Abstract
BackgroundThe major structural protein of coronaviruses, the membrane (M) protein, can elicit the formation of protective antibodies, but little information is available about the M protein of porcine epidemic diarrhea virus (PEDV). Identification of epitopes on the PEDV M protein will be helpful in the elucidation of the antigenic properties of this protein.ResultsOne hybridoma cell line secreting anti-M protein monoclonal antibody (McAb) was generated and designated 4D4. To map the epitopes on the PEDV M protein, a total of 17 partially overlapping fragments covering the C-terminus of M protein were expressed as fusion proteins with a 6×His tag or a GST tag. A linear motif, 193TGWAFYVR200, was identified by enzyme-linked immunosorbent assay (ELISA) and western blot (WB) analysis using McAb 4D4. The motif 195WAFYVR200 was the minimal requirement for reactivity, as demonstrated by removing amino acids individually from both ends of the motif 193TGWAFYVR200. The result of WB analysis showed that the 4D4-defined epitope could be recognized by PEDV-positive serum, but not transmissible gastroenteritis virus (TGEV)-positive serum. Furthermore, this epitope was highly conserved among different PEDV strains, as shown by alignment and comparison of sequences.ConclusionA McAb, 4D4, directed against the M protein of PEDV, was obtained, and the 4D4-defined minimal epitope sequence was 195WAFYVR200. The McAb could serve as a candidate for development of a McAb-based antigen capture ELISA for detection of PEDV. The epitope identified provides a basis for the development of epitope-based differential diagnostic techniques and may be useful in the design of epitope-based vaccines.
Highlights
The major structural protein of coronaviruses, the membrane (M) protein, can elicit the formation of protective antibodies, but little information is available about the M protein of porcine epidemic diarrhea virus (PEDV)
The truncated M gene, encoding the C-terminus of the M protein, was expressed as a His6-fusion protein and a GST-fusion protein in E. coli BL21(DE3), respectively. Both of the fusion proteins, tM-His6 and GST-tM, could react with porcine antiPEDV serum as shown by western blot (WB) analysis, which implies that they had similar antigenicity to the native M protein of PEDV
Production and characterization of M protein-specific monoclonal antibody (McAb) The tM-His6 protein was used as an immunogen to prepare the McAb, and the GST-tM protein was used as a coating antigen to establish the indirect enzyme-linked immunosorbent assay (ELISA) for screening the antibody-secreting hybridoma cell lines
Summary
The major structural protein of coronaviruses, the membrane (M) protein, can elicit the formation of protective antibodies, but little information is available about the M protein of porcine epidemic diarrhea virus (PEDV). PED was first reported in England in 1971 [2] and the virus was identified in Belgium and United Kingdom for the first time [3]. Since it has become prevalent in many swineraising countries and it is one of the most important viral causes of diarrhea, resulting in heavy economic losses to the swine industry, mainly in European and. The M protein of coronavirus can stimulate the production of alpha-interferon (α-IFN) [19]
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