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

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。

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

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 IgG1kappa, IgG1lambda, and IgG2kappa, respectively, and the affinity constant were 3.0x10(9), 2.8x10(9), 1.9x10(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 high-titer and high-specific have been established. It can be used to deeply study EHEC O157:H7 pathopoiesis mechanism.