Production And Characterization Of Monoclonal Antibody Against Recombinant Protein MgPa' From Mycoplasma Genitalium

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 SDS-PAGE and 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 that can stably secrete mAbs were got. Positive clones screen respective antibodies were injected to the abdominal cavitise of BALB/c mice and then the ascites was collected; The antibodies titers of ascites was examined, and purified by salt fractionation. The isotypes of mAbs was checked with Mouse Monoclonal Antibody Isotyping Kit; The isotype of mAb secreted by 12E7 and 4B1 strain was IgG2b, the isotype of other four mAb all belonged to IgG1. The mAbs secreted by six clones respectively were proved can specifically react with recombinant MgPa'by compeititive ELISA and four of which can specifically react with recombinant MgPa'by Western-blot.Conclusions:1. Six clones secreting monoclonal antibodies were prepared and every monoclonal antibody was proved can react with recombinant MgPa'.2. The mAbs against recombinant MgPa'belonged to IgG, which all can specifically react with MgPa'.