NF-κB荧光素酶报告基因系统的构建及验证

Construction and verification of NF-κB luciferase reporter gene system

摘要:

为了定量检测NF-κB的活化效果及筛选与NF-κB活化调控相关的药物，通过去除逆转录病毒载体pQCXIP原有的CMV启动子，并分别插入NF-κB增强子序列及荧光素酶NanoLuc报告基因序列，构建了一种新的含有NF-κB增强子序列和NanoLuc (NLuc) 报告基因序列的表达载体，并进一步建立受NF-κB调控的稳定表达NLuc荧光素酶的细胞系。酶切鉴定及测序结果表明成功构建了重组质粒pQCXIP-NF-κB-NLuc；NF-κB信号通路的刺激物肿瘤坏死因子TNF-α作用于构建的稳定表达NLuc的细胞系后出现特异性的荧光素酶反应，且该酶反应与TNF-α的刺激呈良好的时间、剂量依赖性，该结果表明受NF-κB调控的稳定表达NLuc荧光素酶的细胞系构建成功。实例验证中，NF-κB抑制剂雷公藤甲素对此细胞系NLuc荧光素酶表达的抑制呈剂量效应。综上，本实验构建的受NF-κB调控的稳定表达NLuc荧光素酶的报告基因系统可用于NF-κB的活化效果的定量检测及筛选与NF-κB活化调控相关的药物，具有研究和应用价值。

Abstract:

To quantify the transcriptional activity of NF-κB and to screen drugs related to the regulation of NF-κB activation, we constructed a recombinant plasmid through deleting the original CMV promoter of retrovirus vector pQCXIP and inserting the NF-κB enhancer and NanoLuc luciferase sequence into the vector. Then, using the recombinant plasmid we constructed a cell line in which the expression of NanoLuc luciferase (NLuc) was regulated by NF-κB. The inserted sequences were verified by restriction endonuclease digestion and sequencing. Tumor necrosis factor-α (TNF-α), an NF-κB activator, acted on the constructed NLuc cell line and leaded to the specific luciferase reaction. The luciferase reaction showed a fine time and dose dependence to the TNF-α stimulation, indicating the successful construction of the NF-κB regulated NLuc-expressing cell line. Besides, the NF-κB inhibitor, triptolide, reduced the expression of NLuc in a dose-dependent way. The constructed reporter system in this study could be applied in the quantification of the NF-κB transcriptional activity and in the NF-κB regulation-related drug screening.