Animal Immunological Evaluation Of A New Kind Of Brucella Submit Vaccine BP26

ObjectiveTo observe the biologic activity and immune protective effect of Brucella recombinant protein BP26 after combined with adjuvants (aluminum or CpG) in mice and guinea pigs , and offer some evidence from laboratory for researching a new kind of Brucella vaccine.Methods1. Immunological evaluation of the mice immunized with recombinant protein BP26:①The Balb/c mice were immunized with Brucella protein BP26 combined with adjuvants (aluminum or CpG);②lymphocytes proliferation test was performed to observe lymphocytes proliferation by MTT test;③Splenic lymphocytes were isolated for enzyme-linked immunosorbent spot assay to detect the secreted specific interferon-gamma(IFN-γ);④Splenic lymphocytes were isolated for enzyme-linked immunosorbent spot assay to detect the secreted specific interleukin-4( IL-4);.⑤The lymphocytes in peripheral blood was grouped by the FCM assay;⑥the antibody titers of IgG, in the serum were measured by ELISA;⑦the antibody titers of IgG1, IgG2a in the serum were measured by ELISA;⑧the immune protection against the attack of Brucella 104M in the immunized mice was evaluated.2. Immunological evaluation of the guinea pigs immunized with recombinant protein BP26:①The guinea pigs were immunized with Brucella protein BP26 combined with adjuvants (aluminum or CpG);②The DTH were tested on those guinea pigs at 4 and 8 weeks post-last immunization;③And the antibody titer of IgG in the serum was measured by ELISA.Results1. Results of the experiment on Balb/c mice immunized with recombinant protein BP26:①The results of lymphocytes proliferation test: The SIs of lymphocytes proliferation test of vaccine groups were higher than those of control groups (p<0.05).②The results of enzyme-linked immunosorbent spot assay to detect the secreted specific interferon-gamma(IFN-γ) : The secreted specific IFN-γfrom isolated splenic lymphocytes after having been stimulated in vitro with BP26 and Br.PPD remarkably increased in groups immunized by BP26 and that combined with adjuvants;the changes were significantly different between these three groups and control groups (P<0.05).③The results of enzyme-linked immunosorbent spot assay to detect the secreted specific interleukin-4(IL-4): The secreted specific IL-4 from isolated splenic lymphocytes after having been stimulated with BP26 remarkably increased in group immunized by BP26+Al.④The results of grouping the lymphocytes in peripheral blood by Flow cytometry assay: The percent of CD4~+ lymphocytes in BP26 and BP26+Al groups is higher than that in NS and Al groups, p<0.05. While, in BP26+CpG group, the percent of CD8~+ lymphocytes is higher than that in CpG group, the value of CD4~+/CD8~+ was lower than that in CpG group,p<0.05.⑤The results of the antibody titers of IgG: IgG titers of BP26 group and BP26+ CpG group could reach 1:3200, while, BP26+Al group could reach 1:29405.⑥The results of the antibody titers of IgG1and IgG2a: IgG1and IgG2a titers of BP26+Al group were the highest in all groups. IgG1 titer could reach 1:67558, and the IgG2a titer could reach 1:8445. And , IgG1/ IgG2a>1.⑦The results of Brucella attacking test : The numbers of spleen bacteria in BP26 group and BP26+CpG group were less than NS group and CpG group, p<0.05. 2.Results of the experiment on guinea pigs immunized with recombinant protein BP26:①The results of guinea pigs skin test: The skin test results showed that the red areas diameters of BP26,BP26+Al and BP26+CpG groups were more than 10mm, while that of NS group could not be detected.②The results of IgG titers in guinea pigs serum by ELISA assay : IgG antibody levels of BP26,BP26+Al and BP26+CpG groups were higher than those of control groups (p<0.05). The A450 values of BP26,BP26+Al and BP26+CpG groups were between 1.46-2.41, while the A450 values of control groups were 0.4-0.6.Conclusions1. The results showed that BP26 protein could activated the cellular immunity and humoral immunity in mice.2. Among the vaccine groups ,the dectected results of cellular immunity and humoral immunity in the groups with adjuvants were higher than BP26 group without any adjuvant.The cellular immunity in BP26+CpG grous was enhanced ,while the humoral immunity in BP26+Al was enhanced.3. The protection efficiency in BP26+CpG was the best among vaccine groups.4. BP26 protein activated the cellular immunity and humoral immunity in guinea pigs.