摘要
黄曲霉毒素B1(aflatoxinB1,AFB1)是一种毒性强、污染广的真菌毒素,建立高效、准确、快速的AFB1检测方法具有重要意义。利用噬菌粒-辅助噬菌体展示系统构建单链抗体(singlechainvariablefragment,scFv)文库,应用“淘选-洗脱”的策略,是筛选高亲和配体的常用方法之一。同时结合同源建模和分子对接等计算机辅助手段,分析得到抗体与抗原的结合位点与关键氨基酸,为在基因水平改造抗体提供基础。从AFB1-BSA免疫小鼠的脾细胞内扩增重链可变区和轻链可变区,将组合成的scFv片段插入噬菌粒pCANTAB5e中,构建了噬菌体展示单链抗体文库,以不同浓度的AFB1-OVA作为抗原,从文库中筛选到一株亲和力较好的抗AFB1单链抗体scFv,其亲和常数为8×105L/mol;根据同源建模和分子对接发现,与抗原AFB1结合时,scFv中Tyr33、Ser52和Tyr102起关键作用,分别以π-π共轭键、氢键和范德华力与AFB1结合。
Aflatoxin B1 (AFB1) is a mycotoxin which has highly toxic and highly polluted.It is of great significance for AFB1 to establish an efficient,accurate and rapid detection method.Phagemid/helper phage system is one of the most common used system for single-chain antibody fragment(scFv) libraries construction.The “panning-elution” strategy is an effective method for screening ligands with high affinity.Combined with homology modeling and molecular docking,the key amino acid binding sites between antibody and antigen were analyzed to provide the basis for genetically modifying antibodies.In this study,the heavy chain variable regions and light chain variable regions were amplified from the spleen cells of AFB1-BSA immunized mice.Then assembled scFv fragment was inserted into the phagemid pCANTAB5e to construct a phage display single-chain antibody library.Using different concentrations of AFB1-OVA as coating antigens,anti-AFB1 scFv was isolated from this library,which affinity constant is 8×10 5L/mol.According to homology modeling and molecular docking,Tyr33,Ser52,and Tyr102 play a key role in binding with AFB1 under π-π conjugated bonds,hydrogen bonds,and van der Waals forces,respectively.
作者
庞倩
陈晶
王小红
王佳
PANG Qian;CHEN Jing;WANG Xiao-hong;WANG Jia(College of Food Science and technology,Huazhong Agricultural University,Wuhan 430070,China)
出处
《中国生物工程杂志》
CAS
CSCD
北大核心
2018年第12期41-48,共8页
China Biotechnology
基金
国家自然科学基金(31601539)
湖北省自然科学基金(2016CFB143)资助项目
