人源细胞色素P450 2E1与细胞色素P450氧化还原酶的共表达

Co-expression of human cytochrome P450 2E1 and cytochrome P450 oxidoreductase

目的利用大肠埃希菌(E.coli)表达系统异源表达细胞色素P450 2E1(cytochrome P450 2E1,CYP2E1),并与细胞色素P450氧化还原酶(cytochrome P450 oxidoreductase,POR)实现共表达,使其具有代谢活性,为药物代谢的研究提供单一性的酶源。方法以人肝组织RNA为模板,RT-PCR扩增CYP2E1基因,插入pCW空载体,构建重组表达质粒pCW-2E1,转化E.coli DH5α,IPTG诱导表达,表达产物经Western blot鉴定;利用双启动子原理构建CYP2E1与POR共表达的重组质粒pCW-2E1-POR,采用对氨基苯酚法检测CYP2E1重组酶的代谢活性,并对不同培养时间(18、28、38 h)的代谢活性进行比较。结果质粒pCW-2E1经PCR鉴定,证明构建正确;表达的重组人CYP2E1蛋白相对分子质量约56 000,主要以可溶性形式表达。质粒pCW-2E1-POR经PCR及酶切鉴定,证明构建正确;导入质粒pCW-2E1-POR的重组菌提取的蛋白样品具有明显的代谢活性,培养18、28、38 h后,代谢活性均值分别为(0.195±0.004)、(0.189±0.003)和(0.192±0.004)nmol/(min·mg),差异无统计学意义(P>0.05),但与导入质粒pCW-2E1的重组菌提取的蛋白样品的代谢活性相比,差异有统计学意义(P<0.05)。结论成功构建了具有代谢活性的CYP2E1重组酶,且3个培养时间段之间的代谢活性无明显差异。

Objective To express cytochrome P450 2El （CYP2E1） in E. coli and co-express with cytochrome P450 oxido - reductase （POR） to render the expressed product metabolic activity and provide a single enzyme source for study on drug metabolism. Methods CYP2E l gene was amplified by RT-PCR using human liver tissue RNA as a template, and inserted into empty vector PCW. The constructed recombinant plasmid pCW-2E1 was transformed to E. coli DH5α for expression under induction of IPTG. The expressed product was identified by Western blot. Recombinant plasmid pCW-2E1-POR for coexpression of CYP2E1 and POR was constructed based on double promoter principle. The metabolic activity of recombinant CYP2E1 was determined by p-amino benzene method, of which those cultured for 18, 28 and 38 h were compared. Results PCR proved that recombinant plasmid pCW-2E1 was constructed correctly. The expressed recombinant human CYP2E1 protein, with a relative molecular mass of about 56 000, mainly existed in a soluble form. PCR and restriction analysis proved that recombinant plasmid pCW-2E1-POR was constructed correctly~ The metabofic activities of protein samples extracted from recombinant E. coli in which plasmid pcW-2E1-POR was introduced were （0. 195 ± 0. 004）, （0. 189 ± 0. 003） and （0. 192 ± 0. 004） nmol/（min·mg） after culture for 18, 28 and 38 h respectively, which showed no significant difference （P 〉 0. 05 ）. However, the metabolic activities showed significant difference with that of protein extracted from recombinant E. coli in which plasmid pCW-2E1 was introduced （P 〈 0. 05 ）. Conclusion Recombinant CYP2E1 with metabolic activity was successfully constructed, of which the metabolic activities at three time points showed no significant difference.