pQE-hbFGF表达系统的构建及其蛋白质的表达和纯化  

Construction of expressive system pQE-hbFGF and its expression and purification

作　　者：刘秋英[1] 王一飞[1] 熊盛[1] 胡红梅 钱垂文[1] 张美英[1] 冉延超[1] 袁茵[1] 吴志聪[1] 

机构地区：[1]暨南大学生物医药研究开发基地,广东广州510632

出　　处：《中国药学杂志》2005年第4期308-310,318,共4页Chinese Pharmaceutical Journal

基　　金：国家自然科学基金小额探索项目 (3 0 3 71661) ;广东省自然科学基金团队项目 (0 3 92 13 ) ;广州市科技攻关项目 (2 0 0 3Z3 E0 40 1) ;国家自然科学基金面上项目 (3 0 40 0 0 71)

摘　　要：目的　构建pQE hishbFGF表达载体 ,通过一步纯化得到 6×HishbFGF蛋白质。方法　用PCR扩增目的基因hbFGF ,连接到pQE4 0载体上 ,转化到Top10菌株筛选重组子 ,经测序后将质粒转化到表达菌M15上 ,经IPTG诱导表达 ,超声波破菌后通过镍离子螯合层析一步纯化得 6×HishbFGF蛋白质 ,ELISA鉴定产物 ,用MTT法检测产物促细胞增殖的生物活性。结果　hbFGF基因插入pQE载体中 ,在M15中 6×HishbFGF的表达量达到 2 0 % ,且为可溶性表达。经一步纯化后得到N端带 6个组氨酸的hbFGF蛋白 ,纯度为 96 % ,6×HishbFGF具有免疫原性及促进NIH3T3细胞增殖的活性。结论　 6×HishbFGF蛋白质在M15中可溶性地高效表达 ,并经一步亲和层析得到纯度高活性高的产物 ,降低了生产成本 。OBJECTIVE: To construct pQE-hishbFGF expression vector, obtain 6 × HishbFGF by single purification step of affinity chromatography. METHODS: The hbFGF gene was amplified by polymerase chain reaction, and cloned into pQE40 vector. Recombinant plasmid was sequenced and was transformed to Escherichia coli M15. 6 × HishbFGF expressed after IPTG induction was purified by Ni-NTA affinity column. Recombinant 6 × HishbFGF was identified by ELISA, and its biological activity was determined by MTT test. RESULTS: hbFGF gene was cloned into pQE vector. Recombinant protein was accounted for about 20% of total bacterial protein after 4 h of induction. The purity of 6 × HishbFGF reached 96% after affinity chromatography. HishbFGF bound with hbFGF antibody and stimulated the proliferation of the NIH-3T3 fibroblasts. CONCLUSION: HishbFGF was successfully expressed. High purified recombinant protein was obtained by one affinity column. It reduced the cost of production and underlay the base of large scale production.

关 键 词：人碱性成纤维细胞生长因子 pQE表达系统 可溶性表达 镍离子螯合层析 

分 类 号：Q81[生物学—生物工程]