The identification of N-terminal labeled GFP-TFF3 fusion protein in mammalian fibroblasts

Objective To construct the expression vector of pEGFP-N1-TFF3 fusion protein and explore the expression and localization of pEGFP-N1-TFF3 fusion protein in COS7 cells.Methods Total RNA was extracted from human epithelial cells.The hTFF3(286bp-462bp) gene sequence was amplified from the cDNA of HT29 cells by polymerase chain reaction(PCR) method and subcloned into pEGFP-N1 vector by EcoRⅠand XhoⅠ.After DNA sequencing was employed,the recombinant plasmid was transfected into HEK293 cells,and recombinant protein was identified by Western blot.Then the localization of recombinant protein in COS 7 cells was observed by laser scanning confocal microscopy.Results The hTFF3 gene was successfully cloned into pEGFP-N1 vector,then confirmed by enzyme digestion and sequencing.GFP-TFF3 fusion protein was detected by Western blot,corresponding to a molecular weight about 34kDa.GFP-TFF3 fusion protein was more located in the nucleus,less in the cytoplasm of COS 7 cells.Conclusion The eukaryotic expression plasmid of pEGFP-N1-TFF3 was successfully constructed and the expression of its fusion protein was in the nucleus and cytoplasm of COS 7 cells.