CAJ | 학술논문

利用Cry2 Aa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Cry2Aa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Cry2Aa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0.0211μg/ml,线性检测范围为0.0478~22.6910μg/ml (Y=23.09 lgx+48.692,R2=0.9963)。噬菌体展示肽库筛选方法为快速、准确地检测Cry2 Aa毒蛋白提供了新的途径。
이용Cry2 Aa독소단백대서균체전시수궤십이태고진행4륜사선,병종제4륜사선산물중수궤도선20개단극륭,진행단극륭 ELISA、PCR확증、DNA전영급측서,감정저사극륭여 Cry2Aa독소적결합활성。결과현시,저20개극륭균능여Cry2Aa독소발생특이성결합,병추도출8조불동적서렬。도취양성치최고적극륭GT(안기산서렬위GTPWHHHRHLIV)건립료기우십이태적Cry2Aa독단백적간접경쟁ELISA검측방법。해방법적억제중농도(IC50)위1.1390μg/ml,최저검측한IC10위0.0211μg/ml,선성검측범위위0.0478~22.6910μg/ml (Y=23.09 lgx+48.692,R2=0.9963)。서균체전시태고사선방법위쾌속、준학지검측Cry2 Aa독단백제공료신적도경。
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.