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目的构建核因子I-B基因(NFIB)真核表达重组质粒,应用于其高表达调控肝细胞中蛋白磷酸酶2A(PP2A)的B亚基基因转录的功能学验证。方法逆转录聚合酶链反应(RT-PCR)获得人NFIB mRNA片段,克隆入真核绿色荧光蛋白表达载体(pEGFP-N1),构建pEGFP-NFIB重组质粒后,分别给予250、500、1 000 ng转染永生化人正常肝L02细胞建立NFIB高表达细胞模型(相应设为250、500和1 000 ng剂量组),对照组为250 ng pEGFP-N1转染的L02细胞,荧光定量聚合酶链反应检测NFIB mRNA,激光共聚焦和免疫印迹法(WB)检测核因子-1(NF-1)蛋白水平。电泳迁移率实验分析与NF-1相结合的DNA序列的特异性,荧光素酶报告基因检测PP2A-B55δ亚基基因(PPP2R2D)启动子区的转录活力,RT-PCR和WB检测细胞内PPP2R2D mRNA和PP2A-B55δ蛋白水平。结果双向测序证实pEGFP-NFIB重组表达质粒构建成功。与对照组比较,各剂量组L02细胞中NFIB mRNA相对水平增高(P<0.01),增强型绿色荧光蛋白、NFI-B蛋白表达水平均升高(P<0.05)。NF-1可与PPP2R2D基因启动子区(2R2Dp)-462G>A不同多态性序列结合,NFIB高表达使L02细胞中2R2Dp-462G启动子活力下降(P<0.05),PPP2R2D mRNA和PP2A-B55δ蛋白水平均升高(P<0.05)。结论成功构建人NFIB真核表达重组质粒,应用于人肝细胞中证实NF-1靶向PPP2R2D的功能学调控作用。
Objective To construct eukaryotic expression recombinant plasmid of nuclear factor I-B gene (NFIB) for functional verification of transcription of subunit B of protein phosphatase 2A (PP2A) with high expression and regulation in hepatocytes. Methods Human NFIB mRNA fragment was obtained by reverse transcription-polymerase chain reaction (RT-PCR), cloned into eukaryotic green fluorescent protein expression vector (pEGFP-N1), and constructed recombinant plasmid pEGFP-NFIB, Transfection of immortalized human normal liver L02 cells established NFIB overexpression cell model (corresponding to 250, 500 and 1 000 ng dose groups), control group 250 ng pEGFP-N1 transfected L02 cells, fluorescence quantitative polymerase chain reaction NF-|ÊB mRNA, laser confocal microscopy and Western blotting were used to detect the level of nuclear factor-1 (NF-1). The electrophoretic mobility was used to analyze the specificity of the DNA sequence binding to NF-1. The transcription activity of PP2A-B55δ subunit gene (PPP2R2D) promoter region was detected by luciferase reporter assay. The intracellular PPP2R2D mRNA was detected by RT-PCR and WB And PP2A-B55δ protein levels. Results Two-dimensional sequencing confirmed that pEGFP-NFIB recombinant plasmid was successfully constructed. Compared with the control group, the relative levels of NFIB mRNA in L02 cells in each dose group increased (P <0.01), and the expression levels of enhanced green fluorescent protein and NFI-B protein increased (P <0.05). NF-1 could bind to different polymorphic sequences of 2R2Dp-462G> A in PPP2R2D gene. The high expression of NFIB could decrease the activity of 2R2Dp-462G promoter in L02 cells (P <0.05), while the expression of PPP2R2D mRNA and PP2A-B55δ Protein levels were elevated (P <0.05). Conclusion The eukaryotic expression recombinant plasmid of NFIB was successfully constructed and used in human hepatocytes to confirm the functional regulation of NF-1 targeting PPP2R2D.