重组γ干扰素的表达与纯化

Expression and purification of recombinant interferon-gamma

目的 探讨γ干扰素在大肠杆菌中的表达及纯化方法 ,为下一步批量生产提供实验依据。方法 应用DNA重组技术把中国人淋巴细胞mRNA反转录产物IFN -γcDNA克隆到原核表达质粒 pBV2 2 0上 ,构建出pBVIFN高效表达载体 ,转化大肠杆菌 plyss。通过温度诱导 ,高效表达IFN -γcDNA。然后 ,经过包涵体溶解、复性、浓缩及DEAE离子交换层析 ,使γ干扰素纯化。结果 IFN -γcDNA表达水平占菌体可溶性蛋白的 40 4% ,包涵体纯度为 70 % ,总生物活性为 1 2 7× 10 9U ,比活性达 1.31× 10 9U/L蛋白 ,活性回收率为 77.7%。

Objective To research interferon-gamma expression and purification methods in E.coli and provide experimental basis for further lots of production. Methods Using DNA recombinant technology,IFN-γ cDNA,which reversed transcription from Chinese lymphocyte mRAN, was cloned to the prokaryotic expression plasmid pBV220 and was constructed pBVIFN of efficient expression vector, then transferred into E.coli plyass.IFN-γ can be expressed efficiently by termoinduction. By the inclusion bodies dissolution, renaturation,concentration and DEAE ion exchange chromatography IFN-γ was purified. Results The IFN-γ expression yield was 40 4% of bacterial soluble proteins. The purification of the inclusion bodise was 70%. The total biological activity was 1 27×10 9 U.The specific activity was 1 31×10 9 U/L.The activity recovery rate was 77.7%. Conclusion The experiment method will lay a foundation for lots of production of the interferon-gamma.