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

作　　者：林婧楠 赵景壮[1] 刘淼[1] 任广明[1] 卢彤岩[1] 徐黎明[1] LIN Jing-Nan;ZHAO Jing-Zhuang;LIU Miao;REN Guang-Ming;LU Tong-Yan;XU Li-Ming(Heilongjiang River Fishery Research Institute,Chinese Academy of Fishery Sciences,Harbin 150070,China;College of Fishery and Life Science,Shanghai Ocean University,Shanghai 201306,China)

机构地区：[1]中国水产科学研究院黑龙江水产研究所,哈尔滨150070 [2]上海海洋大学水产与生命学院,上海201306

出　　处：《水生生物学报》2019年第5期977-982,共6页Acta Hydrobiologica Sinica

基　　金：中央公益性事业单位基本科研业务费专项经费(2018GH16)资助~~

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SVCVshlj1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。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 recombinant 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.

关 键 词：鲤春病毒血症病毒 糖蛋白 酵母表面展示系统 流式细胞仪 

分 类 号：Q786[生物学—分子生物学]