CYP3A29稳定表达HepG_2细胞株的建立及其硝苯地平代谢活性鉴定

目的建立稳定表达Bama小型猪CYP3A29的HepG2细胞株。方法通过总RNA提取、经RT-PCR得到Bama小型猪CYP3A29的基因,并将此基因克隆至亚克隆载体PMD18-T上得到重组质粒pMD-3A29;以测序正确的重组质粒pMD-3A29为模板,采用PCR扩增CYP3A29基因并在其3′添加组氨酸标签,扩增产物经BamHI/XhoI双酶切,将带有组氨酸标签的CYP3A29基因定向克隆至pcDNA3.1(+)中,并测序验证;将测序正确的CYP3A29基因通过脂质体转染至HepG2细胞,G418筛选10代,经RT-PCR及Western-blot分析及探针药物硝苯地平对重组细胞进行活性鉴定。结果与HepG2相比,HepG2-CYP3A29细胞株具有极显著的硝苯地平氧化活性。结论成功建立了稳定表达CYP3A29的HepG2细胞株,可用于相关药物代谢研究。

Establishment of a HepG2 cell line stably expressing the CYP3A29 isoenzyme and identification of its nifedipine metabolic activity

To establish a HepG2 cell line,which stably expressing CYP3A29 of Bama miniature pig in order to evaluate the drug metabolic characteristics of this isoenzyme,the gene for this system was obtained through total RNA extraction and RT-PCR assay.the gene was subcloned into plasmid PMD18-T,designated as pMD-CYP3A29.This gene was then amplified by PCR,and cloned into eukaryotic expression vector pcDNA3.1(+),and the recombinant plasmid was designated as pcDNA-CYP3A29.Sequencing was used to confirmed the correctness of the gene of this gene.the expressed gene was then transfected into HepG2 cells by lipid-media transfection and the transformants were screened by G418 for 10 generations;the expression of CYP3A29 was identified by RT-PCR、West-blot and the metabolic activity of the transformant HepG2-CYP3A29 was verified by nifedipine oxidation.In comparison with HepG2,the transformant HepG2-CYP3A29 showed remarkable oxidative activity.It is apparent that the cell line stably expressing CYP3A29 isoenzyme was successfully established,and it may be used for the metabolic study of related drugs.