交叉保护性抗LPS多克隆抗体的制备与LPS多表位模拟肽的筛选  

Preparation of polycolonal anti-LPS antibody with cross reactivity and screening of mimotopes of LPS from phage displayed peptide library

作　　者：郑山根[1] 刘北一[1] 朱平[1] 富宁[1] 

机构地区：[1]第一军医大学免疫教研室,广州510515

出　　处：《中华微生物学和免疫学杂志》2003年第11期903-906,共4页Chinese Journal of Microbiology and Immunology

基　　金：国家自然科学基金资助项目 (3 0 0 70 769)

摘　　要：目的　获得具交叉保护性的抗细菌脂多糖 (LPS)多克隆抗体与模拟LPS多表位的噬菌体展示环肽克隆。方法　制备具交叉反应性的兔抗鼠伤寒沙门菌抗血清 ,鉴定抗血清对大肠杆菌和铜绿假单胞菌攻击小鼠的交叉保护性。以亲和纯化的多克隆抗体为靶 ,亲和筛选噬菌体随机环七肽库。双夹心ELISA和竞争抑制ELISA鉴定阳性噬菌体克隆。结果　兔抗血清能与多种来源的LPS反应 ,对用大肠杆菌和铜绿假单胞菌攻击的小鼠有显著的保护性。用亲和层析纯化的多克隆抗体为靶分子进行 3轮筛选 ,随机挑选 4 6个克隆 ,其中 2 0个克隆显示与多抗结合。鼠伤寒沙门菌LPS可抑制阳性噬菌体克隆与多抗结合 ,所有克隆的IC50 (达到 5 0 %抑制率的LPS浓度 )为 12 5ng ml。挑选其中10个克隆测序并推导氨基酸序列 ,其中 5个克隆具X QFYP X A保守序列 ;3个克隆具LFTFAHY序列 ;2个克隆具YQYYPAA序列 ,所有序列非极性氨基酸含量平均值为 80 .0 %。结论　获得能与多种LPS反应且具交叉保护作用的兔多克隆抗体 ,筛选得到可模拟鼠伤寒沙门菌LPS多表位噬菌体展示环七肽。Objective To prepare polyclonal anti-LPS antibody with cross protection against LPS from different bacteria and to screen mimotopes of LPS from cyclic 7-mer phage peptide library. Methods Rabbits were immunized with S.typhimuri . The antisera were tested for the reaction to LPS from different bacteria by ELISA and for protection of mice infected by E.coli and P.pyocyanea . Mimotopes of LPS were screened from cycle 7-mer phage displayed library by biopaning using purified antibody against S.typhimurium LPS, and the antigenicity of selected clones were identified by ELISA. Results Antisera can react with different LPS and protect mice from infection by E.coli and P.pyocyanea . After 3 round of screening, 20 of 46 clones were identified as positive which can bind to polyclonal antibody. The IC 50 of all positive clones was about 125ng/ml of LPS of S.typhimurium . 10 positive clones were sequenced and the amino acid sequences were deduced, in which 5 clones showed sequence as X-QFYP-X-A, 3 clones as LFTFAHYA and 2 clones as YQYYPAA. The average content of hydrophobic amino acid was 80.0%. Conclusion Polyclonal anti-LPS antibody with cross reactivity and protection was obtained, and mimotope clones of LPS were screened from phage displayed peptide library. These phage clones can bind to anti-LPS antibody like LPS, which suggests that the peptide displayed on phage can mimic epitope of LPS.

关 键 词：交叉保护性 抗LPS多克隆抗体 制备 LPS多表位模拟肽 筛选 

分 类 号：R725.5[医药卫生—儿科]