[The study on immune responses in BALB/c mice by immunization with the transgenic Alfalfa(Medicago sativa) containing Eg95-EgA31 fusion gene of Echinococcus granulosus].
OBJECTIVE
To investigate the immune responses and the protection induced by the transgenic Alfalfa (Medicago sativa) containing Eg95-EgA31 fusion gene of Echinococcus granulosus against Eg protoscoleces.


METHODS
The leaf protein was extracted from the transgenic alfalfa by heat-coagulation method, its concentration was prepared for 20 g/L. BALB/c mice were immunized intranasally or orally with the leaf protein once per 3 days for 2 months.At the same time, the leaf protein transfected with pBI-121 blank vector and the normal leaf protein without foreign antigen was served as control. The mice were then challenged intraperitoneally with Eg protoscoleces (50 protoscoleces per mouce)on week 8 after the last vaccination and sacrified on week 24 postinfection to count the rate of reduced hydatid cyst. The specific antibody (IgE, IgG and its subclasses)in the sera collected from the eyeballs was evaluated by ELISA. Splenocytes were separated and cultured in vitro with EgAg, ConA or LPS stimulus.The substes of CD4(+); and CD8(+); T cells were measured by FCM. The splenocytes' proliferation was determined by MTT method.Then the cells were collected, stained by PI and Annexin V-FITC, and analyzed by FCM to get the splenocytes' apoptotic rate.The supernatant was collected to measure the level of IL-12, IL-10, IFN-gamma and TNF-alpha by ELISA.


RESULTS
Compared with the control group, The hydatid cyst weight in the oral immunization group decreased by 64.1%; the splenocytes' apoptotic rate got obviously lower than that in the control group; the splenocytes' proliferation increased significantly, the CD4(+); subsets and the ratio of the CD4(+);/CD8(+); did so, and the similar trend about the specific antibody titer and the level of cytokines could be seen in this group.


CONCLUSIONS
Apoptosis of splenocytes may be inhibited in mice by immunization with the transgenic alfalfa, splenocytes' proliferation and Th1 response can be induced in the mice against the challenge of Eg protoscoleces. CD4(+); T cell and the specific antibody(IgG, IgG2b and IgE) may play important roles in the protection induced by the transgenic alfalfa vaccine.
Ye
Yan-ju
YJ
Institute of Infectious and Parasitic Diseases, the First Affiliated Hospital, Chongqing Medical University, Chongqing 400016, China. YeYanju.happy@163.com
Li
Wen-gui
WG
D000818:Animals; D000909:Antibodies, Helminth / Q000097:blood / Q000276:immunology; D000947:Antigens, Helminth / Q000008:administration & dosage / Q000235:genetics / Q000276:immunology*; D004195:Disease Models, Animal; D004443:Echinococcosis / Q000276:immunology* / Q000469:parasitology; D048209:Echinococcus granulosus / Q000235:genetics / Q000276:immunology*; D015801:Helminth Proteins / Q000008:administration & dosage / Q000235:genetics / Q000276:immunology*; D006801:Humans; D007114:Immunization; D008297:Male; D000455:Medicago sativa / Q000235:genetics* / Q000276:immunology; D051379:Mice; D008807:Mice, Inbred BALB C; D010940:Plant Proteins / Q000008:administration & dosage / Q000235:genetics / Q000276:immunology; D030821:Plants, Genetically Modified / Q000235:genetics* / Q000276:immunology; D015202:Protein Engineering; D011897:Random Allocation; D011993:Recombinant Fusion Proteins / Q000008:administration & dosage / Q000235:genetics / Q000276:immunology; D014612:Vaccines / Q000008:administration & dosage / Q000235:genetics / Q000276:immunology
D000909:Antibodies, Helminth; D000947:Antigens, Helminth; C446034:EG95 protein, Echinococcus granulosus; C401864:EgA31 protein, Echinococcus granulosus; D015801:Helminth Proteins; D010940:Plant Proteins; D011993:Recombinant Fusion Proteins; D014612:Vaccines