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目的构建pcDNA3.1(+)-MEK1真核表达载体,体外转染人肝癌MHCC97H细胞,观察MEK1在肝癌细胞中的表达及其对ERK通路活性的影响。方法应用RT-PCR扩增出MEK1基因插入pcDNA3.1(+)中,形成重组载体pcDNA3.1(+)-MEK1。经测序确认后,利用LipofectamineTM 2000将重组载体转染MHCC97H肝癌细胞,使用含G418的培养基进行筛选;将肝癌细胞分为不转染组、转染空载体组和转染重组载体组,运用Western-blot方法检测MEK1、p-MEK1、ERK1/2、p-ERK1/2在各组细胞中的表达并绘制各组细胞的生长曲线对比其增殖能力。结果重组载体pcDNA3.1(+)-MEK1酶切鉴定电泳条带大小正确,测序结果经Blast比对与人MEK1基因开放性读码框100%吻合;重组载体载体转染肝癌细胞后MEK1表达水平较不转染及转染空载体载体组细胞显著升高,且磷酸化的MEK1及ERK1/2水平也明显升高,转染重组载体载体的肝癌细胞增殖能力得到显著增强。结论成功构建了真核表达载体pcDNA3.1(+)-MEK1,过表达MEK1蛋白可提高肝癌细胞MAPK/ERK通路活性。
Objective To construct pcDNA3.1 (+) - MEK1 eukaryotic expression vector and in vitro transfect human hepatocellular carcinoma cell line MHCC97H to observe the expression of MEK1 in hepatocellular carcinoma cells and its effect on ERK pathway activity. Methods The gene of MEK1 was amplified by RT-PCR and inserted into pcDNA3.1 (+) to form recombinant vector pcDNA3.1 (+) - MEK1. After confirmed by sequencing, the recombinant plasmids were transfected into MHCC97H hepatoma cells with LipofectamineTM 2000 and screened by medium containing G418. The hepatoma cells were divided into non-transfected group, empty vector transfected group and recombinant vector transfected group. -blot method was used to detect the expression of MEK1, p-MEK1, ERK1 / 2 and p-ERK1 / 2 in each group of cells, and the growth curve of each group of cells was compared to compare their proliferation ability. Results The recombinant plasmid pcDNA3.1 (+) - MEK1 was digested by restriction endonucleases to confirm the size of the electrophoresis band. The sequencing result was 100% identical to the open reading frame of human MEK1 gene by Blast. The expression level of MEK1 in the transfected hepatoma cells The number of phosphorylated MEK1 and ERK1 / 2 cells was significantly higher than that of the cells transfected with empty vector. The proliferation of hepatoma cells transfected with recombinant vector was significantly enhanced. Conclusion The eukaryotic expression vector pcDNA3.1 (+) - MEK1 was successfully constructed and the overexpression of MEK1 protein could enhance the activity of MAPK / ERK pathway.