| ObjectTo construct and determine the expression of the recombinant Bb(pGEX-Sj14-3-3) vaccine against Schistosoma Japonicum(sj);To dynamically investigate the immune responses induced by the rBb(pGEX-Sj14-3-3) vaccine in BALB/c mice immunized orally and intranasally respectively,It could provide an valuable vaccine for the control of Schistosomiasis.MethodsThe gene encoding Sj14-3-3antigen was amplified by RT-PCR from template of the total RNA extracted from adult worms of Sj,then cloned into pGEX-1λT to construct the recombinant plasmid pGEX-Sj14-3-3,and the plasmid pGEX-Sj14-3-3was transformed into F.coliBL21(DE3),and then electroporated into Bifidobacteria bifidum(Bb) to construct recombinant Bb(pGEX-Sj14-3-3) vaccine.BL21(pGEX-Sj14-3-3) was induced with isopropyl-β-D-thiogalactopyranosid(IPTG),then the expressed products were identified by SDS-PAGE and Western blotting.To investigate the dynamic change of immune response in mice after immunized with the rBb(pGEX-Sj14-3-3) vaccine.96BALB/c mice were randomly divided into two groups with48mice per group.Mice were orally and intranasally immunized with the vaccine respectively. On weeks0,2,4,6,8,10,12,14,16,18,20and22after vaccination,the levels of serum IgG,IgG subclass,IgE and IgA were detected by ELISA.Proliferative response of splenocytes were determined by methyltetrazolium(MTT).The percentage of T-Lymphocyte subsets and the rates of splenocytes apoptosis were tested by flow cytometry(FCM).The production of IFN-γ,IL-12,TNF-α and IL-10were quantified by ELISA in the supernatant of cultured splenocytes.Results399bp Sj14-3-3coding gene was successfully amplified by RT-PCR,and the recombinat plasmid pGEX-Sj14-3-3was identified by using BamH Ⅰand EcoR Ⅰ.The construction of rBb(pGEX-Sj14-3-3) vaccine was identified successfully by PCR.The expressed protein of40kDa was detected by SDS-PAGE in E.coli BL21(DE3) induced by IPTG.The level of expressed protein came to the highest in3～7h,and the rate was about21%of the total bacterial proteins. Western blot confirmed that the protein could be recognized specifically by the sera from rabbit infected with Schistosomajaponicum. The results of dynamic study were showed as follows:Compared with0week,the level of IgG,IgG1,IgG2a,IgG2b,IgG3,IgE and IgA of the orally immunized mice achieved peaks in the8,6,6,4,8,10and6th week respectively.However,and the level in the intranasal immunization group peaked in the4,6,4,4,8,10and8th week respectively.The proliferation and the rates of apotosis of splenocytes came to peaks in the4th week in both groups.The percentage of CD4+cells peaked in the8th week,but the percentage of CD8+cells in the14th week.Meanwhile,in the intranasally immunized group,the percentage of CD4+cells also peaked in the8th week,while the percentage of CD8+cells in the4th week.The values of IFN-y,IL-12,TNF-a and IL-10reached the highest level in the8,8,6and4th week respectively in the orally immunized group,while they achieved the highest values respectively in the2,2,4and4th week after intranasal immunization.Conclusion1.The recombinant plasmid pGEX-Sj14-3-3and Bb(pGEX-Sj14-3-3) vaccine were constructed successfully.2.The plasmid pGEX-Sj14-3-3was able to express40kDa Sj14-3-3/GST fusion protein in E.coli BL21(DE3) induced by IPTG,Western blot confirmed the expressed protein showed antigenic specificity. 3.The recombinant Bb(pGEX-Sj14-3-3) vaccine could induce effective humoral and cellular immune responses in BALB/c mice immunized orally and intranasally. |
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