关键词: 肝细胞, CYP2E1基因, 慢病毒载体, 转染, 过表达

Abstract:

OBJECTIVE: To construct the lentivirus vector with CYP2E1 gene overexpression，lentivirus was packaged，then transduced into normal human liver cells (L02 cells)，finally the CYP2E1 overexpressing cells (CYP2E1 OE cells) were constructed and identified. METHODS：Primers were designed according to cDNA sequence of CYP2E1 gene from GenBank. The gene was amplified using PCR and ligated into the lentiviral vector pLVX-acGFP-C1. 293FT cells were transfected with the recombinant vector，viral supernatant was collected，L02 liver cells were then transfected. After puromycin screening，L02 cells with CYP2E1 gene transfection were constructed. Finally，gene sequencing，real-time fluorescent quantitative PCR and western blot assays were performed for identification. RESULTS：The sequence contained in the recombinant vector was exactly the same as CYP2E1 gene from GenBank. CYP2E1 gene expression level in L02 cells with CYP2E1 gene transfection was 273 times higher than that of normal L02 cells. CYP2E1 protein level in L02 cells with CYP2E1 gene transfection was 3.2 times higher than that of normal L02 cells. CONCLUSION：CYP2E1-overexpressing cell strain was successfully constructed by using lentiviral vector，this cell strain expressed CYP2E1 with high efficiency，and could become a useful tool for metabolism research of environmental or occupational pollutants.

Key words: liver cells, CYP2E1 gene, lentivirial vector, transfection, overexpression