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 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.
Huang
Chienjin
C
Graduate Institute of Veterinary Microbiology, College of Veterinary Medicine, National Chung Hsing University, 250 Kuo Kuang Road, Taichung 40227, Taiwan, ROC. cjhuang@dragon.nchu.edu.tw
Chien
Maw-Sheng
MS
Hu
Ching-Mei
CM
Chen
Chung-Wei
CW
Hsieh
Pei-Chen
PC
D000818:Animals; D000914:Antibodies, Viral / Q000097:blood; D015153:Blotting, Western; D006691:Classical Swine Fever / Q000175:diagnosis; D006692:Classical Swine Fever Virus / Q000276:immunology* / Q000302:isolation & purification; D003001:Cloning, Molecular; D003470:Culture Media / Q000737:chemistry; D019281:Dimerization; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D015870:Gene Expression; D005822:Genetic Vectors; D006031:Glycosylation; D008562:Membrane Glycoproteins / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification; D008970:Molecular Weight; D010843:Pichia / Q000235:genetics / Q000378:metabolism*; D012313:RNA / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification; D012260:Ribonucleases / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism; D013552:Swine; D014759:Viral Envelope Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification
D000914:Antibodies, Viral; D003470:Culture Media; C466501:E(rns) protein, classic swine fever virus; D008562:Membrane Glycoproteins; D011994:Recombinant Proteins; D014759:Viral Envelope Proteins; D012313:RNA; D012260:Ribonucleases