rhHGFα重组质粒的构建及其在大肠杆菌中的表达

Construction of Recombinant rhHGFα Plasmid and its Expression in E.coli

目的 :建立人肝细胞生长因子α链 (rhHGFα)高效原核表达体系 ,分离纯化rhHGFα蛋白 ,检测其生物活性。方法 :以pRC CMV hHGF为模板 ,扩增hHGFαcDNA继以XhoⅠ切为两个片段 ,亚克隆入pBSKS,测序后 ,将上述两个片段与pBV2 2 0连接 ,构建重组质粒。转化大肠杆菌 ,筛选高表达菌株。分离包涵体 ,8mol L尿素溶解 ,稀释复性后纯化重组蛋白 ,MTT法检测活性。结果 :测序证实所克隆的hHGFα基因序列正确 ,筛选出高效表达菌株E .coliBY ,SDS PAGE显示在相对分子量 5 2 0 0 0处出现特异的目的蛋白表达带 ,表达量约占全菌蛋白的 2 5 %。表达产物以不溶性的包涵体 (IB)形式存在 ,复性后具有刺激原代培养成年大鼠肝细胞生长的作用。结论 :以大肠杆菌为宿主 ,成功表达了hHGFα蛋白 ,为进行hHGFα结构与功能研究及中试生产奠定了基础。

Objectives:To construct an engineerd E.coli strain expressing rhHGFα and develop a procedure for isolation and purification of rhHGFα and determine its biological activity.Methods: The plasmid pRC/CMV-hHGF was used as template to amplify the gene of interest. The product of PCR was digested with XhoⅠ and two fragments were obtained and then subcloned into pBS+ KS vector. These two fragments were cloned into expression vector pBV220 after sequencing and recombinant plasmid was introduced into E.coli JM109,DH5α,BL21(DE3). Strains highly expressing rhHGFα were screened. The inclusion body was denatured and renatured after dissolved in 8mol/L urea solution to purify rhHGFα protein. MTT method was used to determine its activity.Results: The sequence of rhHGFα gene was correct. Strain E.coli BY highly expressing rhHGFα was obtained. Molecular weight of the rhHGFα determined by SDS-PAGF was 52?000. rhHGFα was expressed as inclusion body and the expression level accounted for 25% of the total bacteria protein. The recombinant protein showed biological activity of enhancing the growth of adult rat hepatocyte in primary culture.Conclusion: A prokaryotic expression system of rhHGFα has been established. This work lays the foundation on the research between structure and its function of hHGFα and large-scale production.;