Application Of The Gene Encoding Outer Membrane Protein B Of Rickettsia Rickettsii

Rickettsia rickettsii is an obligate intracellular gram-negative bacterium, the etiologic agent of Rocky Mountain spotted fever (RMSF). Human beings are susceptible to the pathogen. The major outer membrane protein B (rOmpB), the most abundant surface-exposed protein, is one of the principal rickettsial candidate antigens for developing diagnostic agents and subunit vaccine.Firstly, a real-time quantitative polymerase chain reaction (PCR) assay using species-specific probe was developed for the quantitation and monitoring of R. rickettsii. The assay is based on TaqMan 3'-minor groove binder (MGB) chemistry using the outer membrane protein B gene (ompB) as the target. The assay amplified R. rickettsii but other members of the genus rickettsia did not produce positive reactions, nor did other members of the order rickettsiales or any non-rickettsial bacterium. The assay had a sensitivity of less than ten target copies per reaction as determined by serial dilutions of a plasmid containing a target sequence. This quantitative assay has been successfully applied to the monitoring of replication of rickettsiae in cells and the quantitation of rickettsial loads in experimentally infected animal blood and tissues.Secondly, the gene encoding 120kDa surface antigen of R. rickettsii were cloned and expressed. To identify the immunostimulatory regions of the 120kDa protein, six small fragments of the gene were subcloned and expressed. In western blotting analysis, the 120kDa and P5 recombinant proteins reacted to immunoserum to R. rickettsii. To study the immunogenicity of the recombinant proteins, the proteins purified by affinity chromatograph were used to immunize BALB/c mice. After two booster injections, the IgG titers to R. rickettsii were detected in the sera from the immunized mice by IFA. The antigen-specific spleen cell proliferative responses were detected by MTT method. The animals immunized with 120kDa protein and P5 exhibited profound humoral and cellular immune responses, including much higher IgG titers, much higher levels of gamma interferon (TFN-γ), interleukin-2 (IL-2), interleukin-4 (IL-4), interleukin-10 (TL-10) and much stronger proliferation of spleen cells. Seven days after challenge of 10 infection dose of R. rickettsii, immunized mice were euthanized and their spleens and livers were collected. By real-time PCR assay, the rickettsial loads of spleens and livers of mice immunized with whole-cell antigen (WCA), 120kDa recombinant protein or P5 protein were significantly lower than that of mice immunized with PBS. The data indicate that 120kDa recombinant protein and P5 protein are good immunogen for eliciting immunoresponses against R. rickettsii.The purified 120kDa protein and P5 protein were recognized by sera from animals infected with agents of the genus rickettsia, not Coxiella burnetii and Orientia tsutsugamushi., suggesting the two antigens are specific for genus rickettsia. The results of enzyme-linked immunosorbent assay (ELISA) show that the expressed P5 protein can replace WCA or 120kDa recombinant protein as a suitable ELISA diagnostic agent. The sensitivity and specificity of the P5-coated IgG indirect ELISA relative to immunofluorescent antibody assay testing were 100% and 93.4%, respectively. These results indicate that the expressed P5 protein is an attractive source of antigen for development of an ELISA for the detection of antibodies to R. rickettsii or other agents of genus rickettsia.