以“王林”苹果为试材,采用RT-PCR的方法克隆获得Mal d 1基因,并将该基因连接到原核表达载体pColdⅡ上,经测序确定所构建的重组载体pCold-Mal d 1开放阅读框正确。将获得的重组质粒pCold-Mal d 1转化大肠杆菌BL21(DE3)菌株,经IPTG 0.1mmol·L~(-1),15℃,24h冷诱导表达,SDS-PAGE电泳检测,证明Mal d 1蛋白获得了稳定而高效的表达,所表达蛋白是大小约为22.4kD的融合蛋白。经镍柱纯化后获得相对单一的蛋白,可用于免疫组织化学,蛋白印记检测等。 The Mal d 1 gene was cloned by RT-PCR and the gene was ligated into prokaryotic expression vector pCold Ⅱ. The recombinant plasmid pCold-Mal d 1 Open reading frame is correct. The recombinant plasmid pCold-Mal d 1 was transformed into E.coli BL21 (DE3) strain, which was induced by 0.1 mmol·L -1 IPTG, induced by cold at15 ℃ for24h and detected by SDS-PAGE electrophoresis. It was proved that Mal d 1 protein Obtained a stable and efficient expression, the expressed protein is about 22.4kD size of the fusion protein. After purification by nickel column to obtain a relatively single protein, can be used for immunohistochemistry, Western blot detection.