| ObjectSchistosomiasis japonica is one of serious parasitic diseases which is prevented in plan in our country,and to develop new vaccines which could be used in clinical to prevent Sj and could cover the shortages of the past ones at the same time is necessary. To amplify Sj32 gene and transformed it into Bb to construct recombinant Bb(pGEX-Sj32) vaccine.And Trying to investigate the immunologic mechanisms of this vaccine by observing the dynamic change of immune response on BALB/c mice after immunized with rBb,so as to provide a new vaccine for control of schistosomiasis.MethodsSj32 gene was amplified by PCR from template of plasmid pET28a-Sj32 which was extracted from recombinant bacterium BL21(pET28a-Sj32) stored by our laboratory,and then the Sj32 gene and the vector pGEX-1λT were connected by gene recombination technology to construct pGEX-Sj32.The recombinant plasmid pGEX-Sj32 was transformed respectively into Bb to construct restructured Bb (pGEX-Sj32) vaccine and into BL21(DE3) to study the expression of pGEX-Sj32 under the environment of IPTG induction. SDS-PAGE and Western blot were used to identifiy the expressed product.BALB/c mice were immunized with Bb(pGEX-Sj32) vaccine through per os(PO) and intranasal (IN) respectively, and then dynamic changes of immune response during 0～20 weeks after immunization was observed. Enzyme-linked immunosorbent assay (ELISA) was performed to detect the levels of IgG,IgG subclass,IgE and IgA.Double antibody sandwich ELISA method was used to detect the levels of cytokines(TNF-a, IL-10 and IL-12) in splenocyte cultures and in serum,splenocyte proliferation during 0-20 week after immunized was analyzed by MTT colorimetric assay, subsets of T(CD4+and CD8+) cells in spleen and apoptosis of splenocytes were measured by flow cytometry (FCM), respectively.Results1.The gene Sj32 of 1270 bp in length was amplified by PCR which confirmed by agarose gel electrophoresis.The restriction endonuclease digestion proved that gene Sj32 was successfully connected with vector, and the recombinant plasmids pGEX-Sj32 was successfully constructed-PCR showed the recombinant Bb(pGEX-Sj32) vaccine was successfully preparated.SDS-PAGE confirmed that recombinant plasmids pGEX-Sj32 could express the protein of 58 kDa under induction of IPTG in BL21 and highly expressed in 5-7h after induction.The recombinant proteins can be identified by serum from rabbits infected with Schistosomajaponicum.2.Except for IgE reached peak on the 14th week, all mice developed significant peaks of IgG, IgG1, IgG2a, IgG2b, IgG3 and IgA on the 8th week in PO group and those appeared on the 12th(IgG2b and IgG3) week and on the 8th(IgG,IgG2a and IgA) week in IN group, respectively. The level of IL-10, IL-12 and TNF-awhich detected in serum peaked on the 14th,6th and 8th week respectively in group PO, and got the maximum on the 8th,6th and 8th week in group IN, respectively. The maximum proliferation efficiencies of both PO and IN group got on week 6 after immunization, while the apoptotic rates appears on 6th week without any stimulation as well as on 8th week with ConA. Rates of CD4+T and CD8+T cells subsets reached to the peak on 6th and 8th week in PO group after immunization while on 8th and 12th week in IN group, respectively. The level of IL-10, IL-12 and TNF-ain splenocyte supernatant peaked on the 10th,6th and 8th week in PO group, while got the maximum on the 12th, 8th and 8th week in IN group, respectively.Conclusion1.The gene Sj32 was connected with vector and then the recombinant plasmids pGEX-Sj32 were successfully constructed.2.Transforming pGEX-Sj32 into Bb and successfully constructed the recombinant Bb(pGEX-Sj32)vaccine.3.The recombinant plasmids pGEX-Sj32 in BL21 could express recombinant protein of 58 kDa which has strong immunogenicity.4.The rBb(pGEX-Sj32) vaccine may induce mice to produce immune response effectively. |
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