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KMID : 1094720070120060690
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Biotechnology and Bioprocess Engineering
2007 Volume.12 No. 6 p.690 ~ p.695
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Surface displayed expression of a neutralizing epitope of spike protein from a Korean strain of porcine epidemic diarrhea virus
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Park Seung-Moon
Cho Dong-Ha
Kim Kang-Ju
Mo Ae-Young
Lim Jung-Gu
Chung Hea-Jong
Kim Tae-Geum
Yang Moon-Sik
Kim Dae-Hyuk
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Abstract
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The neutralizing epitope (K-COE) of the spike protein from a Korean strain of porcine epidemic diarrhea virus (PEDV) has been shown to prevent and foster an immune response to PED, when orally adjusted. The cell surface of the budding yeast,Saccharomyces cerevisiae, was engineered to anchor the K-COE on the outer layer of the cell, and consequently, the altered yeast was applied as a dietary complement for animal feed, with immunogenic functions. In this study, the K-COE gene (K-COE) of the Korean strain of PEDV with the signal peptide of rice amylase 1A (Ramy 1A), was fused with the gene encoding the carboxyterminal half (320 amino acid residues from the C terminus) of yeast α-agglutinin, a mating associated protein that is anchored covalently to the cell wall. The glyceraldehyde-3-phosphate dehydrogenase (GPD) promoter was selected in order to direct the expression of the fusion construct, and the resulting recombinant plasmid was then introduced intoS. cerevisiae. The surface display of K-COE was visualized via confocal microscopy using a polyclonal antibody against K-COE as the primary antibody, and FITC (fluorescein isothiocyanate)-conjugated goat anti-mouse IgG as the secondary antibody. The display of the K-COE on the cell surface was further verified via Western blot analysis using the cell wall fraction after the administration of α-1,3-glucanase/PNGase F/β-mannosidase treatment.
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KEYWORD
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surface display, porcine epidemic diarrhea virus, Saccharomyces cerevisiae
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