抗AFB_1单域重链抗体文库淘选方法的研究  

Research on Panning Methods of Single-Domain Heavy-Chain Antibody Fragments for Aflatoxin B_1 from Non-Immune Library

作　　者：刘夏[1] 许杨[1,2] 涂追[1] 何庆华[2] 

机构地区：[1]食品科学与技术国家重点实验室,南昌大学,江西南昌330047 [2]南昌大学中德联合研究院,江西南昌330047

出　　处：《食品与生物技术学报》2011年第6期950-955,共6页Journal of Food Science and Biotechnology

基　　金：食品科学与技术国家重点实验室目标导向资助项目(SKLF-MB-201002);国家中小企业创新基金项目(09C26213604449)

摘　　要：比较两种从噬菌体展示文库中淘选抗黄曲霉毒素B1单域重链抗体的方法,并为淘选针对小分子物质的单域重链抗体提供参考。采用固相淘选技术,以黄曲霉毒素B1人工抗原为靶分子,淘选天然单域重链抗体库,分别采用Gly-HCl法和AFB1竞争法洗脱结合噬菌体,对洗脱物进行滴度测定,以回收率和富集度为指标,对两种洗脱方法进行比较。采用phage-ELISA法鉴定噬菌体克隆,并对阳性克隆进行交叉反应分析以及序列测定。滴度测定结果显示,Gly-HCl法回收率比竞争法高5～10倍。分别随机挑取20个单克隆进行phage-ELISA鉴定,其中,Gly-HCl洗脱法未获得阳性克隆,AFB1竞争洗脱法得到8个阳性克隆。序列测定结果显示,这8个克隆均编码单域重链抗体。The objective of this study was tocompare the efficiency of two elute strategies in biopanning of single-domain heavy-chain antibody（HCAbs） phage display library against low molecular hapten,Aflatoxin B1（AFB1）.For this,the artificial antigen AFB1-BSA and AFB1-OVA were used as ligand to screen the binding phages from an Alpaca non-immune library by antigen-coated solid phase selection.The binding phages were directly eluted with Gly-HCl or competitively eluted with AFB1 solution.The eluants were tittered separately.It was found that the recovery rate of Gly-HCl is 5 to 10 times higher than that of competition method.Twenty colons randomly picked from each eluant were characterized by phage-ELISA.Eight positive colons were isolated from the eluant by the competition method;however,no positive colons were isolated by Gly-HCl method.The sequencing data show that all the colons are encoding single-domain HCAbs.

关 键 词：黄曲霉毒素B1 单域重链抗体 噬菌体展示 天然文库 

分 类 号：R392.1[医药卫生—免疫学] Q511[医药卫生—基础医学]