集胞蓝细菌PCC 6803中CcmK2蛋白多克隆抗体的制备与检测
Preparation and detection of polyclonal antibody to CcmK2 protein from Synechocystis sp. PCC 6803

CcmK2蛋白是集胞蓝细菌PCC 6803羧酶体外壳蛋白的主要组分，在羧酶体的组装与功能行使方面扮演重要角色。为解析其功能，本研究构建了原核表达载体pET28a CcmK2,并在大肠杆菌BL21(DE3)中诱导表达带有6个组氨酸标签的CcmK2重组蛋白。通过金属亲和层析技术，将CcmK2重组蛋白纯化后免疫家兔，制备抗CcmK2多克隆抗体。基于Dot blot分析的结果显示：该多克隆抗体能够有效检测蓝细菌CcmK2蛋白；进一步的抗体特异性分析显示，CcmK2多克隆抗体与其他羧酶体外壳蛋白存在微弱的抗原抗体反应；最后，通过Western blot分析证实该多克隆抗体能够特异而灵敏地检测集胞蓝细菌PCC 6803中CcmK2的丰度。
CcmK2,as one of the most abundant carboxysome shell proteins in Synechocystis sp. PCC 6803,plays an important role in the assembly and biogenesis of carboxysome.In this study,the prokaryotic expression vector pET28a CcmK2 was constructed and high yield recombinant CcmK2 protein with six histidine tags was expressed by inducing in Escherichia coli BL21 (DE3).In order to produce the polyclonal antibody against rabbit,the highly performed CcmK2 protein was purified with metal affinity chromatography.Result of Dot blot analysis showed that the polyclonal antibody could effectively detect cyanobacterial CcmK2 protein.Result of antibody specificity test showed that CcmK2 polyclonal antibody could weakly reacted with other carboxysome shell proteins.Western blot analysis confirmed that this antibody was able to specifically and sensitively detect the abundance of CcmK2 in cyanobacterium PCC 6803.The polyclonal antibody generated in this study will lay a foundation for further studying the abundance of CccmK2 in cyanobacterium PCC 6803.