Construction Of Novel Vaccines For Brucellosis And Evaluation Of Their Immune Protective Effection

Objective To develop novel candidate vaccines for brucellosis in order to lay a foundation for safe and effective prevention to the brucellosis.Methods The DNA fragments encoding the Brucella's protective antigens 0MP16, L7/L12, BCSP31 and P39, and the DNA fragments encoding the fusion protective antigens OMP16-L7/L12, BCSP31-L7/L12 and P39-L7/L12 were cloned and inserted into the prokaryotic expression vector pET32a(+) respectively, and transformed into E. Coli. BL21(DE3). Subsequently all the corresponding proteins were expressed separately in the presence of IPTG, and purified by using affinity chromatography. Meanwhile all the cloned DNA fragments were inserted into the eukaryotic expression vector pcDNA3.1(+) in sense orientation, and transfected into COS-7 cells, and the expression of the target proteins in the cells were identified by immunocytochemistry. At last Balb/c mice were immunized separately with the monovalent and bivalent candidate DNA and protein vaccines, and the immune response was assayed. And the immune protection against the attack of toxic strains of B.abortus 544 in the immunized mice was evaluated.Results(1) All the cloned DNA fragments except P39 DNA were consistent with the corresponding sequences reported in Genbank. All the recombinant proteins including 0MP16, L7/L12, BCSP31, P39, OMP16-L7/L12, BCSP31-L7/L12 and P39-L7/L12 were expressed in both prokaryotic and eukaryotic cells. All the proteins expressed by the prokaryotic system were purified.(2) Immunized Balb/c mice with the purified proteins produced high level specific antibodies, their titers could reach 1:6400-1:25600, and the titers of the antibodies induced by the recombinant bivalent proteins were higher than that induced by the recombinant monovalent proteins, which indicated that the recombinant proteins could stimulate effective humoral immune response, but not the cellular immunity. However the immuneresponse triggered by the proteins could not protect against the attack of A544 in the immunized mice.(3) Immunized Balb/c mice with the candidate monovalent DNA vaccines produced both effective specific humoral and cellular immune responses.The level of IgG2a-specific antibody was higher than that of IgGl-specific antibody,which indicated that a typical T-helper I-dominated immune response was induced. T lymphocyte proliferation assay showed that the ConA-induced proliferation of spleenic T lymphocytes in the immunized mice was increased; ELISPOT assay showed that the number of the specific IFN-y-producing CD8+ T cells was much more than that of the control group; Flow eytometry analysis showed that the ratio of CD4+/CD8+ was lower than that of the control group. The results indicated that the specific CD8+ T cell response was elicited by the candidate monovalent DNA vaccines. The immune protection assay displayed that all the candidate monovalent DNA vaccines could induce effective immune protection against the attack of A544 in the immunized mice.(4) Immunized Balb/c mice with the candidate bivalent DNA vaccines could also produce functional immune response and immune protective effection, which was better than that elicited by the candidate monovalent DNA vaccines.(5) The predominant protective antigens, L7/L12 and P39, were identified by the in vivo experiment in mice, and the DNA vaccines were better than the corresponding recombinant protein vaccines. The bivalent candidate DNA vaccines were more excellent than the monovalent DNA vaccines.Conclusions The prepared novel candidate protein vaccines could elicited humoral immune response, but could not induce effective immune protection in mice.While the prepared candidate DNA vaccines could stimulate both humoral and cellular immune response, which could protect against the attack of A544 in mice.The preliminary date in the present study might pave the way for the further study on developing effective prevention methods to the infection of brucellosis based on vaccine strategy.