关键词: aquaporin-4, 转染, FRT细胞, 载体

Abstract: Abstract:Objective  To construct the  eukaryotic expression vector of pEGFP-aquaporin-4 and observe the expreesion and location of aquaporin-4 in FRT cells  for its EGFP fusion protein as a reporter by fluorescence microscope and to  provide experimental  basis for further study on  aquaporin-4.Methods The full-length coding sequence of aquaporin-4 was amplified by PCR,and subcloned into pEGFP-N1 for the expression in FRT cells.The FRT cells were transfected with aquaporin-4 using Lipofectamine 2000 Transfection Reagent with different concentrations.The expression of the fusion protein was detected by Western blotting and its location was observed by fluorescence microscope.The osmotic water permeability of the FRT cells was measured using a calcein fluorescence quenching method.Results The results of electrophoresis after restriction enzyme cleavage and sequencing indicated that the EGFP-aquaporin-4 eukaryotic expression vectors were identified.The results of fluorescencemicroscope and Western blotting confirmed the FRT cells expressed aquaporin-4 after EGFP-aquaporin-4 transfection.The osmotic water permeability of the  FRT cells transfeced with  recombinant eukaryotic expression vectors of EGFP-aquaporin-4 was 1.65 times higher than the control. Conclusion The recombinant eukaryotic expression vector of EGFP-aquaporin-4  is established successfully and it is proved that aquaporin-4 can express on FRT plasma membranes by Lipofectamine 2000 transfection.The FRT cells transfeced with aquaporin-4 display high osmotic water transport performance across cell  membranes.

Key words: aquaporin-4, transfection, FRT cells, plasmid