【摘要】 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。更多还原

【Abstract】 Objective To prepare monoclonal antibody(mAb) against vacuolating cytotoxin antigen(VacA) of H.pylori.Methods Recombinant VacA protein was expressed by genetically engineered strain of E.coli DH5α/pQE30-v,and then was purified by Ni2+-NTA affinity chromatography.The antigenicity of the recombinant protein was confirmed by Western blotting.Rabbits were immunized with the recombinant protein while the VacA antibody level in the rabbits was detected for confirmed the immunogenicity by ELISA.The splenocytes of the VacA protein-immunized mice were fused with myeloma cells Sp2/0.Hybridoma cells were selectively cultured by HAT medium and screened by indirect ELISA.The titer of monoclonal antibody secreted by hybridoma cells was detected,classified,and typed.Results Four hybridoma cell lines stably secreting specific mAbs against VacA were obtained and identified.Identification of subclass showed that mAbs were IgG2b,IgM,and IgG1,and all the light chains of mAbs were κ.Western blotting showed that IgG1mAb had a specific affinity for the recombinant VacA.Conclusion Four hybridoma cell lines secreting specific monoclonal antibody against VacA of Helicobacter pylori are established and the mAbs against VacA are successfully obtained,which might play an important role in preparing the VacA test kit and could afford data in exploring the mechanisms of the VacA cytotoxicity.更多还原