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目的 研究含双启动子表达载体在真核细胞中的表达活性。方法 构建分别含有CMV及CMV SV40启动子的pC LacZ及pCS LacZ真核表达载体。用阳离子脂质体Lipofectamine将其转染至TJ90 5真核细胞中。对转染后 2 4～ 72h的细胞提取物进行 β 半乳糖苷酶活性检测 ;并与转染标准pSV gal载体的细胞提取物的酶活性进行比较分析。结果 TJ90 5细胞中转染pCS LacZ的 β 半乳糖苷酶活性明显高于其它两种载体 (P <0 .0 5 ) ,且转染后 48h是酶活性达高峰的时间。结论 两种启动子同时启动的基因表达效率较高 ,这为今后基因治疗中提高外源基因表达效率提供了一条有益的探索之路。
Objective To study the expression activity of double promoter expression vector in eukaryotic cells. Methods The eukaryotic expression vectors pC LacZ and pCS LacZ containing CMV and CMV SV40 promoters were constructed. It was transfected into TJ90 5 eukaryotic cells using Lipofectamine as a cationic liposome. The β-galactosidase activity of the cell extracts from 24 h to 72 h after transfection was measured and the enzyme activity of cell extracts transfected with the standard pSV gal vector was compared. Results The β-galactosidase activity of pCS LacZ transfected TJ905 cells was significantly higher than that of the other two vectors (P <0.05), and the peak of enzyme activity reached the peak 48 hours after transfection. Conclusions Both promoters have higher gene expression efficiency at the same time, which provides a useful way to improve the efficiency of foreign gene expression in gene therapy in the future.