尿激酶型纤溶酶原激活物生物传感器的构建及鉴定

doi:10.13481/j.1671-587x.20150605

Abstract

Abstract:

Objective To construct the eukaryotic expression vector urokinase-type plasminogen activator (uPA) biosensor which was the composition of the fusion protein enhanced cyan fluorescent protein-uPA (substrate)-yellow fluorescent protein variant (ECFP-uPA substrate-linker-YPet). Methods By the template Src-biosensor,the YPet primers were designed by Primer Premier 5.0 software,and the restriction enzyme sites,uPA substrate gene sequence and linker were added in its 5' end.With the intermediate vector pDM^TM-18T,an eukaryotic expression vector which contained a fusion protein of ECFP-uPA substrate-linker-YPet was constructed by genetic engineering.Then the uPA biosensor was transfected into 293T cells.The transfection efficiency and 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 MetaFlour FRET 4.6 software. Results The uPA biosensor vector was confirmed by the fragment of PCR and double restriction enzyme digestion.The transfection efficiency was nearly 40%.The immunofluorescence detection results displayed that uPA biosensor fusion protein expressed in the 293T cells membrane and the FRET of uPA biosensor in the living 293T cells was observed after incubation with the recombinant human uPA(rhuPA). Conclusion uPA biosensor is successfully constructed and it could be used as a molecular probe to study the temporal and spatial variation of uPA in living cells.

Key words: urokinase-type plasminogen activator, biosensor, fluorescence resonance energy transfer, osteoarthritis

CLC Number:

CUI Ganghua, RAO Feng, WANG Yan, CAO Weiwei, LIU Wei, WANG Weishan, SHI Chenhui. Construction and identification of urokinase-type plasminogen activator biosensor plasmid[J].Journal of Jilin University Medicine Edition, 2015, 41(06): 1124-1129.

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Construction and identification of urokinase-type plasminogen activator biosensor plasmid

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