Surface displayed expression of a neutralizing epitope of spike protein from a Korean strain of porcine epidemic diarrhea virus

Abstract

^Äëíê~Åí= 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, p~ÅÅÜ~êçãóÅÉë=ÅÉêÉîáëá~É, 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 (hJ`lb) of the Korean strain of PEDV with the signal peptide of rice amylase 1A (o~ãóN^), 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 (dma) promoter was selected in order to direct the expression of the fusion construct, and the resulting recombinant plasmid was then introduced into pK=ÅÉêÉîáëá~É. 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. © KSBB

Surface-Displayed Expression of a Neutralizing Epitope of ApxIIA Exotoxin inSaccharomyces cerevisiaeand Oral Administration of It for Protective Immune Responses against Challenge byActinobacillus pleuropneumoniae

“…The cells and primary antibody were incubated at room temperature for 2 h. After the cells were washed, the second antibody, fluorescein isothiocyanate (FITC)-conjugated goat antimouse IgG (1:50; Sigma) was reacted with them at room temperature for 2 h. After washing, the cells were observed by confocal laser scanning microscopy (Carl Zeiss, Zena, Germany). 27) Western blot analysis. Preparation of the cell-wall fraction (CWF) was performed as described by Pitarch et al, 28) with some modifications.…”

“…In brief, the cells were grown for 3 d, harvested, washed twice with lysis buffer (10 mM Tris-HCl, pH 7.4, 1 mM phenylmethylsulfonyl fluoride), and homogenized 3 times in a bead beater (Biospec Products, Bartlesville, OK) for 1 min, with 3-min cooling intervals on ice. 27) The homogenate was observed by microscopy to verify that the cells were broken, and then was centrifuged (10;000 Â g, 10 min). The resulting supernatant was filtered through a cellulose filter (diameter, 0.4 mm) to prepare cell-free extract (CFE).…”

“…Immunofluorescent labeling of cells was carried out as previously described. 26,27) Cultivated yeast cells (48 h) were washed twice with PBS/BSA solution after centrifugation at 3;000 Â g for 5 min at room temperature. They were suspended in the same solution, and 100 ml of cell suspension (10 7 cells/ml) was placed on a glass slide and air-dried for immunostaining and observation.…”

“…The growth rate of the selected transformants was similar to that of the control strain, and appeared to be similar to those of other previous studies. 20,27) In addition, no morphological abnormalities were observed in the selected transformants, suggesting that expression of the target gene product and surface anchoring of the recombinant protein had no harmful effects on cell metabolism and architecture respectively.…”

Heterologous expression of cholera toxin B subunit in Saccharomyces cerevisiae

“…2). Several studies have also shown a wide variation in the expression level of heterologous genes in S. cerevisiae when using episomal 2 μ oribased plasmids, possibly due to the variations in the plasmid copy number between different transformants [26,28,37,38]. Thus, transformant TYEGCTB-3, which expressed the highest levels among the 20 transformants, was selected and employed for further studies.…”