作　　者： ; ; ; ; ;

机构地区： 广东药学院生命科学与生物制药学院生物制药研究所

出　　处： 《广东药学院学报》 2010年第3期305-309,共5页

摘　　要： 目的构建单链T细胞抗原受体(scTCR),借助酵母展示载体对其进行表达和展示,并确定优化的展示时间点。方法利用融合PCR(fusion PCR)通过linker将TCR分子两条链的可变区连接在一起,构建成scTCR,并克隆入展示载体pYD1;转化酵母细胞后,通过激光共聚焦显微镜观察scTCR的表达情况,并利用流式细胞仪测定其表达曲线;洗脱酵母细胞表面的蛋白质,通过点杂交检测进一步确定scTCR的表达。结果 scTCR被成功地构建和克隆;转化酵母细胞后,激光共聚焦检测表明其分布在酵母细胞的表面,流式细胞检测表明scTCR在诱导24 h左右在酵母细胞表面的展示比例达到峰值;对酵母表面洗脱蛋白的点杂交检测进一步表明了scTCR的表达和展示。结论所构建的scTCR能够有效地表达并展示在酵母细胞表面,为下一步构建scTCR酵母展示库并确定最佳的表达诱导和筛选时机奠定了良好的基础。 Objective To construct the single chain T cell receptor（scTCR） and display it on the surface of yeast cells,and to identify the optimal time point for the display of scTCR.Methods Two variable regions of TCR were connected through the linker by fusion PCR to construct scTCR,which was consequently cloned into the yeast display vector pYD1.The expression of scTCR was determined by confocal laser scanning microscope（CLSM）,and the expression curve was produced based on the changes in expression level detected by flow cytometry（FCM）.The display of scTCR on the surface of yeast cells was confirmed by dot blot assay after treating yeast cells with elution buffer.Results scTCR was successfully constructed and cloned into the pYD1.The display of scTCR on the surface of yeast cells was identified by CLSM,and the expression peak appeared at 24 h after induction.With the eluted protein from yeast surface,dot blot assay further confirmed the expression and display of scTCR.Conclusion scTCR constructed by fusion TCR could express and display on the surface of yeast cells,which laid the foundation for the construction of yeast display scTCR library and the determination of optimal time point for induced expression and clone scanning.

关 键 词： 酵母展示 单链 细胞抗原受体 构建

领　　域： [生物学]