P53结合位点在NOD8启动子基因克隆调节及其对NOD8基因中的作用

The role of P53 binding element in the regulation of NOD8 gene

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。

To investigate the role of P53 binding element in the regulation of NODS gene,the promoter region of this gene containing P53 consensus was amplified by PCR from human genomic 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.Mutagenesis of the constructed vector pEGFP-C2-NOD8wt to delete the P53 binding site was carried out by using the Quik hange site-directed mutagenesis kit.The constructed plasmids pEGFP-C2-NOD8wt and mpEGFP-C2-NOD8 were transiently transferred into cell line HEK293 by JetPeiTM and the GFP expression was observed under the condition of inversion fluorescence microscope.After the HEK293 were treated with PFT-α,an inhibitor of P53,at different concentrations for 24 h,the recombinant plasmid pEGFP-C2-NOD8 was transiently transferred into cell line HEK293 by JetPeiTM,and the GFP expression was observed.It was found the constructed pEGFP-C2-NOD8wt plasmids and mpEGFP-C2-NOD8 were the same as the design confirmed by restriction digestion and sequence analysis.The GFP expression of constructed pEGFP-C2-NOD8wt was stronger than that of mpEGFP-C2-NOD8wt（P0.01）.PFT-α could inhibit the GFP expression of pEGFP-C2-NOD8wt in a concentration dependent manner（P0.05）.In the end,the GFP expression vector driven by human NOD8 gene promoter containing P53 consensus site and the site of deleted plasmid were successfully constructed.The result of the cell transient transfection indicated that the GFP expression of recombinant plasmid mpEGFP-C2-NOD8,deleted the P53 binding site,was obviously weaken in HEK293 cells.In a dose-dependent manner,PFT-α down-regulated the expression of GFP in cells transfected with pEGFP-C2-NOD8wt plasmids via inhibiting the function of P53.These results indicate that P53 binding element may play a positive role in regulation of NOD8 gene,thus establishing a favourable base for further study on the mechanism of NOD8 gene regulation.