The Role Of NF-κB Binding Element In Regulation Of NOD2 Gene

ObjectiveTo construct a specific GFP expression vector drived by promoter of human NOD2 gene and detect its transient expression in eukaryotic cells,to investigate the role of NF-κB binding element in regulation of NOD2 gene.MethodsFour DNA promoter regions of NOD2 containing the NF-κB consensus site were amplified by PCR from human genome DNA and correctly connected to the vector pEGFP-N3 which had cut out promoter by restriction enzyme to obtain the GFP expression vector driven by human NOD2 gene promoter:pEGFP-N3-NOD2(617 bp) wt,pEGFP-N3-NOD2(747 bp) wt,pEGFP-N3-NOD2(1136 bp) wt, pEGFP-N3-NOD2(1387 bp) wt.The constructed plasmids were transiently transferred into cell line HEK293,Hela and ECV304 by Lipofectamine^TM 2000,GFP expression was observed under the inversion fluorescence microscope.Mutagenesis of the constructed vector pEGFP-N3-NOD2(617 bp) wt to deletion the NF-κB binding site was carried out by using the QuikChange site-directed mutagenesis kit. The recombinant plasmid mpEGFP-N3-NOD2 was transiently transferred into cell line Hela and HEK293 by Lipofectamine^TM 2000,the GFP expression was observed.ResultsThe constructed pEGFP-N3-NOD2wt plasmids and mpEGFP-N3-NOD2 were the same as the design confirmed by restriction digestion and sequence analysis.The results of the cell transient transfection indicated that green fluorescence of different length NOD2 promoter expressed by recombinant plasmids in HEK293,Hela and ECV304 cells could be observed under the inversion fluorescence microscope.The GFP expression of vectors driven by different length human NOD2 gene promoter which contained the NF-κB consensus site showed the same intensity(P＞0.05) and the GFP expression of constructed pEGFP-N3-NOD2wt is stronger than that of mpEGFP-N3-NOD2(P＜0.05).Conclusion(1) The GFP expression vector driven by human NOD2 gene promoter which contains the NF-κB consensus site and the site deleted plasimid have been successfully constructed;(2) The GFP expression of vectors driven by different length human NOD2 gene promoter which contain the NF-κB consensus site shows the same intensity,which demonstrates that they have same priming efficiencies;(3) The GFP expression of recombinant plasmid mpEGFP-N3-NOD2,deleted the NF-κB binding site,is obviously weaken than that of pEGFP-N3-NOD2wt in Hela and HEK293.The results indicate that NF-κB 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.