pSecTag-EGFP真核表达载体的构建及其转染鸡胚胎成纤维细胞条件的优化

Construction of eukaryotic expression vector pSecTag-EGFP and the study of transfecting chicken embryonic fibroblast cells

目的构建含报告基因增强绿色荧光蛋白(EGFP)的分泌型真核表达载体pSecTag-EGFP,用以研究pSecTag/HygroB转染鸡胚胎成纤维细胞的转染效率,为目的基因的成功表达奠定基础。方法用PCR方法克隆得到EGFP基因,通过酶切、连接、转化构建pSecTag-EGFP分泌型真核表达载体,设计优化实验,通过脂质体介导的方法转染鸡胚胎成纤维细胞,在荧光显微镜下直接观察该基因的表达。用B radford法检测不同组合的细胞培养液中的EGFP蛋白的相对含量。结果脂质体介导的方法能有效转染鸡胚胎成纤维细胞,实验中EGFP蛋白相对表达量随质粒量的增加而明显升高(P<0.01)。其中脂质体2μl、质粒1.0μg时目的蛋白相对表达量最高。结论pSecTag-EGFP质粒构建成功,并成功优化了转染条件。

Objective： To construct secretion eukaryotic expression vector pSecTag-EGFP and study the transfection efficiency in chicken embryonic fibroblast cells,in establishing the foundation for the aim gene expression in chicken embryonic fibroblast cells.Methods： EGFP gene was cloned and secretion eukaryotic expression vector was constructed by the method of digestion ligation and transformation.An optimum experiment was designed and chicken embryonic fibroblast cells with pSecTag-EGFP was transfectted.The things of the EGFP gene expression was observed under fluorescence microscope.Results： It was efficient to transfect chicken embryonic fibroblast cells by the method of lipofectamine mediation.The expression levels of protein were significantly different.The optimum condition of transfection was with 2μl lipofectamine and 1μg expression vector in medium.Conclusion： pSecTag-EGFP eukaryotic expression vectoris constructed successfully,and an optimum condition of transfection is found.