首页 | 本学科首页   官方微博 | 高级检索  
     

重组人肝细胞生长因子α在大肠杆菌中的表达及活性检测
引用本文:杨利军 杨涛 牛勃 程牛亮 王惠珍 赵建滨. 重组人肝细胞生长因子α在大肠杆菌中的表达及活性检测[J]. 中国生物制品学杂志, 2005, 18(1): 19-22
作者姓名:杨利军 杨涛 牛勃 程牛亮 王惠珍 赵建滨
作者单位:山西医科大学生物化学与分子生物学教研室 太原030001(杨利军,杨涛,牛勃,程牛亮,王惠珍),山西医科大学生物化学与分子生物学教研室 太原030001(赵建滨)
基金项目:山西省高等学校科技研究开发项目 (2 0 0 413 16)
摘    要:目的 建立人肝细胞生长因子α链 (rhHGFα)高效原核表达体系。方法 以pRC/CMV hHGF为模板 ,扩增hHGFαcDNA ,继以XhoI酶切为 386和 95 4bp 2个片段 ,亚克隆入pBSKS,DNA测序后 ,将上述两个片段与pBV2 2 0连接 ,构建重组质粒。转化E .coliJM10 9、DH5α和BL2 1(DE3) ,筛选高效表达菌株。分离包涵体 ,8mol/L尿素溶解 ,梯度复性后利用反相和凝胶过滤层析纯化重组蛋白 ,经MTT法检测活性。结果 所克隆的hHGFα基因序列正确 ,筛选出高效表达菌株BY ,经SDS-PAGE显示在相对分子质量 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 (IBs)形式存在 ,复性后具有刺激原代大鼠肝细胞生长的作用。结论 已成功表达了rhHGFα蛋白 ,为研究rhHGFα结构与功能及中试生产奠定了基础。

关 键 词:肝细胞生长因子  原核表达  活性
修稿时间:2003-12-22

Expression of Recombinant Human Hepatocyte Growth Factor α in E. coli and Activity of Expressed Product
YANG Li-jun,YANG Tao,NIU Bo,et al. Expression of Recombinant Human Hepatocyte Growth Factor α in E. coli and Activity of Expressed Product[J]. Chinese Journal of Bilogicals, 2005, 18(1): 19-22
Authors:YANG Li-jun  YANG Tao  NIU Bo  et al
Abstract:Objective To construct a prokaryotic system for high expression of human hepatocyte growth factor α(rhHGFα).Methods hHGFα cDNA was amplified using pRC/CMV-hHGF as a template, digested with Xho I into two fragments at lengths of 386 and 954 bp respectively, and subcloned into plasmid pBS KS.After DNA sequencing, insert the two fragments into plasmid pBV220.Transform the recombinant plasmid into E.coli and screen the bacterial strain in which rhHGFα is highly expressed.Separate the expressed protein existing in a form of inclusion body, dissolve with 8 mol/L urea, re-naturalize and purify by reverse and gel filtration chromatography and detect for activity by MTT method.Results The cloned hHGFα gene showed a correct sequence, and an E.coli strain BY for high expression of rhHGFα was screened.SDS-PAGE showed a specific protein band with a relative molecular weight of 52 000.The expressed product existed in a form of insoluble inclusion body and contained about 25% of total somatic protein.After renaturation, the expressed protein showed stimulating effect on the growth of primary rat hepatocytes.Conclusion rhHGFα protein was successfully expressed in E.coli.It laid a foundation of study on the structure and function as well as pilot production of hHGFα.
Keywords:Hepatocyte growth factor  Prokaryotic expression  Activity
本文献已被 CNKI 维普 万方数据 等数据库收录!
设为首页 | 免责声明 | 关于勤云 | 加入收藏

Copyright©北京勤云科技发展有限公司  京ICP备09084417号