论文部分内容阅读
目的制备抗重组幽门螺杆菌致细胞空泡毒素抗原(VacA)的单克隆抗体(mAb)。方法用基因工程菌pQE30-v-DH5α大量表达重组蛋白VacA,经Ni2+-NTA树脂纯化后,Western blot鉴定抗原性,免疫家兔后ELISA法检测血清VacA抗体鉴定其免疫原性。用重组VacA免疫Balb/c小鼠。取免疫鼠脾细胞与骨髓瘤SP2/0细胞融合,HAT选择性培养和间接ELISA进行筛选,并检测所分泌抗体的效价和分析Ig类别。结果获得4株能稳定分泌VacA mAb的杂交瘤细胞,能分泌IgG2b、IgM和IgG1 3类抗体,轻链均为κ型。其中,IgG1 mAb经Western blot鉴定能与重组VacA发生特异性反应。结论应用纯化的重组VacA,成功获得了能稳定分泌幽门螺杆菌VacA单克隆抗体的杂交瘤细胞,并制备了单克隆抗体。为进一步研制检测VacA的试剂盒及探讨VacA的致病机制奠定了基础。
Objective To prepare a monoclonal antibody (mAb) against recombinant vacuolar toxin (VacA) of recombinant Helicobacter pylori. Methods Recombinant protein VacA was expressed in large quantities using genetically engineered bacteria pQE30-v-DH5α. After purified by Ni2 + -NTA resin, the antigenicity was identified by Western blot. Immunization of rabbits with VacA was detected by ELISA. Balb / c mice were immunized with recombinant VacA. The immunized murine spleen cells were fused with myeloma SP2 / 0 cells, HAT selective culture and indirect ELISA were screened, and the titer of the secreted antibodies and the Ig class were analyzed. Results Four hybridoma cells that could secrete VacA mAb were obtained, secreting IgG2b, IgM and IgG1 3 antibodies, and all light chains were κ. Among them, IgG1 mAb identified by Western blot with recombinant VacA specific reaction. Conclusion The purified recombinant VacA was successfully used to obtain hybridoma cells secreting monoclonal antibodies against VacA of H. pylori and monoclonal antibodies were prepared. It laid the foundation for the further development of the kit for testing VacA and the pathogenesis of VacA.