荧光素酶
NF-κB
化学
αBκ
细胞培养
分子生物学
重组DNA
细胞生物学
转染
生物化学
生物
基因
信号转导
遗传学
作者
Zhilan Guo,Luyang Che,Jingzhe Li,Zhenxiao Sun,Changzhen Liu
出处
期刊:PubMed
日期:2016-10-25
卷期号:32 (10): 1465-1473
标识
DOI:10.13345/j.cjb.160185
摘要
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.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。.
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