人胰岛素样生长因子-1基因的克隆及其表达

目的获得大量高纯度、具有生物学活性的人胰岛素样生长因子 1(hIGF 1) ,构建hIGF 1原核表达载体 ,并进行表达与纯化。方法以pUCIGF质粒为模板 ,PCR扩增了hIGF 1基因。经适当酶切后 ,构建表达载体pRSET IGF ,转入大肠杆菌BL2 1(DE3)进行表达 ,并经阴离子交换色谱、疏水色谱和凝胶过滤纯化。结果带有重组质粒pRSET IGF的大肠杆菌经IPTG诱导后 ,以可溶性形式表达 7.8kD大小的蛋白 ,其表达量占菌体总蛋白量的10 %～ 2 0 % ,纯化后目的蛋白纯度达 95 %以上 ,Western印迹表明重组蛋白具有hIGF 1抗原活性。结论构建了pRSET IGF重组质粒 ,并成功地在大肠杆菌中获得可溶性表达 ,为获得大量基因工程产品奠定了基础

Cloning and expression of human insulin-like growth factor-1 gene in E.coli

PurposeThe aim is to get sufficient quantity of pure biologically human Insulin like growth factor 1(hIGF 1), and hIGF 1 expression vector was constructed and expressed in E.coli. MethodsThe hIGF 1 DNA fragment was obtained from the pUCIGF plasmid by PCR amplification. After being digested by restriction enzyme, the DNA fragment was cloned into a prokaryotic expression vector pRSET B and hIGF 1 expression vector pRSET IGF was constructed. After it was expressed in the host cells E.coli BL21(DE3),the recombinant protein was purified by anion chromatography, hydrophobic chromatography and gel filtration.ResultsSDS PAGE analysis suggested the bacteria containing the recombinant plasmid produced a protein of 7.8 kD as induced by IPTG. The recombinant hIGF 1 was expressed in soluble form, consisting of 10%～20%of the total bacterial proteins. After purification, the target protein could be purified up to 95%. Western blot indicated that the protein could react with antibodies against hIGF 1. ConclusionThe results demonstrate that hIGF 1 expression vector has been successfully constructed and could be expressed in soluble form in E.coli.