抗生殖支原体MgPa’重组蛋白单克隆抗体的制备及鉴定-病原及免疫专业毕业论文.docx

PAGE PAGE 10 株杂交瘤细胞中 12E7、4B1 分泌的 mAb 为 IgG2b,其它 4 株分泌的 mAb 均为 IgG1。 结论： （1）筛选出 6 株稳定分泌抗 MgPa’ mAb 的杂交瘤细胞株，经鉴定 6 株杂交 瘤细胞分泌的 mAb 均能识别重组 MgPa’。 （2）获得的抗重组 MgPa’的 mAb 均为 IgG 类抗体，且能识别 MgPa’抗原的 天然表位，具有较好的特异性。 关键词：生殖支原体；单克隆抗体；黏附素蛋白（MgPa’）；诊断；应用 本课题受以下课题资助： 国家自然科学基金 Production and characterization of monoclonal antibody against recombinant protein MgPa’ from Mycoplasma genitalium ABSTRACT Objective: The recombinant protein encoded by the immuno-dominant epitope of MgPa’ gene（3 223~4 092bp）of Mycoplasma genitalium was expressed and purified，and then the antigenicity of MgPa’ was analyzed. SP2/0 cells and spleen cells from BALB/c mouse immunized with recombinant MgPa’ of M. genitalium were fused by hybridization technology; The cell lines which stably secrete monoclonal antibodies against recombinant MgPa’ was screened and then identified and purified. And then monoclonal antibodies were evaluated as a method for M. genitalium diagnosis to establish a foundation for the study on diagnosis of M. genitalium. Methods: Recombinant MgPa’ was purified with Ni-NTA-His affinity chromatography and protein concentration was determined by BCA assay, the product was then analyzed with SDSand Western-blot. The BALB/c mice were immunized with purified recombinant MgPa’. The mouse’s spleen cells were fused with SP2/0 cells and then the positive clones were screened with ELISA. Mouse ascites were induced, and the mAbs were produced and accumulated. The titer and isotype of the antibodies were identified with indirect ELISA, and then the specificity of monoclonal antibodies was evaluated by competitive ELISA and Western-blot. Results: A recombinant protein mainly existed in the pattern of inclusion body with an estimated Mr of 37kDa was obtained after expression and purification. The recombinant protein MgPa’ was proved can specifically react with Anti-His mAb by Western-blot. The BALB/c mice were immunized with the purified recombinant MgPa’. Six clones tha