基于CDR2和CDR3区随机突变筛选抗黄曲霉毒素B1单域重链抗体的研究

Screening of Anti-aflatoxin B1 Single Domain Heavy Chain Antibody Based on Random Mutation of CDR2 and CDR3 Regions

基于抗原-抗体特异性反应的免疫学方法是黄曲霉毒素B1的常用检测方法。为制备针对AFB1的抗体,综合参考已报道的噬菌体文库筛选的抗AFB1单域重链抗体(variable domain of heavy chain of heavy chain antibody,VHH)序列,合成一条经密码子优化[适于大肠杆菌(Escherichia coli,E.coli)表达]的高同源性序列。在抗AFB1 VHH的CDR2和CDR3区引入部分随机突变,构建噬菌体抗体库。采用phage-ELISA技术,以AFB1O-OVA为包被抗原,淘选单域重链抗体库,经过4轮筛选,获得15株能与AFB1特异性结合的阳性克隆。以结合力最高的1株克隆为材料,扩增相应的VHH基因,构建表达质粒p ET-22b-VHH。在E.coli BL21(DE3)中表达VHH,经间接竞争ELISA分析,获得的抗AFB1 VHH的灵敏度约为10μg/m L。

Immunoassay,based on antigen-antibody reaction,was one of the common methods employed to detect AFB1. In order to prepare antibody against AFB1,A codon-optimized DNA sequence（ suitable for expression in Escherichia coli（ E. coli）） of high homology was synthesized according to comprehensive screening of a large number of anti-AFB1 VHH sequences obtained through phage display library. A phage antibody library was constructed by introducing random mutations in part of CDR2 and CDR3 regions of the synthesized anti-AFB1 VHH. Anti-AFB1 VHH of high affinity was screened through phage-ELISA,using AFB1-OVA as coating antigen. After four rounds of screening,15 clones capable of binding to AFB1 were obtained. VHH gene of the clone with highest binding ability was amplified,and applied to construct expression plasmid p ET-22b-VHH. The corresponding VHH protein was expressed in E. coli BL21（ DE3）. According to indirect competitive ELISA,the sensitivity of anti-AFB1 VHH obtained was about 10μg / ml.