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目的对在大肠杆菌中表达的重组人肝再生增强因子(rhALR)进行分离纯化及生物学活性分析。方法以大肠杆菌DH5α发酵表达重组人肝再生增强因子,经包涵体洗涤、变性、目的蛋白复性,以离子交换、分子筛等技术纯化,并进行一系列检定。结果目的蛋白以包涵体形式存在,经变性、复性及柱层析纯化,纯度大于98%,SDS-PAGE测定相对分子质量为30000,MOLDI-TOF-MS测定其相对分子质量为30780·522,N-末端15个氨基酸与理论序列一致。Western blot证实rhALR正确表达,等电点5·85~6·85,氨基酸含量与理论值基本符合。动物实验证实rhALR具有明显的促进CCl4毒性肝损伤小鼠肝细胞修复活性。结论经柱层析纯化的rhALR具有促进小鼠CCl4肝损伤肝细胞修复的活性。
Objective To isolate, purify and characterize the recombinant human augmenter of liver regeneration (rhALR) expressed in Escherichia coli. Methods Recombinant human augmenter of liver regeneration was expressed in E. coli DH5α and purified by inclusion body washing, denaturation and protein renaturation, and purified by ion exchange and molecular sieve techniques. A series of assays were performed. Results The target protein was expressed in inclusion bodies and purified by denaturing, refolding and column chromatography. The purity of the target protein was more than 98%. The relative molecular mass was 30,000 by SDS-PAGE and the relative molecular mass was 30780.522 by MOLDI-TOF-MS. N-terminal 15 amino acids consistent with the theoretical sequence. Western blot confirmed rhALR correctly expressed, isoelectric point of 5 · 85 ~ 6 · 85, amino acid content and the theoretical value basically consistent. Animal experiments confirmed rhALR has a significant promotion of CCl4 toxic liver injury in mice liver cell repair activity. Conclusion rhALR purified by column chromatography has the activity of promoting hepatic cell repair by CCl4 liver injury in mice.