The Cloning Of The Promoter Region Of Tie-2 Gene And The Expression Activity In HUVEC

Objective:To construct an eukaryotic expression vector of PEGFP-Nl-Tie-2 promoter which is under the control of the Tie-2 Promoter and to investigate the expression activity of this gene in Human Umbilical Vein Endothelial CellsMethods:Four DNA fragments which contain the 5'upstream sequences of Tie-2 gene coding region were isolated from human peripheral blood DNA genome by polymerase chain reaction(PCR) and then were subcloned into the eukaryotic expression vector PEGFP-N1.After confirming the correct of the recombinants,they were transformed into TOP 10 bacteira in order to get a lot of recombinants containing Tie-2 promoter.The recombinants were transfected into the four different cells:HUVEC,HELA,HEK293T and HepG2.The green fluorescent protein (GFP) was observed by fluorescence microscopy, and the different expresson activity of Tie-2 promoter was judged by comparison of expressed levels of GFP in various cell lines.Resμlts:It was successfμLly constructed the recombinant of expression vector containing Tie-2 promoter confirming by pcr,restriction endonucleas, and Sequencing.The GFP was found in HUVEC by transfecting two recombinant plasmids including the fragments of (-917bp-123bp) and (-1197bp-123bp) after 59 hours but none of others, moreover, none singal of GFP was found in cells of HELA,HEK293T and HepG2 transfected by all of the four recombinant plasmids.Conclusions:The recombinant eukaryotic expression vector was successfμLly constructed, driven by the promoter of Tie-2,which showed activity and specificity only in Human Umbilical Vein Endothelial Cells. This woμLd provide the way for gene targeted therapy...