CYP2 B6基因表达质粒的构建及在大肠杆菌中的表达  

Construction of Recombinant Prokaryotic Expression Plasmids of CYP2 B6 Gene and Expression in Escherichia coli

作　　者：张宝[1] 霍霞[1] 徐锡金[1] 齐宗利[1] 郑谨[1] 彭琳[1] 

机构地区：[1]汕头大学医学院共聚焦实验室广东省免疫病理学重点实验室

出　　处：《汕头大学医学院学报》2007年第1期1-4,共4页Journal of Shantou University Medical College

基　　金：中国博士后科学基金(2005038187);国家自然科学基金(30600733)资助

摘　　要：目的:构建CYP2 B6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CYP2 B6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CYP2 B6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(+)-CYP2 B6未见目的蛋白表达;pET-28b(+)-CYP2 B6有目的蛋白表达,约占蛋白总量的5%;而pET-32a(+)-CYP2 B6可以高效地表达CYP2 B6,在低浓度(50μmol/L)IPTG诱导3 h后,所表达的CYP2 B6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CYP2 B6。结论:成功地使CYP2 B6基因在原核系统中高效表达。Objective： To construct the expression plasmids of CYP2B6 gene, and to express CYP2B6 protein in Escherichia coli[ Rosetta2（ DE3）pLysS ]. Methods： The CYP2B6 gene was amplified by PCR technique from the template of the plasmid containing CYP2B6 gene, then was inserted into plasmids of pET-22b（ ＋ ）, pET-28b（ ＋ ）, and pET-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 the target 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 he approximately 30% after 3 hours induction in the concentration of 50μmol/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.

关 键 词：CYP2B6 大肠杆菌 表达 

分 类 号：Q75[生物学—分子生物学]