双启动子报告基因表达载体的构建及其在真核细胞中表达活性测定

Construction of reporter gene vector containing two promoters and its expressing activity assay in eukaryotic cells

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 expressing activity of vector containing two promoters in eukaryotic cells.Methods Reporter gene expressing vectors pC LacZ and pCS LacZ containing CMV promoter and CMV SV40 promoters were constructed respectively.β galactosidase activity transfected with these two vectors in TJ905 cells were assayed.The vectors constructed were transfected into TJ905 cells with cationic liposome,lipofectamine.Preparation of extract from the cells 1 to 3 days post transfection and β galactosidase activity assays were performed.The enzyme activity of the cell extract transfected with standard pSV gal vector was compared with that of transfected with the two vectors constructed.Results The activity of β galactosidase with pCS LacZ was significantly higher than that with the two other vectors( P <0.05).The peak of the enzyme activity occurred 48 hours post transfection.Conclusion This will provide a beneficial exploring way in promoting gene expressing efficiency in futrue gene therapy.