%0 Journal Article
%T 人胰岛素原基因在大肠杆菌中的表达和纯化<br>Expression and purification of human proinsulin gene in Escherichia coli
%A 张晓玮
%A 彭振英
%A 陈高
%A 郑玲
%A 于金慧
%A 毕玉平
%A 边斐&lt
%A br&gt
%A ZHANG Xiao-wei
%A PENG Zhen-ying
%A CHEN Gao
%A ZHENG Ling
%A YU Jin-hui
%A BI Yu-ping
%A BIAN Fei
%J 山东大学学报（理学版）
%D 2015
%R 10.6040/j.issn.1671-9352.0.2014.573
%X 摘要： 将人胰岛素原基因序列(human proinsulin, PI)经密码子优化后与TrxA蛋白融合表达,构建原核表达载体TrxA-PI转入不同大肠杆菌表达菌株BL21(DE3)、TransB(DE3)和Rosetta-gami(DE3)中。SDS-PAGE显示融合蛋白在3种表达菌株中均有表达,在Rosetta-gami(DE3)中表达量最高,是普通大肠杆菌BL21(DE3)表达量的10倍。通过优化诱导温度等发酵条件,可溶性重组融合蛋白TrxA-PI在Rosetta-gami(DE3)菌株中表达量为3.5 g/L,比未优化时提高约10倍。TrxA-PI用肠激酶酶切后,利用HisTrap FF柱分离纯化PI,经Western-blot检测重组PI具有免疫原性。<br>Abstract: The human proinsulin (PI) gene sequence was optimized using the Escherichia coli preferred codons. The reformed PI gene was amplified, the fused expression vector (TrxA-PI) was constructed and transformed into three E.coli expression strains BL21(DE3),TransB(DE3) and Rosetta-gami(DE3). SDS-PAGE analysis showed that the expression level of the fused protein was the highest in Rosetta-gami (DE3) strain, which was about 10 times higher than in BL21(DE3) strain. The production of soluble fused protein was 3.5 mg/mL after optimization of the fermentation conditions. The purified recombinant PI was obtained after enterokinase digestion and HisTrap FF column purification, and its antigen activity was measured using Western-blot analysis
%K 胰岛素原
%K 密码子优化
%K 大肠杆菌表达
%K &lt
%K br&gt
%K proinsulin
%K &lt
%K em&gt
%K E.coli&lt
%K /em&gt
%K expression
%K preferred codons
%U http://lxbwk.njournal.sdu.edu.cn/CN/10.6040/j.issn.1671-9352.0.2014.573