细胞色素P450 2C9药物代谢酶体外酶学活性检测新方法的建立  

A Novel in vitro Functional Analysis Method for CYP2C9 Protein

作　　者：王双虎[1,2] 戴大鹏[3] 胡利明[1,2] 耿培武[1,2] 蔡剑平[3] 胡国新[2] 

机构地区：[1]温州医学院研究生院,325035 [2]温州医学院药理教研室 [3]卫生部北京医院/卫生部北京老年医学研究所/卫生部老年医学重点实验室,100730

出　　处：《医学研究杂志》2013年第2期23-26,共4页Journal of Medical Research

基　　金：科技部重大新药创制;心脑血管疾病新药临床评价技术平台研究基金资助项目(2012ZX09303008)

摘　　要：目的建立一种基于COS-7细胞的细胞色素P450 2C9(CYP2C9)体外酶学活性检测的新方法。方法以野生型CYP2C9*1基因的全长cDNA为模板,通过定点诱变方法获得突变型CYP2C9*2、*3及*13的cDNA全长,分别与分泌型荧光素酶Gluc内参基因一起导入pIRES载体的A、B多克隆位点,构建成双表达载体pIRES-Gluc-CYP2C9。利用Lipofectamine 2000瞬时转染COS-7细胞,48h后弃培养基,加入含100μmol/L Luciferin-H荧光底物的新鲜培养基,继续培养8h后利用Promega CYP2C9活性试剂盒及NEB Gluc检测试剂盒分别检测CYP2C9各型及内参Gluc的活性,用Western blot检测两种蛋白的表达量。结果本研究成功构建了pIRES-Gluc-CYP2C9*1及其突变体*2、*3、*13的表达载体,通过检测CYP2C9特异性荧光素底物Luciferin-H的分解产物的荧光信号与分泌型荧光素酶Gluc的内参荧光信号的比值作为CYP2C9的体外酶学活性值,研究结果显示突变体CYP2C9*2、*3、*13的体外酶学活性分别为野生型CYP2C9*1的43.12%、8.15%和1.49%。结论本研究成功建立了一种基于COS-7细胞的CYP2C9体外酶学活性检测的新方法,该方法操作简便、实验周期短,适于新突变体CYP2C9的快速活性检测及其代谢新药物或抑制剂的相关性研究。Objective To construct a novel in vitro CYP2C9 functional analysis method using COS - 7 cell so as to simplify the anal- ysis process and improve the reliability. Methods Full - length cDNA fragments of variants CYP2C9 ＊ 2, ＊ 3 and ＊ 13 were obtained by PCR site - directed mutagenesis using the cDNA of wild type CYP2C9＊ 1 as the DNA template. The products and open reading frame （ORF） of internal control Glue were inserted into the multiple cloning sites A and B of dual - expression vector plRES separately. Resul- ting plasmid was named pIRES - Gluc - CYP2C9 and transfected into COS - 7 cell using lipofeetamine 2000 reagent. Forty - eight hours later, the culture medium was discarded and replaced with fresh medium containing Lueiferin - H （ 100tzmol/L） and the cells were incu- bated at 37℃ for additional 8 hours in CO2 incubator. Then the catalytic activities of CYP2C9 variants and internal control protein Gluc were detected separately using the corresponding Promega or NEB assay kit, and the protein expression level in transfected ceils was ana- lyzed by Western blot method. Results Using Luciferin - H in Promega kit as the substrate, as compared with wild type CYP2C9 ＊ 1, the catalytic activities of variants CYP2C9 ＊ 2, ＊ 3, ＊ 13 were 43. 12% , 8. 15% and 1.49% separately after calibration. Conclusion In vitro functional analysis results showed that the metabolic properties of typical CYP2C9 variants in our system were similar to that of tradi- tional detection system using microsomes as the starting meterial. Our novel in vitro CYP2C9 functional analysis method is simple, time - saving and reliable.

关 键 词：CYP2C9 Western BLOT 体外活性分析 

分 类 号：R969[医药卫生—药理学]