| Abstract | ObjectiveTo observe dynamically the changes of the immune responses induced in BALB/c mice immunized intragastrically or intranasally with the leaf protein extracted from the transgenic alfalfa(Medicago sativa) containing Eg95-EgA31 fusion gene of Echinococcus granulosus(Eg);To study the protective immunity and immune mechanism in BALB/c mice immunized by the leaf protein and challenged with Eg protoscoleces.MethodsThe leaf protein was extracted from the transgenic alfalfa containing Eg95-EgA31 fusion gene of Eg by heat-coagulation method,its concentrat–ion was prepared for 20μg/μl.Meanwhile, leaf protein extracted from the transgenic alfalfa containing pBI121 and normal alfalfa served as control.To study the dynamic changes of the immune responses induced in BALB/c mice by immunization with the transgenic alfalfa,88 female BALB/c mice were randomly divided into 2 groups,44 in each group.The two Groups were immunized intragastrically (100μl)and intranasally (10μl)respectively with the leaf protein containing Eg95-EgA31 fusion antigen. 5% sodium bicarbonate (60μl) were given to the mice of intragastrical immunization to neutralize stomach acid 30 min ahead of immunization. All mice were immunized once per 3 days for 2 months.Serum antibody titers of IgG, IgG1,IgG2a, IgG2b,IgG3 and IgE were detected by ELISA on the 0th week,2th week,4th week,6th week,8th week,10th week,12th week,14th week,16th week,18 th week and 20th week post immunization. Meanwhile, the levels of IFN-γ,IL-12,TNF-αand IL-10 produced by splenocytes with or without Eg-Ag and ConA(LPS) stimulation were examined by ELISA too.The proliferation of splenocytes were measured by MTT method, the number of CD4+ and CD8+ subsets of splenocytes were detected by FCM at the above-mentioned time.To study protective immunity and immune mechanism in BALB/c mice after immunization with the leaf protein and challenged with Eg protoscoleces.32 female BALB/c mice were divided into 4 groups randomly.Group A and B were immunized intragastrically(100μl)and intranasally (10μl) respectively with the leaf protein containing Eg95-EgA 31 fusion antigen,group C was vaccinated intranasally by 10μl leaf protein with pBI121,group D was given intragastrically 100μl normal leaf protein. 5% sodium bicarbonate (60μl)were given to the mice of intragastrical immunization to neutralize stomach acid 30 min ahead of immunization. All mice were immunized once per 3 days for 2 months.The mice in all groups were challenged intraperitoneally with Eg protoscoleces(50 protoscoleces per mouse) on the 8th week after the last vaccination and sacrified on the 24th week after infection.The weight of hydatid cysts was measured and the weight-reducation rate was calculated. Serum antibody titers of IgG, IgG1,IgG2a, IgG2b,IgG3 and IgE were respectively detected by ELISA.Sandwich ELISA kits were used to examine the levels of IFN-γIL-12,TNF-αand IL-10 produced by splenocytes with or without Eg-Ag and ConA (LPS)stimulation. The proliferation of splenocytes was measured by MTT method, the counts of CD4+ and CD8+ subsets of splenocytes were detected by FCM and the apoptotic rates of splenocytes with or without ConA stimulation were examined by Annexin V-FITC kits.ResultsThe dynamic observation showed that in the oral immunization group,the antibody titers of IgG,IgG2a,IgG2b,IgG1,IgG3 and IgE increased significantly from week 2 to week 14,week 2 to week 14,week 4 to week 20, week 6 to week 12, week 6 to week 12,and week 4 to week 16, respectively,peaking on week 4,week 4,week 6,week 8,week 8 and week 10,respectively;the splenocytes proliferation increased significantly from week 4 to week 10,peaking on week 6;the percentages of the CD4+ and CD8+ subsets increased significantly from week 6 to week 10 and week 4 to week 12,respectively,peaking on week 6 and week 8,respectively;the levels of IL-12,IFN-γ,TNF-αand IL-10 in the splenocytes culture supernatant increased significantly from week 4 to week 6,week 2 to week 8,week 2 to week 6 and week 4 to week 12,respectively,peaking on week 4,week 2,week 2 and week 8,respectively.In the intranasal immunization group,the antibody titers of IgG,IgG2a,IgG2b,IgG1,IgE and IgG3 increased significantly from week 2 to week 14,week 2 to week 18,week 4 to week 10,week 6 to week 14,week 6 to week 12 and on week 8,respectively, peaking on week 4,week 4,week 6,week 12,week 6 and week 8,respectively ;the splenocytes proliferation increased significantly from week 4 to 12, peaking on week 6;the percentages of the CD4+ and CD8+ subsets increased significantly from week 4 to week 6 and week 4 to week 10,respectively, peaking on week 6 and week 8,respectively;the values of IL-12, IFN-γ,TNF-αand IL-10 increased significantly from week 4 to week 6, week 2 to week 10,week 4 to week 10,and week 6 to week 16,respectively, peaking on week 6,week 4,week 6 and week 6 respectively.On the 24th week after mice challenged with protoscoleces,we found that as compared with group D,the hydatid cysts weight of group A decreased by 64.1%;the splenocytes proliferation of group A increased significantly;the percentages of CD4+ and CD8+subsets and the ratio of CD4+/CD8+ of group A increased significantly;the antibody titers of IgG,IgG2b and IgE of group A increased significantly;the levels of IFN-γ,IL-12 and TNF-αof group A increased significantly;while the level of IL-10 and the splenocytes apoptotic rate of group A decreased significantly. There was no obvious difference between group B and group D about the average weight of hydatid cysts.As compared with group D,the splenocytes proliferation of group B increased significantly;the percentage of CD4+subset and the ratio of CD4+/CD8+ of group B increased significantly;the antibody titers of IgG,IgG2b and IgE of group B increased significantly;the values of IFN-y and TNF-a of group B increased significantly;while the splenocytes apoptotic rate of group B decreased significantly. As compared with group B, the splenocytes proliferation of group A increased significantly;the percentage of CD4+ subset of group A increased significantly;the level of IgG of group A increased significantly;the values of IFN-y and IL-12 of group A increased significantly;the splenocytes apoptotic rate of group A decreased significantly;but there was no significant difference about the average weight of hydatid cysts between the two groups.No obvious difference could be found about the results between group C and group D.Conclusion1.The effective immune response could be induced in mice by oral or intranasal immunization with the leaf protein extracted from the transgenic alfalfa containing Eg95-EgA31 fusion antigen. 2.The protective immunity against Eg protoscoleces could be induced in mice by oral immunization with the leaf protein extracted from transgenic alfalfa containing Eg95-EgA31 fusion antigen,and the immune response of Thl type played an important role in the protection. |
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