基于全合成纳米抗体库的黄曲霉毒素B1抗体制备

Preparation of Aflatoxin B1 Antibody Based on Fully Synthesized Nanobody Library

目的利用全合成纳米抗体库筛选制备黄曲霉毒素B1(aflatoxinB1,AFB1)的纳米抗体。方法制备AFB1完全抗原并依托全合成纳米抗体库进行4轮筛选,将捕获的高亲和力抗体基因转化至原核表达系统表达,分析纯化后的抗体特异性及亲和力,并构建抗体G5-AFB1复合三维结构,分析二者的结合特点。结果在AFB1完全抗原制备基础上筛选,随着轮数的增加,特异性噬菌体明显富集,最终获得一株阳性单克隆G5,经鉴定,其半数效应浓度(effec-tive concentration 50%,EC50)为0.953μmol/L,半数抑制浓度(half maximal inhibitory concentration,IC50)为43μmol/L。另外,G5-AFB1复合物不仅形成了稳定的氢键,也具备较好的疏水和静电作用。结论通过全合成纳米抗体库可以筛选出良好的AFB1抗体,为AFB1检测与治疗提供了良好基础,也证实了噬菌体展示技术以及全合成纳米抗体库在小分子生物毒素抗体制备中具有较大的发展潜力。

Objective To prepare nanobodies of aflatoxin B1(AFB1)by using fully synthesized nanobody library.Methods The complete antigen of AFB1 was prepared and screened in 4 rounds relying on the fully synthesized nanobody library,and the captured high-affinity antibody gene was transformed into the prokaryotic expression system for expression to analyze the specificity and affinity.In addition,the three-dimensional structure of the antibody G5-AFB1 was constructed to analyze the binding characteristics of the two.Results On the basis of AFB1 complete antigen preparation,specific phage was obviously enriched with the increase of rounds,and finally a positive monoclonal G5 was obtained,which was identified as having effective concentration 50%(EC50)of 0.953μmol/L and an half maximal inhibitory concentration(IC50)of 43μmol/L.In addition,the G5-AFB1 complexes not only formed a stable hydrogen bond,but also possessed better hydrophobicity and electrostatic effects.Conclusion The ability to screen good AFB1 antibodies by fully synthetic nanobody library provides a good basis for AFB1 detection and treatment,which confirms that phage display technology as well as fully synthesized nanobody library have a large potential for development in the preparation of small molecule biotoxin antibodies.