细胞色素P4503A46基因的异源表达及药物代谢活性分析

Heterologous Expression and Metabolic Activity Analyses of Cytochrome P4503A46

为了克隆巴马小型猪细胞色素P4503A46基因,建立P4503A46稳定表达肝癌细胞株(Hep G2-P4503A46),并探究其药物代谢活性,通过RT-PCR从肝组织中钓取基因P4503A46,将基因与真核表达载体pc DNA3.1连接并转染Hep G2细胞,经G418筛选10代,获得稳定表达重组细胞株Hep G2-P4503A46,以P4503A特异性代谢底物硝苯地平(NF)探针药物,将探针药物与重组细胞在优化的细胞浓度、药物浓度、孵育时间等条件下进行体外孵育,通过高效液相色谱仪检测其药物代谢(抑制)动力学参数,并将对照组进行比较分析。结果表明,本研究成功从人Bama小型猪肝组织克隆了P4503A46基因,并通过G418筛选,成功建立了细胞稳定表达株,其硝苯地平代谢活性显著高于阴性对照组(P<0.01);因此,成功克隆、并建立了小型猪P4503A46的稳定表达细胞株(Hep G2-P4503A46),该细胞株具有显著的硝苯地平代谢活性。

To establish stably expressing recombinant cell line of P4503A46（ Hep G2-P4503A46）,and the drug metabolic activity of Hep G2-P4503A46 was detected. Methods： Sus scrofa P4503A46 gene was cloned from Bama miniature pig liver and the stably expressing cell line Hep G2-P4503A46 was obtained under the selection of gentamycin（ G418）,the nifedipine（ NF） was incubated with the recombinant cell lines of Hep G2 and Hep G2-P4503A46 in optimal conditions,the high performance liquid chromatograph（ HPLC） was utilized to detect the metabolites after the incubation ended,significant metabolic activity was observed（ P〈0. 001）. Results showed that the gene of Sus scrofa P4503A46 was cloned,and the cell line heterologously expressing the P4503A46 was established successfully,the activity analysis demonstrated that the cell line of P4503A46 had the metabolic activities of nifedipine. Conclusion： stable cell line of P4503A46 was produced successfully,and significantly metabolic activity to nifedipine was observed（ P〈0. 001）.