The Application Research Of Fluorescent Protein And Its Mimics In The Cell Membrane

Fluorescent proteins(FPs)had been developed as one of the most important tools in the field of biological sciences.Furtherly,Green fluorescent protein(GFP)is already widely used as a tracer in life science and medical research.GFP has a barrel structure consisting of eleven ?-strands,with its core chromophore covalently bonded an alpha helix running through the center.According to the research of fluorescent protein,especially its luminescence principle and chromophore,the researchers had developed many new systems based on GFP.Such as the bimolecular fluorescence complementation(BiFC)with split-GFP,and the GFP mimics by mimicking the bioluminescence principle of GFP.In this paper,we design and expend the research of the cell membrane imaging application based on the BiFC of split-GFP system and a red fluorescent protein(RFP)mimics synthesized by our team.The resrarch work is as follows:(1)We had constructed the recombinant plasmid of split-GFP thought the molecular cloning technology and cold shock expression vectors with the cDNA of sfGFP.Besides,we obtained the sfGFPl-9 and sfGFPl-10 through prokaryotic expression and purification steps in vitro.They can be combined with small synthetic peptide fragments(S10-11 or S11)and release fluorescence.The experiment could lay the groundwork for the development of the new analytic sensing method.(2)Our lab had exploited a RFP mimics called RMFP,consisting of small molecule named DFHBFSI,which is similar to the RFP chromophore,and a G-quadruplex(G4)sequence of DNA,namely NG16.RMFP has excellent photo-physical merits,such as large Stokes shift(93 nm),superior anti-photobleaching property,two-photon fluorescent property,etc.Then,we managed to apply RMFP to cell imaging systems based on cholesterol or membrane proteins aptamer.In addition,we successfully realized the membrane imaging with three kinds of membrane proteins(tyrosine kinase-7(PTK7),hepatocyte growth factor receptor(HGFR)and epithelial cell adhesion molecule(EpCAM)),and developed a membrane protein-specific probe(MPP).(3)In order to further prove that our PTK7-MPP is the specific-target to cell membrane surface,we build PTK7 eukaryotic expression recombinant plasmid and enhanced GFPs(EGFPs)plasmid with a pDisplayTM a mammalian expression vector that allows display of EGFPs on the extracellular side of cell plasma membrane.The result of fluorescence co-localization experiment shows that the Manders' overlap coefficient between EGFP Channel and MPP channel is 0.90.At the same time,we achieved a 2-hour fluorescence imaging of the cell membrane with RMFP,which is of great significance to the research of cell membranes.In addition,PTK7-MPP was collected at depth of approximately 100 ?m with tissue section by two-photon imaging.