong Agricultural University,Wuhan 430070,PR China [2]Key Laboratory of Preventive Veterinary Medicine in Hubei Province,The Cooperative Innovation Center for Sustainable Pig Production,Wuhan 430070,China [3]Hubei Topgene Biotechnology Co.,Ltd.Wuhan Branch,Wuhan,Hubei 430070,China [4]Shenzhen Institute of Nutrition and Health,Huazhong Agricultural University,Shenzhen,Guangdong 518000,China [5]Shenzhen Branch,Guangdong Laboratory for Lingnan Modern Agriculture,Agricultural Genomics Institute at Shenzhen,Chinese Academy of Agricultural Sciences,Shenzhen,Guangdong 518000,China

出　　处：《Animal Diseases》2024年第2期127-137,共11页动物疾病（英文）

基　　金：the National Natural Science Foundation of China(32072920);the Fundamental Research Funds for the Central Universities(2662022DKPY007);the HZAU-AGIS Cooperation Fund(SZYJY2022024).

摘　　要：This is the first report on the screening,expression,and recognition mechanism analysis of single-chain fragment variable(scFv)against phenylethanolamine A(PEAA),a newly emergedβ-adrenergic agonist illegally used as a feed additive for growth promotion.The PEAA-specific scFv scFv,called scFv-32,was screened from hybridoma cell lines by phage display and was found to be optimally expressed in the E.coli system.The ic-ELISA results revealed an IC_(50)value of 10.34μg/L for scFv-32 and no cross-reactivity with otherβ-adrenergic agonists.Homology modeling and molecular docking revealed the key binding sites VAL178,TYP228,and ASP229.One hydrogen bond,two pisigma bonds,and one pi-pi bond maintain the formation of the antibody‒drug complex.Alanine scanning mutagenesis of the three predicted key binding sites showed that the mutants completely lost their recognition activity,which confirmed the accuracy of the theoretical analysis.These results are valuable for the preparation of scFvs and the analysis of the molecular recognition mechanism of antigen-antibodies.

关 键 词：Phenylethanolamine A SCFV Recognition mechanism Homology modeling Molecular docking 

分 类 号：S85[农业科学—兽医学]