重组人胰岛素样生长因子1的原核可溶性表达及活性鉴定

目的:原核可溶性表达人胰岛素样生长因子1(hIGF-1)并进行生物活性测定。方法:PCR扩增hIGF-1核酸序列,克隆至融合表达载体pET-DsbC中,转入大肠杆菌BL21(DE3)进行诱导、表达和纯化,对融合蛋白DsbC-hIGF-1分别进行酶切、Western印迹和生物活性测定。结果:融合蛋白DsbC-hIGF-1在原核系统内经IPTG诱导,主要以可溶性形式表达,其表达量占菌体总蛋白量的30%～35%,Western印迹和MTT法测定结果证明重组DsbC-hIGF-1蛋白具有较强的抗原活性和明显的促NIH/3T3细胞增殖作用。结论:融合蛋白DsbC-hIGF在大肠杆菌中以可溶性表达形式存在并具有类似天然hIGF-1的生物活性,这对进一步研究hIGF-1的结构和功能,开发相应的基因工程产品具有重要意义。

Soluble Expression and Biological Activity Detection of Recombinant Human Insulin Like Growth Factor 1 in Prokaryotic System

Objective： To obtain human insulin like growth factor 1（hIGF-1） expressed in solubility in prokaryotic system and detect its biological activity.Methods： hIGF-1 gene was obtained by PCR and was cloned into pET-DsbC fusion vector.After induction,expression and purification,the antigenicity and biological activity of the product were examined by Western blot and MTT assay.Results： DsbC-hIGF-1 was expressed in solubility in Escherichia coli and its expression quantity was about 30%～35% to bacterial total protein with IPTG induction,which also showed a strong antigenic ability and stimulate proliferation of NIH/3T3 cells evidently.Conclusion： The results suggest that the DsbC-hIGF-1 protein has the similar biological activity to wide type hIGF-1 and could express in solubility in E.coli.It provides a futher investigation of the IGF-1 structure and function and build the foundation for the large scale production of IGF-1.