Prokaryotic expression of truncated S2 subunit of porcine epidemic diarrhea virus and identification of its linear B cell epitopes containing regions

Abstract:

[Background] Porcine epidemic diarrhea (PED) caused by porcine epidemic diarrhea virus (PEDV) has caused significant economic losses in pig industry. PEDV S protein can induce the host to produce neutralizing antibodies. [Objective] To prokaryotically express the truncated S2 (aa: 961?1 382) of PEDV CV777 vaccine strain and prepare polyclonal antibodies with which to identify regions containing linear B cell epitopes on the truncated S2. [Methods] The codon-optimized DNA fragment encoding the truncated S2 of PEDV (s2t) was inserted into pET-28a. The resulting pET-28a-S2t was then transformed into Escherichia coli BL21(DE3) for expression under the induction of IPTG. Polyclonal antibody was prepared by immunizing New Zealand white rabbits with the truncated S2 purified by cutting out the aimed band from the SDS-PAGE. Serial 16-mers, overlapping 8 aa each other and covering the whole sequence of the truncated S2, were GST-fusion expressed in E. coli BL21. Positive 16-mers were identified by western blot (WB) using prepared polyclonal serum as the primary antibody. linear B cell epitopes containing regions were located. [Results] The truncated S2, about 50 kD, mainly existed in form of inclusion body, of which the expression level reached the maximum at 4 h post induction. The purified truncated S2 could be recognized by porcine anti-PEDV serum in WB analysis. The titer values of the polyclonal serum ranged from 1:25 600 to 1:102 400. Immunohistochemical and indirect immunofluorescence analyses indicated that the polyclonal antibodies showed specific reactivity with PEDV DR13 in Vero cells. Eleven positive 16-mers were identified from 52 GST-fused 16-mers by WB using the prepared serum. All the 11 16-mers could be recognized by porcine anti-PEDV serum in WB analysis. The identified positive 16-mers formed 4 linear B cell epitopes containing regions on the truncated S2 (aa: 969?984, 1 065?1 096, 1 225?1 280, and 1 361?1 382). [Conclusion] The preparation of high titer polyclonal antibodies against truncated S2 of PEDV and the identification of linear B cell epitopes containing sequences on it laid a foundation for understanding the structure and function of S protein, as well as developing effective PEDV diagnostic methods.

Key words:PEDV, S protein, Polyclonal antibodies, Linear B cell epitope