肠出血性大肠杆菌O157：H7志贺毒素ⅡA1亚单位单克隆抗体的制备和生物学特性鉴定  

Preparation and biological characterization of monoclonal antibody against shiga toxin Ⅱ A1 subunit of enterohemorrhagic E. coli O157∶H7

作　　者：罗萍[1] 陈洪章[1] 曾明[2] 郭鹰[1] 张卫军[1] 毛旭虎[1] 刘璐[1] 曾浩[1] 邹全明[1] 

机构地区：[1]第三军医大学医学检验系临床微生物学及免疫学教研室,重庆400038 [2]中国药品生物制品检定所,北京100000

出　　处：《第三军医大学学报》2008年第8期698-701,共4页Journal of Third Military Medical University

基　　金：国家高技术研究发展计划(“863”计划)(2006AA02Z405);全军医学科研“十一五”科技攻关项目(06G078)~~

摘　　要：目的制备出高效价的抗志贺毒素ⅡA1亚单位(STX2A1)的单克隆抗体。方法以重组GST-STX2A1融合蛋白作为免疫抗原,免疫BALB/c小鼠,运用细胞杂交瘤技术制备,以天然STX2毒素为筛选抗原,采用间接ELISA法筛选产生针对STX2A1的单克隆抗体细胞株;以体内诱生法产生腹水,并采用Protein A-Sepharose柱对其纯化,快速定性试纸鉴定McAb的Ig亚类,采用间接ELISA相加实验鉴定抗原识别表位。结果获得3株产生针对STX2A1的单克隆抗体细胞株STX2-1A3、STX2-1E10、STX2-3A7,Ig亚类分别为IgG1λ型、IgG1κ型和IgG3κ,亲和力常数分别为5.76×109、1.21×109和3.97×108。结论成功制备3株稳定分泌抗STX2A1的杂交瘤细胞株,产生的单克隆抗体特异性好,亲和力高,能与天然STX2A亚单位反应。Objective To prepare high-titer monoclonal antibodies against STX2A1 subunit of enterohemorrhagic E. coli （EHEC） O157： H7. Methods BALB/c mice were immunized with GST-STX2A1 fusion protein and the spleen cells of BALB/c mice which were not immunized were used as feeder cells. Hybridoma technique, natural STX2A protein and ELISA test were used to prepare and screen the hybridoma cell lines of monoclonal antibodies against STX2A1. The ascites developed by injecting the hybridoma cells into abdominal cavity of the BALB/c mice and was purified with Protein A-Sepharose. The subclasses and isotypes were identified by mouse monoclonal antibody isotyping kit. The antigenic epitopes that can be recognized by STX2-1A3, STX2-1E10 and STX2-3A7 were analyzed by the ELISA additivity test. Results Three hybridoma cell strains were obtained and named as STX2-1A3, STX2-1E10 and STX2-3A7, respectively, all of which produced monoclonal antibodies specifically against STX2A1. The isotypes of the monoclonal antibodies were IgG1λ, IgG1κ, and IgG3κ and the affinity constant was 5.76× 10^9, 1.21× 10^9 and 3.97× 10^8, respectively. Conclusion We have successfully prepared three hybridoma cell strains which secrete high-titer and highly specific monoclonal antibodies against STX2A1. Our study provides a basis for researching the early diagnosis, prevention and cure of the disease induced by EHEC O157： H7.

关 键 词：O157:H7 STX2A1 单克隆抗体 生物学特性 

分 类 号：R378.21[医药卫生—病原生物学] R392-33[医药卫生—基础医学]