The Construction of MIC3-EGFP Fusion Protein in Eukaryotic Expression Vector and Its Expression in COS-7 Cells

Objective:The eukaryotic expression vector pEGFP-C2-MIC3 was constructed for determination of expression and lo- cation of enhanced green fluorescentprotein (EGFP) gene in COS-7 cells.Method:MIC3 ORF was amplified by PCR from pcD- NA3-MIC3 and was inserted into the eukaryotie expression vector plasmid pEGFP-C2.There combinant plasmid pEGFP-C2-MIC3 was identified by PCR,double enzyme digestion and DNA sequencing.There combinant plasmid was transfected into COS-7 cell lines by lipofectamine.The transient expression and cellular localization of the fusion proteins were analyzed in the transfected COS-7 cells by fluorescent-microscopy.Results:There combinant plasmid pEGFP-C2-MIC3 was confirmed with PCR double enzyme digestion and DNA sequenceing.The results revealed that the MIC3-EGFP fusion protein was expressed in cytoplasm aftert 24h transfection.Conclu- sions:The recombinant plasmid MIC3-EGFP was successfully constructed and expressed in COS-7 cells.The fusion protein was located in cytoplasm.