脑膜炎大肠埃希菌侵袭素IbeA结合多肽的初步研究  

Study of phage displayed-peptides binding to invasion protein IbeA of meningitic E. coli K1

作　　者：曹虹[1] 陈丽丹[1] 林琳[1] 刘北一[2] 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系,广州510515 [2]南方医科大学基础医学院,广州510515 [3]南方医科大学生物技术学院,广州510515

出　　处：《免疫学杂志》2006年第6期618-622,共5页Immunological Journal

基　　金：广东省自然科学基金重点项目(C020094)

摘　　要：目的脑膜炎大肠埃希菌(E.coli)侵袭素IbeA通过与脑微血管内皮细胞表面受体结合,从而介导细菌穿透血脑屏障引起新生儿细菌性脑膜炎。为通过肽库筛选获得与IbeA蛋白结合的多肽,建立噬菌体随机肽库筛选IbeA结合多肽模体的方法。方法首先制备靶分子,用PCR扩增出全长ibeA基因序列,克隆至表达载体pET28a中,转化大肠埃希菌BL21(DE3),经IPTG诱导表达出带有His-tag的目的蛋白,经变性复性后通过Ni2+-NTA亲和层析柱得到纯度达90%的重组蛋白。然后以固相化IbeA重组蛋白为靶亲和筛选噬菌体线性12肽库。结果经过3轮淘选,得到能和IbeA结合的24个重组噬菌体克隆;挑选15个亲和力高的克隆进行DNA测序,对比分析所得的短肽序列。固相Fmoc法合成短肽,并进行结合实验、阻断实验和竞争抑制实验。结论以重组表达的IbeA融合蛋白为靶筛选得到的重组噬菌体十二肽克隆,能够和IbeA蛋白结合,为IbeA蛋白结合多肽。结果提示所筛选的阳性噬菌体克隆及其合成肽可能模拟IbeA受体表位的结构。Objective To obtain the recombinant lbeA protein and use it as a target for screening phage displayed-peptides binding to lbeA. Methods The fidl-length ibeA gene was subcloned into pET28a （ ＋ ） vector, and then expressed as a recombinant protein with an N- terminal histidine tag. After Ni2. -NTA resin affinity chromatography, the IbeA protein was used as solid phase molecule for biopanning the phage libraries binding to IbeA from Ph. D-12 phage display peptide library. Results Total of 24 positive phage clones were picked out randomly to detect ligand binding by ELISA assay, and 15 of them with high binding ability were sequenced. A common amino acid sequence was found and synthesized. The synthetic peptide was analyzed by ELISA, block assay, and competitive inhibition assay. Conclusion The results show that the positive 12-mer peptide phage clone screened by using IbeA protein as target could binding to recombinant IbeA protein specifically and could mimic the surface structure of IbeA receptor.

关 键 词：脑膜炎大肠杆菌 IbeA蛋白 表达 噬菌体展示肽库 结合多肽 

分 类 号：R378[医药卫生—病原生物学]