AIM:To investigate the effect of NF-κB binding element deletion on transcriptional regulation of (NOX1).METHODS:pGL3-Basic vector inserted with the NOX1 proximal promoter, and the same vector inserted with the NOX1 proximal promoter in the absence of the positive NF-κB binding element, were constructed.After cloning, diges-tion and purification, NOX1 proximal promoter (≈1 415 bp) was inserted into the multicloning site of the pGL3-Basic vec-tor and then sequenced ( pGL3-NOX1-1415) .NF-κB binding elements in the NOX1 promoters were predicted by Alibaba 2.1 software.The positive element was deleted by overlapping PCR.The deletion mutant was inserted into the pGL3 vector in the same way (pGL3-NOX1-1327).The plasmids were transfected into A549 cells, and then the cells were stimulated with TNF-α.The luciferase activity was monitored on MD SpectraMax M5 enzyme-labeled instrument.RESULTS:The se-quences of pGL3-NOX1-1415 and pGL3-NOX1-1327 were identified to be correct.Compared with control group, the lucif-erase activity was significantly higher in the cells transfected with pGL3-NOX1-1327 (P<0.05), but it was significantly lower than that in the cells transfected with pGL3-NOX1-1415 (P<0.05).CONCLUSION: NF-κB plays an essential role in the transcriptional regulation of NOX1 in TNF-α-induced A549 cells.Activated NF-κB binds to specific elements in NOX1 promoter regions to control the transcription.%目的：构建含NADPH氧化酶1（NOX1）近端启动子区的pGL3萤光素酶报告基因载体及缺失NF-κB结合元件的相应载体，分别测定其相应活性，探讨NF-κB结合元件缺失对NOX1启动子区转录活性的影响。方法：采用PCR法克隆NOX1启动子区序列（1415 bp），将目的片段与pGL3萤光素酶载体分别双酶切、纯化后进行连接（pGL3-NOX1-1415），测序鉴定；应用Alibaba 2．1软件分析NOX1近端启动子区，获取NF-κB结合元件；重叠PCR法将含该元件的启动子区域（88 bp）缺失，并构建相应载体（pGL3-NOX1-1327）。将两载体分别与pRL-TK内参质粒瞬时转染进入A549细胞，采用TNF-α（10μg/L）刺激细胞24 h，萤光酶标仪检测A549细胞的萤光素酶活性。结果：测序鉴定结果提示pGL3-NOX1-1415及NF-κB结合元件缺失的pGL3-NOX1-1327载体构建成功；细胞实验显示，TNF-α刺激后，转染pGL3-NOX1-1415的A549细胞萤光素酶活性明显强于对照组（P＜0．05），而转染NF-κB结合元件缺失的pGL3-NOX1-1327的细胞萤光素酶活性明显低于转染pGL3-NOX1-1415组（ P＜0．05）。结论：TNF-α诱导A549细胞NOX1基因活化与NF-κB密切相关，NF-κB参与了TNF-α诱导的NOX1基因启动子的转录调控。
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