Cry2Aa毒素结合十二肽的筛选与鉴定

Screening and identification of Cry2Aa-binding peptides from a phage 12-mer peptide library

利用Cry-2Aa毒素蛋白对噬菌体展示随机十二肽库进行4轮筛选，并从第4轮筛选产物中随机挑选20个单克隆，进行单克隆ELISA、PCR扩增、DNA电泳及测序，鉴定这些克隆与Cry2Aa毒素的结合活性。结果显示，这20个克隆均能与c巧也Aa毒素发生特异性结合，并推导出8条不同的序列。挑取阳性值最高的克隆GT（氨基酸序列为GTPWHHHRHLIV）建立了基于十二肽的Cry-2Aa毒蛋白的间接竞争ELISA检测方法。该方法的抑制中浓度（IC50）为1｜1390μg／ml，最低检测限IC10为O．0211μg／ml，线性检测范围为0．0478～22.691Oμg／ml（Y=23．09lgx＋48．692，R^2=0．9963）。噬菌体展示肽库筛选方法为快速、准确地检测Cry2Aa毒蛋白提供了新的途径。

A phage displayed random 12-mer peptide library (Ph. D.-12TM Phage Display Peptide Library) is screened with Cry2Aa toxin. After 4 rounds of panning, 20 single colonies were selected randomly,and the positive clones are identified by monoclonal phage competitive enzyme-linked immunosorbent assay (ELISA), and PCR,DNA electrophoresis and sequencing. Totally 8 peptides, which could recognize Cry2Aa specifically have been confirmed. The peptide ( amino acid sequence:GTP-WHHHRHLIV) holding better binding ability was employed to develop an indirect competitive ELISA for the Cry2Aa detection. The peptide-based indirect competitive ELISA showed that the IC50 was 1. 139 0μg/ml, the minimum detection limit was 0. 021 1μg/ml, and the linear detection ranged from 0. 047 8 to 22. 691 0μg/ml. With this new detection method, Cry2Aa can be detec-ted rapidly and accurately.