CYP2B6基因表达质粒构建及在大肠杆菌中表达.docVIP

CYP2B6基因表达质粒构建及在大肠杆菌中表达 　【摘要】 目的：构建CYP2B6基因原核表达质粒，在大肠杆菌［Rosetta2(DE3)pLysS］中高效表达重组蛋白。方法：以质粒为模板，用PCR技术扩增出CYP2B6基因，并将其分别插入pET_22b(+)、pET_28b(+)和pET_32a(+)质粒中，经PCR、测序鉴定后，转化Rosetta2(DE3)pLysS，并用异丙基_β_D_硫代半乳糖苷(IPTG)诱导表达。结果：经PCR、测序鉴定，构建了CYP2B6基因表达质粒；采用考马斯亮蓝染色法检测重组蛋白表达，pET_22b(+)_CYP2B6未见目的蛋白表达；pET_28b(+)_CYP2B6有目的蛋白表达，约占蛋白总量的5%；而pET_32a(+)_CYP2B6可以高效地表达CYP2B6，在低浓度(50μmol/L)IPTG诱导3h后，所表达的CYP2B6蛋白约占细菌总蛋白的30%；质谱分析结果进一步证实所表达的蛋白为CYP2B6。结论：成功地使CYP2B6基因在原核系统中高效表达。 【关键词】 CYP2B6 大肠杆菌 表达 ［Abstract］Objective: To construct the expression plasmids of CYP2B6 gene, and to express CYP2B6 protein in Escherichia coli［Rosetta2(DE3)pLysS］. Methods: TheCYP2B6genewasamplifiedbyPCRtechniquefromthetemplate of the plasmid containing CYP2B6 gene, then was inserted into plasmidsofpET_22b(+),pET_28b(+),andpET_32a(+)respectively, and identified with PCR and sequencing. The recombinant expression plasmids containing CYP2B6 gene were transformed into Rosetta2(DE3)pLysS and the target protein expression was induced by isopropyl_β_D_thiogalactopyranoside(IPTG). Results: The recombinant plasmids of pET_22b(+)_CYP2B6, pET_28b(+)_CYP2B6, and pET_32a(+)_CYP2B6 were obtained and identified by PCR and sequencing. Commassie Blue staining method was used to detect thetarget protein expression. The pET_22b(+)_CYP2B6 could not express target protein, the pET_28b(+)_CYP2B6 could express CYP2B6 protein accounting for about 5% of total protein; The target protein was greatly expressed by pET_32a(+)_CYP2B6 and the expression level of CYP2B6 protein was to be approximately 30% after 3hours induction in the concentration of 50 μmol/L IPTG. The expressed protein was CYP2B6 protein by the mass spectrometry results. Conclusion: CYP2B6 is successfully constructed, which can highly express objective protein in Rosetta2(DE3)pLysS. ［Key Words］CYP2B6; Escherichia coli; expressionCYP450(cytochrome P450) 　　系统是机体对内、外源性物质，尤其是药物进行生物转化的重要酶系。CYP450是一个超家族，目前已确定的人类CYP450分属于18个家族，42个亚家族［1］。在人体药物代谢过程中，主要有CYP1、CYP2和CYP3 3个家族。CYP2B6是CYP2