Studies On Construction And Immune Mechanism Of Recombinant Bb(pGEX-OprF-I) Vaccine Of Pseudomonas Aeruginosa

ObjectiveTo construct the Bifidobacterium bifidum-vectored outer membrane protein F-I vaccine of Pseudomonas aeruginosa and study its protection and immune mechanism on mice.MethodsThe Genomic DNA was extracted from Pseudomonas aeruginosa PA01 strain,acted as a temple,the OprF and OprI antigen gene was respectively amplified by PCR.The OprF-I fusion gene was obtained by gene SOEing,ligated to pGEX-1?T to obtain pGEX-OprF-I,and then electroporated into E.coli BL21(DE3).After IPTG induction,SDS-PAGE and Western blot were used to analyze the expression of recombinant bacteria.The recombinant plasmid was electroporated into Bifidobacterium bifidum to construct rBb(pGEX-OprF-I)vaccine.The vaccine was identified by double enzyme digestion and PCR,and then analyze and identify its induced expression products.Mice were immunized with rBb(pGEX-OprF-I)vaccine by intragastric administration and sacrificed two weeks after challenge with PA01 strain.The Serum antibodies were detected by routine ELISA.The lung tissues were separated to count lung bacteria loads.The spleens were separated to prepare splenocytes suspension.The proliferation,subpopulation and apoptosis of splenocytes were detected by MTT assay and FCM respectively,and the changes of spleen cytokines were detected by PCR.ResultsAgarose gel electrophoresis confirmed that the 1050 bp OprF gene,194 bp OprI gene and 1289 bp OprF-I fusion gene were successfully amplified.The plasmid pGEX-OprF-I was constructed successfully and electroporated into BL21(DE3).The Mr about 68×10~3 fusion protein was expressed by recombinant bacteria by SDS-PAGE analysis,and the fusion protein had antigen specific by western blot.The pGEX-OprF-I successfully transformed into Bb.The rBb(pGEX-OprF-I)vaccine could express a fusion protein with Mr about 68×10~3,and the protein was specifically recognized by the serum of Pa-infected mice.The number of lung bacterial colonies in the vaccine group was lower than that in the control groups,and the serum IgG and its subclass levels increased.The proliferation activity and CD4~+T cell percentage of splenocytes rose,but the apoptosis rate decreased.DNA extracted from the splenocytes of vaccine group was used as a temple to amplify IFN-?,IL-12,IL-4,IL-17and Foxp3 gene fragments by PCR.ConclusionThe rBb(pGEX-OprF-I)vaccine was constructed successfully,which may produce humoral and cellular immune responses and protect mice against Pa infection.