The Role Of P53 Binding Element In Regulation Of NOD8.

Objective:To construct a specific GFP expression vector drived by promoter of human NOD8 gene and to detect its transient expression in eukaryotic cells,to investigate the role of P53 binding element in regulation of NOD8.Methods:Four DNA promoter region of NOD8 containing the P53 consensus site was amplified by PCR from human genome DNA and correctly connected to the vector pEGFP-C2 which had cut out promoter by restriction enzyme to obtain the GFP expression vector driven by human NOD8 gene promoter:pEGFP-C2-NOD8(520bp) wt,pEGFP-C2-NOD8(760bp) wt.The constructed plasmids were transiently transferred into cell line HEK293 K562 and Hela cell by lipofectamine^TM 2000,to observe the GFP expression under the condition of inversion fluorescence microscope.Mutagenesis of the constructed vector pEGFP-C2-NOD8(7 60bp) wt to deletion the P53 binding site was carried out by using the QuikChange site-directed mutagenesis kit.The recombinant plasmid mpEGFP-C2-NOD8 was transiently transferred into cell line HEK293 by lipofectamine^TM 2000,the GFP expression was observed.Results:The constructed pEGFP-C2-NOD8wt plasmids and mpEGFP-C2-NOD8 were the same as the design confirmed by restriction digestion and sequence analysis.The results of the cell transient transfection indicated that different strength of green fluorescence expressed by recombinant plasmids in HEK293 K562 and Hela cellcould be observed under the condition of inversion fluorescence microscope.The GFP expression vectors driven by different length human NOD8 gene promoter which contains the P53 consensus site were different intensity(P＜0.05) and the GFP expression of constructed pEGFP-C2-NOD8(760bp)wt is stronger than that of the pEGFP-C2-NOD8(520bp)wt.The GFP expression of K293 cell is the strongest of all.The GFP expression of constructed pEGFP-C2-NOD8wt is stronger than that of mpEGFP-C2-NOD8wt(P＜0.05).Conclusion:(1) The GFP expression vector driven by human NOD8 gene promoter which contains the P53 consensus site and the site deleted plasimid were successfully constructed;(2) The GFP expression vectors driven by different length human NOD8 gene promoter which contain the P53 consensus site were different intensity,demonstrated that they have different priming efficiencies;(3)The GFP expression of recombinant plasmid mpEGFP-C2-NOD8, deleted the P53 binding site,was obviously weaken in HEK293 and K562 cell than that of pEGFP-C2-NOD8wt.The results indicate that P53 binding element may play a positive role in regulation of NOD8 gene,which establishes favourable bases for further study on the mechanism of NOD8 gene expression and regulation.