含有纯化标签的重组人表皮生长因子的构建及表达

Construction and expression of recombinant human epidermal growth factor with a purification tag

目的:高效表达和制备有活性的人表皮生长因子(hum an ep iderm al growth factor,hEGF)。方法:用DNA重组技术构建EGF基因并插入表达载体pQE-40,与载体上的6×H is标签融合,获得的表达质粒pQE-EGF转化E.coliM15[pREP4],得工程菌M15[pQE-EGF]。MTT法测定活性,培养工程菌M15[pQE-EGF]并诱导目的蛋白表达,镍离子螯合亲和层析纯化重组EGF。结果:所构建的EGF序列正确,重组EGF在大肠杆菌中以包涵体形式存在,表达量占菌体总蛋白的21.2%,经镍离子亲和层析一步纯化后的重组EGF纯度达90%,得率为94%。复性后的重组EGF具有促Hela细胞生长活性。结论:以E.coliM15[pREP4]/pQE-40系统表达EGF具有技术路线简单、目的蛋白得率高、成本低等优点,是一种制备活性hEGF的有效手段。

Objective：To prepare active human epidermal growth factor（hEGF）. Methods：An artificial egf gene was constructed with recombinant DNA technology and inserted into pQE -40 to get a expression plasmid pQE -EGF, in which egf was fused with 6 × His tag. Plasmid pQE -EGF was transformed into E. coli M15 [ pREP4]. The resulted recombinant bacteria, M15 [ pQE - EGF], was cultured and induced. Recombinant EGF produced in E. coli plasma was purified with Ni^2＋ -NTA chelating chromatography and its bioactivity was determined with MTr assay. Results： The sequence of egf was correct and its protein product formed into inclusion body in E. coli to a level of 21.2%. After purified with Ni^2＋ -NTA chelating chromatography, rhEGF reached a purity of 90% with recovery rate of 94%. The refolded rhEGF exhibited proliferation - stimulate activity against Hela cell. Conclusion：E. coli M15 [ pREP4]/pQE-40 is a simple, efficient and cost system to produce EGF.