NF-κB结合位点在NOD2基因调控中的作用

发布时间：2018-05-15 23:37

本文选题：NOD2启动子 + NF-κB结合位点　；参考：《暨南大学》2008年硕士论文

【摘要】： 目的构建含有NF-κB结合位点的人NOD2基因启动子驱动的绿色荧光蛋白表达载体和缺失NF-κB结合位点的人NOD2基因启动子驱动的绿色荧光蛋白表达载体,观察其在真核细胞中表达情况,探讨NF-κB结合位点在NOD2基因调控中的作用。方法以人基因组DNA为模板,PCR扩增含有NF-κB结合位点的四段不同长度的人NOD2基因启动子序列,以切除启动子的pEGFP-N3作为框架结构,将这四段序列片段进行酶切并定向克隆入表达载体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载体pEGFP-N3-NOD2(617bp)wt、pEGFP-N3-NOD2(747 bp)wt、pEGFP-N3-NOD2(1136 bp)wt、pEGFP-N3-NOD2(1387 bp)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、Hela细胞及ECV304细胞,在倒置荧光显微镜下观察其能否在NOD2基因启动子的调控下表达报告基因绿色荧光蛋白(greenfluorescent proteins,GFP)。用突变试剂盒将重组质粒pEGFP-N3-NOD2(617 bp)wt中的NF-κB结合位点缺失突变,将构建的突变重组质粒mpEGFP-N3-NOD2瞬时转染Hela细胞及HEK293细胞,观察绿色荧光蛋白的表达情况。结果 pEGFP-N3-NOD2wt和mpEGFP-N3-NOD2经酶切鉴定和序列测定证实重组质粒构建成功,并且NF-κB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HEK293、Hela及ECV304细胞株后,在倒置荧光显微镜下均能看到绿色荧光,含有NF-κB结合位点的不同长度的人NOD2启动子片段驱动的绿色荧光蛋白的表达的强度相同(P＞0.05),其中构建的pEGFP-N3-NOD2wt重组质粒在Hela及HEK293细胞中绿色荧光表达明显强于突变质粒mpEGFP-N3-NOD2的表达(P＜0.05)。结论 (1)成功构建了含有NF-κB结合位点的不同长度的人NOD2基因启动子的重组质粒和含有NF-κB结合位点缺失突变的重组质粒; (2)含有NF-κB结合位点的不同长度的人NOD2启动子片段驱动的绿色荧光蛋白的表达强度相同,说明含有NF-κB结合位点的不同长度人NOD2启动子效率相同; (3)NF-κB结合位点突变重组质粒在Hela细胞及HEK293细胞中绿色荧光表达明显减弱,说明NF-κB结合位点在NOD2基因调控中可能发挥了正调节作用;为进一步研究NOD2基因表达及调控机制奠定了良好的基础。
[Abstract]:Purpose The green fluorescent protein expression vector driven by human NOD2 gene promoter with NF- 魏 B binding site and the green fluorescent protein expression vector driven by human NOD2 gene promoter with missing NF- 魏 B binding site were constructed to observe its expression in eukaryotic cells. To investigate the role of NF- 魏 B binding site in the regulation of NOD2 gene. Method Human genomic DNA was used as template to amplify four fragments of human NOD2 gene promoter with NF- 魏 B binding site. The pEGFP-N3 of the excision promoter was used as the frame structure. The four fragments were digested by enzyme and cloned into the expression vector pEGFP-N3. To construct a green fluorescent protein vector pEGFP-N3-NOD2N 617bpwtTN 747bpN3-NOD2N 747 bEGFP-N3-NOD2N 1136 BEGFP-N3-NOD2N 1136 bpwtpEGFP-N3-NOD2N 1387 bpwtand, the constructed recombinant plasmid was transfected into HEK293 cells and ECV304 cells by liposome Lipofectamine~(TM)2000, and the recombinant plasmid pEGFP-N3-NOD2N was transfected into Hela cells of HEK293 cells and ECV304 cells by liposome Lipofectamine~(TM)2000, and the recombinant plasmid pEGFP-N3-NOD2N was transfected into Hela cells and ECV304 cells by liposome Lipofectamine~(TM)2000, and the recombinant plasmid pEGFP-N3-NOD2N was constructed. The expression of the reporter gene green fluorescent protein (GFP) was observed under inverted fluorescence microscope under the regulation of promoter of NOD2 gene. The deletion of NF- 魏 B binding site in recombinant plasmid pEGFP-N3-NOD2(617 bp)wt was detected by mutation kit. The recombinant plasmid mpEGFP-N3-NOD2 was transiently transfected into Hela cells and HEK293 cells to observe the expression of green fluorescent protein (GFP). Result PEGFP-N3-NOD2wt and mpEGFP-N3-NOD2 were identified by restriction endonuclease digestion and sequencing. The recombinant plasmid was successfully constructed and NF- 魏 B binding site mutation was successful. The results of cell transfection showed that the green fluorescence could be observed under inverted fluorescence microscope after transfection of the recombinant plasmid into HEK293 Hela and ECV304 cell lines. The expression intensity of the green fluorescent protein driven by human NOD2 promoter fragment with different length of NF- 魏 B binding site was the same as that of the mutant plasmid mpEGFP-N3-NOD2 (P > 0.05). The green fluorescence expression of the constructed pEGFP-N3-NOD2wt recombinant plasmid was significantly stronger than that of the mutant plasmid mpEGFP-N3-NOD2 in Hela and HEK293 cells (P < 0.05). Conclusion 1) Recombinant plasmids containing different lengths of human NOD2 gene promoter with NF- 魏 B binding sites and recombinant plasmids with deletion mutation of NF- 魏 B binding sites were successfully constructed. (2) the expression intensity of green fluorescent protein driven by human NOD2 promoter fragment with different length of NF- 魏 B binding site was the same, which indicated that the efficiency of human NOD2 promoter with different length of NF- 魏 B binding site was the same. The expression of green fluorescence in Hela cells and HEK293 cells decreased obviously, indicating that NF- 魏 B binding sites may play a positive role in the regulation of NOD2 gene. It lays a good foundation for further study of NOD2 gene expression and regulation mechanism.
【学位授予单位】：暨南大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R346

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