Preliminary Studies On Construction And Immune Mechanism Of Recombinant Bb-OprH Vaccine Of Pseudomonas Aeruginosa

ObjectiveTo construct the recombinant Bb-OprH vaccine of Pseudomonas aeruginosa,to study its protection and immune response after the BALB/c mice were immunized with the recombinant vaccine and challenged with PAO1 strain.MethodsThe PCR amplified gene was cloned into pGEX-1?T to obtain the recombinantplasmidpGEX-OprH;thenthepGEX-OprHwas electroporated into E.coli BL21(DE3).The target protein expressed was analyzed by SDS-PAGE and its immunogenecity was identified by Western blot.BALB/c mice were immunized intragastrically with the rBb vaccine and killed two weeks after the challenge.The pulmonary bacterial loads were counted.ELISA was used to detect the serum antibodies.MTT assay was applied to detect the proliferation of splenocytes.Flow cytometry was employed to identify T lymphocyte subsets and apoptotic rate of splenocytes.PCR was used to analyze the changes of spleen cytokines and Treg cell.ResultsPCR successfully amplified the 660 bp OprH gene.PCR identification confirmed that the OprH gene was cloned successfully into vector pGEX-1?T.The recombinant plasmid transformed E.coli BL21(DE3)successfully.SDS-PAGE showed that the recombinant bacteria expressed a fusion protein with Mr of approximately 47 kDa and Western blot indicated that the fusion protein could be recognized by the sera of mice infected with Pa.After immunization with Bb-OprH vaccine and challenge with PAO1 strain,the pulmonary bacterial loads in the mice of the vaccine group decreased,their sera antibody levels of IgG,IgG2a,IgG2b,IgG3 and IgE increased,the proliferation of splenocytes rose up and the number of CD4~+T cell subsets did so,but the apoptosis of splenocytes decreased.The differences were significant compared with the control group.PCR amplified successfully the splenocytes IL-2,IFN-?,IL-12,IL-17 and Foxp3genes from the DNA abstracted from the splenocytes of the vaccine group,but not of the control group.ConclusionThe recombinant Bb-OprH vaccine was constructed successfully.The vaccine may induce the mice to produce a protective humoral and cellular immune response and enhance their resistances to Pseudomonas aeruginosa.