Construction of Eukaryotic Expression Vector for Fusion Gene of Granulysin Active Peptide 9ku-GLS and Enhanced Green Fluorescent Protein and Its Expression in Murine Melanoma B16 Cells

Objective To construct a eukaryotic expression vector for fusion gene of granulysin active peptide(9ku-GLS)and enhanced green fluorescent protein(EGFP)and observe its expression in murine melanoma B16 cells.Methods 9ku-GLS gene was amplified by PCR and insert into plasmid pBudCE4.1.The constructed recombinant plasmid pBudCE4.1 /S9K was identified by re-striction analysis and sequencing and subcloned to plasmid pEGFP-C1.B16 cells were transfected with the constructed recombinant plasmid pEGFP-C1 /S9K and the expressed fusion protein was identified by fluorescent microscopy and RT-PCR.Results Restric-tion analysis proved that recombinant plasmid pEGFP-C1 /S9K was constructed correctly.Green fluorescence was observed in the B16 cells 24 h after transfection with pEGFP-C1 /S9K,and the target gene fragment at a length of 267 bp was amplified by RT-PCR.Conclusion The eukaryotic expression vector for fusion gene of 9ku-GLS and EGFP was successfully constructed and expressed in B16 cells.