CYP2B6基因表达质粒的构建及在大肠杆菌中的表达.doc

CYP2B6基因表达质粒的构建及在大肠杆菌中的表达
(作者:___________单位: ___________邮编: ___________)
【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 binant 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 binant 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 expres