重组人胰岛素样生长因子1的制备  

Preparation of Recombinant Human Insulin-like Growth Factor 1

作　　者：梁东春[1] 左爱军[1] 吴亚锋[1] 郭刚[1] 张镜宇[1] 

机构地区：[1]天津市内分泌研究所,300070

出　　处：《天津医药》2005年第7期434-436,共3页Tianjin Medical Journal

基　　金：天津市自然科学基金重点项目(项目编号:033801611)

摘　　要：目的:建立人胰岛素样生长因子1(IGF-1)基因大肠杆菌高效表达系统及rhIGF-1的初步纯化方法。方法:将人IGF-1基因克隆入原核表达质粒载体pBV220,重组质粒转化大肠杆菌DH5α,温度诱导表达,WesternBlot对表达产物进行鉴定。超滤离心纯化rhIGF-1,纯化产物行高效液相色谱分析。H3-TdR掺入法测定所制备rhIGF-1的促细胞增殖活性。结果:成功构建了重组质粒pBV-IGF-1,聚丙烯酰胺凝胶电泳可见与预期7.5ku大小相符的蛋白条带,WesternBlot证实该条带即为rhIGF-1。超滤离心一步纯化后rhIGF-1纯度即可达95%以上。H3-TdR结果显示,所制备的rhIGF-1可促进体外培养成肌细胞增殖。结论:成功建立了rhIGF-1的制备及纯化方法,表达产物经体外试验证实具有促细胞增殖的生物活性。Objective To construct high expression system of recombinant human insulin-like growth factor 1 gene and make a basic for the industrial production of rhIGF-1. Methods Recombinant plasmid pBV-IGF-1 was constructed and transformed into E.coli DH5α. Then the transforming was induced to express by increasing the cultural temperature from 30 ℃ to 42 ℃. Expressing product was analyzed by SDS-PAGE rhIGF-1 was verified by Western Blot and its biological activity was detected by H3-TdR incorporation. Results SDS-PAGE and Western Blot proved that there was rhIGF-1 expressed in E.coli DH5α. The result of H3-TdR incorporation showed that this rhIGF-1 had the funcation of enhancing cell proliferation in vitro. Conclusion High expression system of recombinant human insulin-like growth factor 1 was constructed successfully.

关 键 词：人胰岛素样生长因子1 制备 Western rhIGF-1 聚丙烯酰胺凝胶电泳 高效液相色谱分析 PBV220 温度诱导表达 细胞增殖活性 成肌细胞增殖 纯化方法 大肠杆菌 表达产物 BLOT 表达系统 质粒载体 原核表达 DH5Α 质粒转化 纯化产物 

分 类 号：R394.8[医药卫生—医学遗传学] R735.7[医药卫生—基础医学]