| Coxiella burnetii is an obligate intracellular bacterium and the etiological agent of Q fever. It is an air-borne infectious agent and recognized as an important agent of biological threats. In present study, the protective antigens of C. burnetii, including 27kDa, 30kDa, 34kDa outer membrane protein, and heat shock protein 60 (Hsp60) B subunit (HSPB), were studied in order to choose better protective antigens for construction of the molecular vaccine against Q fever.1. Cloning and sequencing of genes encoding 27kDa, 30kDa, 34kDa protein, and HSPBThe genes encoding 27kDa, 30kDa, 34kDa protein, and HSPB were amplified from the genomes of Xinqiao strain isolated from China and Henzerling strain isolated from Italy by PCR, and the genes were cloned and sequenced. The gene sequences of Xinqiao and Henzerling strain were compared with that of Nine Miles isolated from United States. The similarities of gene sequences of 27kDa, 30kDa, 34kDa protein, and HSPB were more than 99. 1% and the similarities of their amino acid sequences deduced from gene sequences were more than 97. 4% among the 3 strains. The results suggest that these antigens are very conservative among the strains of C. burnetii.By using the software of gene analyses, the B and T cellular epitopes of 27kDa, SOkDa, 34kDa protein, and HSPB were predicted. The results showed that there were several B and T cellular epitopes in the deduced amino acid sequence of each protein, suggesting these protective antigens may efficiently induce the immune response in bodies.2. Construction of the recombinant prokaryotic expression plasmids and the immunization of mice with the recombinant proteinsThe genes encoding 27kDa, SOkDa, 34kDa protein, and HSPB were linked to the prokaryotic expression vector pQE30, constructing recombinant plasmids pQE/27, pQE/30, pQE/34, and pQE/hspB. E. coli cells transformed with these recombinant plasmids expressed the recombinant proteins, respectively, induced by IPTG.. The 27kDa, 30kDa, 34kDa, and HSPB recombinant protein reacted to the serum from the mice infected with C. burnetii, suggesting that these proteins had the capabilities to induce humoral immune response in bodies.27kDa, 30kDa, 34kDa, and HSPB recombinant protein were purified by affinity chromatograph and they were used to immunize the mice (BABL/c). The sera and spleen cells were collected from immunized mice 28 days after boost immunization. The specific humoral and cellular immune levels of the immunized mice were evaluated by ELISA to detect the sera and by lymphocyte proliferation assay to to examine the spleen cells,respectively. The results demonstrated that these recombinant proteins stimulated mice to produce specific humoral and cellular immunity.The mice were challenged with 10 ID50 C. burnetii 28 days after boost immunization and the lungs, livers, and spleens from the mice were examined 7 days after challenge. The pathological changes in lungs and the organisms of C. burnetii in spleens were found the mice immunized with 27kDa protein. The mild pathological changes in lungs and a few organisms of C. burnetii in spleens were observed in the mice immunized with SOkDa, 34kDa, or HSPB protein. However, the severe pathological changes in lungs, livers, and spleens and severe infection of C. burnetii in spleens were recognized in mice unimmunized. These results suggest that 30kDa, 34kDa, and HSPB protein are better than 27kDa protein in immunogenicity. Although the antibody levels of the mice immunized with SOkDa protein was lower than that of mice immunized with 27kDa, 34kDa, or HSPB protein, their levels of lymphocyte proliferation and the abilities against the challenges of C. burnetii were higher than that of the mice immunized with other proteins. This result indicates that the specific cellular immunity is much more important than the humoral immunity in immunity against C. burnetii.The 30kDa protein gene and 34kDa protein gene were fused into one and the fused gene was recombined with pQE/J?0to construct the pQE/30-34 plasmid. The...
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