重组人肝细胞生成素的纯化及活性研究

人肝细胞生成素 ( human hepatopoietin,h HPO)是一种新型肝再生调控因子 .在大肠杆菌中表达的的重组 h HPO( rh HPO)是以包涵体的形式存在的 ,其表达量为菌体总蛋白的 2 0 % .包涵体经各种溶液洗涤后 ,用 8mol/L尿素裂解 ,裂解上清经凝胶过滤、复性和离子交换柱层析得到电泳纯的 rh HPO,经还原型 SDS- PAGE测定其分子量为 1 5k D.纯化 rh HPO的 N端氨基酸序列与其c DNA推导序列完全一致 ;纯化产物的氨基酸组成分析结果亦与 rh HPO氨基酸组成的理论值吻合 .生物学活性研究表明 ,rh HPO在体外具有刺激原代培养肝细胞增殖作用

Studies on Purification and Activity of Recombinant Human Hepatopoietin

Hepatopoietin(HPO)is a noval cytokine which specifically stimulates hepatic cell proliferation. Recombinant human hepatopoietin (rhHPO), overexpressed in E.coli, was found to accumulate incytoplastic inclusion bodies. After washing, the inclusion bodies were dissolved in 8 mol/L urea. Under the denatured states, rhHPO was purified by size exclusion chromatography, then it was renatured by diluting and dialysis. The renatured rhHPO was then separated by ion exchange chromatography with continuous gradient of increasing sodium chloride up to 500 mmol/L. The final purified rhHPO was of high purity(96%) and it exhibited the mass of molecule 15 kD analyzed by reduced SDS PAGE. The sequence of 16 amino acid residues from NH 2 terminus of the purified rhHPO was found to consist with that of rhHPO deduced from cDNA. The composition of amino acids of the protein was also analyzed, the result also consisted well with the theoretic values of amino acids of rhHPO. The renatured rhHPO could produce the significant increase of 3 H TdR into primary hepatocytes in culture, which demonstrated that the rhHPO might be used in clinical trial for liver regeneration in the treatment of hepatic disease.