金黄色葡萄球菌功能蛋白的酵母表面展示

华南理工大学学报（自然科学版） ›› 2007, Vol. 35 ›› Issue (11): 105-109.

金黄色葡萄球菌功能蛋白的酵母表面展示

向柱方¹林影^1†王小宁¹赵树进²韩双艳¹

1.华南理工大学生物科学与工程学院，广东广州 510640; 2. 广州军区广州总医院，广东广州 510010

收稿日期:2006-12-05 出版日期:2007-11-25 发布日期:2007-11-25
通信作者: 林影(1962-) ，女，教授， E-mail: feylin @ scut.edu. cn E-mail:raindrop506@ 126. com
作者简介:向柱方(1980-) ，女，博士生，主要从事分子生物学研究.
基金资助:
国家重大科技攻关项目(2004BA711 A20)

Yeast Surface Display of Functional Protein of Starphylococcus aureus

Xiang Zhu-fang¹ Lin Ying¹ Wang Xiao-ning¹ Zhao Shu-jin² Han Shuang-yan¹

1. School of Biological Science and Engineering , Soutb China Univ. of Tech. , Guangzhou 510640 , Guangdong , China;2. General Hospital of Guangzhou Military Command , Guangzhou 510010 ， Guangdong , China

Received:2006-12-05 Online:2007-11-25 Published:2007-11-25
Contact: 林影(1962-) ，女，教授， E-mail: feylin @ scut.edu. cn E-mail:raindrop506@ 126. com
About author:向柱方(1980-) ，女，博士生，主要从事分子生物学研究.
Supported by:
国家重大科技攻关项目(2004BA711 A20)

摘要/Abstract

摘要： 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(5αcchαromyces 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纽中.重纽菌经培养，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Westem 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.

关键词: 酿酒酵母, 金黄色葡萄球菌蛋白zz, 免疫荧光染色, 流式细胞仪

Abstract:

In this paper , the ZZ domain from Stαphylococcus aureus was first successfully displayed on the cell sur-face 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 plCZZ was constructed by amplifying the geneencoding ZZ on pEZZ18 template via PCR and by inserting the ZZ gene into shuttle vector plCAS via restriction enzyme digestion. Moreover , the constructed plCZZ was introduced in Saccharomyces cerevisiae MT8-l. 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 westem blot. All the results show that the rabbit IgG isolated by cells displaying ZZ is purer than that of standard sample.

Key words: Saccharomyces cerevisiae, ZZ domain from Staphylococcus αureus, immunofluorescence labeling, flow cytometry

向柱方林影王小宁l 赵树进韩双艳. 金黄色葡萄球菌功能蛋白的酵母表面展示[J]. 华南理工大学学报（自然科学版）, 2007, 35(11): 105-109.

Xiang Zhu-fang Lin Ying Wang Xiao-ning Zhao Shu-jin Han Shuang-yan. Yeast Surface Display of Functional Protein of Starphylococcus aureus[J]. Journal of South China University of Technology (Natural Science Edition), 2007, 35(11): 105-109.

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