Prokaryotic Expression And Immune Effect Of Major Epitope Regions Of Type A Foot-and-Mouth Disease Virus Capsid Protein

Foot-and-mouth disease(FMD)is an acute and highly contagious animal disease caused by foot-and-mouth disease virus(FMDV),which mainly infects cloven-hoofed animals such as pigs,cattle and sheep.FMD outbreaks can lead to great economic losses and negative social influence.Therefore,FMD is one of the most important animal disease listed for prevention and control every year in our country.At present,the comprehensive measures with vaccination are adopted in China for FMD control,among these inactivated vaccines are the most widely used weapon against FMD.However,there is the biosafty risk of virus escape from vaccine production plant.So,it is neccesary to develop new vaccine for future FMD control.Epitope based vaccine is one of the most promising direction of new FMD vaccine development.In 2013,FMDV type A/Sea/97 virus strain was introduced in China and caused great economic losses.In order to explore the main epitope regions of FMDV serotype A capsid proteins and their immunogenicity,we expressed prokaryotically the epitope regions in capsid proteins of FMDV type A and evaluated their immunological effects in pigs.This study will provide some valuable clues for development of epitope-based vaccine against FMDV type A.According to the previously reported antigenic sites of FMDV type A,a series of antigenic site regions were selected in this study to design epitope-based protein vaccine.These epitope regions included: amino acids 70~81 and 118~140 in VP2,131~157 and 188~212 in GH loop of VP1 from A/QH/CHA/2013 strain;amino acids 43~48 of VP1 and 55~72 of VP3 reference to A/WH/CHA/09;and one T cell epitope in 3A and a universal T cell epitope were also involved to design the epitope-based protein molecular.All the epitopes were linked with flexible amino acids,and a 6×His tag was introduced at C-terminal.The two prokaryotic expression vector named pET28a-ANX2 and pET28a-ANX3 were constructed to express two multiple epitope proteins named ANX2 and ANX3.Epitope protein ANX2 was designed as a reverse the amino acids sequence compared with ANX3.The expression of ANX2 and ANX3 were identified by SDS-PAGE and Western blot.ANX2 and ANX3 proteins were expressed and purified for vaccine preparation.Two purified epitope proteins were separately emulsifed with ISA201 adjuvant supplemented with CpG-ODNs enhancer to form the vaccines.Mice and pigs were vaccinated with ANX2 and ANX3 proteins vaccine to analyze the humoral immune and cellular immune responses,and the immune swine were challenged with the epidemic A/Sea/97 virus strain at 28 days after vaccination to evaluate the protective effect of the vaccine.The results showed that the multi-epitope proteins were mainly expressed in inclusion bodies,and the molecular weight of two proteins were about 41 kDa and 39 kDa respectively.The results of Western blot showed that two proteins both can specifically react with positive serum of FMDV type A.Mice and swine produced FMDV type A specific antibody and increased level of IFN-? and IL-4 cytokines responses after vaccination.Protective effects were observed after challenge with virulent type A virus,and ANX2 showed better effects than ANX3,indicating this a reverse the amino acids sequence displayed the epitope more like natural conformation and could enhance the immune effects.This research accumulate some useful data for development of epitope-based vaccine against FMDV type A.