The role of NF-κB binding element in regulation of NOD2 gene
- VernacularTitle:NF-κB结合位点在NOD2基因调控中的作用
- Author:
Liping SUN
;
Chaofeng HU
;
Jikai ZHANG
- Publication Type:Journal Article
- Keywords:
NOD2 promoter regions (genetics);
NF-κB binding site;
Gene regulation;
Deletion mutation
- From:
Chinese Journal of Microbiology and Immunology
2008;28(7):606-609
- CountryChina
- Language:Chinese
-
Abstract:
Objective To investigate the role of NF-KB binding element in regulation of NOD2. Methods Promoter region of NOD2 containing the NF-κB binding site was amplified by PCR from human genome DNA and correctly connected to the vector pEGFP-N3 which had been cut out promoter by restriction enzyme to obtain the GFP expression vector driven by human NOD2 gene promoter. The constructed plasmids were transiently transferred into cell line HeLa by LipofectAMINETM2000 and the GFP expression was ob- served by the inversion fluorescence microscope. The NF-κB binding site in the constructed vector pEGFP- N3-NOD2wt was deleted by the QuikChange site-directed mutagenesis kit. The recombinant plasmid mpEG- FP-N3-NOD2 was transiently transferred into cell line HeLa by LipofectAMINETM2000, and the GFP expres- sion was observed by the inversion fluorescence microscope. Results The constructed pEGFP-N3-NOD2wt plasmids and mpEGFP-N3-NOD2 were the same as the design confirmed by restriction digestion and se- quence analysis. The results of the cell transient transfection indicated that different strength of GEP ex- pressed by recombinant plasmids in HeLa cells could be observed. The GFP expression of constructed mpEGFP-N3-NOD2 was lower than that of pEGFP-N3-NOD2wt. Conclusion The GFP expression vector driven by human NOD2 gene promoter which contains the NF-κB binding site, and the site deleted plasmid were successfully constructed. The GFP expression of recombinant plasmid mpEGFP-N3-NOD2, deletion of the NF-KB binding site, was obviously weaken in HeLa. The results indicate that NF-KB binding element may play a positive role in regulation of NOD2 gene, which establishes favourable bases for further study on the mechanism of NOD2 gene expression and regulation.
