Identification Of Linear B-Cell Epitope On VP2 Of FMDV

Foot and mouth disease(FMD) is a acute,fever and highly contagious viral vesicular disease affecting cloven hoofed animals by FMDV. The highly susceptible.animals such as cattle,sheep,goats and pigs It is considered to be one of the reported animal diseases. It needs analyze the structure and function of viral antigen to select the suitable peptides be as subunits vaccine for desease prevention diagnosis of foot-and-mouth. In this study, above all,the cloning and expression of vp2 gene of FMDV serotype Asia1in Escherichia coli. Then Western blot showed that it could be distinguished by sera of viral infected.In oeder to map the antigenic epitopes on VP2 protein of serotype Asia1, a set of partially overlapping fragments of 44aa spanning the protein were designed. overlapping of fifteen amino acids.The totals of 7 pairs of primers were synthesized. All the forward primers contained a BamHI restriction site, and reverse primers contained a termination codon and an XhoI restriction site. PCR products were cloned into expression vector pET-32a and then these fragments confirmed by sequencing. Each fragment was expressed in Escherichia coli Rosetta(DE3)pLysS.Then western blot reactivity of these short peptide fused protein to viral infected sera were surveyed. The results showed that VP2 linear B-cell epitope is located in the N-terminal F1(1~44aa). We also found that serotype O, serotype A, serotype C FMD standard serum and infected SAT2 FMD rehabilitation period bovine serum could also identify the epitope fusion protein.the short peptide protein F1 was dissected into six overlapping fragments,overlapping of five amino acids, a pair of oligonucleotide fragments were synthesized for each short peptide. After Phosphorylation, annealing, the products were cloned into expression vector pET-32a.The same way further identified linear B cell epitope on VP2 serotype Asia1is located in the 6~15 amino acid of N-terminal region.On this basis, In oeder to map the antigenic epitopes of VP2 protein serotype O, Amino acid sequence alignment of VP2 protein between serotype Asia1and serotype O. The result showed that at one of its N-terminal 31 amino acid sequence is exactly the same.Then a set of partially three overlapping fragments spanning the protein VP2 (32aa~218aa) of serotype O were designed. The totals of 3 pairs of primers were synthesized. All the forward primers contained a EcoRI restriction site, and reverse primers contained a termination codon and an HindIII restriction site. Western blot reactivity of these short peptide fused protein to standard serum were surveyed.Noone could be recognized by standard serum..So, the linear B cell epitope on VP2 serotype O is also located in the 6~15 amino acid of N-terminal region as serotype Asia1.And this linear B cell epitope district is also belongs to other serotypes of foot and mouth disease virus.The results from the study may also be useful for diagnosis of FMDV infection based on antigenic epitope or developing a new strategy for vaccine design.