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

作　　者：武爱华[1,2] 张霄[1] 刘媛[1,2] 赵扬[1] 赵岩岩[1,2] 张存政[1] 谢雅晶[1] 刘贤金[1,2] 

机构地区：[1]江苏省农业科学院食品质量安全与检测研究所/农业部食品质量安全监控重点开放实验室,江苏南京210014 [2]南京农业大学植物保护学院,江苏南京210095

出　　处：《江苏农业学报》2015年第4期929-941,共13页Jiangsu Journal of Agricultural Sciences

基　　金：国家"973"项目(2012CB722505);国家自然科学基金项目(3111343;31201535)

摘　　要：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.

关 键 词：Cry2Aa毒素 噬菌体展示肽库:ELISA 

分 类 号：Q785[生物学—分子生物学]