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[目的]研究CPNE5的A结构域(AD)对肿瘤坏死因子α(TNF-α)激活核因子κB(NF-κB)转录活性的调控作用。[方法]用PCR法扩增AD序列,将其构建EGFP-N1载体上,在真核细胞中表达融合蛋白AD-GFP。以连有κB序列和表达水平受κB序列调控的荧光素酶基因的质粒为报告基因载体,将EGFP-N1、AD-GFP质粒分别与报告基因共转染到HEK293细胞中,经TNF-α处理后,用双荧光素酶报告基因系统测定荧光素酶的活性,检测它们对NF-κB转录活性的影响。[结果]构建了AD-GFP质粒;A结构域能够显著抑制NF-κB转录活性(P<0.05);AD对NF-κB转录活性的抑制具有剂量依赖性。[结论]CPNE5的A结构域能够剂量依赖性抑制NF-κB的转录激活活性。
[Objective] To investigate the regulatory effect of A domain (CPAD) of CPNE5 on the transcriptional activity of nuclear factor kappa B (NF-κB) activated by tumor necrosis factor α (TNF-α). [Method] The AD sequence was amplified by PCR and then constructed into EGFP-N1 vector. The fusion protein AD-GFP was expressed in eukaryotic cells. The recombinant plasmids of EGFP-N1 and AD-GFP were co-transfected into HEK293 cells with reporter plasmids, respectively. The plasmids carrying the luciferase gene with κB sequence and expression level regulated by κB sequence were co-transfected into HEK293 cells with TNF-α Afterwards, luciferase activity was measured using a dual luciferase reporter system and their effect on NF-κB transcriptional activity was examined. [Results] Construction of AD-GFP plasmid; A domain could significantly inhibit the transcriptional activity of NF-κB (P <0.05); AD inhibited the transcription of NF-κB in a dose-dependent manner. [Conclusion] The A domain of CPNE5 can inhibit the transcriptional activation of NF-κB in a dose-dependent manner.