Establishment of a HepG2 cell line stably expressing the CYP3A29 isoenzyme and identification of its nifedipine metabolic activity / 中国人兽共患病学报

ABSTRACT

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.