Construction and verification of NF-κB luciferase reporter gene system
(2.Beijing Key Laboratory of Research of Chinese Medicine on Prevention and Treatment for Major Diseases, Experimental Research Center, China Academy of Chinese Medical Sciences, Beijing, China 100102)
(3.Beijing Key Laboratory of Research of Chinese Medicine on Prevention and Treatment for Major Diseases,Experimental Research Center,China Academy of Chinese Medical Sciences, Beijing ,China 100700)
(4.Department of Orthopedics, Chinese People's Liberation Army General Hospital, Beijing, China 100853)
【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.
【Keywords】 NF-κB; NanoLuc luciferase; reporter gene system; NF-κB; NanoLuc luciferase; reporter gene system;
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