Evalution Of Recombinant Protein TpF1of Treponema Pallidum For Serodiagnosis Of Syphilis

Objective: To further investigate the diagnostic potential and biological functionof the recombinant protein TpF1, we constructed a recombinant vector expressingrecombinant protein TpF1of Treponema pallidum，expressed in and purified fromE.coli as His-tag fusion protein, and analyzed the immunoreactivity andimmunogenicity of recombinant protein.Methods: Treponema pallidum Nichols strain and clinical isolates were propagatedand isolated. The genomic DNA was extracted from Treponema pallidum Nicholsstrain. The TpF1gene was amplified by PCR from a preparation of T. pallidumgenomic DNA and cloned into the expression vector pET28a. The recombinantprotein TpF1was expressed in E.coli and purified by Ni2+-NTA purification system.To further determine the immunoreactivity and immunogenicity of recombinantprotein TpF1, sera obtained from recombinant protein TpF1immunized rabbits andTp Nichols strain and Tp clinical isolates infected rabbits were examined byWesternblot assay and indirect enzyme-linked immunosorbent assay. The sensitivityand specificity of recombinant protein TpF1were assessed by screening human serafrom patients with syphilis, cross-reactive infections (Leptospiral spp. and Borreliaburgdorferi) and uninfected.Results: TpF1gene was successfully amplified from the T. pallidum Nichols straingenome, and then cloned into expression vector pET28a. The soluble recombinantprotein TpF1were purified from E.coli as a20kDa His fusion protein. Anti-sera werecollected recombinant protein TpF1immunized rabbits and determined with a hightiter (>1:25,600) by TpF1-based ELISA. The specific reactivities of recombinantprotein TpF1were determined by Westernblot assay with sera obtained fromrecombinant protein TpF1immunized rabbits and Tp Nichols strain and Tp clinicalisolates infected rabbits. The sensitivities and specificities of TpF1-based ELISA were 98%and100%respectively for sera from patients with syphilis, cross-reactiveinfections (Leptospiral spp. and Borrelia burgdorferi) and uninfected.Conclusion：1. TpF1gene was successfully cloned into expression vector pET28a and expressed asa20kDa recombinant protein.2. Recombinant protein TpF1could induce a strong humoral immune response inexperimentally syphilis infection.3. Recombinant protein TpF1showed high sensitivity (98.8%) and specificities (100%)for detection of syphilis. These data indicate that the recombinant protein TpF1is apotential candidate of diagnostic antigens for syphilis.