Identification of proteins specified by porcine epidemic diarrhoea virus

Abstract

Up to now, little was known about the proteins of porcine epidemic diarrhoea virus (PEDV). Using (i) metabolic labelling (ii) antisera directed against synthetic peptides and monoclonal antibodies, and (iii) eukaryotic and prokaryotic expression systems, we have started to identify and characterize these proteins. The nucleocapsid protein (N) of PEDV was identified as a phosphoprotein with a relative mobility (Mr) of 57 k. No additional phosphoproteins were detected. At least three glycoproteins were virion associated. The 180/200 k band most probably represented the surface glycoprotein (S), whereas the 27 k protein was the membrane protein (M). The 21 k species could not yet be identified. A rabbit anti-M protein antiserum was generated by using synthetic peptides corresponding to the C-terminus of M. The specificity of this serum was reconfirmed by transient expression of the M-gene in Vero cells followed by immunofluorescence. In order to generate antisera against the putative gene products of ORF3 and of the internal N reading frames (I-1, I-2, I-3), additional anti-peptide sera were raised. The putative ORF3 product which had been tagged by a 6 Histidine tail, was expressed in E. coli and purified by nickel chelate affinity chromatography before 2-dimensional polyacrylamide gel electrophoresis and immunostaining with a rabbit anti-peptide serum directed against the N-terminus of the ORF3 product. Transient expression of the I-1 and I-3 reading frames was used to confirm the specificity of the corresponding anti-peptide sera. The immunological tools presented in this paper are now being used to identify the putative corresponding gene products specified by PEDV.