细菌外膜蛋白A通用抗原表位预测及其抗体磁珠捕获病原菌效果的初步评价  

Bioinformatics design of bacterial universal outer membrane protein A,anti-polypeptide antibody preparation and evaluation of its recognition of foodborne pathogens

作　　者：王磊 柯跃华 栗业 李怡璇 杨瑞馥 高波[1] 韩延平 WANG Lei;KE Yue-hua;LI Ye;LI Yi-xuan;YANG Rui-fu;GAO Bo;HAN Yan-ping(State Key Laboratory of Pathogen and Biosecurity,Institute of Microbiology and Epidemiology,Academy of Military Medical Sciences,Academy of Military Sciences,Beijing 100071,China;No.32277 Troops of PLA,Hami,Xinjiang 839108,China;Center for Disease Control and Prevention of PLA,Beijing 100071,China)

机构地区：[1]军事科学院军事医学研究院微生物流行病研究所,病原微生物生物安全国家重点实验室,北京100071 [2]解放军32277部队,新骝哈密839108 [3]解放军疾病预防控制中心,北京100071

出　　处：《军事医学》2021年第5期336-339,353,共5页Military Medical Sciences

基　　金：全军后勤科研计划重点项目(BWS18J012,BWS17J030)。

摘　　要：目的利用革兰阴性细菌外膜蛋白A高度保守的特性,设计和制备可识别多种食源性革兰阴性细菌的免疫磁珠,并对该免疫磁珠的识别和富集效果进行评价。方法利用革兰阴性外膜蛋白A序列高度保守的特性,从国际生物基因数据库(NCBI)提取该蛋白的氨基酸序列进行同源比对;使用抗原表位预测工具"BepiPred"预测具有免疫原性的抗原表位;将预测的多肽序列分两组混合免疫大耳白兔制备多克隆抗体,并利用间接ELISA法对抗体进行评价,挑选出优质抗体偶联磁珠捕捉细菌,利用平板培养计数的方法检验抗体的有效性和广谱性。结果经间接ELISA筛选,制备的多抗可识别阪岐克洛诺杆菌、肠出血性大肠埃希菌O157∶H7、弗氏志贺菌、肠炎沙门菌、鼠伤寒沙门菌、小肠结肠炎耶尔森菌6种常见食源性致病菌,识别灵敏度为25μg/ml;使用该抗体制备的500 nm羧基免疫磁珠对6种细菌的捕捉率为0.47%~2.03%。结论与传统的免疫磁珠相比较,该项目制备的多抗免疫磁珠具有广谱性的特点,可提高检验效率,是一种具有应用前景的检测思路。Objective To obtain immunomagnetic beads coupled with a polyclonal antibody and detect a variety of Gram-negative bacteria.Methods By using National Coalition Building Institute(NCBI)and"BepiPred"software,12 polypeptide fragments derived from OmpA conserved among Gram-negative bacteria were shown to potentially possess immunogenicity as antigenic epitopes.The polyclonal antibodies were prepared by animal immunization and evaluated by ELISA assays.High quality antibodies were chosen for magnetic bead coupling.The captured bacteria were cultivated and identified to test the effectiveness and spectrum width of the anti-polypeptide antibodies.Results The polyclonal antibody could identify 6 food-borne bacterial pathogenic species by ELISA assays,including Cronobacter sakazakii,Enterohemorrhagic E.coli O157∶H7,Shigella flexneri,Salmonella enteritidis,S.typhimurium and Yersinia enterocolitica.The sensitivity was 25μg/ml.The ability of 500 nm carboxyl immunomagnetic beads coupled with the antibody to capture the six kinds of bacteria was 0.47%-2.03%.Conclusion Compared with the traditional monoclonal antibody,the polyclonal antibody has a broader spectrum and is able to improve the detection efficiency.The strategy based on universal antigens provides a new promising idea for bacterial enrichment prior to detection.

关 键 词：革兰阴性细菌 外膜蛋白A 免疫磁珠 食源性致病菌 快速检测 

分 类 号：R446[医药卫生—诊断学]