Construction And Immunogenicity Of Recombinant Pichia Pastoris Expressing Multi-epitopes Of Type Asia I And O Foot-and-Mouth Disease Virus

Foot-and-mouth disease (FMD) caused by foot and mouth disease virus (FMDV) is a highly contagious and economically devastating disease, which always inflict cloven-hoofed animals. FMDV is the prototype member of the Aphthovirus genus of the family Picornaviridae. There are 7 serotypes and more than 80 subtypes of FMDV, so it brought great difficulties to control it. Because of following risky factors such as the incomplete inactivation of the virus and escape of live virus, new type of vaccine which should be safer and more effective are under intensive study worldwide. In this paper we selected VP1 gene of serotype Asia I and O of FMDV and non-strcture protein 2A gene as the major antigen , and other epitopes gene which composed of the cholera toxin B subunit gene, Th2 cell epitopes and the box constructed with T cell andB cell epitope gene.We amplified VP1-2A gene of Asia I/JL/05 and O/NY/00 of FMDV by PCR .Then, VP1-2A gene of Asia I/JL/05 and O/NY/00 of FMDV was ligated into pMD18-T vector. The recombinant clone, pMD18-T-VP1-2A, were sequenced. The nucleotide sequence of VP1-2A was compared and analyzed with the Asia I/JL/05 and O/NY/00 of FMDV. Then we obtained CTE multiple epitope fragment by restricted digestion of pMD18-T-CTE plasmid of type Asia I and O FMDV. Further the VP1-2A and CTE fragment was ligated into pPIC9K expression vector. Then, it was transformed into Pichia pastoris GS115 and was induced with methanol to express recombinant VP1-2A and CTE fusion protein. The expression of target protein was detected by SDS-PAGE and Western blot assay. Mice and guinea pigs were inoculated with these recombinant vaccines. And the FMDV specific antibody, T lymphoproliferation and T lymphocyte subpopulation were detected.The results showed that VP1-2A gene of serotype Asia I and O of FMDV was successfully amplified and cloned. Sequencing results showed that the VP1-2A gene of Asia I of FMDV shares 100% homology in nucleotide sequence, and the VP1-2A gene of O of FMDV shares 99.7% homology in nucleotide sequence. Recombinant expression plasmid pPIC9K-VP1-2A-CTE was constructed by inserting of serotype Asia I and O of FMDV VP1-2A and CTE gene into yeast expression vector pPIC9K. Secondly, plasmid pPIC9K-VP1-2A-CTE of serotype Asia I and O of FMDV was transformed into GS115 cells by electroporation.SDS-PAGE results showed that expression product could be secreted into media and existed in dimer form, production of target protein could reach 75 mg and 80 mg per litter.And the Western blot results showed that expression product could be detected by FMDV antiserum, which proved the good antigen specificity of expressed protein. Mice and guinea pigs were inoculated intramuscularly with recombinant vaccines and the inactivated vaccine. The FMDV specific antibody, T lymphoproliferation and cytokine concentration results showed that in all recombinant vaccines inoculated groups, could be induced specific humoral and cellular immune responses. In mice, the inactivated vaccine had the highest immunogenicity. Comparing with GS115/pPIC9K group and PBS group the GS115/pPIC9K-VP1-2A-CTE of serotype Asia I and O of FMDV group could induce not only approximately same FMDV antibody level, but also higher cellular immune responses. In guinea pigs, the GS115/pPIC9K-VP1-2A-CTE of serotype Asia I and O of FMDV group had higher cellular immune responses than in mice.but only had weaker humoral responses than inactivated vaccine.In conclusion, we selected VP1 gene of serotype Asia I and O of FMDV and non-strcture protein 2A gene as the major antigen , and CTE multi-epitopes fagment as the second antigen, and expressed them in the expression system of Pichia. The protein has good immunogenicity.It could be as the candidate vaccines to contol foot and mouth disease.