【作者】 孙丽萍；

【导师】 胡巢凤；

【作者基本信息】 暨南大学 ， 病理生理学， 2008， 硕士

【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^TM2000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。更多还原

【Abstract】 ObjectiveTo construct a specific GFP expression vector drived by promoter of human NOD2 gene and detect its transient expression in eukaryotic cells,to investigate the role of NF-κB binding element in regulation of NOD2 gene.MethodsFour DNA promoter regions of NOD2 containing the NF-κB consensus site were amplified by PCR from human genome DNA and correctly connected to the vector pEGFP-N3 which had cut out promoter by restriction enzyme to obtain the GFP expression vector driven by human NOD2 gene promoter:pEGFP-N3-NOD2（617 bp） wt,pEGFP-N3-NOD2（747 bp） wt,pEGFP-N3-NOD2（1136 bp） wt, pEGFP-N3-NOD2（1387 bp） wt.The constructed plasmids were transiently transferred into cell line HEK293,Hela and ECV304 by Lipofectamine^TM 2000,GFP expression was observed under the inversion fluorescence microscope.Mutagenesis of the constructed vector pEGFP-N3-NOD2（617 bp） wt to deletion the NF-κB binding site was carried out by using the QuikChange site-directed mutagenesis kit. The recombinant plasmid mpEGFP-N3-NOD2 was transiently transferred into cell line Hela and HEK293 by Lipofectamine^TM 2000,the GFP expression was observed.ResultsThe constructed pEGFP-N3-NOD2wt plasmids and mpEGFP-N3-NOD2 were the same as the design confirmed by restriction digestion and sequence analysis.The results of the cell transient transfection indicated that green fluorescence of different length NOD2 promoter expressed by recombinant plasmids in HEK293,Hela and ECV304 cells could be observed under the inversion fluorescence microscope.The GFP expression of vectors driven by different length human NOD2 gene promoter which contained the NF-κB consensus site showed the same intensity（P＞0.05） and the GFP expression of constructed pEGFP-N3-NOD2wt is stronger than that of mpEGFP-N3-NOD2（P＜0.05）.Conclusion（1） The GFP expression vector driven by human NOD2 gene promoter which contains the NF-κB consensus site and the site deleted plasimid have been successfully constructed;（2） The GFP expression of vectors driven by different length human NOD2 gene promoter which contain the NF-κB consensus site shows the same intensity,which demonstrates that they have same priming efficiencies;（3） The GFP expression of recombinant plasmid mpEGFP-N3-NOD2,deleted the NF-κB binding site,is obviously weaken than that of pEGFP-N3-NOD2wt in Hela and HEK293.The results indicate that NF-κB binding element may play a positive role in regulation of NOD2 gene,which establishes favourable bases for further study on the mechanism of NOD2 gene expression and regulation.更多还原