Secreted expression of the classical swine fever virus glycoprotein Erns in yeast and application to a sandwich blocking ELISA

Erns is an envelope glycoprotein of classical swine fever virus (CSFV) with RNase activity. The purpose of this study was to produce an active Erns for further applications using the yeast secreted expression system. The Erns gene was cloned into the expression vector pGAPZαC which was introduced into Pichia pastoris. Expression of Erns protein in culture supernatant was confirmed by Western blot analysis using both the monoclonal antibody against CSFV Erns and CSFV-positive swine serum. The yeast-expressed Erns (yErns) was shown to have N-linked glycosylation and to form homodimer of 74 kDa molecules. All monomer, homodimer, and deglycosylated forms of yErns demonstrated intrinsic ribonuclease activity and a clear preference for uridine-rich sequence. A direct sandwich blocking enzyme-linked immunosorbent assay (ELISA) based on the yErns was developed with a high sensitivity and specificity. The yErns which possesses enzymatic activity and retains antigenicity may provide a useful material for developing a diagnostic kit.