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重组粒细胞 巨噬细胞集落刺激因子 /白细胞介素 3(GM CSF/IL 3)融合蛋白是一种很有发展潜力与应用前景的重组药物 ,它在大肠杆菌中是以包含体形式表达的。针对这一重组蛋白包含体的洗涤、变性、复性以及最终的纯化过程 ,进行了深入地研究 ,重点分析和比较了不同条件及因素对于重组蛋白包含体变性及复性过程的影响。实验结果表明 ,8mol/L尿素及 10mmol/L的DTT可以使包含体充分溶解。在复性过程中 ,采用分步逐渐降低尿素浓度的方法 ,可以有效地提高复性的效率。与此同时 ,在复性溶液中加入氧化型及还原型的谷胱甘肽有助于提高变性蛋白质中二硫键的正确配对。复性好的蛋白质用DEAE离子交换层析进行分离 ,最后获得具有生物活性的高纯度的重组蛋白 ,最终的蛋白质回收率在 30 %以上。经还原、非还原电泳及反相HPLC检测 ,所纯化的蛋白质的纯度达 95 %以上。基本建立和完成了实验室规模的重组GM CSF/IL 3融合蛋白的纯化工艺 ,为这一重组药物进一步的开发与应用打下坚实的基础。
Recombinant granulocyte-macrophage colony-stimulating factor / interleukin-3 (GM CSF / IL 3) fusion protein is a recombinant drug with great potential and application prospects. It is expressed in inclusion bodies in Escherichia coli. In order to investigate the process of washing, denaturation, renaturation and final purification of the inclusion body of recombinant protein, the effects of different conditions and factors on the process of inclusion body degeneration and renaturation were analyzed and compared. The experimental results show that 8mol / L urea and 10mmol / L DTT can make the inclusion body fully dissolved. In the process of refolding, the method of gradually reducing urea concentration step by step can effectively improve the efficiency of renaturation. In the meantime, the addition of oxidized and reduced glutathione in the renaturation solution helps to improve the correct disaggregation of disulfide bonds in denatured proteins. The renatured protein is separated by DEAE ion exchange chromatography, and finally the highly active recombinant protein with high biological activity is obtained. The final protein recovery rate is above 30%. After the reduction, non-reducing electrophoresis and reverse phase HPLC detection, the purity of the purified protein was more than 95%. The purification and purification of recombinant GM CSF / IL 3 fusion protein on a laboratory scale has been basically established and completed, laying a solid foundation for the further development and application of this recombinant drug.