重组人肝细胞生长因子α的纯化与活性测定

Purification and activity determination of recombinant human hepatocyte growth factor-α

目的纯化重组人肝细胞生长因子α(rhHGFα)包涵体,复性并测定其活性。方法大肠杆菌中表达的rhHGF琢以包涵体形式存在,表达量约占菌体总蛋白的25%。离心分离包涵体,用8mol/L尿素溶解,梯度稀释复性。经SephadexG75凝胶过滤和POROSHQ阴离子交换纯化,噻唑蓝法(MTT)测定活性。结果rhHGF琢纯度达到90%以上,其刺激肝细胞生长的活性达到rhHGF标准品的80%。结论建立了纯化rhHGF琢的技术路线,rhHGF琢具有刺激原代培养大鼠肝细胞生长的作用。

Objective To purify recombinant human hepatocyte growth factorαchain(rhHGFα),renature and determine its activity.Methods rhHGFαproduced as inclusion body in E.coli with25%expression level.The rhHGFαprotein was renatured after inclusion body was isolated and dissolved in8mol/L urea solution and purified by Sephadex G75and POROS HQ chromatography.MTT method was used to determine its activity.Results The product purity was up to90percent and over,its activity reached80percent compared with standard rhHGFα.Conclusion A purification technique was established and rhHGFαshowed a biological activity in stimulating the growth of rat hepatocyte in primary culture.