【摘要】 将含有前导肽的人神经生长因子基因(proNGF)克隆在原核表达载体pET15b中,转化大肠杆菌BL21(DE3)pLysS,经IPTG诱导实现了目标融合蛋白的高效表达。SDS-PAGE分析表明表达蛋白占全菌总蛋白的20%左右,表达蛋白主要以包涵体的形式存在。用6mol/L的盐酸胍溶解包涵体后,通过Ni2+-NTA柱纯化,获得纯化的目标融合蛋白,电泳谱带扫描分析表明蛋白纯度可达90%以上。Western blotting检测显示,表达产物有较强的免疫学活性。经肠激酶作用后得到proNGF非融合蛋白,分子量为27kD,100mL表达菌液可获得13.1mg proNGF蛋白。用透析复性的方法将目的蛋白重折叠,复性率为18%,在重折叠过程中前导肽发挥了一定的积极作用。用PC12细胞进行生物活性鉴定,结果显示复性后的proNGF蛋白具有良好的生物活性。更多还原

【Abstract】 Nerve growth factor(NGF) promotes neuronal survival and differentiation and stimulates neurite outgrowth.NGF is synthesized as a precursor-proNGF in vivo.In this paper,a pET-proNGF prokaryocyte expression vector was constructed and transformed into E.coli BL21(DE3)pLysS.The proNGF was expressed in the form of non-active aggregated monomer in E.coli after induction with IPTG.SDS-PAGE revealed the proNGF expression product had a Mr.30.2 kD.Western blotting analysis showed that the protein had good antigenicity.Fusion protein was successfully purified by Ni2+-NTA affinity chromatography and cleaved by Enterokinase and 13.1 mg proNGF was obtained from 100 mL cell culture in a typical experiment.The protein was dialyzed in a redox system containing reduced and oxidized glutathione.RP-HPLC was used to analysis the result of the refolding.The refolded proNGF protein can induce neurite outgrowth of PC12 cells,which indicated that pro-form of NGF we obtained had biological activity.更多还原