Identification Of Linear B-cell Epitope On Nonstructural Protein 3A Of FMDV Serotype Asia1

Foot-and-Mouth Disease (FMD) caused by Foot-and-Mouth Disease Virus (FMDV) is a highly contagious and acutely infected disease of cattle, pigs, sheep, goats and wild ruminant species. It has an economically devastating impact on affected countries, where it creates great losses to productivity and considerable economic losses to the husbandry industry. Whenever the disease has happened; it is must reported to the OIE. The antibodies against polyprotein 3ABC have been proved to be the most reliable marker of FMDV past or present infection. Some research groups reported that antibodies against 3A, part of 3ABC protein, existed in some vaccinated cloven-hoofed animals and influenced the specificity of assay based on 3ABC. 3A protein of FMDV is an important nonstructural protein, which is able to induce protective immune response in target animals and can be used as specific serological diagnosis tool. Therefore, it is necessary to find more specific infection-related epitopes on 3A.In order to identify epitopes on nonstructural protein 3A of FMDV serotype Asia1, 3A was dissected into fifteen overlapping fragments. The totals of fifteen pairs of primers were synthesized. 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) pLys S. Short fusion peptides were then detected by Western blot and ELISA with positive serum against FMDV serotype Asia1. The results showed that 3A linear B-cell epitope is located in A8 and A11. In order to further map the antigenic epitopes on nonstructural protein 3A of serotype Asia1, thirteen overlapping fragments spanning the A8 and A11 were designed and expressed in Escherichia coli Rosetta (DE3) pLys S. Then Western blot reactivity of these short peptide fused protein to viral infected sera were surveyed. This strategy screened and identified two regions of 3A that are Asia1 serotype-specific epitopes, which named amino acid residues A8-3(V90NEYIDKA97) and A11-12-Z7(A109EKNPLE115).According to the consequences (226 sequences from A, O, C and Asia1) of sequence alignment by MEGA4, the epitope of VNEYIDKA contains amino acid substitution at position 95(Asp) of the 3A coding region. We have constructed mutant namely amino acid residues A8-3-E(VNEYIEKA) and A8-3-F(VNEYIFKA) to further confirm on epitope, according to structure , hydrophilicity, hydrophobicity and polarity. Amino acid substitution of epitope mutant of A8-3-E(Asp95â†'Glu) is exist in FMDV serotype Asia1 and other serotypes. The epitope mutant of A8-3-F(Asp95â†'Phe) is not exist in.So, the linear B cell epitope on 3A serotype Asia1 is also precisely located and epitope mutant constructed. Indirect ELISA depended on A8-3 and A11-12-Z7 can be used in bovine serum testing. The results from the study may also be useful for understanding of the antigenic structure of FMDV and have great potential for development of specific serological diagnosis.