Expression ,Purification Of Tp0319 Outer Membrane Recombinant Protein From Treponema Pallidum And Characterization Of Its Immunocompetence

Objective:To construct a recombinant vector containting the gene encoding immuno- dominant epitope of Tp0319 outer membrane protein of Treponema pallidum (T. pallidum), and to express and purify Tp0319 outer membrane recombinant protein and analyze the immunoreactivity and immunogenicity of recombinant protein, to find a new antigen for the exploiting diagnosis of syphilis.Methods:The immuno-dominant epitope of Tp0319 gene was chosen by computer analysis , then was amplified from T. pallidum complete genome by polymerase chain reactions (PCR), cloned into the expression vector pQE32 to generate recombinant plasmid pQE32/Tp0319, then expressed in E. coli M15, and analyzed via SDS-PAGE and Western blot. The recombinant protein was purified with Ni-NTA affinity chromatography, Purified protein was analyzed via SDS-PAGE, and protein concentration was determined by the BCA protein assay kit. The immunoreactivity of recombinant protein was evaluated by Indirect ELISA, Serum T. pallidum-antibody was detected with indirected-ELISA based on the recombinant protein. Zealand rabbits were immunized with the recombinant protein, antibodies to anti-Tp0319 in sera were detected with indirected ELISA.Results:The size of PCR amplification product was about 824bp. Restriction enzyme digestion analysis and sequencing showed that the inserted target gene was Tp0319, compared with gene reported by GenBank, it had 100% similarity; and the results of BLAST confirmed that the sequence was Tp0319. A fusion protein with molecular weight about 30kDa was attained after expression and purification. Western blot proved that the recombinant protein can specifically react with T. pallidum IgG positive sera. Indirect ELISA was successfully developed to detect the antibody to T. pallidum in human sera, the sensitivities and the specificities of the diagnostic reagent detected by 80 control sera was 100%(40/40),100%(40/40) respectively. While detecting uninfected and infected T. pallidum human sera, the sensitivities of ELISA was 90.7% compared with the results of the TPPA tests, and the specificities was 100% when the results of the indirect ELISA test was compared with those of the TPPA test. The concordance of results between the ELISA test and the TPPA was 95.3%. Specific humoral response were elicited by recombinant protein in zealand rabbit and the specific antibody titer was more than 1:10 240 after 3 vaccinations.Conclusion1. Prokaryotic expression vector pQE32/Tp0319 was constructed successfully and Tp0319 with molecular weight about 30 kDa was well attained.2. Tp0319 recombinant protein showed excellent immunongenicity and could induce the humoral responses in zealand rabbits efficiently.3. The expressed recombinant protein showed better immunoreactivity, could specifically react with T. pallidum IgG positive sera, and the results lay the foundation for development of quick diagnostic kit applying to detect T. pallidum.