海洋哈维氏弧菌溶血素蛋白在酵母菌细胞表面上的展示及其活性的测定

为制备以酒精酵母为载体的基因工程疫苗,参照其Genbank中基因序列设计一对引物,PCR扩增得到预期长度的产物,双酶切插入用于酒精酵母表面展示的穿梭质粒载体pYD1,转化大肠杆菌TOP10,提取阳性质粒转化酒精酵母菌株EBY100,诱导表达后,用免疫荧光和流式细胞仪检测外源蛋白的表达,结果测得最佳诱导时间为36～48h,诱导培养后约30.0%左右的酵母细胞表达外源蛋白;同时以EBY100和转空质粒的酵母菌株为对照,测得诱导培养后的阳性酵母转化株的细胞对牙鲆鱼血细胞具有溶血活性,以此酵母活细胞分设高中低三个浓度组,腹腔注射养殖牙鲆幼鱼,检测其毒性,结果表明此重组酵母对牙鲆是安全的.该研究为下一步鱼用活载体疫苗免疫效果的研究奠定了基础.

Yeast surface display of marine vibrio harveyi hemolysin and activity assay of the displayed protein

To make a vaccine using Saccharomyces cerevisiae as the live carrier,the gene encoding hemolysin was cloned into plasmid pYD1 which allows regulated expression,secretion and detection of expressed proteins on surface of Saccharomyces cerevisiae cells.The construct was propagated in E.coli TOP10 and then was transformed into the yeast strain EBY100.The display of the hemolysin protein on the yeast surface was conformed either by hemolytic activity assay against flounder erythrocytes or by fluorescent staining with antibody.The results indicated that the activity of the fusion protein reached the highest level after induced with 2.0% galactose for 36 hours.Fluorescence Activated Cell Sorting(FACS) analysis showed that 30% of the cells was anchored with the fusion protein.Then the lethal effect on young flounders of the yeast cells displaying the hemolysin was examined and they were found safe to the fish,which laid a foundation for the succeeding research work on the immunological effect of the live vaccine.