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首页> 外文期刊>Biotechnology and bioprocess engineering >Surface Displayed Expression Of Aneutralizing Epitope Of Spike Protein From a Korean Strain Of Porcine Epidemic diarrhea Virus
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Surface Displayed Expression Of Aneutralizing Epitope Of Spike Protein From a Korean Strain Of Porcine Epidemic diarrhea Virus

机译：猪流行性腹泻病毒韩国株的穗蛋白中和表位的表面展示表达

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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 (Ramy1A), 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 into S. 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.
机译：口服调整后，韩国流行性猪腹泻病毒株（PEDV）的刺突蛋白的中和表位（K-COE）已显示可预防和促进对PED的免疫反应。芽胞酵母酿酒酵母的细胞表面经过工程改造，可将K-COE锚定在细胞的外层，因此，改变后的酵母可作为具有免疫原性功能的动物饲料的饮食补品。在这项研究中，将带有水稻淀粉酶1A（Ramy1A）信号肽的韩国PEDV株的K-COE基因（K-COE）与编码羧基末端一半（C末端的320个氨基酸残基）的基因融合）酵母α-凝集素，这是一种与交配相关的蛋白质，共价锚定在细胞壁上。选择3-磷酸甘油醛脱氢酶（GPD）启动子以指导融合构建体的表达，然后将得到的重组质粒引入酿酒酵母中。 K-COE的表面展示可通过共聚焦显微镜观察，使用抗K-COE的多克隆抗体作为一抗，结合FITC（异硫氰酸荧光素）的山羊抗小鼠IgG作为二抗。在施用α-1，3-葡聚糖酶/ PNGase F /β-甘露糖苷酶处理后，使用细胞壁级分通过蛋白质印迹分析进一步证实了K-COE在细胞表面上的显示。

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