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为探讨P53结合位点在NOD8基因调控中的作用,采用PCR技术从人基因组DNA中扩增含有P53结合位点人NOD8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-C2中,构建含有人P53结合位点的人NOD8基因启动子驱动的绿色荧光蛋白载体pEGFP-C2-NOD8;并构建P53结合位点缺失突变载体pEGFP-C2-NOD8,将构建的质粒经阳离子聚合物JetPeiTM介导瞬时转染HEK293细胞,在倒置荧光显微镜下观察绿色荧光蛋白表达。用不同浓度的p53抑制剂PFT-α(pifithrinalpha,PFT-α)预处理HEK293细胞24 h后,将重组质粒pEGFP-C2-NOD8wt瞬时转染HEK293细胞,观察绿色荧光蛋白的表达。结果表明pEGFP-C2-NOD8wt和mpEGFP-C2-NOD8经酶切鉴定和序列测定证实重组质粒构建成功。细胞转染后,缺失P53结合位点的NOD8启动子驱动的绿色荧光蛋白荧光强度明显低于含P53结合位点的重组质粒;且不同浓度PFT-α预处理HEK293细胞后重组质粒绿色荧光蛋白表达下降呈剂量依赖性。结论是P53结合位点突变重组质粒在HEK293细胞中其绿色荧光表达明显减弱;PFT-α可以通过抑制P53表达使重组质粒pEGFP-C2-NOD8wt在细胞中绿色荧光表达呈剂量依赖性减弱。说明P53结合位点在NOD8基因调控中发挥了正调节作用。
In order to investigate the role of P53 binding site in the regulation of NOD8 gene, the promoter sequence of NOD8 gene with P53 binding site was amplified from human genomic DNA by PCR and cloned into the expression vector pEGFP-C2 To construct the green fluorescent protein vector pEGFP-C2-NOD8 driven by the NOD8 gene promoter of human P53 binding site and construct the pEGFP-C2-NOD8 deletion mutation vector of the P53 binding site, and the constructed plasmid was treated with a cationic polymer HEK293 cells were transiently transfected with JetPeiTM, and the expression of green fluorescent protein (GFP) was observed under an inverted fluorescence microscope. HEK293 cells were pretreated with different concentrations of PFT-α (PFT-α) for 24 hours. The recombinant plasmid pEGFP-C2-NOD8wt was transiently transfected into HEK293 cells to observe the expression of green fluorescent protein (GFP). The results showed that recombinant plasmid pEGFP-C2-NOD8wt and mpEGFP-C2-NOD8 were confirmed by restriction enzyme digestion and sequence analysis. After transfection, the fluorescence intensity of green fluorescent protein driven by NOD8 promoter with deletion of P53 binding site was significantly lower than that of recombinant plasmid with P53 binding site. After pretreatment of HEK293 cells with different concentrations of PFT-α, the expression of green fluorescent protein Decline in a dose-dependent manner. Conclusions: The green fluorescent protein expression of P53-binding mutant in HEK293 cells was significantly weakened. PFT-α could inhibit the expression of green fluorescent protein pEGFP-C2-NOD8wt in a dose-dependent manner in a dose-dependent manner. P53 binding sites play a positive regulatory role in NOD8 gene regulation.