绿色荧光蛋白与Wee1 Hu融合基因的构建及其真核表达

 构建Wee1Hu与增强型绿色荧光蛋白 (GFP)融合基因表达载体 ,并对其在真核细胞的表达及生物学效应进行研究 .应用基因工程技术构建重组载体 ,脂质体转染胰岛 β细胞株 ,流式细胞仪和免疫沉淀 Western印迹检测融合蛋白的表达 ,共聚焦显微镜分析融合蛋白在活细胞内的分布 ,3 D结构模建分析其结构特点 ,并用MTT法检测其生物学活性 .结果显示融合基因在瞬时或稳定转染的真核细胞中均获表达 ,融合蛋白主要分布在胞核区 ,融合蛋白中Wee1Hu的空间构象与天然Wee1Hu完全相同 ,表达融合蛋白的胰岛β细胞可避免其被细胞毒T淋巴细胞 (CTL)杀伤 .结果表明GFP Wee1Hu融合蛋白中 ,可发绿色荧光的分子标签GFP未能影响Wee1Hu的结构及其生物学活性 ;Wee1Hu可通过调控细胞周期而阻断CTL介导的细胞凋亡

Construction and Expression of Recombinant Gene for Fusion of Wee1Hu and Green Fluorescent Protein

To construct the fusion gene of Wee1Hu and green fluorescent protein(GFP),and study the biological characteristics of Wee1Hu and the expression of the fused Wee1Hu\|GFP in islet cells,by using the standard cloning technology,the Wee1Hu\|GFP fusion gene was constructed successfully.The fused gene was transfected into islet cell line with LipofectAMINE.The expression of the fused protein was determined by flow cytometry(FCM) and immunoprecipitation\|Western blot,respectively.Confocal scanning microscope was used to evaluate the distribution of green fluorescence in the transfected cell.The expression of the fusion gene was observed both in transiently and stably transfected islet cell lines.The distribution of the fluorescence into the intranuclear domain was confirmed by means of the confocus scanning microscopy.The structure of the fused protein was the same as the natural Wee1Hu.The fusion protein could protect cells against the killing by cytotoxic T lymphocyte(CTL),observed with MTT assay.These data suggested that the GFP tag did not interfere with the natural assembly and the biological activity of Wee1Hu molecule.The forced expression of Wee1Hu protein during the cell cycle blocked apoptosis mediated by CTL.