中国明对虾过氧化物还原酶基因在大肠杆菌中的重组表达、产物纯化及活性测定  

Expression,Purification and Activity Analysis of Fenneropenaeus chinensis Peroxiredoxin Gene in E.coli

作　　者：张庆利[1] 李富花[1] 张继泉[1] 蒋昊[1] 相建海[1] 

机构地区：[1]中国科学院海洋研究所实验海洋生物学重点实验室

出　　处：《中国生物工程杂志》2008年第2期47-52,共6页China Biotechnology

基　　金：国家“973”计划(2006CB101804);国家“863”计划(2006AA100311)资助项目

摘　　要：采用RT-PCR扩增编码中国明对虾Prx成熟肽的基因,并克隆到大肠杆菌表达载体pCRT7/NT TOPO TA中进行体外重组表达。重组质粒转化大肠杆菌BL21(DE3)pLysS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,结果表明融合蛋白的4个肽段与中国明对虾Prx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,最后获得了具有较高过氧化物酶活性的重组Prx。The cDNA encoding peroxiredoxin （Prx） of Fenneropenaeus chinensis protein was amplified by RT -PCR and cloned into prokaryotic expression vector pCR TT/NT TOPO TA. The recombinant peroxiredoxin gene was expressed in E. coli and the recombinant protein was expressed as inclusion body after IPTG induced. LC - ESI - MS analysis showed that four peptide fragments of the recombinant protein were identical to the corresponding sequence of F. chinensis Prx. The fusion protein was purified using the immobilized-metal affinity chromatography. After refolded, the purified recombinant protein was shown high reducibility of H202 in the presence of dithiothreitol.

关 键 词：中国明对虾 过氧化物还原酶 重组表达 纯化 活性 

分 类 号：Q789[生物学—分子生物学]