Construction of a specific green fluorescent protein expression vector derived from promotor of human nucleotide-binding oligomerization domain-2 gene

To construct a specific green flurescent protein(GFP)expression vector derived from human nucleotide-binding oligomerization domain-2(NOD2) gene,4 DNA fragments of NOD2 gene promoter from human genomic DNA were amplified by PCR and correctly connected to vector pEGFP-N3 which had been cut out by restriction enzymes.Four sequences of the constructed plasmid pEGFP-N3-NOD2 were analyzed after identification with Vsp I and Pst I restriction digestion and PCR.The recombinant plasmid was transfected to cell line HEK293T by lipofectamineTM2000 and the HEK293T cells was treated with TNF-α for 24 hrs within 24 hrs after transfection or the cells were let untreated with TNF-α.Whether the GFP expression derived from human NOG2 gene promoter was present,it was determined by use of the minitor of inverted fluorescent microscopy.It was found that the sequences of 4 constructed target plasmids pEGFP-N3-NOD2(617 bp),(747 bp),(1 136 bp),(1 387 bp)had been proved to be inserted to the recombinant plasmids as demonstrated by restriction enzyme digestion and sequence analysis.The HTK293T cells transfected with these 4 target plasmids could develop green fluorescence,in which the recombinant plasmid pEGFP-N3-NOD2(1387 bp)developed the strongest fluorescence after stimulation with TNF-α.It is apparent that the GFP expression vector derived from human NOD2 gene promoter is succesively constructed,thus establishing a favorable basis for further study on the mechanism of NOD2 gene expression and regulation.