Cloning And Expression Of The Gene Encoding 120-kDa Surface Protein And Study Of The Antigenicity Of The Recombinant Proteins Of Rickettsia Prowazekii

Rickettsia prowazekii is an obligate intracellular gram-negative bacterium, the etiologic agent of epidemic typhus. The objects of the study are to clone and express the gene fragments encoding N-end region and the C-end region of R. prowazekii 120kDa surface antigen and to analyze their antigenicity.Two gene fragments encoding the N-end region and the C-end region of the 120kDa surface protein antigen were amplified from the genome of R. prowazekii by PCR. T he g ene fragments w ere c loned i nto p rokaryotic e xpression v ector p QE30, respectively. E. coli cells were transformed with the recombinant plasmid pQE30/N and pQE30/C; and the transformants were induced to express the recombinant proteins by IPTG.The 26-kDa recombinant protein N (rpN) expressed in the pQE30/N-transformed cells and the 67-kDa recombinant protein C (rpC) expressed in the pQE30/C-transformed cells were identified in SDS-PAGE analysis. Both recombinant proteins reacted to immunoserum to R. prowazekii. However, rpN cross-reacted to immunoserum to R. rickettsii and rpC cross-reacted to immunosera to other agents of Rickettsia genus in immunoblot analysis, but they did not cross-react to the immunosera to other bacteria used in this study. In ELISA analysis, rpN and rpC cross-reacted to immunosera to the agents of Rickettsia genus but immunosera to Coxiella burnetii and Orientia tsutsugamushi. These results suggest that rpN and rpC are antigens specific to Rickettsia genus.R. prowazekii and R. rickettsii were detected by IFA with the immunoserum to rpN or to rpC, but other rickettsial agents were not detected by IFA with the immunosera to the recombinant protein. It indicates that the specific immunosera may be useful to identify R. prowazekii and R. rickettsii.To study the immunogenicity of the recombinant proteins, the recombinant proteins rpN and rpC purified by affinity chromatograph were used to immunize BALB/c mice. After a booster injection, the IgGs to the recombinant proteins rpN and rpC were detected in thesera from the immunized mice by IFA and ELISA. The antigen-specific spleen cell proliferative responses were demonstrated in the immunized mice by MTT method. The animals immunized with rpN and rpC exhibited profound humoral and cellular immunoresponses, including much higher levels of specific IgG antibodies, much higher levels of gamma interferon (IFN-y) and interleukin-2 (IL-2), and much stronger proliferation of splenic cells in response to homogenous antigen as compared with unimmunized mice. The results suggest that the recombinant proteins rpN and rpC are the protective antigenscapable to stimulate mice to produce specific humoral and cellular immunoresponses. In conclusion, the data presented in this paper suggest that the recombinant proteins rpNand rpC having the antigenic properties of 120-kDa surface protein antigen ofR. prowctzekii are the efficient antigens in eliciting humoral and cellular immunity against R. prowozekii and the useful antigens in serological diagnoses for rickettsial diseases.