Abstract:

Abstract:Objective To construct the fusion gene eukaryotic expression vector pEGFP-N1-SUMO-3,and to study the expression and localization of GFP-SUMO-3 fusion protein in human prostate cancer cell line LNCaP.Methods The encoding region of SUMO-3 was obtained by PCR,and cloned into eukaryotic expression vector pEGFP-N1.Then the pEGFP-N1-SUMO-3 plasmid was transfected into LNCaP cells by liposome.The expression of the fusion protein was detected by Western blotting,and the subcellular localization  was observed by fluorescence microscope.Results The recombinant plasmid  pEGFP-N1-SUMO-3 was identified by BamHⅠ and XhoⅠ double digestion.As expected,it showed two bands of 4 700 and 312 bp.The recombinant plasmid also showed right sequence by the full length sequencing.The pEGFP-N1-SUMO-3 plasmid was transfected into prostate cancer LNCaP cells.A specific protein expression band at molecular weight of 39 000 was detected with  using GFP-antibody by Western blotting method.When observed by fluorescence micro
scope,the GFP-SUMO-3 fusion protein was mainly located in the nucleus of LNCaP cells.Conclusion The recombinant plasmid pEGFP-N1-SUMO-3 is successfully constructed,and GFP-SUMO-3 fusion protein is successfully expressed in mammalian cells and mainly located in the nucleus.

Key words: GFP-SUMO-3 fusion protein;recombinant protein;LNCaP cells;gene expression