脆性X智力低下蛋白的克隆表达与纯化

目的 探讨脆性X智力低下蛋白(FMRP)的克隆表达与纯化条件.方法 用分子克隆的方法构建重组质粒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,Western-blot鉴定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79 000的蛋白;该蛋白为FMRP,且可与特定RNA相互作用.结论 在原核系统中表达得到了纯度和活性都较为理想的FMRP蛋白,为相关功能性研究奠定了基础.

Cloning, expression and purification of fragile X mental retardation protein

Objective Explore the conditions of the cloning,expression and purification of FMRP.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.Recombinant FMRP was purified by affinity chromatography,verified by Western-blot,and 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 79 000 was purified and confirmed to be FMRP.This protein retained the RNA binding ability of FMRP.Conclusion We successfully expressed recombinant hFMRP with high purity and activity in E.coli,which provided a reliable material to study the function of FMRP.