双报告基因真核表达载体的构建及其功能鉴定

Constructing and Functional Analysis of the Expression Vector with Double Reporter Genes

目的:构建绿色荧光蛋白和海肾荧光素酶共同高效表达的双报告基因真核表达载体。方法:将增强型绿色荧光蛋白基因和海肾荧光素酶基因以昆虫病毒T2A序列相连接而后克隆进入pcDNA3.1(-)质粒,构建双报告基因真核表达载体。将该载体转染至COS-7细胞,通过荧光显微镜观察、照度计定量分析检测绿色荧光蛋白和海肾荧光素酶生物活性,Western Bolt检测T2A序列自剪切效率。结果:双报告基因真核表达载体能够同时表达非融合的绿色荧光蛋白和海肾荧光素酶,与单独表达载体产物具有相似的生物活性和表达效率。结论:双报告基因真核表达载体建立成功,为基因表达调控等相关领域研究提供辅助工具。

Objective： To construct an expression vector with double reporter genes of green fluorescent protein and renilla luciferase. Methods： Enhanced green fluorescent protein and renilla luciferase gene were linked by T2 A sequences of insect virus. Then the sequences were cloned into pcDNA3.1（-） plasmid to obtain the expression vector. The vector was transfected into Cos-7 cell line. The morphological changes of Cos-7 cells were observed under microscope. Illuminometer was used to analyze the bioactivity of green fluorescent protein and renilla luciferase. Western blot assay was used to detect self-shear efficiency. Results： The expression vector was constructed successfully which could express unfused green fluorescent protein and renilla luciferase. The bioactivity and expression efficiency of the vector were equal to each single expression vector. Conclusion： The constructed eukaryotic expression vector with double reporter genes will provide an alternative and helpful method for gene expression regulation studies.