传染性胰脏坏死病毒VP2蛋白酵母表面展示系统的建立

doi:10.16535/j.cnki.dlhyxb.2018-235

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大连海洋大学学报 ›› 2019, Vol. 34 ›› Issue (5) : 662-668. DOI: 10.16535/j.cnki.dlhyxb.2018-235

传染性胰脏坏死病毒VP2蛋白酵母表面展示系统的建立

贺文斌1、2、3，徐黎明2，赵景壮2，刘淼2，任广明2，卢彤岩2，尹家胜2*

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Establishment of yeast surface display system for VP2 protein in infectious pancreatic necrosis virus

HE Wen-bin 1, 2, 3, XU Li-ming2，ZHAO Jing-zhuang2, LIU Miao2, REN Guang-ming2, LU Tong-yan2, YIN Jia-sheng2*

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摘要

为了将传染性胰脏坏死病毒(Infectious pancreatic necrosis virous，IPNV)的主要保护性抗原及IPNV检测和疫苗开发的主要靶基因——VP2蛋白展示于酵母细胞表面，本研究中基于IPNV VP2基因序列设计引物，以IPNV ChRtm213的RNA为模板进行PCR扩增，然后将扩增产物连接到酵母表面展示载体pYD1上构建了重组质粒pYD1-VP2，将重组质粒转化至酵母EBY100感受态细胞中，得到含有重组质粒的酵母菌EBY100/pYD1-VP2，再向EBY100/pYD1-VP2中加入半乳糖进行VP2蛋白的诱导表达，并采用蛋白免疫印迹和细胞免疫荧光技术对VP2蛋白的酵母表面展示情况进行了分析。结果表明：经半乳糖诱导后VP2蛋白在酵母细胞表面获得了成功表达；细胞免疫荧光分析显示，重组酵母诱导表达后出现特异性绿色荧光，并且在一定时间内荧光强度与诱导时间成正相关，诱导24、36、48 h组及对照组(48 h)各组之间荧光酵母比例有显著性差异(P<0.05)。研究表明，IPNV VP2蛋白已经被酵母细胞高效表达并且成功展示于酵母细胞表面，本研究结果可为IPNV口服活载体疫苗的研制奠定基础。

Abstract

The VP2 gene， as the most important surface antigen of infectious pancreatic necrosis (IPN), was amplified by RT-PCR with specific PCR primers based on IPNV ChRtm213 VP2 gene, and the PCR product was ligated to pYD1 vector and the reliable plasmid was named as pYD1-VP2. The Saccharomyces cerevisiae strain EBY100 was transformed with pYD1-VP2 plasmid using electroporation, and then induced by galactose to express VP2 protein on yeast cell surface. The Western Blotting and immunofluorescence assay revealed that VP2 expression and a specific VP2 protein were observed in the induced EBY100/pYD1-VP2 cell which presented a specific green fluorescence on the cell surface, with positive correlated relationship between fluorescence intensity and induction time, with significant differences in proportion of fluorescent yeast among 24 h, 36 h, and 48 h, and control groups. The findings indicated that the VP2 protein was successfully displayed on the yeast cell surfaces and that provided the basis with oral living vector vaccine of IPNV.

导出引用

贺文斌, 徐黎明, 赵景壮, 刘淼, 任广明, 卢彤岩, 尹家胜. 传染性胰脏坏死病毒VP2蛋白酵母表面展示系统的建立[J]. 大连海洋大学学报, 2019, 34(5): 662-668 https://doi.org/10.16535/j.cnki.dlhyxb.2018-235

HE Wen-bin, XU Li-ming, ZHAO Jing-zhuang, LIU Miao, REN Guang-ming, LU Tong-yan, YIN Jia-sheng.

Establishment of yeast surface display system for VP2 protein in infectious pancreatic necrosis virus

[J]. Journal of Dalian Fisheries University, 2019, 34(5): 662-668 https://doi.org/10.16535/j.cnki.dlhyxb.2018-235

中图分类号： S941.41

基金

中央公益性事业单位基本科研业务费专项(2016HY-ZD0504)；中国博士后科学基金资助项目(198920)；国家自然科学基金资助项目(31802345)

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2019-10-10
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2019-10-10

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