Expression And Purification Of Membrane Immunogens Of Treponema Pallidum In Pichia Pastoris And Preparation Of Monoclonal Antibody

Syphilis is a chronic, complex, sexually transmitted disease of humans caused by the bacterium Treponema pallidum. At present, clinical diagnosis of syphilis include darkfield microscopy, PCR detection and serology assay. In recent years, some test methods like ELISA based on several recombinant antigens expressed in E.coli are applied largely at home and abroad. Of these antigens, 47 kDa, 17 kDa and 15 kDa lipoproteins on the surface of cell wall membrane are proved strongly antigenic, have been applied for serologic tests. Also, monoclonal antibodies against these three antigens were prepared and used for clinical diagnosis.In this research, we firstly express above referred three antigens in pichia pastoris, which has many advantages compared with E.coli such as promoter strongly induced by methanol, secretive expression and post-translational modifications, etc. Genes of T. pallidum three membrane antigens (47 kDa, 17 kDa and 15 kDa) were amplified by PCR with the template of the genomic DNA of Nichols strain and cloned into plasmids pPICZ B and pPIC9K respectively, recombinant plasmids were transformed into Pichia pastoris GS115 and recombinant proteins expressive strains BTP47-GS115, BTP17-GS115, BTP15-GS115 and KTP47-GS115, KTP17-GS115, KTP15-GS115 were obtained by genes of antigens integrating to the genomes.Recombinant antigens were expressed by the methanol induction and confirmed by the western blot assay. Fusion antigens with 6xHis tag were induced by methanol in strains of BTP47-GS115, BTPI7-GS115 and BTP15-GS115, purified usingNi-NTA Agarose, fusion protein yields were 4.8 mg/L, 6.6 mg/L and 25 mg/L of cell culture for His-BTP15, His-BTP17 and His-BTP47, respectively. These recombinant antigens then were evaluated by ELISA assay with serum from syphilis patient and healthy blood donors, all three antigens showed strong sensitivity and specificity.Also, strains of KTP47-GS115, KTP17-GS115 and KTP15-GS115 were induced and confirmed by the same methods, recombinant proteins were secreted to the medium with high expressive levels. KTP47 and KTP17 were expressed over two hundred milligrams per liter of cultures, KTP15 was expressed nearly one hundreds of milligrams per liter of cultures. All of that have the potential for scale-up production and further application.To obtain monoclonal antibodies (mAbs) against 47 kDa antigen, BALB/c mice were immunized by immunogen His-BTP47. Splenocytes of immunized mice and Sp2/0 cells were fused conventionally, and indirect ELISA based on KTP47 antigen was applied to screen for positive mAb-secreting hybridoma cells. At last, three hybridoma cell lines secreting mAbs of IgGl subclass were obtained, the light chain of both mAbs is k chain.