重组人肝再生增强因子的纯化及生物学活性分析

Purification and Biological Activity of Recombinant Human Augmenter of Liver Regeneration

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30 000,MOLDI-TOF-MS测定其相对分子质量为30 780.522,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 separate, purify and study the biological activity of recombinant human augutenter of liver regeneration（rhALR） expressed in E. coli. Methods Incubate the recombinant E. coli strain pBV220-rhALR/DHSα by fermentation and induce the expression of rhALR. Treat the expressed product in a form of inclusion body by washing, de-naturalization and re-naturalization,then purify by ion exchange and molecular sieve chromatography and perform control tests. Results The expressed product reached a purity of more than 98% after purification, and its relative molecular weight was proved as 30 000 by SDS-PAGE and 30 780. 522 by MOLDI-TOF-MS. The sequence of 15 amino acids at N-termlnus of the expressed product was identical to that in theory. Western blot proved that rhALR with a pI of 5.85-6. 85 was correctly expressed, and its amino acid content was basically consistent with the theoretical value. Animal test proved that rhALR promoted the restoration of liver cells of CCl4 -intosicated mice significantly. Conclusion The rhALR purified by column chromatography showed significant activity of rhALR in promoting the restoration of liver cells of CCl4-intosicated mice.