Expression And Purification Of TP 0993 Outer Membrane Recombinant Protein Form Treponema Pallidum And Its Immunocompetence

Objective:To construct a recombinant vector containing the gene encoding TP 0993 outer membrane protein of Treponema pallidum (T. pallidum), and to express and purify TP 0993 outer membrane recombinant protein and analyze the immunoreactivity and immunogenicity of recombinant protein.,and find a new antigen for the exploriting diagnosis of Treponema pallidum.Methods:The genes sequence of TP 0993 gene was obtainde from Genbank, then was amplified from T. pallidum complete genome by polymerase chain reactions (PCR), subcloned into the expression vector pET28a(+) to generate recombinant plasmid pET28a(+)/TP 0993, then expressed in E.coli Rosseta, and analyzed by SDS-PAGE and Western-Blot. The fusion protein was purified with Ni-NTA affinity chromatography, and analyzed using SDS-PAGE,The protein concentration was determined by the BCA protein assay kit. The immunoreactivity of fusion protein was evaluated by Indirect ELISA , serum T. pallidum-antibody was detected with indirected-ELISA based on the fusion protein. Zealand rabbits were immunized with the fusion protein, antibodies to TP 0993 fusion protein in sera were detected with indirected ELISA.Results:The size of PCR amplification product was about 950 bps. Restriction enzyme digestion analysis and sequencing showed that the inserted target gene was TP 0993, compared with gene reported by Genbank, it had 99% similarity; and the result of BLAST confirmed that the sequence was TP 0993. A fusion protein with molecular weight near 34KDa 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. While detecting uninfected and infected T. pallidum human sera, the sensitivities of ELISA was 88.3% compared with the results of the TPPA , and the specificities was 85.8% when the results of ELISA was compared with those of the TPPA . The concordance of results between the ELISA and the TPPA was 86.5%. Specific humoral response were elicited by recombinant protein in New zealand rabbit and the specific antibody titer was above 1: 640 after immunization for 3 times .Conclusion1. Prokaryotic expression vector pET28a(+)/TP 0993 was constructed successfully and TP 0993 with molecular weight near 34 KDa was well attained.2. TP 0993 recombinant protein showed excellent immunongenicity and can induce humoral responses in zealand rabbits efficiently.3. The expressed recombinant protein showed excellent immunoreactivity, can 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...