噬菌体展示技术筛选可特异结合阪崎肠杆菌的多肽

Screening of Enterobacter sakazakii-binding Peptides by Phage Display Technique

利用噬菌体展示七肽库,通过优化筛选条件,得到阪崎肠杆菌的特异结合多肽。经过4轮筛选,得到的噬菌体单克隆进行测序,用酶联免疫吸附法(ELISA)鉴定阳性单克隆,并评价阳性单克隆的特异性。结果表明:优化吐温-20体积分数、结合时间和洗脱时间,提高了筛选效率,有助于获得结合肽共有序列;ELISA法鉴定出一个与阪崎肠杆菌特异结合的阳性单克隆E2,编码七肽序列QNDGTPR;特异性实验结果表明:E2与其他4株参考菌株细菌没有显著结合力,具有良好的特异性。综上,利用噬菌体展示技术,可成功筛选到阪崎肠杆菌特异结合多肽QNDGTPR。

In this study, we used a phage-displayed random peptide library to identify phage clones, and the displayedpeptides could specifically and strongly bind to the cell surface of Enterobacter sakazakii. The capability of the phage clonesto interact specifically with Enterobacter sakazakii was demonstrated by using enzyme-linked immunosorbent assay （ELISA）.We assessed the selectivity of phage-bacteria binding by comparing the binding capability of the selected clones to the targetbacteria and a panel of other bacterial species. Tween-20 concentration, and binding and elution time were optimized toenhance the stringency of selection or elution and to obtain a consensus binding sequence. After four rounds of screening,positive phage clone DNAs were sequenced and their specific binding activity was identified by ELISA. The affinity ofthe phage clones encoding QNDGTPR peptides was significantly higher than that from the control group. No phage clonesignificantly bound to other selected bacterial species. Overall, Enterobacter sakazakii-binding peptide QNDGTPR wasscreened and obtained by a random peptide library and the peptide may be used to develop novel diagnostic probes.