β-连环素融合绿色荧光蛋白真核表达质粒的构建及表达  

Construction and Identification of Mammalian Cell Expression Vector for β-Catenin Fused to Green Fluorescent Protein

作　　者：张爱红[1] 徐雅飞[2] 王建枝[2] 

机构地区：[1]同济大学高等技术学院医学技术与护理系,上海200070 [2]华中科技大学同济医学院病理与病理生理系,武汉430030

出　　处：《同济大学学报（医学版）》2006年第2期70-73,77,共5页Journal of Tongji University(Medical Science)

基　　金：国家自然科学基金资助项目(39925012);国家杰出青年科学基金资助项目(30100057);国家"973"研究基金资助项目(G1999054007)

摘　　要：目的构建β-连环素融合绿色荧光蛋白真核表达质粒,为进一步研究-βcaten in在神经细胞内与其它蛋白质如tau、PS1的相互作用以及在阿尔茨海默病发病机制中的作用提供一种有用的工具。方法用亚克隆的方法将β-连环素基因插入到真核表达载体pEGFP-C1,重组质粒pEGFP-cat瞬时转染野生型鼠成神经瘤细胞株,并在荧光倒置显微镜下观察转染效率。结果酶切鉴定和测序结果证实编码-βcaten in的重组质粒构建成功,免疫荧光技术和免疫印记结果显示-βcaten in-GFP融合蛋白在真核细胞中获得表达。结论成功构建β-连环素融合绿色荧光蛋白真核表达质粒,并在鼠成神经瘤细胞中表达了-βcaten in-GFP融合蛋白。Objective To construct the expression vector of β-catenin fused to green fluorescent protein which is capable of expressing mammalian ceils and to provide a useful tool for investigating the interaction of （-catenin with other molecules like tau, PSI, especially the role of （-catenin involved in the mechanism of pathogenesis of Alzheimer disease. Methods β-catenin in PCDNA1 vector was subcloned into the green fluorescent protein vector pEGFP-C1. The recombinant plasmid pEGFP-cat was then transfected into mouse neuroblastoma ceils （N2a）, followed by observation of the cells by fluorescent microscope. Results It was identified by enzyme digestion and sequencing confirmed successful construc- tion of the recombinant plasmid. The expression of β-catenin-GFP fusion protein was comfirmed by immunofluorescence studies and Western blot. Conclusion pEGFP-cat fusion gene vector is successfully constructed and the fusion protein can be expressed in N2a ceils.

关 键 词：Β-CATENIN基因 绿色荧光蛋白 成神经瘤细胞 融合蛋白 

分 类 号：Q786[生物学—分子生物学]