PEPCK不同启动子调控报告基因表达效率的比较

Comparison of the Expression Efficiency of Two Different Recombinant Firefly Luciferase Reporter Plasmids pGL4-PEPCK-Luc2

目的构建两种不同的磷酸烯醇式丙酮酸羧激酶(PEPCK)基因启动子调控报告基因表达质粒,比较该启动子在各种细胞系中的表达效率。方法采用PCR克隆大鼠PEPCK启动子1323 bp和560 bp两条片段,将两个PEPCK启动子片段插入pGL4.26-Luc2报告质粒中,将重组质粒转染到肝脏细胞系和脂肪细胞系,以非脂肪或肝脏细胞系做对照,比较PEPCK驱动荧光素酶表达效率。结果通过酶切鉴定及基因测序,证明pGL4.26-PEPCK-Luc2荧光素酶表达质粒构建成功,且能够在体外表达荧光素酶。结论两种同片段的PEPCK启动子在各细胞系中的表达效率差异无显著性。

Objective To construct two different luciferase reporter plasmid pGL4.26-PEPCK-Luc2,and to evaluate the expression efficiency of the phosphoenolpyruvate carboxykinase（PEPCK） gene promoters in several cell lines in vitro.Methods Two PEPCK promoter fragments 1323 bp and 560 bp were obtained from rat genomic DNA by PCR,and the fragments were inserted in pGL4.26-Luc2 luciferase reporter plasmid.Then the recombinant plasmid was transfected into hepatoma and adipocyte cell lines to compare the PEPCK driven luciferase expression efficiency.Results Restriction enzyme digestion and nucleotide sequencing conformed that the coupling site of the recombinant was correct without base mutation and deletion.The luciferase was expressed in the cell lines transfected with different plasmids pGL4.26-PEPCK-Luc2.Conclusion The expression efficiency of the two different PEPCK promoters in various cell lines has no significant difference.