Expression And Purification Of Gpd Outer Membrane Recombinant Protein Of Treponema Pallidum And Characterization Of Its Immunocompetence

Objective:To construct a recombinant vector containing gene encoding outer membrane protein Gpd 1 ~ 353 amino acid residues of Treponema pallidum (T. pallidum), and to express and purify Gpd outer membrane recombinant protein and analyze the immunoreactivity and immunogenicity of recombinant protein., to find a new antigen for the exploiting diagnosis of T. pallidum.Methods:The immuno-dominant epitope of Gpd gene was chosen by computer analysis , then was amplified from T. pallidum complete genome by polymerase chain reactions (PCR), subcloned into the expression vector pET28b(+) to generate recombinant plasmid pET28b(+)/Gpd, then expressed in E. coli RIL, and analyzed by SDS-PAGE and Western-Blot. The fusion protein was purified with Ni-NTA affinity chromatography, Purified protein was analyzed by SDS-PAGE, and protein concentration was determined by the BCA protein assay kit. The immunoreactivity of fusion protein was evaluated by detecting Serum T. pallidum-antibody with indirected-ELISA based on the fusion protein. Zealand rabbits were immunized with the fusion protein, antibodies to anti-Gpd in sera were detected with indirected ELISA.Results:The size of PCR amplification product was about 1059bp. Restriction enzyme digestion analysis and sequencing showed that the inserted target gene were Gpd, compared with gene reported by GenBank, it had 100% similarity; and the results of BLAST confirmed that the sequence was Gpd. A fusion protein with molecular weight...