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  

Construction and evaluation of recombinant plasmids expressing CYP3A4 and GSTA1

作　　者：常彬霞[1] 游绍莉[2] 李保森[1] 貌盼勇[3] 辛绍杰[2] 

机构地区：[1]解放军第302医院非感染肝病诊疗中心,北京100039 [2]解放军第302医院肝衰竭诊疗中心,北京100039 [3]解放军第302医院试验技术保障中心,北京100039

出　　处：《临床肝胆病杂志》2012年第9期697-700,共4页Journal of Clinical Hepatology

基　　金：军队临床高新技术重大项目(2010gxj097)

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。Objective To create a recombinant plasmid that is capable of expressing the drug metabohsm enzymes, cytochrome P450 tamlly 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 pBudCFA. 1 expression vector, which supports simultaneous expression of two genes. The recombinant plasmid was designated as pBudCFA. 1 - CYP3A4 - GSTA1 and confirmed by se- quencing 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.

关 键 词：细胞色素P450酶系统 谷胱甘肽转移酶 质粒 同工酶类 

分 类 号：R96[医药卫生—药理学]