资源描述
食品与药品Food and Drug 2019 年第 21 卷第 5期337论著.重组人骨形成蛋白-2基因工程菌株的构建及蛋白复性纯化张秀华1，刘飞1,2* *，刘英梅1陈勉1，刘霞二刘金明3,凌沛学1,2，王凤山2*(1.山东省药学科学院山东省生物药物重点实验室山东省多糖类药物工程实验室多糖类药物发酵与精制国家地方联合工程实验室博士后科研工作站，山东 济南250101； 2.山东大学 药学院，山东 济南250101； 3.浙江赛灵特医药科技有限公司，浙江 杭州311100)摘要：目的 构建一株高效表达人骨形成蛋白2( hBMP-2 )的基因工程菌株，并通过复性、纯化获得有生物 活性的rhBMP-2。方法采用基因工程法将hBMP-2基因片段与pET-28a(+)质粒载体相连，构建大肠杆菌表达系 统。工程菌发酵后，超声破碎收集包涵体，包涵体经裂解后通过稀释复性法进行复性，SDS-PAGE检测复性情况，并采用HiTrap Heparin HP纯化，C2C12细胞检测活性。结果rhBMP-2在大肠杆菌中以无活性的包涵体 形式表达，1 L发酵罐高密度发酵rhBMP-2包涵体湿重可达20 g/L。采用稀释法复性，蛋白复性率大于 40 %， 亲和层析纯化后，二聚体蛋白纯度达 95 %以上，且能诱导C2C12细胞碱性磷酸酶的表达，具有同市售产品相 同的细胞活性。结论成功构建了高效表达rhBMP-2的基因工程菌株，复性方法简单且无需对包涵体进行纯 化，蛋白复性浓度高，纯化方法简便，大大降低了工业化生产成本。关键词：人骨形成蛋白2;大肠杆菌；包涵体；复性；纯化；生物活性中图分类号：R915文献标识码：A文章编号：1672-979X( 2019)05-0337-06DOI : 10.3969/j.issn.1672-979X.2019.05.001Construction of Genetic Engineering Strains of rhBMP-2 and Its Purification andRen aturati onZHANG Xiu-hua, LIU Fei1,2, LIU Yin g-mei, CHEN Miari, LIU Xia1 , LIU Jin-mi ng, LING Pei-xuB2, WANG Fen g-shain(1. Key Laboratory of Biopharmaceuticals, Engin eeri ng Laboratory of Polysaccharide Drugs, Natio nal-local Joint Engin eeri ng Laboratory of Polysaccharide Drugs, Postdoctoral Scien ti?c Research WorkstatiojnSha ndong Academy of Pharmaceutical Sciences Jinan 250101, China; 2. School of Pharmaceutical SciencesShandong University, Jinan 250101,China; 3. Zhejiang Sealant Pharmaceutical Technology Co Ltd., Hangzhou311100, China) Abstract: Objective To con struct a gen etic engin eeri ng strain express ing huma n bone morphoge netic protein 2 (hBMP-2) ef?cie ntly, and obtai n the biologically active protein by ren aturati on and puri?cati on. Methods The hBMP- 2 gene fragme nt was lin ked with pET-28a(+) plasmid vector by gen etic engin eeri ng to con struct thE. coli expressi on system. After ferme ntatio n of engin eeri ng strai n, i nclusio n bodies were collected by ultras onic fragme ntati on. After decompositi on, in clusi on bodies were refolded by diluti on method. The ren aturatio n was detected by SDS-PAGE. The target protein was puri?ed by HiTrap Heparin HP column, and its activity was tested using C2C12 cells. Results rhBMP-2 was expressed as in active in clusi on bodies in E. coli with a high concen trati on (20 g/L). With a simple收稿日期：2019-03-05 基金项目 山东省级重点实验室专项建设计划(SDKL2017023)，山东省自然科学基金(ZR2015QL007，ZR2017BH051)，泰山学者工程专项作者简介：张秀华，硕士，主管药师，研究方向：生物药物研究E-mail: wfzxh0318。*通讯作者：刘飞，硕士，副研究员，研究方向：生物药物研究 E-mail: lfshwu；王凤山，博士，教授，研究方向：多糖类药物和生物技术药物E-mail: fswang
展开阅读全文