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標題: Secreted expression of the classical swine fever virus glycoprotein E-rns in yeast and application to a sandwich blocking ELISA
作者: Huang, C.J.
Chien, M.S.
Hu, C.M.
Chen, C.W.
Hsieh, P.C.
關鍵字: classical swine fever virus;glycoprotein;Pichia pastoris;RNase;blocking ELISA;protein e-rns;pichia-pastoris;methylotrophic yeast;pestivirus;identification;ribonuclease;diagnosis;immunity;vaccine;cells
Project: Journal of Virological Methods
期刊/報告no:: Journal of Virological Methods, Volume 132, Issue 1-2, Page(s) 40-47.
E-rns is an envelope glycoprotein of classical swine fever virus (CSFV) with RNase activity. The purpose of this study was to produce an active E-rns for further applications using the yeast secreted expression system. The E-rns gene was cloned into the expression vector pGAPZ alpha C 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 E-rns and CSFV-positive swine serum. The yeast-expressed E-rns (yE(rns)) was shown to have N-linked glycosylation and to form homodimer of 74 kDa molecules. All monomer, homodimer, and deglycosylated forms of yE(rns) demonstrated intrinsic ribonuclease activity and a clear preference for uridine-rich sequence. A direct sandwich blocking enzyme-linked immunosorbent assay (ELISA) based on the yE(rns) was developed with a high sensitivity and specificity. The yE(rns) which possesses enzymatic activity and retains antigenicity may provide a useful material for developing a diagnostic kit. (c) 2005 Elsevier B.V. All rights reserved.
ISSN: 0166-0934
DOI: 10.1016/j.jviromet.2005.08.020
Appears in Collections:微生物暨公共衛生學研究所

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