摘要
采用 PCR 方法扩增编码中国明对虾线粒体锰超氧化物歧化酶(MnSOD)成熟肽的基因,并克隆到大肠杆菌表达载体 pCR^RT7/NT TOPO^R TA 中进行体外重组表达。重组质粒转化大肠杆菌 BL21(DE3)pLvsS 后,经 IPTG 诱导表达产生包涵体形式的目的蛋白。对重组蛋白进行 LC-ESI-MS 分析的结果表明,融合蛋白的三个肽段与中国明对虾线粒体MnSOD 相应肽段完全一致。将重组蛋白通过金属螯合柱进行纯化,进而透析、复性,最后获得了活性高达2373U/mg 的重组蛋白。中国明对虾线粒体 MnSOD 的成功表达,为深入研究其在中国明对虾免疫反应和抗氧化胁迫中的作用奠定了基础。
The cDNA encoding mature mitochondrial manganese superoxide dismutase （MnSOD） of Fenneropenaeus chinensis protein was cloned and expressed in E. coli. The recombinant protein was expressed as an inclusion body after the induction with isopropy-β-D-thiogalactosic （ IPTG）. The LC-ESI-MS analysis showed that three peptide fragments of the recombinant protein were identical to the corresponding sequence of F. chinensis mitochondrial MnSOD. The fusion protein was purified using the immobilized-metal affinity chromatography. The enzyme activity of the refolded recombinant protein was as high as 2373 U/mg. The fusion protein is useful for studies on the function of mitochondrial MnSOD in immune defense and against oxidative stress of F. chinensis.
出处
《高技术通讯》
CAS
CSCD
北大核心
2008年第8期868-873,共6页
Chinese High Technology Letters
基金
863计划(2006AA09Z424)
973计划(2006CB101804)资助项目
关键词
中国明对虾
锰超氧化物歧化酶
重组表达
纯化
活性分析
fenneropenaeus chinensis, MnSOD, recombinant expression, purification, activity analysis
