一种新型双荧光素酶报告基因表达载体的构建

Construction of a novel dual luciferase reporter gene vector

目的:构建一种新型的双荧光素酶报告基因表达载体。方法:设计并扩增海肾荧光素酶基因hRLUC,将其插入到双酶切的含有萤火虫荧光素酶报告基因(Firefly)的载体pGL4.10中,得到pGL4.10-hRLUC;使用lipofectamine3000将pGL4.10和pGL4.10-hRLUC分别转染入HEK293细胞,检测细胞中荧光素酶活性,以未转染细胞作对照。结果:使用PCR方法获得hRLUC表达单元(2 350 bp);双荧光素酶报告基因表达载体pGL4.10-hRLUC经酶切和测序鉴定,插入正确。转染pGL4.10组荧光素酶活性相对值为(220.311±2.082);转染pGL4.10-hRLUC的细胞中荧光素酶活性低于pGL4.10转染细胞,高于未转染细胞(P<0.05)。结论:成功构建了双荧光素酶报告基因(hRLUC和Firefly)表达载体。

Aim： To construct a novel dual luciferase reporter gene vector. Methods： The rennet luciferase gene hRLUC was designed and amplified,then was inserted into pGL4. 10 to obtain pGL4. 10-hRLUC. pGL4. 10 and pGL4. 10-hRLUC were transfected into HEK293 cells using lipofectamine3000,and then the luciferase activity was detected. The cells without transfection were the control. Results： The hRLUC expression unit（ 2 350 bp） was amplified by PCR. The double luciferase reporter gene expression vector pGL4. 10-hRLUC was identified by enzyme digestion and sequencing. The luciferase activity of cells transfected with pGL4. 10-hRLUC was lower than those transfected with pGL4. 10（ 220. 311 ± 2. 082）,higher than those without transfection（ P〈0. 05）. Conclusion： The novel double luciferase（ hRLUC and Firefly） reporter gene vector is successfully constructed.