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

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 VspⅠand NheⅠrestriction digestion,PCR and sequence analysis.Recombinant plasmids were transferred into cell line HEK293,K562 and HeLa by lipofectamineTM2000.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 further study on the mechanism of NOD8 gene expression and regulation.