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林婧楠, 赵景壮, 刘淼, 任广明, 卢彤岩, 徐黎明. 鲤春病毒血症病毒糖蛋白酵母表面展示系统的建立[J]. 水生生物学报, 2019, 43(5): 977-982. DOI: 10.7541/2019.116
引用本文: 林婧楠, 赵景壮, 刘淼, 任广明, 卢彤岩, 徐黎明. 鲤春病毒血症病毒糖蛋白酵母表面展示系统的建立[J]. 水生生物学报, 2019, 43(5): 977-982. DOI: 10.7541/2019.116
LIN Jing-Nan, ZHAO Jing-Zhuang, LIU Miao, REN Guang-Ming, LU Tong-Yan, XU Li-Ming. ESTABLISHMENT OF YEAST EXPRESSION SYSTEM FOR SPRING VIRERNIA OF CARP VIRUS GLYCOPROTEIN[J]. ACTA HYDROBIOLOGICA SINICA, 2019, 43(5): 977-982. DOI: 10.7541/2019.116
Citation: LIN Jing-Nan, ZHAO Jing-Zhuang, LIU Miao, REN Guang-Ming, LU Tong-Yan, XU Li-Ming. ESTABLISHMENT OF YEAST EXPRESSION SYSTEM FOR SPRING VIRERNIA OF CARP VIRUS GLYCOPROTEIN[J]. ACTA HYDROBIOLOGICA SINICA, 2019, 43(5): 977-982. DOI: 10.7541/2019.116

鲤春病毒血症病毒糖蛋白酵母表面展示系统的建立

ESTABLISHMENT OF YEAST EXPRESSION SYSTEM FOR SPRING VIRERNIA OF CARP VIRUS GLYCOPROTEIN

  • 摘要: 糖蛋白(Glycoprotein, G)作为鲤春病毒血症病毒(Spring Virernia of Carp Virus, SVCV)主要的抗原蛋白, 已成为现阶段SVCV病毒检测、抗体制备以及疫苗研制的热点。为了对其进行酵母表面展示, 研究以SVCV-shlj1分离株基因组为模板, 通过RT-PCR技术, 体外扩增获得SVCV表面糖蛋白的基因开放阅读框(1530 bp)片段, 将其克隆至酵母表面展示载体pYD1, 构建重组质粒pYD1-G。利用电转化方法将重组质粒pYD1-G导入酿酒酵母EBY100感受态细胞, 经YNB选择培养基筛选和菌液PCR的鉴定, 挑选出阳性转化子(命名为EBY100-pYD1-G), 对其进行2%半乳糖诱导。利用细胞免疫荧光和流式细胞仪检测G蛋白的酵母表面展示情况。细胞免疫荧光结果显示, 诱导后的酵母细胞EBY100-pYD1-G能产生特异性红色荧光, 且随着诱导时间的增加, 红色荧光的酵母细胞所占比例不断增加, 各组之间差异显著(P< 0.05)。流式细胞仪检测结果显示, 酵母细胞的荧光强度与诱导时间呈正比, 其中诱导48h与72h的酵母细胞荧光强度不存在显著差异, 基本趋于稳定不变的状态。因此, 选取诱导48h为酵母表面展示的最佳诱导时间。上述研究结果表明SVCV的G蛋白已经成功展示于酿酒酵母细胞表面, 研究为鲤春病毒血症酵母口服疫苗的研发奠定了前期基础。

     

    Abstract: In this study, open reading frame (ORF) of glycoprotein (1530 bp) was amplified by using RNA extracted from Spring Virernia of Carp Virus (SVCV). The SVCV G ORF was cloned into pYD1 vector to construct a recombi-nant plasmid pYD1-G and then transformed into competent yeast cells EBY100, and positive colonies were screened by colony PCR. The expression of G gene was induced by 2% glucose and detected by cell immunofluorescence and flow cytometry. The immunofluorescence staining observed increased EBY100-pYD1-G signal of induced yeast cells with increased induction time. Flow cytometry analysis observed significantly increased fluorescence intensities in prolonged induced EBY100-pYD1-G cells (P<0.05). These results indicated the SVCV G protein has been successfully expressed and localized on the surface of yeast cell. This study laid a foundation for the novel oral vaccine development against SVCV infection in carps in future.

     

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