NF-κB结合位点突变对人NOD2基因启动子调控的影响

The influence of NF-κB binding site mutation on the regulation of human NOD2 gene promoter

探讨NF-κB结合位点突变对人NOD2基因启动子调控的影响。采用PCR技术从人基因组DNA中扩增含有NF-κB结合位点的人NOD2基因启动子序列,并定向克隆入已切除启动子的表达载体pEGFP-N3中,构建含有NF-κB结合位点的人NOD2基因启动子驱动的绿色荧光蛋白(green fluorescent proteins,GFP)载体pEGFP-N3-NOD2wt,并构建NF-κB结合位点2个碱基缺失突变的载体,将构建的重组质粒瞬时转染HEK293细胞,在倒置荧光显微镜下观察绿色荧光蛋白的表达情况。结果显示重组质粒转染HEK293细胞后,在倒置荧光显微镜下均能看到绿色荧光,其中pEGFP-N3-NOD2wt重组质粒在HEK293细胞中荧光表达较强,而突变质粒mpEGFP-N3-NOD2荧光强度明显减弱。说明NF-κB结合位点是驱动NOD2基因转录的重要元件,且在NOD2基因启动子的调控中发挥了正调节作用,为进一步研究NOD2基因表达及调控机制提供了新的思路。

To investigate the impact of NF-κB binding site mutation on the regulation of NOD2 gene promoter,promoter region of human NOD2 gene containing the NF-κB consensus site from human genomic DNA was amplified by PCR and correctly connected to the vector pEGFP-N3 which had cut out promoter to obtain the GFP expression vector driven by human NOD2 gene promoter.The constructed plasmids were transiently transferred into cell line HEK293 to observe the GFP expression under inversion fluorescence microscope.Mutagenesis of the constructed vector pEGFP-N3-NOD2wt to delete two-base of the NF-κB binding site was carried out by using the QuikChange site-directed mutagenesis kit.The mutation plasmid mpEGFP-N3-NOD2 was transiently transferred into cell line HEK293,and the GFP expression was observed under inversion fluorescence microscope.In the end,the constructed pEGFP-N3-NOD2wt plasmids and mutation of NF-κB binding site were the same as the design confirmed.The results of the cell transient transfection indicated that different strength of green fluorescence expressed by recombinant plasmids in HEK293 cells could be observed under the condition of inversion fluorescence microscope.The GFP expression of the mutant plasmid mpEGFP-N3-NOD2,deleted two-base of the NF-κB binding site,was obviously weaken in HEK293 than that of the recombinant plasmid pEGFP-N3-NOD2wt.These results indicate that the two bases play an important role in NF-κB binding element and indirectly indicate that NF-κB binding element may play a positive role in regulation of NOD2 gene,thus establishing a favorable basis for further study on the mechanism of NOD2 gene expression and regulation.