nestin启动子驱动微管蛋白-EGFP载体的构建及其在神经干细胞中的应用

Construction of nestin promoter driven Tubulin-EGFP vector and its application in neural stem cells

目的:构建巢蛋白nestin启动子驱动的微管蛋白表达载体,并在斑马鱼及大鼠神经干细胞中正确表达,为实现神经干细胞/神经前体细胞中微管骨架的动态观察和研究提供工具。方法:设计引物扩增斑马鱼基因组DNA中nestin基因上游启动子-952 bp片段并克隆测序;通过PCR获得斑马鱼EGFP-α-Tubulin融合蛋白序列,构建pzNestin-EGFP-α-Tubulin重组表达质粒;通过显微注射将目的质粒导入斑马鱼胚胎并在共聚焦显微镜下观察并拍摄;采用电转法将目的质粒导入大鼠神经干细胞,在荧光显微镜下观察。结果:在荧光显微镜下均观察到斑马鱼胚胎和大鼠神经干细胞中的EGFP荧光蛋白信号。结论:成功构建了pzNestin-EGFP-α-Tubulin质粒,在斑马鱼胚胎和大鼠神经干细胞中均能表达。

Objective:This study aims to construct a vector of nestin promoter driven Tubulin-EGFP protein to be expressed in zebrafish embryos and rat neural stem cells for dynamic observation of the remodeling of microtubule in neural stem cells/neural precursor cells.Methods:A nestin promoter fragment(-952 bp)was obtained by PCR from zebrafish genomic DNA,and then cloned and sequenced.The sequence coding EGFP-α-Tubulin fusion protein of zebrafish was harvested by PCR and ligated into the vector with nestin promoter.The pzNestin-EGFP-α-Tubulin recombinant plasmid was constructed and verified by DNA sequencing.The plasmid was microinjected into zebrafish embryos,and EGFP protein signals expressed were observed and photographed under a confocal microscope.The plasmid was transfected into rat neural stem cells by electroporation,and the EGFP protein expression signals were observed under a fluorescence microscope.Results:Fluorescence signals of EGFP-fused protein were observed under fluorescence microscope in zebrafish embryos as well as in rat neural stem cells.Conclusion:The pzNestin-EGFP-α-Tubulin plasmid was successfully established and expressed in both zebrafish embryos and rat neural stem cells.