Secreted expression of the classical swine fever virus glycoprotein E(rns) in yeast and application to a sandwich blocking ELISA

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 pGAPZalphaC which was int...

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Bibliographic Details
Published in:Journal of virological methods 2006-03, Vol.132 (1-2), p.40-47
Main Authors: Huang, Chienjin, Chien, Maw-Sheng, Hu, Ching-Mei, Chen, Chung-Wei, Hsieh, Pei-Chen
Format: Article
Language:English
Subjects:
Animals
Antibodies, Viral - blood
Blotting, Western
Classical Swine Fever - diagnosis
Classical swine fever virus - immunology
Classical swine fever virus - isolation & purification
Cloning, Molecular
Culture Media - chemistry
Dimerization
Enzyme-Linked Immunosorbent Assay - methods
Gene Expression
Genetic Vectors
Glycosylation
Membrane Glycoproteins - biosynthesis
Membrane Glycoproteins - genetics
Membrane Glycoproteins - immunology
Membrane Glycoproteins - isolation & purification
Molecular Weight
Pichia - genetics
Pichia - metabolism
Recombinant Proteins - biosynthesis
Recombinant Proteins - genetics
Recombinant Proteins - immunology
Recombinant Proteins - isolation & purification
Ribonucleases - genetics
Ribonucleases - immunology
Ribonucleases - isolation & purification
Ribonucleases - metabolism
RNA - metabolism
Swine
Viral Envelope Proteins - biosynthesis
Viral Envelope Proteins - genetics
Viral Envelope Proteins - immunology
Viral Envelope Proteins - isolation & purification
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Description
Summary: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 pGAPZalphaC which was introduced into Pichia pastoris. Expression of E(rns) 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.
ISSN:0166-0934
1879-0984
DOI:10.1016/j.jviromet.2005.08.020