内源性血管生成抑制因子Arresten的克隆表达  

Cloning and expression of human endogenous angiogenesis Inhibitor arresten

作　　者：吕懿[1] 郑金平[1] 陈显久 唐海英[3] 

机构地区：[1]山西医科大学卫生毒理学教研室,太原030001 [2]分子生物学教研室 [3]营养学与食品卫生学教研室

出　　处：《中国医药》2009年第10期740-742,共3页China Medicine

基　　金：山西省科学技术发展计划项目（042082）;山西省高校产业化项目（20080017）

摘　　要：目的构建内源性血管生成抑制因子Arresten的原核表达体系，并于大肠杆菌中初步表达。方法从健康产妇胎盘组织中提取总RNA，经反转录．聚合酶链式反应扩增出Arresten基因，将基因克隆人克隆载体（pMD19-T）中，测序确认。酶切后插入表达载体pBV221，转入大肠杆菌JM109进行温控诱导表达，经蛋白质免疫印迹技术（Western-blot）检测Arresten蛋白的表达。结果酶切鉴定证实Arresten基因正确地插入表达载体中。经SDS-聚丙烯酰胺凝胶电泳（SDS-PAGE）分析，重组体Arresten在大肠杆菌中获得表达，分子量约为26000，表达量约占菌体总蛋白的30％。经Western blotting检测表明该异源重组蛋白具有添加的亲和纯化标签多聚组氨酸标签抗原活性。结论成功构建了有效的原核表达体系pBV221-Arresten。Objective To construct prokaryotic expression recombinant of human endogenous angiogenesis inhibitor arresten and to express primarily recombinant arresten in Escherichia coli. Methods Human Arresten gene was amplified by RT-PCR using total RNA extracted from placenta tissue of healthy puerperal, and cloned into the pMD19-T Simple Vector. The sequence of Arresten gene was analyzed. Digested with restriction endonuclease and inserted into plasmid pBV221. The constructed recombinant Arresten was transformed into E. coli JM 109 and expressed primarily. The Western blotting method was used to examine whether the protein was expressed. Results Restriction endonuclease analysis indicated that the Arresten gene was inserted into the expression vector ssuccessful- ly. SDS-PAGE analysis indicated that expressed product was about 26 000 and its amount was about 30% of total bacterial proteins. Western blotting indicated that the protein could react with His-tag antibody. Conclusion The recombinant of pBV221-Arresten is an effective prokaryotic expression recombinant.

关 键 词：ARRESTEN 克隆表达 原核表达重组体 

分 类 号：R34-33[医药卫生—基础医学]