RESUMO
Vp1 gene of O type foot-and-mouth diseases virus and M. tuberculosis HSP70 were expressed in methylotrophic yeast Pichia pastoris expression system. The results of cellular immune responses and humoral immune response were examined after BALB/c mice were immunized with fusion protein expressed in methylotrophic yeast Pichia pastoris. The genes was cloned into the vector pPICZalpha-A by routine molecular technique. The plasmid fusion (pPICZalphaA-vp1-HSP70) was created that HSP70 located downstream of VP1 gene of O type foot-and-mouth disease virus. Vp1 was expressed by fusing to the amino terminus of M. tuberculosis hsp70 in yeast Pichia pastoris. The recombined fusion plasmid was transformed into methylotrophic yeast Pichia pastoris X-33 by electrophoration. The recombinant transformants were selected by Zeocin and induced by the addition of methanol every 24h. The expressived product analyzed by SDS-PAGE and Western blotting. The result indicated that the fusion protein(vp1-HSP70) has specific antigenicity. Mice were inoculated transcutaneous three times at a two-weeks interval with fusion protein, PBS and conventional inactivated vaccines. To evaluate the prophylaxtic efficacy of fusion protein, Titers of antibodies was detected by ELISA and proliferation of lymphocytes were determined by MTT. The results indicated that fusion protein could elicit specific humoral immune and cellular immune responses. Compared with conventional inactivated vaccines, fusion protein elicited slightly lower FMDV antibody level but stronger T cell proliferation.