筇竹CBF1基因的原核表达和多克隆抗体的制备

Prokaryotic Expression of Qiongzhuea tumidinoda CBF1 Gene and Preparation of Its Polyclonal Antibody

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。

C-repeat binding factor 1 gene （CBF1） was amplified by RT-PCR from Qiongzhuea tumidinoda Hsueh leaves and cloned into prokaryotic expression vector pET32a （＋） . After identification by enzyme digestion and sequencing, the expression of recombinant plasmid carried CBF1 gene was transformed into Rosetta2 （DE3） E. coli. Through induced with IPTF, the expression of recombinant protein was analyzed by SDS-PAGE. The results showed that the protein was highly expression in E. coli, and the molecular weight of the recombinant protein was 45 kD. After purification with Ni2^-NTA affinity chromatography, the immune reactivity of recombinant CBF1 protein was identified with positive antiserum against nature CBF1 protein specifically by Western-blotting analysis. Antibodies against recombinant CBF1 protein was obtained by subcutaneous injection of rabbit with purified CBF1 recombinant protein, which is specific to the total protein of Qiongzhuea tumidinoda Hsueh leaves induced by low temperature. Our results indicate that the recombinant protein can be used for immunohistochemistry and western blot detection.