Construction and evaluation of recombinant plasmids expressing CYP3A4 and GSTA1
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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CYP3A4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CYP3A4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。
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关键词:
- 细胞色素P450酶系统 /
- 谷胱甘肽转移酶 /
- 质粒 /
- 同工酶类
Abstract: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 pBudCE4.1-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.
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Key words:
- cytochrome P-450 enzyme system /
- glutathione transferase /
- plasmids /
- isoenzyines
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