细胞色素P450 3A4及谷胱甘肽S转移酶A1重组质粒的构建及表达

摘要

目的 构建重组质粒pBudCE4.1 -细胞色素P450 3A4( CYP3A4) -谷胱甘肽S转移酶A1(GSTA1),使CYP3 A4和GSTA1可在真核细胞C3A中表达,并且增加其表达量.方法 从含有GSTA1和CYP3A4的开放阅读框(ORF)克隆中扩增GSTA1和CYP3A4基因;将片段GSTA1以及载体pBuDCE4.1双酶切后连接并转化,质粒命名为pBuDCE4.1 - GSTA1;将片段CYP3A4以及质粒pBuDCE4.1 - GSTA1双酶切后连接并转化,所得重组质粒命名为pBudCE4.1 - CYP3A4 - GSTA1;测序验证重组克隆中目的基因片段的序列信息;构建稳定细胞系,测定CYP3 A4活性及GSTA1的表达情况.结果 酶切及测序验证双表达重组质粒pBudCE4.1 - CYP3A4 - GSTA1构建成功,CYP3A4及GSTA1在转染细胞系中的表达量增多.结论 构建成的双表达重组质粒pBudCE4.1 - CYP3A4 - GSTA1符合应用要求.%Objective To create a recombinant plasmid that is capable of expressing the drug metabolism enzymes, cytochrome P450 family member 3A4 (CYP3A4) and glutathione S - transferase alpha 1 , (GSTA1) in eukaryotic cells. Methods The CYP3A4 and GSTA1 genes were amplified by PCR. The fragments were cloned by means of digestion and ligation into the pBudCE4. 1 expression vector, which supports simultaneous expression of two genes. The recombinant plasmid was designated as pBudCE4. 1 - CYP3A4 - GSTA1 and confirmed by sequencing and basic local alignment search tool (BLAST) analysis. After transfection into the human hepatoma -derived HepG2 cell line, C3A, the activity of CYP3A4 and expression of GSTA1 were assessed by midazolam (MDZ) 1 ' - hydroxylation and 4 - hydroxylation assay and immunohistochemistry, respectively. Results The recombinant pBudCE4, I -CYP3A4 - GSTA1 plasmid was constructed successfully and expressed GSTA1 and CYP3A4 in mammalian cells in vitro. Conclusion The recombinant pBudCE4. 1 - CYP3A4 - GSTA1 plasmid can increase expression of the drug metabolizing enzymes, CYP3A4 and GSTA1, in mammalian liver cells. Future studies may develop this tool into a novel therapy to improve metabolic activity and liver detoxification of drugs in humans.