Expression & Immune Responses Of The Fusion Protein Of Two Epitopes Of FMDV VP1 And The Fusion Protein Including Two Epitopes Of FMDV VP1 And Enterotoxins Of E.coli

It is usually thought that the peptide aa21-40 of VP1 of food-and-mouth disease( FMDV) Type O is related with cellular immunity and the peptide aal41-160 is related with humoral immunity. According to the sequences of epitopes of peptides aa21-40 and aal41-160 of VP1 of FMDV Type O and biased codons in E.coli, the nucleotides fragment (198bp) containing the peptides aa21-40 and aal41-160 codons was synthesized. The recombined expression vector r2020-2020 was correctly constructed , which expressing fusion protein of tandem sequence of aa21-40^aa141-160 aa21-40^aa141-160 peptides. The fusion protein was successfully expressed in BL₂1(DE₃) RIL and confirmed by SDS-PAGE and Western Blot. The molecular weight of the fusion protein is about 18kD. The fusion protein was purified and used to vaccinate rabbits and guinea pigs, whose immune responses were then observed. The fusion protein could induce T cells proliferation of vaccinated guinea pigs , which were observed when MTT was added to the spleen T cells stimulated by FMDV. At the same time,the fusion protein could elicit high level of neutralizing antibody, which was demonstrated when the sera of guinea pigs were analysed by the means of immunoassay. It could be concluded that the fusion protein could activate cellular immune-response and humoral immune-response simultaneously and it has potential to be FMDV vaccine.The enterotoxins produced by Enterotoxigenic Escherichia coli (ETEC) are the main diarrhea-causing pathogen, and they were divided into two groups: heat-labile toxin(LT) and heat-stable toxin(ST). In this study, the recombined expression vector r2020-LTB-2020-STI was correctly constructed , which expressed fusion protein of tandem sequence of 2020-LTB-2020-STI peptides. The fusion protein was successfully expressed in BL₂1(DE₃) RIL at high lever and confirmed by SDS-PAGE, and the molecular weight of the fusion protein is about 45kD. The purified fusion protein couldbe recognized by CTB antibody specially. The purified protein was then used to vaccinate guinea pigs, rabbits and Bald/c mice 6-8 weeks old, whose immune responses were then observed. The fusion protein could induce proliferation of spleen T cells of vaccinated guinea pigs when stimulated by FMDV antigen, and elicit high level of neutralizing antibody in rabbits against FMDV. It could be concluded that the fusion protein could activate cellular immune-response and humoral immune-response simultaneously. At the same time, the vaccinated mice could survive challenge of 1.5MLD Escherichia coli C83902, and the sera of vaccinated rabbits could neutralize the STI tonxin, moreover, the fusion protein had no STI toxicity, which demonstrated that the fusion protein have LTB and STI immunities. So that, it can be concluded that the fusion protein have the potent to be efficient FMDV and ETEC vaccine.