人肝细胞生长因子β链在E.coli中的表达、纯化及活性测定

目的 为获得重组人肝细胞生长因子β(rhHGF-β)的高效表达体系,分离纯化rhHGF-β,以期研究其在生物学和医学中的作用。方法 从人胎肝组织中提取总RNA,采用RT-PCR技术,对HGF-β基因加工修饰,与载体PBV220重组,构建rhHGF-β的表达载体,转化大肠杆菌,筛选rhHGF-β的高效表达菌株。对rhHGF-β进行粗纯化,透析复性后MTT法检测生物活性。结果 获得了rhHGF-β完整的cDNA序列,重组克隆的酶切结果与设计一致。目的 蛋白的表达量占全菌蛋白的30％,粗纯化后纯度达90％,复性产物能刺激大鼠肝细胞的增殖。结论 建立了rhHGF-β的表达体系,获得了具有生物活性的rhHGF-β,为进一步大量制备和研究HGF的生物学功能提供了重要的实验数据。

Expression of Recombinant Human Hepatocyte Growth Factor-β(hHGF-β) in E. coli and Purification and Activity of Expressed Product

Objective To establish a high expression system for recombinant human hepatocyte growth factor-β(hHGF-β), purify hHGF-β and study the application of it in biology and medicine. Methods Extract total RNA from human fetal liver tissue, amplifty by RT-PCR, insert into vector PBV220, transform to E. coli, and select highly expressed clones. The expressed rhHGF-β was primarily purified and detected for biological activity by MTT method after renaturation by dialysis. Results A full length cDNA sequence of rhHGF-β was obtained.The result of enzyme digestion of recombinant clone was identical to that designed. The expressed product contained 30% of total somatic protein and reached a purity of 90% after primary purification. After renaturation, the expressed product stimulated the proliferation of hepatocytes in rats. Conclusion An expression system for rhHGF-β was established,and the rhHGF-β with biological activity was expressed.lt provided an important basis for the large-scale production, as well as study of the biological function of HGF.