抗肠出血性大肠杆菌O157:H7 EspA单克隆抗体的制备及其特性鉴定  

Preparation and characterization of monoclonal antibody against Enterohemorrhagic Escherichia coli O157:H7 EspA

作　　者：余抒[1] 罗萍[1] 陈洪章[1] 李海侠[1] 毛旭虎[1] 

机构地区：[1]第三军医大学临床微生物学教研室,重庆400038

出　　处：《细胞与分子免疫学杂志》2007年第7期657-659,共3页Chinese Journal of Cellular and Molecular Immunology

基　　金：军队杰出人才基金资助项目(04J009);军队"十一五"攻关项目(06G078)

摘　　要：目的:制备抗肠出血性大肠杆菌O157:H7EspA单克隆抗体(mAb)。方法:以重组EspA蛋白为免疫原免疫BALB/c小鼠,运用杂交瘤技术并且联合间接ELISA筛选只针对EspA的杂交瘤细胞株。以Ig类与亚类鉴定试剂盒鉴定mAb的Ig亚类,ELISA、Western blot及免疫荧光技术鉴定抗体的免疫学特性。结果:获得3株稳定分泌抗EspA杂交瘤细胞的mAb,Ig亚型分别为IgG1κ型、IgG2aκ型、IgG2bκ型。Western blot和免疫荧光分析表明,3株杂交瘤细胞制备的mAb腹水都能特异地结合重组EspA蛋白和识别O157:H7的EspA成分。结论:成功地制备了抗肠出血性大肠杆菌O157:H7EspA的mAb,并证明了该mAb的生物学活性,为深入研究肠出血性大肠杆菌O157:H7的致病机制提供新的手段。AIM： To prepare hybridoma cell lines were obtained by fusing Sp2/0 with spleen cells from BALB/c mice immunized with killed enterohemorrhagic E. coli O157 ： H7 EspA （EHEC O157：H7 EspA）. METHODS： The subclass isotype and the specificity of monoclonal antibodies （mAb） were determined and identified by ELISA, Western blot and immune fluorescence staining, RESULTS： Isotype of 3 mAb was IgGIK, IgG1λ, and IgG2κ, respectively, and the affinity constant were 3, 0 × 10^9, 2.8 × 10^9, 1.9× 10^9. As demonstrated by Western blot, these 3 mAb specifically reacted with EspA protein and EHEC O157： H7. Useing immune fluorescence staining, EHEC O157：H7 could adhere to the membrace of Hela cell. CONCLUSION： Three hybridoma cell lines can stably secrete anti-EspA mAb with hightiter and high-specific have been established, It can be used to deeply study EHEC O157：H7 pathopoiesis mechanism.

关 键 词：EspA 单克隆抗体 特性鉴定 

分 类 号：R392.11[医药卫生—免疫学]