小鼠精胺氧化酶的原核表达与鉴定

Prokaryotic Expression and Identification of Mouse Spermine Oxidase

目的:原核表达和分离纯化小鼠精胺氧化酶(SMO)。方法:采用RT-PCR法从小鼠胚胎干细胞(ES细胞)RNA中克隆小鼠SMOcDNA,构建SMO原核表达质粒并转染大肠杆菌BL21(DE3)菌株,经IPTG诱导,将表达的小鼠SMO重组蛋白在变性条件下经Ni-NTA树脂亲和层析纯化和透析复性。结果:在大肠杆菌中高表达出小鼠SMO重组蛋白;纯化并透析复性后的重组SMO具备快速氧化特异性底物精胺的酶活性。结论:建立了原核表达和纯化有活性小鼠SMO的实验方法。

Objective： To establish a method for prokaryotic expression and purification of the mouse spermine oxidase （SMO）. Methods： Mouse SMO cDNA was amplified by RT-PCR from RNA of ES cells. The SMO cDNA was then cloned into prokaryotic expression plasmid and transformed into E.coli BL21 （DE3）. The expression of SMO in host ceils was induced by IPTG and resulted recombinant SMO protein was purified under denature condition by Ni-NTA resin and dialyzed to recover the enzyme activity. Results： Mouse recombinant SMO was successfully expressed in E.coli by IPTG induction. Purified and dialyzed SMO has high ability to oxidize specific substrate spermine. Conclusion： A method for prokaryotic expression and purification of the mouse SMO was successfully established.