Recombinant 56-Kilodalton Major Outer Membrane Protein Antigen of Orientia tsutsugamushi Shanxi and Its Antigenicity

Abstract

ABSTRACT The gene encoding the 56-kDa protein of Orientia tsutsugamushi Shanxi was amplified by a nested PCR and cloned into the expression vector pQE30. The 56-kDa protein of O. tsutsugamushi Shanxi (Sxh56) was expressed as a fusion protein with the His 6 -binding protein of Escherichia coli by deleting the signal peptide-encoding sequence from the 5′ end of the open reading frame. The recombinant protein formed inclusion bodies when expressed in E. coli M15. The recombinant protein was examined for reactivity with mouse sera against three antigenic prototypes of O. tsutsugamushi by an immunoblot assay. The recombinant Sxh56 reacted only to polyclonal antiserum to O. tsutsugamushi Gilliam in an enzyme-linked immunosorbent assay (ELISA) and in an immunoblot assay. Recombinant Sxh56 was purified by Ni-nitrilotriacetic acid affinity chromatography and injected into mice to evaluate its ability to stimulate immune responses. High levels of immunoglobulin G and T-cell proliferation appeared in mice immunized with the recombinant protein. The recombinant Sxh56 was used in an ELISA to evaluate the ability of the method to detect antibodies to O. tsutsugamushi in human and animal sera. Thirty sera from mice infected with O. tsutsugamushi Gilliam or Shanxi and 55 sera from normal mice were detected in the ELISA with recombinant Sxh56, and the sensitivity and specificity were 96.67 and 100%, respectively. One hundred fifty-one positive sera and 412 negative sera to O. tsutsugamushi Gilliam were detected in an indirect immunofluorescence assay with the recombinant protein, and the sensitivity and specificity were 96.36 and 88.08%, respectively. These results strongly suggest that the recombinant Sxh56 is a suitable type-specific immunodiagnostic antigen and vaccine candidate.