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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 effect of NF-κB binding site on the regulation of human NOD2 gene promoter. The promoter sequence of human NOD2 gene containing NF-κB binding site was amplified from human genomic DNA by PCR and cloned into the expression vector pEGFP-N3 of the excised promoter to construct a recombinant plasmid containing NF-κB binding site The human NOD2 gene promoter-driven green fluorescent protein (GFP) vector pEGFP-N3-NOD2wt was constructed and a 2-base deletion mutation vector of NF-κB binding site was constructed. The constructed recombinant plasmid was transiently transfected into HEK293 Cells were observed under inverted fluorescence microscope, the expression of green fluorescent protein. The results showed that the recombinant plasmid transfected HEK293 cells under inverted fluorescence microscope can be seen under the green fluorescence, pEGFP-N3-NOD2wt recombinant plasmid in HEK293 cells showed strong fluorescence, while the mutant plasmid mpEGFP-N3-NOD2 fluorescence intensity was significantly Weakened. These results suggest that NF-κB binding site is an important element that drives the transcription of NOD2 gene and plays a positive regulatory role in the regulation of NOD2 gene promoter, providing a new idea for further studying NOD2 gene expression and regulation mechanism.