Studies On Construction And Immune Mechanism Of Recombinant Bb-Eg95-EgA31 Vaccine Of Echinococcus Granulosus

ObjectiveEg95 and EgA31 coding gene was respectively amplified by RT-PCR from Echinococcus granulosus (Eg) protoscoleces.The Eg95-EgA31 fusion gene was obtained with Gene SOEing,then cloned into Escherichia coli-Bifidobacteria shuttle expression vector pGEX-1λT to construct recombinant plasmid pGEX-Eg95-EgA31.This recombinant plasmid was electroporated into Bifidobacteria bifidum (Bb) and E.coli BL2(DE3) to construct recombinant Bb-Eg95-EgA31 vaccine,with the purpose of exploring the expression of Eg95-EgA31 fusion gene in E.coli BL2(DE3),discussing the dynamics of immune responses induced by the recombinant Bb vaccine in BALB/c mice,and meanwhile investigating the protection against challenge with protoscoleces and immune mechanism in mice after immunization with the recombinant Bb vaccine.This could provide a safe and effective vaccine for the control of cystic echinococcosis(CE).Methods1. The total RNA was extracted from hydatid cyst protoscoleces shattered by ultrasound-breaking,then Eg95 and EgA31 coding gene was respectively amplified by RT-PCR from the total RNA.The Eg95-EgA31 fusion gene was obtained with Gene SOEing,then cloned into Escherichia coli-Bifidobacteria shuttle expression vector pGEX-1λT to construct recombinant plasmid pGEX-Eg95-EgA31.This recombinant plasmid was electroporated into Bifidobacteria bifidum (Bb) and E.coli BL2(DE3) to construct recombinant Bb-Eg95-EgA31 vaccine.The plasmid pGEX-Eg95-EgA31 was transformed into BL21(DE3) by electroporation and the Eg95-EgA31 fusion gene was expressed in the presence of isopropyl-β-D-thiogalactopyranosid(IPTG).The recombinant Eg95-EgA31 fusion protein was analyzed and identified by SDS-PAGE and Western blot.2. To observe the dynamics of humoral and cellular immune responses in mice after immunization with recombinant Bb-Eg95-EgA31 vaccine.BALB/c mice were immunized orally or intranasally by the vaccine.On week 0,2,4,6,8,10,12,14,16,18 and 20 after vaccination,the level of IgG,IgG subclass and IgE in sera was determined by ELISA.The level of IFN-γ,IL-12,TNF-αand IL-10 in splenocyte culture supernatant was measured by ELISA.The level of proliferation in splenocytes was tested by MTT.The percentage of CD₄⁺ and CD₈⁺ T cells in splenocytes was determined by flow cytometry(FCM).3. To explore protection against challenge with protoscoleces and immune mechanism in mice after immunization with the recombinant Bb vaccine.BALB/c mice were immunized subcutaneously, intramuscularly,orally or intranasally by the vaccine,blank vector,Bb or MRS served as control groups,respectively.All mice were challenged with 50 protoscoleces in the 8th week after immunization and killed in the 25th week after challenge.The weight of hydatid cyst was measured and the reduction rate of the weight was calculated.The level of IgG,IgG subclass and IgE in sera was determined by ELISA.The level of IFN-γ,IL-12,TNF-αand IL-10 in splenocyte culture supernatant was measured by ELISA.The level of proliferation in splenocytes was tested by MTT.The percentage of CD₄⁺ and CD₈⁺ T cells in splenocytes was determined by FCM.The apoptotic rate of splenocytes was determined by Annexin V-FITC staining method.Results1. Eg95(471bp) and EgA31(500bp) coding genes,and Eg95-EgA31 fusion gene were successfully amplified by agarose gel electrophoresis analysis.The recombinant plasmid pGEX-Eg95-EgA31 was successfully constructed by restriction analysis.The recombinant Bb-Eg95-EgA31 vaccine of Echinococcus granulosus was successfully constructed by PCR.SDS-PAGE showed that the recombinant Eg95-EgA31 fusion protein was successfully expressed in E.coli BL2(DE3) in the presence of IPTG,and the relative molecular weight of the protein was approximately 62.5kDa.The expression level the protein reached highest at 3～5h after induction,and the expression efficiency of the protein was 18% of the total bacterial proteins.Western blot showed that the expressed fusion protein showed specific antigenicity.2. Dynamic observation showed as follows:Compared with 0 week non immunization group,in the oral immunization group,the level of IgG,IgG1,IgG2a,IgG2b,IgG3 and IgE significantly increased in the 8～10th week,4～8th week,2～20th week,2～20th week,6～12th week,and 10th week after immunization,respectively,and peaked in the 8th,6th,2th,6th,8th and 10th week after immunization,respectively.The level of IFN-γ,IL-12,TNF-αand IL-10 significantly increased in the 2～16th week,2～12th week,2～6th week and 4～12th week after immunization,respectively,and peaked in the 4th,2th,4th and 6th week after immunization,respectively.The level of splenocyte proliferation significantly increased in the 4～10th week after immunization,and peaked in the 6th week after immunization.The percentage of CD₄⁺ T in splenocytes significantly increased in the 4～10th week after immunization,and peaked in the 6th week after immunization.The percentage of CD₈⁺ T in splenocytes had no obvious change.In the intranasal immunization group,the level of IgG,IgG1,IgG2a,IgG2b,IgG3 and IgE significantly increased in the 4～10th week,2～12th week,4～20th week,2～20th week,4～12th week and 10～12th week after immunization,respectively,and peaked in the 10th,8th,6th,10th,8th and 10th week after immunization,respectively.The level of IFN-γ,IL-12,TNF-αand IL-10 significantly increased in the 2～8th week,2～12th week,2～8th week and 6～16th week after immunization,respectively,and peaked in the 2th,2th,4th and 8th week after immunization,respectively.The level of splenocyte proliferation significantly increased in the 4～8th week after immunization,and peaked in the 6th week after immunization.The percentage of CD₄⁺ T in splenocytes significantly increased in the 4～8th week after immunization,and peaked in the 6th week after immunization.The percentage of CD₈⁺ T in splenocytes had no obvious change.3. Protective experiments found that the hydatid cyst weight was reduced by 45.33%,41.33%,70.67% and 62.67% in the subcutaneous,intramuscular,oral or intranasal immunization group,respectively.Compared with the control group,in the immunization groups,the level of IgG,IgG2a,IgG2b and IgG1 significantly increased while the level of IgG3 and IgE significantly decreased.The level of IFN-γ,IL-12 and TNF-αsignificantly increased while the level of IL-10 significantly decreased.The level of splenocyte proliferation significantly increased.The percentage of CD4+ and CD₈⁺ T of splenocytes were significantly increased.The apoptotic rate of splenocytes significantly decreased.Conclusion1. Eg95 and EgA31 coding genes were successfully cloned by RT-PCR.2. The Eg95-EgA31 fusion gene was successfully amplified by Gene SOEing.3. The recombinant plasmid pGEX-Eg95-EgA31 was successfully constructed.4. The recombinant Bb-Eg95-EgA31 vaccine was successfully constructed.5. BL21(pGEX-Eg95-EgA31) could express recombinant Eg95-EgA31 fusion protein in the presence of IPTG.The expression efficiency was 18%,and the expressed fusion protein showed specific antigenicity.6. The recombinant Bb-Eg95-EgA31 vaccine could induce effective immune responses in mice.7. The recombinant Bb-Eg95-EgA31 vaccine could induce protective immune responses in mice,which could be responsible for protection against the challenge with Eg protoscoleces. Intranasal inoculation and oral vaccination are two better immune routes,and the former is better than the latter.