人参Cu/Zn SOD原核可溶性表达条件的优化  

Optimization for Soluble prokaryotic expression of panax ginseng Cu/Zn SOD

作　　者：高云鹏[1] 赵雨[1] 王思明[1] 刘美辰[1] 王佳雯[1] GAO Yunpeng;ZHAO Yu;WANG Siming;LIU Meichen;WANG Jiawen(Traditional Chinese Medicine and Biotechnology Research and Development Center,Changchun University of Chinese Medicine,Changchun 130117,China)

机构地区：[1]长春中医药大学中医药与生物工程研究开发中心,长春130117

出　　处：《长春中医药大学学报》2018年第4期669-672,共4页Journal of Changchun University of Chinese Medicine

基　　金：国家自然科学基金项目"人参皂苷调控TLR/RLR信号通路激活APOBEC3G和BST-2抗病毒功能的分子机制研究";(81503324)

摘　　要：目的在大肠杆菌中高效表达可溶性人参SOD,纯化后检测其活性。方法人参SOD cDNA插入到原核表达载体pET30α中,构建重组表达质粒,转化大肠杆菌BL21感受态细胞,IPTG诱导表达。分别对诱导时间、诱导温度、IPTG浓度以及离子浓度进行条件优化。对表达产物进行SDS-聚丙烯酰胺凝胶电泳和考马斯亮蓝染色检测。检测上清中蛋白的抗氧化活性。结果双酶切结果显示质粒构建成功,经过条件优化,表达的SOD蛋白主要存在于破碎菌体的上清中,表达量约占菌体上清总蛋白的50%,上清酶活达到363.9 U/mg。结论成功在大肠杆菌中高效表达了可溶性人参SOD蛋白,且具有较高的酶活性,为其工业化生产和应用奠定了基础。Objective Expressingand purification soluble ginseng SOD efficiently in Escherichia coli and detect the activity. Methods Ginseng SOD cDNA was inserted into prokaryotic expression vector pET30α to construct recombinant expression plasmid, then the plasmid was transformed into E.coli BL21 competent cells and induced by IPTG. The induction time, induction temperature, IPTG concentration and ion concentration were optimized. SDSpolyacrylamide gel electrophoresis and Coomassie brilliant blue staining were performed on the expressed product. The antioxidant activity of the purified protein was examined. Results The result of double digestion showed that the plasmid was successfully constructed. After the condition was optimized, the expressed SOD protein was mainly found in the supernatant of the disrupted cells. The expressed protein was about 50% of the total soluble bacterial protein. Super natant enzyme activity reaches 363.9 U/mg. Conclusion Successful expression of soluble SOD protein in Escherichia coli and its high activity were established, which laid a foundation for its industrial production and application.

关 键 词：人参 CU/ZN SOD 可溶性表达 原核 

分 类 号：R284.2[医药卫生—中药学]