含人P21启动子双荧光素酶基因载体的构建

目的构建带有EGFP标记含人P21启动子的双荧光素酶报告基因载体。方法从人血白细胞DNA中克隆P21启动子,用于控制firefly荧光素酶的表达;renilla荧光素酶基因和EGFP基因用IRES连接,在CMV启动子控制下表达;上述元件和基因克隆至载体pLL3.7中,应用酶切和测序的方法进行鉴定正确后转染至293FT细胞观察EGFP表达。结果经酶切、测序证实成功构建了带有EGFP标记的含人P21启动子的双荧光素酶报告基因的载体,并成功表达EGFP。结论含人P21启动子的双荧光素酶报告基因的载体成功构建,为下一步的药物筛选打下基础。

Construction of the vector with human P21 promoter and Dual-Luciferase reporter gene

Objective To construct the Dual-Luciferase reporter vector with human P21 promoter and EGFP gene.Methods P21 promoter was amplified by PCR from human blood cell genomic DNA,which was used to control the expression of firefly gene.Renilla luciferase gene in the plasmid was constructed under the control of the CMV promoter,and the EGFP gene was connected to renilla luciferase gene with the IRES.All above elements and gene were cloned into the pLL3.7 vector.The recombinant vector pLL3.7-Dluc was identified by restriction enzyme digestion and sequencing,and then pLL3.7-Dluc was transfected into 293FT cells for expressing EGFP.Results The Dual-Luciferase reporter vector with human P21 promoter gene and EGFP gene was successfully constructed and expressed EGFP protein.Conclusion The recombinant vector pLL3.7-Dluc was constructed successfully,providing the foundation for further study of drug screening.