Primary Studies On Construction And Immune Mechanism Of Recombinant Bb-OprI Vaccine Of Pseudomonas Aeruginosa

Objectives To construct the recombinant Bb-OprI vaccine of Pseudomonas aeruginosa(Pa),and to study the protective effect and immune mechanism of the vaccine in mice by immunization with the vaccine against Pseudomonas aeruginosa.Methods The OprI encoding gene was amplified by PCR and cloned into the prokaryotic expression vector pGEX-1?T to construct the recombinant plasmid pGEX-OprI.The pGEX-OprI was transformed to Escherichia coli BL21(DE3)by eletroporation,and the expression products were analyzed and identified by SDS-PAGE and Western blot.The Bb-OprI vaccine was constructed when the recombinant plasmid were transformed into Bifidobacterium bifidum(Bb)by electroporation,and the expression products were analyzed and identified by SDS-PAGE and Western blot.After intragastric immunization with the recombinant Bb-OprI vaccine to mice,the mice were challenged by PA01 strain of Pa.The lungs and spleens were removed.The bacterial loads in the lung tissue were detected.The serum antibodies were detected by ELISA.The proliferation of splenocytes was detected by MTT assay.The impacts of the vaccine on T lymphocyte subsets and apoptotic rate of splenocytes were identified by flow cytometry.The changes of spleen cytokines and Treg were analyzed by PCR.Results The OprI gene of 194 bp was successfully amplified,double enzyme digestion and PCR identification confirmed that the OprI gene was successfully cloned into pGEX-1?T,and pGEX-OprI was successfully transformed into BL21(DE3).The fusion protein GST-OprI with Mr of approximately 32 kDa was expressed by BL21(DE3).The fusion protein could be specifically identified by the sera of the mice infected with Pa.The pGEX-OprI was successfully transformed into Bb,and the Bb-OprI vaccine expressed a fusion protein GST-OprI with Mr of 32 kDa approximately.The fusion protein could be specifically identified by the sera of the mice infected by Pa.After the vaccination with Bb-OprI vaccine and challenge with PA01 strain,bacterial load in lung tissue of Bb-OprI group decreased.The serum antibodies IgG,IgG2b,IgG3 and IgE levels in vaccine group increased.The proliferation of splenocytes and proportion of CD4+ T cells increased,while the apoptosis of splenocytes decreased.The IL-2,IFN-?,IL-12,IL-4,IL-10,IL-17,and Foxp3 genes of slenocytes of mice could be amplified by PCR in vaccine group,but not in two control groups.Conclusion The recombinant Bb-OprI vaccine was successfully constructed.The recombinant Bb-OprI vaccine may induce BALB/c mice to produce a protection against Pa infection and maintain immune homeostasis through humoral immunity,cellular immunity and immunoregulation.