Preparation Of Fusion Proteins Of Outer Membrane Protein With HspB Of Coxiella Burnetii And Studies On Its Immunogenicity

Coxiella burnetii is an obligate intracellular bacterium that multiplies within the phagolysosomes of eukaryotic cells and causes a worldwide zoonotic disease, Q fever. It is an air-borne infectious agent and recognized as an important agent of biological threats. Inoculation of vaccines against Q fever is a key way to prevent the human infection caused by C. burnetii.Previous studies demonstrated that the outer membrein proteins of C. burnetii with molecular masses of 29kDa (P1), 27kDa (Com1), and 34kDa(SP34) were recognized as immunodominant antigens. Heat shock protein B (HspB) was also an immunodominant antigen of C. burnetii. In addition, HSPs were recognized as immune adjuvants. Therefore, the outer membrein proteins of C. burnetii were fused with HspB to construct fusion proteins and the fusion proteins were analyzed in immunogenicity and immunoprotection in this study.The gene fragments, p1, com1, sp34, and htpB were cloned from C. burnetii genomic DNA and the cloned gene frogments p1, com1, and sp34 were fused with htpB to construct expression plasmids pQE30/p1-htpB, pQE30/com1-htpB, and pET2Sa/htpB-sp34. The fusion proteins, P1-HspB, Com1-HspB, and HspB-SP34 were expressed in E. coli cells transformed with the expression plasmids, respectively. The resulting fusion proteins, P1-HspB, Com1-HspB, and HspB-SP34 were recognized by immunosera to C. burnetii.The purified fusion proteins of C. burnetii were used to immunize BALB/c mice and the specific antibody, cytokines (IL-2 and IFN-γ), and splenic T-cell proliferation of the immunized mice were analyzed. The results showed that the levels of specific antibody and IL-2 and IFN-γof mice immunized with P1-HspB fudsion protein were significantly higher than that of mice immunized with P1 recombinant protein. In addition, the specific antibody levels of mice immunized with Com1-HspB or HspB-SP34 fusion protein were significantly higher than that of mice immunized with Com1 or SP34 recombinant protein.After challenge of C. burnetii, the coxiella loads of spleens of mice were detected by quantitative real-time PCR assay. The coxiella loads of spleens of mice immunized with P1-HspB, Com1-HspB, or HspB-SP34 fusion protein were significantly lower than that of mice immunized with single protein, P1, Com1, or SP34.In conclusion, the data presented in this study suggest that the fusion antigens, P1-HspB, Com1-HspB, and HspB-SP34 of C. burnetii are more efficient antigens in eliciting humoral and cellular immunoresponses against Q fever compared with recombinant proteins P1, Com1, and SP34, and the fused HspB play a key role for their enhanced immunigeinicity and immunoprotectivity. The fusion antigens, P1-HspB, Com1-HspB, and HspB-SP34, may be new canadidates for developing of new molecular vaccine against Q fever.