P53结合位点在NOD8基因调控中的作用
发布时间：2018-09-02 09:47
【摘要】： 目的: 构建含有P53结合位点的人N0D8基因启动子驱动的绿色荧光蛋白表达载体和含有P53结合位点缺失突变的人N0D8基因启动子驱动的绿色荧光蛋白表达载体,观察其在真核细胞表达情况,探讨P53结合位点在N0D8基因调控中的作用。 方法: 以人基因组DNA为模板,PCR扩增含有P53结合位点的两段不同长度的人NOD8基因启动子序列,以切除启动子的pEGFP-C2作为框架结构,将这两段序列片段进行酶切并定向克隆入表达载体pEGFP-C2中,构建含有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(520bp)wt、pEGFP-C2-NOD8(760bp)wt,将构建的重组质粒经脂质体Lipofectamine~(TM)2000介导瞬时转染HEK293细胞K562细胞及Hela细胞,在倒置荧光显微镜下观察其能否在NOD8基因启动子的调控下表达报告基因绿色荧光蛋白(green fluorescent proteins,GFP)。用突变试剂盒将重组质粒pEGFP-C2-3NOD8(760 bp)wt中的P53结合位点缺失突变,将构建的突变重组质粒mpEGFP-C2-NOD8瞬时转染HEK293细胞,观察绿色荧光蛋白的表达情况。 结果: pEGFP-C2-NOD8(760bp)wt和mpEGFP-C2-NOD8经酶切鉴定和序列测定证实重组质粒构建成功,并且P53结合位点突变成功。细胞转染结果表明,构建的重组质粒转染三种细胞后,在倒置荧光显微镜下均能看到绿色荧光,含有P53结合位点的不同长度的人NOD8启动子片断驱动的绿色荧光蛋白的表达的强度不相同(P＜0.05),其中重组质粒pEGFP-C2-NOD8(760bp)转染组荧光强度高于pEGFP-C2-NOD8(520bp)wt转染组;各重组质粒均在HEK293细胞中绿色荧光表达最强,而在HELA细胞中很弱。缺失P53结合位点的NOD8启动子驱动的绿色荧光蛋白的荧光强度明显弱于含P53结合位点的NOD8启动子驱动的绿色荧光蛋白的荧光,上述荧光的强弱用灰度值表示,均经统计学方法分析,P＜0.05,差异有统计学意义。 结论: (1)成功构建了含有P53结合位点的不同长度的人NOD8基因启动子的重组质粒和含有P53结合位点缺失突变的重组质粒;(2)含有P53结合位点的不同长度的人NOD8启动子片断驱动的绿色荧光蛋白的表达的强度不相同;说明在人NOD8基因不同长度的启动子中包含了不同作用的调控位点;(3)P53结合位点突变重组质粒在HEK293细胞中绿色荧光表达明显减弱,说明P53结合位点在NOD9基因调控中发挥了正调节作用;为进一步研究NOD8基因表达及调控机制奠定了良好的基础。
[Abstract]:Aim: to construct a green fluorescent protein expression vector driven by human N0D8 gene promoter with p53 binding site and a green fluorescent protein expression vector driven by human N0D8 gene promoter with p53 binding site deletion mutation. To observe the expression of p53 binding site in eukaryotic cells and to explore the role of p53 binding site in the regulation of N0D8 gene. Methods: human genomic DNA was used as template to amplify two sequences of human NOD8 gene promoter containing p53 binding site. The pEGFP-C2 of the excision promoter was used as the frame structure. The two fragments were digested and cloned into the expression vector pEGFP-C2. A green fluorescent protein vector pEGFP-C2- NOD8 (520bp) wt,pEGFP-C2-NOD8 (760bp) wt, with p53 binding site of human NOD8 gene promoter was constructed. The recombinant plasmid was transiently transfected into HEK293 cell line K562 and Hela cells via liposome Lipofectamine~ (TM) 2000 mediated transfection. The expression of reporter gene green fluorescent protein (green fluorescent proteins,GFP) was observed under inverted fluorescence microscope under the regulation of NOD8 promoter. Mutation kit was used to mutate p53 binding site of recombinant plasmid pEGFP-C2-3NOD8 (760 bp) wt). The recombinant plasmid mpEGFP-C2-NOD8 was transiently transfected into HEK293 cells to observe the expression of green fluorescent protein (GFP). Results: pEGFP-C2-NOD8 (760bp) wt and mpEGFP-C2-NOD8 were identified by restriction endonuclease digestion and sequencing. The recombinant plasmid was successfully constructed and p53 binding site mutation was successful. The results of cell transfection showed that green fluorescence could be observed under inverted fluorescence microscope after transfection of the constructed recombinant plasmid into three kinds of cells. The intensity of green fluorescent protein (GFP) driven by human NOD8 promoter fragment with p53 binding site was different (P < 0. 05). The fluorescence intensity of recombinant plasmid pEGFP-C2-NOD8 (760bp) group was higher than that of pEGFP-C2-NOD8 (520bp) wt transfection group. All the recombinant plasmids expressed the strongest green fluorescence in HEK293 cells, but weak in HELA cells. The fluorescence intensity of green fluorescent protein driven by NOD8 promoter without p53 binding site was significantly weaker than that of NOD8 promoter with p53 binding site. The difference was statistically significant (P < 0.05). Conclusion: (1) Recombinant plasmids with different lengths of human NOD8 gene promoter containing p53 binding sites and recombinant plasmids with deletion mutation of p53 binding sites were successfully constructed; (2) different p53 binding sites were found in the recombinant plasmids containing p53 binding sites. The intensity of the expression of green fluorescent protein driven by human NOD8 promoter fragment was different. The results showed that the promoter of human NOD8 gene had different regulatory sites, (3) the expression of green fluorescence in HEK293 cells was significantly decreased. The results suggest that p53 binding site plays a positive role in the regulation of NOD9 gene, which lays a good foundation for further study on the expression and regulation mechanism of NOD8 gene.
【学位授予单位】：暨南大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R346
本文编号：2218987
[Abstract]:Aim: to construct a green fluorescent protein expression vector driven by human N0D8 gene promoter with p53 binding site and a green fluorescent protein expression vector driven by human N0D8 gene promoter with p53 binding site deletion mutation. To observe the expression of p53 binding site in eukaryotic cells and to explore the role of p53 binding site in the regulation of N0D8 gene. Methods: human genomic DNA was used as template to amplify two sequences of human NOD8 gene promoter containing p53 binding site. The pEGFP-C2 of the excision promoter was used as the frame structure. The two fragments were digested and cloned into the expression vector pEGFP-C2. A green fluorescent protein vector pEGFP-C2- NOD8 (520bp) wt,pEGFP-C2-NOD8 (760bp) wt, with p53 binding site of human NOD8 gene promoter was constructed. The recombinant plasmid was transiently transfected into HEK293 cell line K562 and Hela cells via liposome Lipofectamine~ (TM) 2000 mediated transfection. The expression of reporter gene green fluorescent protein (green fluorescent proteins,GFP) was observed under inverted fluorescence microscope under the regulation of NOD8 promoter. Mutation kit was used to mutate p53 binding site of recombinant plasmid pEGFP-C2-3NOD8 (760 bp) wt). The recombinant plasmid mpEGFP-C2-NOD8 was transiently transfected into HEK293 cells to observe the expression of green fluorescent protein (GFP). Results: pEGFP-C2-NOD8 (760bp) wt and mpEGFP-C2-NOD8 were identified by restriction endonuclease digestion and sequencing. The recombinant plasmid was successfully constructed and p53 binding site mutation was successful. The results of cell transfection showed that green fluorescence could be observed under inverted fluorescence microscope after transfection of the constructed recombinant plasmid into three kinds of cells. The intensity of green fluorescent protein (GFP) driven by human NOD8 promoter fragment with p53 binding site was different (P < 0. 05). The fluorescence intensity of recombinant plasmid pEGFP-C2-NOD8 (760bp) group was higher than that of pEGFP-C2-NOD8 (520bp) wt transfection group. All the recombinant plasmids expressed the strongest green fluorescence in HEK293 cells, but weak in HELA cells. The fluorescence intensity of green fluorescent protein driven by NOD8 promoter without p53 binding site was significantly weaker than that of NOD8 promoter with p53 binding site. The difference was statistically significant (P < 0.05). Conclusion: (1) Recombinant plasmids with different lengths of human NOD8 gene promoter containing p53 binding sites and recombinant plasmids with deletion mutation of p53 binding sites were successfully constructed; (2) different p53 binding sites were found in the recombinant plasmids containing p53 binding sites. The intensity of the expression of green fluorescent protein driven by human NOD8 promoter fragment was different. The results showed that the promoter of human NOD8 gene had different regulatory sites, (3) the expression of green fluorescence in HEK293 cells was significantly decreased. The results suggest that p53 binding site plays a positive role in the regulation of NOD9 gene, which lays a good foundation for further study on the expression and regulation mechanism of NOD8 gene.
【学位授予单位】：暨南大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R346
【参考文献】
相关期刊论文 前1条
1 胡巢凤;参与免疫及炎症反应调控的胞浆蛋白NODs[J];中国病理生理杂志;2004年07期
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