重组人胰岛素样生长因子-1分离纯化工艺的建立

Purification of recombinant human insulin-like growth factor-1 from Escherichia coli

目的 建立重组人胰岛素样生长因子 1(rhIGF 1)大肠杆菌表达后的高效分离纯化工艺 ,以便获得高纯度的重组蛋白。方法 已构建好的表达载体转化大肠杆菌BL2 1,经IPTG诱导进行表达。将离心收集到的菌体用超声破壁 ,破壁菌液离心分离 ,收集上清液 ,进行阴离子交换层析、疏水层析和分子筛纯化 ,得到最终纯品。蛋白纯度用SDS PAGE鉴定 ,ELISA法检测rhIGF 1含量 ,检测纯化后重组蛋白的生物学活性。结果 用大肠杆菌BL2 1表达rhIGF 1以可溶性形式存在胞浆中 ,经 3步柱层析纯化后 ,可获得高纯度的rhIGF 1(纯度 >98% )。该蛋白在体外具有良好的促NIH3 T3 细胞增殖作用。结论 建立了rhIGF 1分离纯化工艺 ,其工艺流程可获得具有生物学活性的高纯度rhIGF 1,为产业化及临床应用奠定基础。

OBJECTIVE: To establish a method for improving the purity of recombinant human insulin-like growth factor-1 (rhIGF-1) from Escherichia coli expressing system. METHODS: The E. coli BL21 was transfected with the constructed recombinant hIGF-1 plasmid. The engineering bacterium was induced to express rhIGF-1 by IPTG. Then bacterium was collected by centrifugation and split by ultrasonic method. The supernatants were purified by anion chromatography, hydrophobic chromatography and gel filtration respectively. The purified products were analyzed by SDS-PAGE, and the quantity of rhIGF-1 was determined by ELISA. RESULTS: The target protein was soluble in the supernatant. By three-step chromatography, rhIGF-1 was purified to 98% of purity. Western blotting indicated that the recombinant protein could react with antibodies against anti-hIGF-1. The effect of the protein stimulating NIH3T3 proliferation showed that rhIGF-1 had good biological activity. CONCLUSION: The processing above can be a scheme to obtain the high purity rhIGF-1 from E. coli.