金黄色葡萄球菌功能蛋白的酵母表面展示

Yeast Surface Display of Functional Protein of Staphylococcus aureus

利用酿酒酵母表面展示系统,成功地将金黄色葡萄球菌功能蛋白ZZ锚定在酵母表面,并进一步用展示有蛋白ZZ的酵母细胞纯化出兔IgG.以pEZZ18为模板,通过PCR技术克隆了ZZ基因,将ZZ基因通过双酶切连接到穿梭载体pICAS,构建了酵母表面展示载体pICZZ,并将其转化至酿酒酵母(Saccharomyces cerevisiae)MT8-1中.核酸电泳结果表明ZZ基因成功整合到了酵母基因组中.重组菌经培养,利用免疫荧光染色方法进行染色,显微镜观察表明ZZ蛋白已经展示在酵母细胞表面,流式细胞仪分析结果证实80.4%的酵母细胞表达了ZZ蛋白.利用展示有蛋白ZZ的酵母细胞吸附兔血清中的IgG,洗脱后进行十二烷基磺酸钠-聚丙烯酰胺电脉,并进行W estern blot免疫印迹,结果表明,此重组酵母细胞纯化的兔IgG比标样兔IgG纯度更高.

In this paper, the ZZ domain from Staphylococcus aureus was first successfully displayed on the cell surface of yeast Saccharomyces cerevisiae by yeast cell-surface display systems. Next, rabbit IgG was purified from the serum by using the cells displaying ZZ. Then, yeast display vector pICZZ was constructed by amplifying the geneencoding ZZ on pEZZ18 template via PCR and by inserting the ZZ gene into shuttle vector pICAS via restriction enzyme digestion. Moreover, the constructed pICZZ was introduced in Saccharomyces cerevisiae MTS-1. It is indicated from the nucleic acid electrophoretic map that ZZ gene is successfully integrated into the genome. Moreover, the microscope images reveal that, after the immunofluorescence labeling of the cultured recombinant cells, ZZ proteins were successfully displayed on the cell surface. This observation is further verified by flow cytometry, which convinces the protein display of 80. 4% of cells. Finally, the IgG from the serum was absorbed by the yeast cells displaying ZZ proteins, and the elution was analyzed by means of SDS-PAGE and western blot. All the results show that the rabbit IgG isolated by cells displaying ZZ is purer than that of standard sample.