The Role Of Sp1 Binding Element In Regulation Of RIP2 Gene

ObjectiveIt is found that there is a special Sp1 binding element in human RIP2 gene promoter through bioinformatics studying. To construct a specific EGFP expression vector drived by promoter of human RIP2 gene and detect its transient expression in eukaryotic cells, furthermore, to coustrust a specific EGFP-RIP2 fusion protein expression vector drived by promoter of human RIP2 gene and detect its transient expression and RIP2 mRNA expression in eukaryotic cells, to investigate the role of Spl binding element in regulation of RIP2 gene.MethodsTwo DNA promoter regions of RIP2 containing the Sp1 binding element were amplified by PCR from human genome DNA and correctly connected to the vector pEGFP-C2 which had been cut out the CMV promoter by restriction enzyme to obtain the EGFP expression vector driven by human RIP2 gene promoter:pEGFP-C2-RIP2(750bp)wt and pEGFP-C2-RIP2(941bp)wt. The constructed plasmids were transiently transferred into cell line HEK293 by JetPeiTM, EGFP expression was observed under the inverted fluorescence microscope. Mutagenesis of the constructed vector pEGFP-C2-RIP2(750bp)wt to deletion the Sp1 binding site was carried out by the QuikChange site-directed mutagenesis kit. The recombinant plasmid mpEGFP-C2-RIP2(740bp) was transiently transferred into cell line HEK293 by JetPeiTM, the EGFP expression was observed. Furthermore, CDS region of RIP2 was amplified by PCR from human cDNA, and directly cloned in the MCS domain of vector pEGFP-C2. The EGFP-RIP2 fusion protein pEGFP-C2-RIP2(750bp+S)wt and mpEGFP-C2-RIP2(740bp+S) were transiently transferred into cell line HEK293 by JetPeiTM, EGFP expression was observed and the RIP2 mRNA expression was detected by RT-PCR.ResultsAll the constructed plasmids 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 RIP2 promoter expressed by recombinant plasmids in HEK293 cells could be observed under the inverted fluorescence microscope. The EGFP expression of vectors driven by different length human RIP2 gene promoters which contained the Spl binding element showed the different intensities, and the EGFP intensity of constructed pEGFP-C2-RIP2(750bp)wt is stronger than that of pEGFP-C2-RIP2(941bp)wt(P<0.05). But the EGFP and RIP2 mRNA expression of vectors driven by wildtype promoter and Sp1 binding element deletion mutation promoter show the same intensity (P>0.05).Conclusion(1) The EGFP expression vector driven by human RIP2 gene promoter which contains the Sp1 binding element and the site deleted plasimid have been successfully constructed;(2) The EGFP-RIP2 fusion protein expression vector driven by human RIP2 gene promoter which contains the Spl binding element and the site deleted plasimid have been successfully constructed;(3) The EGFP expression of vectors driven by different length human RIP2 gene promoter which contain the Sp1 binding element shows the different intensities, which demonstrates that they have different efficiencies;(4) The EGFP and RIP2 mRNA expression of vectors driven by wildtype promoter and deletion mutation promoter show the same intensity. The results could not indicate that Sp1 binding element may play a role in regulation of RIP2 gene.