Analysis of the functional domains on the major surface antigen proteins of Rickettsia japonica

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Rickettsia japonica was isolated and identified by us which is the causative agent of the disease mined Oriental spotted fever (OSF) as a specific spotted fever group (SFG) rickettsiosis in the Oriental faunal region. The major outer membrane proteins rOmp A and rOmp B of R.japonica are known to possess immunogenicity and important roles in the entry into the host cells and in the defense from infection. In this research, Escherichia coli BL2I(DE3) was transformed by expression vector pET-21a-d(+) which was inserted by PCR products of ompA and ompB using primers designed based m the sequences of these genes (DDBJ Accession Numbers ; D28766, AB003681). Recombinant proteins expressed as inclusion bodies with IPTG was solubilized with urea, followed by purification by affinity column chromatography. Single bands w ere obtained in SDS-polyacrylamide gel electrophoresis and Western immunoblotting. The proteins were then dialyzed in the presence of chaperonin GroE and ATP to be refolded to show the soluble nature. These recombinant proteins and monospecific antibodies against rOmp A and rOmp B were examined for their effects m the attachment of rickettsiae to the lust cells. Inhibition of attachment to some extent but not complete was observed by using both the monospecific antibodies and recombinant proteins. Moreover, the importance of the conformations was suggested since the conformations of the recombinant proteins are shown to be different from those of proteins on the organisms. Nine strains of pathogenic rickettsiae isolated from OSF patients were also analyzed phenotypically and genotypically using the rOmp A and rOmp B as marker proteins. It is shown that these strains belong to a single type and that the major surface antigen proteins rOmp A and rOmp B which function on the attachment are useful as markers for typing of SFG rickettsiae.