人G6PD在大肠埃希菌中的表达及体外抑制剂筛选模型的建立

Express human G6PD in E.coli and develop its inhibitor screening assay in vitro

目的表达人6-磷酸葡萄糖脱氢酶(G6PD)并建立其体外抑制剂筛选模型。方法设计一对5′端分别带NdeⅠ、XhoⅠ位点的引物,PCR扩增G6PD cDNA。将cDNA和质粒pET-28a经NdeⅠ+XhoⅠ酶切后连接,连接产物转化大肠杆菌Top10。将鉴定的重组质粒转化大肠杆菌BL21(DE3),用0.4 mmol/L IPTG诱导G6PD表达,镍离子亲和层析柱纯化G6PD。以6-磷酸葡萄糖(G6P)和烟酰胺腺嘌呤核苷二磷酸(NADP+)为G6PD的底物和辅因子,检测340 nm波长处光吸收值(A_(340))的变化,确定G6PD及其抑制剂脱氢表雄酮(DHEA)的最佳浓度,计算模型的可靠性指数—Z′因子。结果构建了重组质粒pET-28a-G6PD,重组菌BL21(DE3)-pET-28a-G6PD经IPTG诱导后,可溶性G6PD表达量为2.5 mg/L,G6PD、DHEA的最适浓度分别为900μg/L、20μmol/L,筛选模型的Z′因子为0.88。结论在大肠杆菌中表达了有活性的G6PD,建立了可靠的体外抑制剂筛选模型。

Objective To express human glucose 6-phosphate dehydrogenase(G6PD)and establish its inhibitor screening model in vitro.Methods G6PD cDNA was amplified by PCR with a couple of primers flanked with Nde I and Xho I site at their 5′terminals,respectively.G6PD cDNA and pET-28a plasmids were digested by Nde I and Xho I,and then linked.The linked products were transformed into Escherichia coli(E.coli)Top10.Recombinant plasmids were identified and transformed into E.coli BL21(DE3),the expression of G6PD was induced by 0.4 mmol/L IPTG,recombinant G6PD was purified by Ni^(2+) affinity column.Glucose 6-phosphate and NADP+were used as substrate and coenzyme of G6PD,respectively.G6PD’s activity was observed by the shift of A_(340) of NADPH.The model was optimized to give the greatest performing concentration of G6PD and dehydroepiandrosterone(G6PD’s inhibitor).Results Recombinant plasmid pET-28a-G6PD was constructed,soluble G6PD was expressed in BL21(DE3)with a yield of 2.5 mg/L via the induction of IPTG.The optimum G6PD concentration was 900μg/L.The high Z′score of 0.88 was achieved using 20μmol/L DHEA as positive control.Conclusion Active G6PD is expressed in E.coli,and this assay is highly suitable for screening of G6PD inhibitor in vitro.