基础医学与临床 ›› 2008, Vol. 28 ›› Issue (7): 692-695.

• 研究论文 • 上一篇    下一篇

脆性X智力低下蛋白的克隆表达与纯化

刘剑 邹柯 朱宁 沈岩   

  1. 中国医学科学院基础医学研究所
  • 收稿日期:2008-04-09 修回日期:2008-04-10 出版日期:2008-07-25 发布日期:2008-07-25

Cloning, Expression and Purification of Fragile X Mental Retardation Protein

Jian LIU, Ke ZOU, Ning ZHU, Yan SHEN   

  • Received:2008-04-09 Revised:2008-04-10 Online:2008-07-25 Published:2008-07-25

摘要: 摘要: 目的 将脆性X智力低下基因(FMR1)克隆到原核表达载体pET22b(+)中进行表达,纯化得到其表达产物FMRP,为脆性X综合征的相关研究提供实验材料。方法 运用分子克隆的方法构建质粒pET22b(+)-FMR1,并将其转入原核表达菌株E.coli BL21(DE3)中诱导表达;用固化Ni2+吸收色谱纯化重组FMRP,并将纯化得到的蛋白与已知的RNA片段进行结合反应。结果 成功构建了重组表达质粒pET22b(+)-FMR1,在E.coli BL21(DE))中表达出相对分子质量约69 000的蛋白;经western-blot鉴定,该蛋白为FMRP,且可与特定RNA相互作用。结论 FMR1基因在原核系统中有较好的表达,得到了纯度较高的蛋白质,且证明了该蛋白具有RNA结合的特性,为相关功能性研究奠定了基础。

关键词: 脆性X智力低下蛋白, pET22b(+), BL21(DE3), RNA结合

Abstract: Abstract Objective Clone the human fragile X mental retardation gene 1 cDNA into the pET22b(+) vector, express and purify FMRP to study its function. Methods The plasmid pET22b(+)-FMR1, constructed by molecular cloning, was transformed into E. coli BL21(DE3) competent cells and induced to express FMRP by IPTG. Then, FMRP was purified by affinity chromatography. Purified FMRP was tested for its RNA binding ability. Results FMR1 cDNA was successfully cloned into pET22b(+) vector and expressed in E. coli BL21(DE3). A protein with Mr 69 000 was purified and confirmed to be FMRP by western-blot. This protein retained the RNA binding ability of FMRP. Conclusion FMR1 gene was successfully expressed in BL21(DE3) cells, and highly purified FMRP with RNA binding ability was obtained, which provided reliable material to study the function of FMRP.