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以筇竹(Qiongzhuea tumidinoda Hsueh et Yi)叶片为试材,采用RT-PCR方法克隆其CBF1基因,并将CBF1连接到原核表达载体pET32a(+)上,经克隆测序确定所构建的重组载体pET32-QZ开放阅读框正确。将重组载体pET32-QZ转化大肠杆菌Rosetta2(DE3)菌株,经IPTG诱导表达,SDS-PAGE凝胶电泳,考马斯亮蓝染色,证明CBF1蛋白得到了高效表达,所表达蛋白是大小约为45 kD的融合蛋白。经镍柱纯化后作为抗原免疫家兔,制备CBF1蛋白特异性抗血清。所制备的多克隆抗体能够与融合蛋白和经冷诱导的筇竹叶片总蛋白在25 kD处出现杂交条带。上述结果表明,表达的目的蛋白可用于免疫组织化学、蛋白质印迹检测。
The leaves of Qiongzhuea tumidinoda Hsueh et Yi were used as materials, the CBF1 gene was cloned by RT-PCR and ligated to prokaryotic expression vector pET32a (+). The recombinant vector pET32- QZ open reading frame is correct. The recombinant vector pET32-QZ was transformed into Escherichia coli Rosetta2 (DE3) strain, induced by IPTG, SDS-PAGE gel electrophoresis and Coomassie brilliant blue staining showed that CBF1 protein was highly expressed. The expressed protein was about 45 kD Fusion protein. After purified by nickel column, the rabbit was immunized with antigen to prepare CBF1 protein-specific antiserum. The prepared polyclonal antibodies were able to show hybridization bands at 25 kD with the fusion proteins and the cold-induced total protein of Tamarix. The above results show that the expressed protein can be used for immunohistochemistry, Western blot detection.