Abstract:

It was to construct an eukaryotic expression vector which is able to simultaneously express cytochrome P450, family 4, subfamily F, polypeptide 2(CYP4F2)and green fluorescent protein(GFP)in eukaryotic cells, and provide a useful tool for investigating the function of CYP4F2 gene. The full length cDNA encoding CYP4F2 was amplified by polymerase chain reaction(PCR)with cDNA of human HepG2 cell as a template, and subsequently inserted into pAcGFP1-C1 plasmid after digestion with the corresponding restriction endonuleases. The recombinant plasmid pAcGFP1-C1-CYP4F2 was confirmed by restriction endonuclease mapping, PCR amplification and DNA sequencing. HK2 cells were transfected with pAcGFP1-C1-CYP4F2 to investigate the expressions of CYP4F2 and GFP in eukaryotic cells. Fluorescence microscopy was employed to observe the expression of GFP, while RT-PCR and Western blot was utilized to analyze the expression of CYP4F2 gene in HK2 cells. Results showed that the expression vector pAcGFP1-C1-CYP4F2 was successfully constructed and resulted in simultaneously cellular expression of both CYP4F2 and GFP when transfected into HK2 cells. Fluorescence microscopy confirmed the GFP expression in the cells, RT-PCR and Western blot revealed that the expression of CYP4F2 and ACE was increased in CYP4F2 gene transfected cells.

Key words: CYP4F2, Eukaryotic expression, HK2 cells, ACE