Expression And Purification Of Recombinant Membrane Proteins Of Treponema Pallidum And Evaluation Of Their Immunocompetence

Background and ObjectiveT. pallidum subsp. pallidum (T. pallidum) is the causative agent of syphilis. The membrane proteins of T. pallidum play an important role in the intercellular contact, surface identification, signal transduction, enzymatic activity and material transportation. Analysis of recombinant antigens of T. pallidum led to the identification of potential candidate antigens for vaccine development and syphilis serodiagnosis, and have significance in intensive study the pathogenesis of syphilis and the interaction of host-pathogen during infection. Previous studies have shown that such research was difficult for many reasons, and the results of research were also disputable. We use genetic engineering technique to produce recombinant membrane proteins Tp0965, Tp0136and Tp0608via vector bacteria E. coli, and then assess their antigenicity and immunoreactivity, also to evaluate the possibility of recombinant Tp0965as coating antigen in detecting syphilis.MethodsT. pallidum subsp. pallidum (Nichols strain) was propagated and isolated and the genomic DNA was extracted. The genes of Tp0965and Tp0608were amplified by PCR from the genomic DNA. The complete gene of Tp0136was synthesized. Then the genes were subcloned into the expression vector pET28a to construct a recombinant plasmid, which were subsequently transfected into E. coli Rosetta for protein expression. These three recombinant proteins were expressed and purified by Ni-NTA affinity chromatography, and identified by using sodium dodecyl sulfate polyacrylamide gel electropheresis (SDS-PAGE) and Western blot. The reactivities of protein Tp0965were examined by immunoblot analysis. New Zealand rabbits were immunized with the recombinant Tp0965and Tp0136, and the polyclonal antibodies in sera of immunized rabbits were examined by indirect enzyme-linked immunosorbent assay (ELISA) to evaluate its immunogenicity. Also, the positive sera of patients with syphilis were examined by Western blot to identify the ability of their immunoreactions. At last, we conduct the indirect ELISA which was coated with recombinant Tp0965as the antigen to detect syphilis serum.ResultsThe E. coli expression vectors of pET28with target genes were constructed successfully, and the recombinant proteins Tp0965,Tp0136and Tp0608were also successfully expressed with a higher purity and concentration. The higher titre of antibodies of ant-Tp0136in immuned rabbits was detected after the second time of immunization, however, only a low titer of antiserum against Tp0965in immune rabbits was detected after the third time of immunization. Western blot showed that there were specific bands appeared in gel which demonstrated the specific reactions of these three recombinant proteins with positive sera of syphilis. Furthermore, immunoblot assay showed that the recombinant protein Tp0965could be recognized by human syphilitic sera of all stages. Indirect enzyme-linked immunosorbent assay showed there were only4of74human syphilitic sera that failed to show reactivity to recombinant antigen Tp0965, and lack of reactivity of Tp0965to all28uninfected sera.ConclusionsThe recombinant membrane protein Tp0965,Tp0136and Tp0608with complete genes could be successfully expressed through E. coli expression system, and the recombinant Tp0965could be expressed with high level, however, the others expressed with lower level. The recombinant Tp0136has strong antigenicity, but Tp0965showed weak antigenicity during the rabbit immune test. All these three proteins showed immunoreactivity with human serum infected with T. pallidum. And the recombination Tp0965could be a candidate antigen which could be used to detect syphilitic serum.