pSecTag-EGFP真核表达载体的构建及其转染鸡胚胎成纤维细胞的研究

Construction of Eukaryotic Expression Vector of pSecTag-EGFP and Research of Chicken Embryonic Fibroblast Cells

目的研究pSecTag真核表达载体转染鸡胚胎成纤维细胞的转染效率,选出该质粒转染鸡胚胎成纤维细胞的最佳转染条件,从而为研究目的基因在鸡胚胎成纤维细胞中成功表达奠定基础。方法用PCR方法克隆得到EGFP基因,通过酶切、连接、转化的方法构建了pSecTag-EGFP分泌型真核表达载体,设计优化试验,通过脂质体介导的方法转染鸡胚胎成纤维细胞,用Bradford检测法检测正交优化不同组合细胞培养液中所表达分泌的增强绿色荧光蛋白的相对含量。结果成功构建pSecTag-EGFP分泌型真核表达载体,并选出该质粒转染鸡胚胎成纤维细胞的最佳转染条件,即脂质体加2μl、质粒加1.0μg。结论脂质体介导的方法能有效转染鸡胚胎成纤维细胞。

Objective To study the transfection efficiency of pSecTag in chicken embryonic fibroblast cells, obtained the optimum condition of transfection, and established the foundation for the target gene expression in embryonic fibroblast cells of chicken. Method EGFP gene was cloned by PCR and constructed secretive eukaryotic expression vector by digestion, ligation and transformation, the relative content of the green fluorescent protein were detected by experimental design optimization. Results The optimum condition of transfection was with 2 μl lipofectamine and 1 μg expression vector in medium. Conclusion This method can effectively transfect the chicken embryo fibroblast.