CYP2C9*58型新突变体的体外酶学活性研究

In vitro Enzymatic Activity Analysis of a Novel CYP2C9 Allelic Isoform CYP2C9*58

目的对CYP2C9基因新突变体(CYP2C9*58型)开展体外酶学功能研究,明确其代谢活性与野生型的相关性。方法以CYP2C9基因cDNA为模板,通过定点诱变方法获得CYP2C9各变异体的cDNA全长,用以构建昆虫表达载体。利用杆状病毒包装试剂盒包装昆虫病毒,侵染sf21昆虫细胞后获得高效表达CYP2C9各型蛋白的微粒体。以甲苯磺丁脲为探针底物药,利用获得的微粒体体外测定各变异体的最大反应速率V max和米氏常数K m,评价其主要酶促动力学特性。结果成功构建了CYP2C9*1、CYP2C9*2、CYP2C9*3和CYP2C9*58型4种CYP2C9昆虫表达载体,Western blot证实该载体可用于稳定表达相应的CYP2C9突变体。CYP2C9*2、CYP2C9*3和CYP2C9*58型变异体的体外酶学活性分别为野生型CYP2C9*1的91.6%,13.5%和23.1%。结论 CYP2C9*58型的酶学活性较野生型明显降低,接近于典型缺陷型突变体CYP2C9*3型,提示携带此突变型的患者在服用经由CYP2C9代谢的相关药物时,药物代谢速度较野生型携带者会有一定程度的降低。

Objective To assess the biological function of a novel CYP2C9 allelic isoform CYP2C9 ＊ 58 in vitro and to study wheth- er the site mutation could influence the metabolic activity of CYP2C9 protein. Methods Full - length cDNA fragments of each CYP2C9 a]lelic isoforms were obtained by PCR site - directed mutagenesis and used for the insect expression vector construction. According to the manufacturer＇s instruction, insect cell microsomes expressing 4 CYP2C9 variants were obtained using Bac- to- Bac Baculovirus Expres- sion System. Then tolbutamide was used as the probing substrate to assess the metabolic characteristics （ Vmax and Km values） of each CYP2C9 variants in vitro. Results We successfully constructed four insect cell expression vectors for CYP2C9 variants and the results of western blot confirmed that they could be used for highly expressing the corresponding CYP2C9 mutant in insect cells. Functional analysis results revealed that the enzymatic activity of CYP2C9 ＊ 2, CYP2C9 ＊ 3 and CYP2C9 ＊ 58 were 91.6% , 13.5% and 23.1% to that of wild- type C^F2C9 ＊ 1 respectively. Conclusion The enzymatic activity of CYP2C9 ＊ 58 is significantly decreased than that of CYP2C9 ＊ 1 and is much closer to that of typical defective variant CYP2C9 ＊ 3. Our data indicate that patients carrying this mutated al- lele might be a slow metabolizer when they take drugs metabolized by CYP2C9 protein.