Construction And Identification Of Urokinase-type Plasminogen Activatorbiosensor Plasmid

Background/Object : To construct the eukaryotic expression vector urokinase-type plasminogen activator(u PA) biosensor which was the composition of the fusion protein enhanced cyan fluorescent protein-u PA(substrate)-yellow fluorescent protein variant(ECFP-u PA substrate-linker-YPet).Methods: By the template Src-biosensor, the YPet primers were designed by Primer Premier5.0 software, and the restriction enzyme sites, u PA substrate gene sequence and linker were added in its 5’ end. With the intermediate vector p DMTM-18 T, an eukaryotic expression vector which contained a fusion protein of ECFP-u PA substrate-linker-YPet was constructed by genetic engineering. Then the u PA biosensor was transfected into 293 T cells. The transfection efficiency and protein expression of fusion proteins were observed after 24 h. Fluorescence resonance energy transfer(FRET) was observed by the inversion fluorescence microscope and measured by the Meta Flour FRET 4.6 software.Results:The uPA biosensor vector was confirmed by the fragment of PCR, double restriction enzyme digestion and DNA sequencing. The transfection efficiency was nearly 40%.Immunofluorescence displayed u PA biosensor fusion protein expression situation in 293 T cells membrane and the FRET of u PA biosensor in the living 293 T cells was observed after incubation with the recombinant human u PA.Conclusion:u PA biosensor is successfully constructed and could be used as a molecular probe to study the temporal and spatial variation of u PA in living cells.