| ObjectSchistosomiasis japonicum is one type of parasitic disease which isharmful to people’s health caused by Schistosoma japonicum (Sj). Becauseof its severely damage, it’s imperative to develop the effective vaccine toprevent and control this disease. This study intends to constructrecombinant Bifidobacterium bifidum (Bb)(pGEX-Sj26GST-Sj32) vaccineagainst Schistosoma japonicum, and to study its dynamic changes of theimmune response after BALB/c mice immunized by the vaccine, so as toprovide a new way for the immune precaution of schistosomiasisjaponicum.MethodsThe Sj26GST-Sj32fusion gene was amplified by PCR from plasmidpET28α-Sj26GST-Sj32which was extracted from recombinant bacteriaBL21(DE3)(pET28α-Sj26GST-Sj32) stored in our laboratory. Then the fusion gene was cloned into Escherichia coli(E. coli)-Bifidobacteria shuttleexpression vector pGEX-1λT to construct recombinant plasmidpGEX-Sj26GST-Sj32. The recombinant plasmid was transformed into E.coli (BL21), and it was extracted and identified by using BamHⅠandEcoRⅠ. Then pGEX-Sj26GST-Sj32was electroporated into Bb toconstruct recombinant Bb(pGEX-Sj26GST-Sj32) vaccine. The plasmidpGEX-Sj26GST-Sj32from recombinant Bb was extracted and identified byPCR. Then the expression products of pGEX-Sj26GST-Sj32induced withisopropyl-β-D-thiogalactoside (IPTG) was examined by SDS-PAGE andWestern-blot.96BALB/c mice were immunized with the recombinant vaccine orallyand intranasally respectively,48mice in each group. Then autopsies weremade periodically (at different time points of week0,2,4,6,8,10,12,14,16,18,20and22after immunization) to separate sera to determine thelevels of antibodies and cytokines by ELISA, and to separate splenocytes todetermine proliferative response using the methyltetrazolium (MTT) assayafter being cultured under activation of SjAWA or concanavalin A(ConA),the stock (non-stimulation) actvation was used as control; CD4+and CD8+Tcells of spleen were determined by flow cytometry (FCM); Aftersplenocytes were cultured under activation of SjAWA andConA,supernatants were collected to determined the levels ofinterleukin-10(IL-10) and IL-12by ELISA, with the stock group as a control; After splenocytes cultured by ConA were collected to determinedthe appoptotic rates of splenocytes by FCM, with the stock group as acontrol.ResultsThe Sj26GST-Sj32fusion gene of1991bp in length was amplified byPCR; The electrophoretogram of the products digested by BamHⅠandEcoRⅠdemonstrated that the Sj26GST-Sj32fusion gene was cloned intothe plasmid pGEX-1λT; The products of fusion gene Sj26GST-Sj32of1991bp in length was also amplified by PCR from the extracted plasmid ofthe recombinant Bb(pGEX-Sj26GST-Sj32) vaccine; SDS-PAGE analysisshowed that the protein with85kDa could be expressed after therecombinant plasmid pGEX-Sj26GST-Sj32was induced by IPTG in E.coliBL21, the level of expressed protein came to the highest in5～7h;Western-blot found that the protein can be recognized by rabbit serainfected with Sj.It’s found that the levels of IgG, IgG1, IgG2a, IgG2b, IgG3, IgE, IgA,IL-10and IL-12from sera in oral group reached to peaks on week6,8,8,4,6,12,6,12and6respectively, while those in intranasal group did so onweek4,2,6,2,4,8,8,6and6respectively; The levels of splenocyteproliferation in per os (PO) group reached to peaks on week4afterimmunization,while the levels in intranasal (IN) group did so on week12;The levels of CD4+T cells subsets in PO group reached to peaks on week4 after immunization, while the levels in IN group did so on week12. Thelevels of CD8+T cells subsets in PO group reached to peaks on week10after immunization, while the levels in IN group did so on week6; TheIL-10levels both in PO and IN group reached to peaks on week6afterimmunization. The IL-12levels in PO group reached to peaks on week6after immunization, while the levels in IN group did so on week12; Theappoptotic rates of splenocytes in PO group reached to peaks on week6after immunization, while that in IN group did so on week12.ConclusionsThe recombinant Bb(pGEX-Sj26GST-Sj32) vaccine of Schistosomajaponicum was constructed successfully; The recombinant vaccine couldinduce effective immune responses of both Th1and Th2types in early timeof immunization. |
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