Construction Of Recombinant Ef(pGEX-PopB) Vaccine Of Pseudomonas Aeruginosa And Its Immune Mechanism

ObjectiveTo construct the Enterococcus faecium-based Pseudomonas aeruginosa prolyl oligopeptidase B(PopB)vaccine and to explore its protective immune mechanism in mice.MethodsThe DNA of P.aeruginosa PA01 strain was used as the template to amplify the PopB gene by PCR.It was introduced into the pGEX-1λT vector,and then the obtained recombinant plasmid pGEX-PopB was subjected to the transformation of E.coli BL21(DE3).The PopB gene was inducted in the presence of IPTG.The products were assessed and identified by SDS-PAGE and Western blot.Subsequently,the recombinant plasmid pGEX-PopB was electroporated to transform the Ef,and the recombinant Ef-PopB vaccine was constructed.Following double enzyme digestion and PCR for identification,the expression level was examined.BALB/c mice received intragastrical administration with recombinant vaccine.14 days later after challenge with PA01 strain,the mice were sacrificed for serum samples.ELISA was performed for detecting relative levels of Ig G,its subclasses and Ig E in mouse sera.The lung tissues were collected,which were subjected to the analysis of bacterial load.The spleens were separated to prepare its cell suspension,and the culture was stimulated with Pseudomonas aeruginosa antigen(Pa Ag)and Concanavalin A(Con A).MTT and FCM assay were performed to detect proliferative,subpopulation and apoptosis of splenocytes vaccinated with the recombinant Ef-PopB.In addition,relative level of CK in splenocytes was detected by PCR.ResultsThe 1200-bp PopB gene was successfully amplified.The PopB gene was introduced into the pGEX-1λT and transformed to E.coli BL21(DE3).Mr of the expressed PopB protein from recombinant bacteria at approximately 66 k Da was determined by SDS-PAGE,and its expression products account for 20%of total bacteria.Western blot results have confirmed its antigenicity.The recombinant plasmid pGEX-PopB was successfully transformed to Ef,and the PopB from recombinant Ef vaccine(pGEX-PopB)Mr was about 66 k Da,which was consistent with expected result.Moreover,it could be specifically recognized by the crude antigen immunized mouse serum of the outer membrane protein Pa.Compared with control group,the bacterial load in the lungs of vaccine group decreased.The serum Ig G and its subgroup,and Ig E were significantly higher in vaccine group than those of controls.The ratio of CD4~+T cells and proliferative activity of splenocytes in vaccine group were more than control.In addition,the apoptotic rate was less than control.Splenocytes in the vaccine group were able to amplify gene fragments of IL-2,IL-4,IFN-γ,IL-10,IL-12 and Foxp3.ConclusionThe rEf(pGEX-PopB)vaccine was successfully constructed.It may induce mixed immune response of Th1 and Th2 and provide effective protection against Pa infected mice.