Construction and verification of NF-κB luciferase reporter gene system.
- Author:
Zhilan GUO
1
;
Luyang CHE
2
;
Jingzhe LI
3
;
Zhenxiao SUN
1
;
Changzhen LIU
3
Author Information
- Publication Type:Journal Article
- Keywords: NF-κB; NanoLuc luciferase; reporter gene system
- From: Chinese Journal of Biotechnology 2016;32(10):1465-1473
- CountryChina
- Language:Chinese
- 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.
