人源蛋白酶体α亚基6(Proteasome subunit alpha 6)在酿酒酵母表面展示  

Display of Proteasome Subunit Alpha 6 on The Cell Surface of Saccharomyces cerevisiae

作　　者：唐语谦[1] 叶茂[1] 林影[1] 韩双艳[1] 郑泓[1] 王小宁[1] 梁世中[1] 

机构地区：[1]华南理工大学生物科学与工程学院,广州510640

出　　处：《生物化学与生物物理进展》2007年第9期984-990,共7页Progress In Biochemistry and Biophysics

基　　金：国家科技攻关计划项目(2004BA711A20).~~

摘　　要：为构建人源蛋白酶体α亚基6(α6)的酵母展示体系,研制其单克隆抗体用于抗体表位分析和研究泛素-蛋白酶体途径,建立绕过重组抗原表达及纯化制备、将展示重组抗原直接应用于抗体检测的方法.在酵母展示表达载体pICAS中引入His.tag标签,将编码α6的基因PSA6_HUMAN克隆到酵母表面展示载体pICAS-H上,用流式细胞仪检测其抗原表位活性,以表面展示α6的重组酵母细胞,结合酶联吸附免疫检测技术,建立酵母(yeast)-ELISA检测技术,应用于检测小鼠单克隆抗体及单抗效价.酵母细胞培养48h后获得抗原α6的高效表面展示,展示的α6具有良好的抗原活性和特异性,将α6的展示酵母用于yeast-ELISA的初步实验结果显示可有效检测和筛选到抗α6抗体.In order to construct the yeast display system of the human proteasome subunit alpha 6 （α6） and obtain its specific monoclonal antibodies for epitope analysis and mechanism investigation of ubiquitin-proteasome pathway, and set up a new rapid efficient way for the preparation of specific monoclonal antibodies （MAbs） without proteantigens which applicated recombinant antigen into detection directly, the gene PSA6_HUMAN coding human proteasome subunit alpha 6 was cloned into a yeast-displaying expression vector, pICAS-H, which had been inserted a His.tag marker for expression level detection. As probed with a His.tag monoclonal antibody and a specific monoclonal antibody generated by hybridoma, a recombinant yeast strain, α6-MT8, was selected by flow cytometry and fluorescence microscopy analysis. Combining with enzyme-linked immunosorbent assay （ELISA）, ‘yeast-ELISA＇ detection was established by basing on Saccharomyces cerevisiae cell surface engineering and applied to examined monoclonal antibody and its valance. The yeast-displaying recombinant antigen α6 with highly specific affinity was expressed efficiently after 48 h cultivation. The ‘yeast-ELISA＇ was demonstrated primarily to detect and screen monoclonal antibody successfully.

关 键 词：泛素-蛋白酶体途径 人源蛋白酶体α亚基6 酵母表面展示体系 抗体检测 

分 类 号：Q78[生物学—分子生物学] R392[医药卫生—免疫学]