人NOD8基因启动子绿色荧光蛋白表达载体的构建与鉴定

Construction of GFP expression vector drived by promoter of human NOD8 gene

目的:构建人NOD8基因启动子的绿色荧光蛋白表达载体。方法:用特定的限制性内切酶位点,以人基因组DNA为模板,PCR扩增含有人NOD8基因启动子不同长度2段序列,并进行酶切以切除启动子的pEGFP-C2作框架结构,插入表达载体pEGFP-C2中,构建含有人NOD8基因启动子驱动的绿色荧光蛋白载体pEGFP-C2-NOD8(520 bp)、pEGFP-C2-NOD8(760 bp),用VspⅠ和NheⅠ双酶切和PCR鉴定重组质粒,再将重组质粒进行DNA序列分析。构建的重组质粒经脂质体(lipofectam ine)TM2000介导转染HEK293、K562和HeLa细胞,转染48 h后在倒置荧光显微镜下观察。结果:pEGFP-C2-NOD8(520 bp)、pEGFP-C2-NOD8(760 bp)分别经酶切鉴定和序列测定证实目的基因已插入重组质粒;细胞转染结果表明,构建的2段重组质粒转染HEK293、K562及HeLa细胞均能表达绿色荧光,其中构建的pEGFP-C2-NOD8(760 bp)重组质粒绿色荧光表达强于pEGFP-C2-NOD8(520 bp)。结论:成功构建2段不同长度的人NOD8基因启动子绿色荧光蛋白表达载体。

Aim：To construct a specific GFP expression vector drived by promoter of human NOD8 gene. Methods： Two DNA segments of NOD8 gene promoter were amplified by PCR from human genome DNA and correctly connected to the vector pEGFP-C2 which had cut out promoter by restriction enzyme. Two sequences of constructed pEGFP-C2-NOD8 plasmids were analysed after identified by Vsp I and Nhe I restriction digestion, PCR and sequence analysis. Recombinant plasmids were transferred into cell line HEK293, K562 and HeLa by lipofectamine ^TM2000. The transfection 48 hours later under the inverted fluorescence microscope to observe. Results： Two constructed pEGFP-C2-NOD8 （520 bp ）, pEGFP-C2-NOD8（760 bp） plasmids were the same as the design confirmed by restriction digestion and sequence analysis. The results of cell transient transfection indicated that the GFP could be observed in HEK293, K562 and HeLa by two plasmids transfection. The GFP expression of constructed pEGFP-C2-NOD8 （760 bp） is stronger than that of pEGFP-C2-NOD8 （520 bp）. Conclusion： The GFP expression vector driven by human NOD8 gene promoter is successfully constructed, which establishes favourable bases for furtherstudy on the mechanism of NOD8 gene expression and regulation.