Identification of B cell linear epitopes containing peptides on the hypervariable region of Porcine Epidemic Diarrhea Virus S protein

Abstract:

[Background] Amino acid sequences alignment of the spike (S) proteins of porcine epidemic diarrhea virus (PEDV) classical and prevalent strains indicates that most of the variation maps to N-terminal region of the S protein. [Objective] The objective of this study is to identify linear B cell epitopes (BCE) containing peptides, especially prevalent strain specific linear BCE containing peptides, on the hypervariable region (S10A, 1?496 aa) of the S protein of PEDV prevalent strain. [Methods] The N-terminal hypervariable part of prevalent PEDV SD2014 S protein (S10ASD2014) was recombinantly expressed in Escherichia coli (E. coli). Polyclonal antibody was prepared by immunizing New Zealand white rabbits with S10ASD2014 purified by cutting out the aimed band from the SDS-PAGE. Serial Glutathione S-transferase (GST)-fusion expressed 16-mers with an overlap of 8 amino acids (aa) covering the entire S10ASD2014 sequence was produced in E. coli, and from which linear BCE containing 16-mers were identified by using western blot with prepared anti-S10ASD2014 serum as the primary antibody. The conserved linear BCE containing 16-mers on S10ASD2014 were identified with polyclonal serum against the counterpart of the S protein of PEDV CV777 vaccine strain (S10ACV777). [Results] The relative molecular mass of the expressed S10ASD2014 was about 72 kD. The purified S10ASD2014 could be recognized by clinical porcine anti-PEDV serum. The polyclonal serum prepared by immunizing the rabbits with the purified S10ASD2014 could react with PEDV DR13 in Vero cells. From 61 16-mers, twenty-eight linear BCE containing 16-mers were identified by western blot using the prepared anti-S10ASD2014 and anti-S10ACV777 serum. Thirteen out of the 28 linear BCE containing 16-mers could be recognized by both of the 2 serum. Homology analysis of the corresponding region of 24 typical strains from different PEDV subgroups showed that 2 positive 16-mers were conserved. [Conclusion] The identification of linear BCE containing peptides, especially the prevalent PEDV S protein-specific 16-mers laid a foundation for the understanding the S protein structure and function, as well as for developing epitope-based diagnostic methods.

Key words:PEDV, Hyper variable region of spike protein, Polyclonal antibodies, B cell linear epitope