产肠毒素大肠杆菌F17菌毛黏附亚基单克隆抗体的制备及其抗原表位分析  

Preparation and epitope analysis of monoclonal antibody against F17 fimbriae adhesion subunit of enterotoxigenic Escherichia coli

作　　者：刘嘉利 侯美佳 赵佳慧 董秀梅[1,2] 杨巍 张萍[1,2] 师东方 LIU Jia-li;HOU Mei-jia;ZHAO Jia-Hui;DONG Xiu-mei;YANG Wei;ZHANG Ping;SHI Dong-fang(College of Veterinary Medicine,Northeast Agricultural University,Harbin 150030,China;The State Northeastern Scientific Observation Station of Ministry of Agriculture and Rural Affairs on Veterinary Etiological Biology,Harbin 150030,China)

机构地区：[1]东北农业大学动物医学学院,黑龙江哈尔滨150030 [2]农业农村部动物疫病病原生物学东北科学观测实验站,黑龙江哈尔滨150030

出　　处：《中国预防兽医学报》2022年第11期1208-1215,共8页Chinese Journal of Preventive Veterinary Medicine

基　　金：国家科技支撑计划项目(2012BAD12B03、2012BAD12B05);现代农业产业技术体系专项资金资助(CARS-36);农业农村部动物疫病病原生物学东北科学观测实验站观测项目。

摘　　要：为获得产肠毒素大肠杆菌(ETEC)F17菌毛黏附亚基G的单克隆抗体(MAb)及其抗原表位信息,本研究通过原核表达系统表达了重组F17-G蛋白(rF17-G-His)并纯化,SDS-PAGE和western blot检测结果显示表达的rF17-G-His大小为53 ku,纯化后条带单一。利用该重组蛋白免疫BALB/c小鼠,利用杂交瘤技术获得了F17-G的MAb并制备腹水,采用间接ELISA、SDS-PAGE、western blot、抗体亚类鉴定试剂盒对所获得的MAb进行鉴定。结果显示,获得了3株可稳定分泌MAb的阳性杂交瘤细胞株F2G、C3G、E5G。3株MAb亚类均为IgG1,轻链为κ链,其中MAb F2G效价最高,上清效价为1:2 048,腹水效价为1:1 000 000。3株MAb均不与其他菌毛反应,具有较强的特异性。其中MAb E5G可以有效抑制ETEC DN1502株黏附肠上皮细胞。对MAb识别的抗原表位鉴定和分析结果显示,MAb F2G识别的抗原表位区域是aa1~aa15:1AAVSFIGSTENDVGP15,MAb C3G识别的表位区域是aa211~aa225:211GTTSLKLQCDAGVTV225,二者均为线性表位。MAb E5G识别的是构象表位,且位于凝集素结构域第89位天冬氨酸。采用软件分析F17-G生物学特性,结果显示,F17-G是稳定的亲水性蛋白,aa1~aa15是较活跃的表位区域,aa1~aa15、aa211~aa225在变体F17-G1、F17-G2中较为保守,aa89在3个变体中均高度保守。综合分析后认为MAb E5G具有预防和治疗携带F17菌毛ETEC引发的疾病的潜力。本研究为相应疾病的新型药物、诊断方法和疫苗的研发提供了有效生物制剂和可靠的生物学信息。To obtain the monoclonal antibody(MAb) and epitopes of the F17-G subunit of enterotoxigenic Escherichia coli(ETEC),the recombinant F17-G protein(rF17-G-His) was expressed by prokaryotic expression system and purified.SDS-PAGE and western blot results showed that the molecular weight of expressed rF17-G-His was 53ku with a single band after purification.After immunization of BALB/c mice with the purified recombinant protein,the MAbs against F17-G were obtain using hybridoma technology and the ascites were prepared.The MAbs were identified using indirect ELISA,SDS-PAGE,western blot,and the antibody subclass identification kit.The results showed that three hybridoma cell lines F2G,C3G and E5G which could steadily secrete MAb were obtained.The three MAbs were IgG1 and the light chain was κ chain.The MAb F2G showed the highest titer,and the supernatant titer was 1:2048 and ascites titer was 1:1000000.The three MAbs did not react with other fimbriae indicating good specificity,and MAb E5G could effectively inhibit the adhesion of bacteria DN1502 to intestinal epithelial cells.The results of identification and analysis of antigen epitope showed that the epitope recognized by MAb F2G was aa1-aa15:1AAVSFIGSTENDVGP15,the epitope region recognized by MAb C3G was aa211-aa225:211GTTSLKLQCDAGVTV225,and MAb E5G recognized aspartic acid at position 89 of lectin domain.The biological characteristics of F17-G were analyzed by software.It showed that F17-G was a stable hydrophile protein,aa1-aa15 was a more active epitope region,aa1-aa15 and aa211-aa225 were more conserved in variant F17-G1 and F17-G2,and aa89 was highly conserved in all three variants.Comprehensive analysis indicated that MAb E5G has a potential to prevent and treat diseases caused by ETEC with F17 fimbriae.This study provides effective biological agents and reliable biological information for the development of new drugs,diagnostic methods,and vaccines.

关 键 词：产肠毒素大肠杆菌 F17菌毛 G亚基 单克隆抗体 抗原表位 

分 类 号：S852.61[农业科学—基础兽医学]