脑膜炎大肠杆菌IbeA蛋白结合肽序列的亲和筛选  

Screening of Peptides Binding to IbeA Protein of Meningitic E. coli

作　　者：曹虹[1] 林琳[1] 刘北一[2] 陈丽丹[1] 杨军[1] 贡树基[1] 李明[3] 

机构地区：[1]南方医科大学公共卫生与热带医学学院微生物学教研室,广州510515 [2]南方医科大学基础医学院免疫学教研室,广州510515 [3]南方医科大学生物技术学院,广州510515

出　　处：《微生物学通报》2006年第5期101-106,共6页Microbiology China

基　　金：东省自然科学基金重点项目(No.C020094)

摘　　要：应用噬菌体展示肽库技术,以重组的脑膜炎大肠杆菌致病蛋白IbeA作为靶分子,经过吸附-洗脱-扩增-再吸附的亲和筛选,随机挑选亲和力强的噬菌体克隆,进行ELISA、竞争抑制实验和序列测定。结果显示,经3轮淘选后,间接ELISA鉴定得到高亲和性结合IbeA蛋白的15个阳性克隆。竞争抑制实验结果表明,游离IbeA蛋白能竞争抑制噬菌体结合肽克隆与固相包被的IbeA蛋白的结合,其抑制作用随游离IbeA蛋白浓度的降低而减弱。测序结果得到5种阳性噬菌体克隆展示肽序列。上述结果提示以脑膜炎大肠杆菌IbeA蛋白为靶筛选所获得的噬菌体12肽克隆,具有特异性,其结合肽序列呈现相对保守性。建立的从噬菌体随机肽库筛选IbeA蛋白结合肽的方法具有方便、灵活和高效可行的特点。The recombinant meningitic E. coli IbeA protein was used as a target to screen specific IbeA bindingpeptide from 12-mer phage display peptide library for three rounds of biopanning. The affinity and specificity of selected polypeptide were evaluated by ELISA, competitive inhibition assay and DNA sequencing. 15 of positive phage clones were of high affinity and specific to target protein. The competitive inhibition assay result showed that the soluble IbeA could block the binding between phage display peptide clone and solid phase IbeA protein, and the competitive inhibition rate dropped gradually as the concentration of IbeA protein fell Sequence analysis showed relatively conservative and diverse sequences in the phage display peptide. These results suggest that the successful selection of IbeA binding-peptide phage clones could provide a convenient, flexible and high throughout screenine for bindine epitope from phage random disolav peptide library.

关 键 词：噬菌体展示肽库 脑膜炎大肠杆菌IbeA蛋白 结合肽 亲和筛选 

分 类 号：R91[医药卫生—药学]