细胞色素P450 3A4基因C239S突变体稳定表达Flp-In^(TM)CHO细胞系的建立

Establishment of Flp-In^(TM)CHO cell line with C239S mutant stably expressing cytochrome P450 3A4

目的构建稳定表达细胞色素P450家族3亚家族A成员4(CYP3A4)的C239S突变体(CYP3A4-C239S)的Flp-In^(TM)CHO细胞系。方法使用Lipofectamine2000转染试剂将pcDNA5/FRT-CYP3A4-C239S突变型重组质粒和pcDNA5/FRT-空质粒转染至Flp-In TM CHO细胞,潮霉素B(500 g/L)进行稳定筛选,采用实时荧光定量PCR(qRT-PCR)、蛋白质免疫印迹(Western blot)检测细胞中CYP3A4-C239S的mRNA和蛋白表达,采用黄曲霉素B1(AFB1)细胞毒性实验检测细胞活性。结果与Flp-In^(TM)CHO-空质粒组相比,Flp-In^(TM)CHO-3A4-C239S组的CYP3A4-C239S mRNA和蛋白表达水平均显著增加(P<0.001),AFB1细胞毒性实验显示,CYP3A4-C239S细胞组对AFB1的毒性敏感度显著增加(P<0.001)。结论成功构建了稳定表达CYP3A4-C239S的Flp-In^(TM)CHO细胞系,且为后续研究CYP3A4突变对药物代谢的影响支撑基础。

Objective To establish Flp-In^(TM)CHO cell line with C239S gene mutant(C239S)stably expressing cytochrome P450 family 3 subfamily A member 4(CYP3A4).Method The pcDNA5/FRT-CYP3A4-C239S mutant recombinant plasmid and pcDNA5/FRT empty plasmid were transfected into Flp-In TM CHO cells using Lipofectamine2000 transfection reagent.Stable screening was performed using hygromycin B(500 g/L),and real-time fluorescence quantitative PCR(qRT-PCR)and Western blot were used to detect the mRNA and protein expression of CYP3A4-C239S in the cells.Cell activity was detected using aflatoxin B1(AFB1)cytotoxicity assay.Result Compared with the Flp-In TM CHO empty plasmid group,the Flp-In^(TM)CHO-3A4-C239S group showed a significant increase in CYP3A4-C239S mRNA and protein expression levels(P<0.001).AFB1 cytotoxicity experiments showed that the CYP3A4-C239S cell group had a significantly increased sensitivity to AFB1 toxicity(P<0.001).Conclusion The Flp-In^(TM)CHO cell line that stably expresses CYP3A4-C239S is successfully constructed,so as to provide a basis for the subsequent research of the impact of CYP3A4 mutations on drug metabolism.