晚期氧化蛋白产物模拟表位的初步研究  

Screening and Characterization of the Mimotopes of Adveanced Oxidation Protein Products from Phage Peptide Library

作　　者：侯晓睿[1] 刘北一[1] 田建伟[2] 陈益国[1] 富宁[1] 

机构地区：[1]南方医科大学免疫学教研室,广东广州510515 [2]南方医科大学南方医院肾内科,广东广州510515

出　　处：《中国实验诊断学》2011年第5期765-768,共4页Chinese Journal of Laboratory Diagnosis

基　　金：国家自然科学基金基金-广东省联合基金重点项目(No.U0932002)

摘　　要：目的利用噬菌体展示肽库筛选技术获得模拟人晚期氧化蛋白产物(advanced oxidation protein products,AOPP)表位的序列,探索该表位的分布与表达。方法以抗晚期氧化蛋白产物多克隆抗体为靶,对噬菌体随机12肽库进行免疫亲和筛选,ELISA鉴定阳性噬菌体克隆特异性;对阳性克隆进行DNA测序并推导其氨基酸序列,利用生物信息学技术检索其分布与相似性。结果经3轮亲和筛选获得18株与靶分子特异结合的阳性噬菌体克隆,阳性率为62.07%。竞争ELISA结果表明,AOPP可有效抑制阳性噬菌体克隆与抗体结合,其中对No.6克隆的IC50达到0.2ug/ml,提示其能模拟AOPP表位。经DNA测序并推导氨基酸序列,得到LXDMLXD保守序列(X为任意氨基酸);发现该序列不存在于正常人与哺乳动物的蛋白分子,但与某些真菌及寄生虫蛋白分子有同源性。结论通过噬菌体肽库筛选技术获得模拟AOPP表位并具良好抗原性的短肽序列;证实该结构不存在于正常人与哺乳动物蛋白分子,正如AOPP并非正常组织蛋白。Objective To screen and characterize the antigenic mimotopes of advanced oxidation protein products（AOPP） from a random 12-mer phage display peptide library,and to explore the expression or distribution of the mimotope sequences to AOPP.Methods The polyclonal antibodies（PcAbs）against AOPP was used as targets to screen the 12-mer phage display peptide library,and the specificity of phage clones were identified by sandwich ELISA and competitive ELISA.The peptide sequences displayed on positive phage clones were determined by DNA sequencing,and analyzed by bioinformatics.Results Eighteen of phage clones displaying mimotopes of AOPP were isolated in positive rate of 62.07%,which showed a specific reactivity with the PcAbs.The reactivity of PcAbs with positive phage clone No.6 could be well inhibited by AOPP with 0.2μg/ml for IC50,indicating that phage No.6 mimics antigenicity of AOPP epitope.The analysis on amino acid sequence revealed that mimotope sequences contained a common motif LXDMLXD,X is random amino acid.The analysis by bioinformatics showed that the conservative sequences are presented in some fungus and proteins of some parasites.Conclusion The mimotopes of AOPP were obtained by using the phage display technology,which are not presented in protein molecules of normal human and mammalian.

关 键 词：噬菌体展示肽库 晚期氧化蛋白产物 表位 模拟位 

分 类 号：R392-33[医药卫生—免疫学]