GnRH及其转运肽基因真核表达载体的构建及表达.pdfVIP

生物技术通报 ． 研究报告 · BloTECHNoLoGY BULLETIN 2008年第 1期 GnRH及其转运肽基因真核表达载体的构建及表达 金元昌 (一湖南科技大学生命科学学院，湘潭 41 1201； 广西大学生命科学与技术学院，南宁 530004) 摘 要： 目的 构建GnRH／TRS与绿色荧光蛋白 (GFP)的融合基因并表达。方法 利用DNA重组技术，将 GnRH／TRS片段克隆至真核表达栽体pEGFP—C1转化DH5 OL，重组体质粒pEGFP—C1一GntLH／TRS用LipoGen脂质体 进行转染至Hela细胞，核酸序列测定和Westem印迹分析基因表达，激光共聚焦荧光显微镜观察活细胞内荧光布局。结 果重组子pEGFP—C1一GnRH／TR成功构建。激光共聚焦荧光显微镜观察结果表明，GnRH／TRS—GFP融合基因的瞬间 和稳定表达均获得了相同结果。结论 GnRH／TRS—GFP融合蛋白具有GFP的自发荧光特性，且不影响GnRH／TRS分子 在细胞内的正确表达。 关键词： GnRH 转运肽 绿色荧光蛋白 Construction and Expression of Eucaryotic Expression of GnRH and Transporter Gene ( Hunan University of Science and Technology，Xiangtan 411201； 2College of Life Science and Biotechnology Guangxi University，Nanning 530004) Abstract： Objective To constructe A fu~on gene composed of green fluorescent protein(GFP)and GnRH／TRS DNA then express．MethodsClone GnRH／TRS into eucaryotic expression vector pEGFP-C1 by recombinantDNA technique．then transform to DH5 OL then pEGFP—C1一GnRH／TR transfect into Hela by LipoGen．Snucleotid sequence and W estem blot analysiswere used to evaluate the expression of the fused gene．Laser—scanning confocal microscopy was used to monitor the distribution of green fluorescence．Results Recombinantplasmid pEGFP-C1-GnRH／TR was successfully constructed Laser—scanning confocal microscopy clearly revealed that GnRH／TRS-GFP can be transiently and stably expressed in mammalian cells．Conclusion The GFP tag does not interfere with the natural assembly and transport of GnR H／TRS molecule Key words： Gonadotropin releasing hormone Transporter Green fluorescent protein 下丘脑以正常的频率和幅度分泌促性腺激素释放激素 (gonadotropin releasing hormone，GnRH)对生殖 功能的启动和维持是必要的，如果GnRH分泌无脉冲性，或其脉冲频