糖蛋白（glycoprotein, G）是鱼类传染性造血器官坏死病毒的主要表面抗原，是病毒检测及疫苗研制的主要靶基因。为了对其进行酵母表面展示，本研究利用本实验室保存的含有IHNV-Sn1203毒株G基因的质粒为模板， PCR扩增富含抗原表位的糖蛋白基因片段后，连接酵母表面展示载体pYD1，构建重组质粒pYD1-G。将pYD1-G转化酿酒酵母EBY100细胞后，利用半乳糖诱导G蛋白的表达。利用细胞免疫荧光、 Western Blotting及流式细胞仪检测酵母表面G蛋白的表达情况。细胞免疫荧光结果显示，转化了pYD1-G重组质粒的酵母细胞表面呈现出特异性荧光； Western Blotting结果显示，经半乳糖诱导后G蛋白在酵母细胞表面获得了成功表达；流式细胞仪检测结果表明，随着半乳糖诱导时间的增加， G蛋白的表达量随之增加，并在诱导48 h时达到峰值。以上研究表明G蛋白在酵母细胞表面获得了成功表达，本研究为以酵母为活载体的新型口服疫苗的研制奠定了基础。

Glycoprotein is the most important surface antigen of infectious haematopoietic necrosis virus (IHNV), and it has been served as the major gene for virus detection and vaccine development. The specific PCR primers for IHNV glycoprotein gene replication were designed based on the plasmid containing IHNV-Sn1203 glycoprotein gene. The PCR product was ligated to yeast surface display vector pYD1 and the recombinant plasmid pYD1-G was transformed into EBY100 cell. The transformed EBY100/pYD1-G cell was induced by galactose and the protein expression was detected by cell immunofluorescence, Western Blotting and flow cytometry. The cell immunofluorescence result showed that the transformed EBY100 cell presented a red fluorescence, and Western blotting analysis obtained a specific glycoprotein band. Both of these two results indicated that the glycoprotein was successfully displayed on the surface of yeast cell. The flow cytometry analysis showed that the glycoprotein expression was increased with the galactose induced time, and the maximum expression was obtained at 48 h. The successful display of glycoprotein on the yeast surface laid a foundation for live oral vaccine development.

Q 785； S 917

中央级公益性科研院所基本科研业务费专项（HSY201410）；黑龙江省自然科学基金（C201462）；黑龙江省应用技术研究与开发计划（GA13B401）