重组人中期因子的原核表达、纯化及生物学活性鉴定  

Prokaryotic expression and purification of recombinant human midkine and determination of its biological activity

作　　者：钱海华[1] 康晓燕[1] 殷正丰[1] 王翠红[1] 李瑾[1] 吴孟超[1] 

机构地区：[1]第二军医大学东方肝胆外科医院分子肿瘤实验室,上海200438

出　　处：《第二军医大学学报》2004年第4期394-397,共4页Academic Journal of Second Military Medical University

基　　金：国家自然科学基金 ( 3 0 2 70 5 2 3 ) ;国家重点基础研究规划 ("973"计划 ) ( 2 0 0 2 CB5 13 10 0 )

摘　　要：目的 :表达和纯化具有生物学活性的重组人中期因子蛋白。方法 :应用 RT- PCR从人肝细胞癌组织总 RNA中扩增中期因子 c DNA,将其克隆到表达载体 p ET- 2 4 a,在大肠杆菌中诱导表达 ,经肝素 -琼脂糖亲和层析柱纯化 ,应用 SDS-聚丙烯酰胺凝胶电泳、Western印迹和细胞增殖试验对纯化产物进行分析鉴定。 结果 :构建的重组质粒 p ET- HMK能在大肠杆菌中高表达 ,诱导表达的蛋白主要存在于包涵体中 ,经肝素 -琼脂糖柱亲和层析后纯化产物呈单一条带 ,特异性抗体反应证实是重组人中期因子 ,细胞增殖试验显示其能有效促进 NIH 3T3细胞生长 ,并且具有剂量依赖性。结论Objective:To express and purify recombinant human midkine (rh MK) protein with biological activity. Methods: The MK cDNA was amplified from tissue of human hepatocellular carcinoma by RT PCR method. rh MK was cloned on a pET 24a vector in E.coli , and was expressed in the bacterial inclusion bodies and purified by heparin affinity chromatography. Results: Overexpression of rh MK led to the formation of insoluble aggregated protein which accounted for about 25% of the total cellular protein. Homogeneous protein was obtained from inclusion bodies after one step purification of heparin affinity chromatography, which was active in cell proliferation assay. Conclusion: rh MK protein obtained with prokaryotic system has significant mitogenic activity.

关 键 词：重组人中期因子 原核表达 纯化 生物学活性 鉴定 

分 类 号：Q78[生物学—分子生物学]