集胞藻6803NdhO蛋白多克隆抗体制备及其初步应用

Preparation and Preliminary Application of Polyclonal Antibody of NdhO Protein in the Cyanobacterium Synechocystis sp.Strain PCC 6803

蓝藻NADPH脱氢酶(NDH-1)是一种重要的光合膜蛋白复合体,参与CO2吸收、围绕光系统I的循环电子传递和细胞呼吸。迄今为止,人们在蓝藻细胞中已鉴定出17种NDH-1复合体亚基(NdhA-NdhQ)。最近,人们还获得了NdhO亚基的缺失突变株。然而,人们对NdhO亚基的研究还不充份,至今仍不清楚它的功能角色。通过PCR扩增出集胞藻6803 NdhO亚基编码基因ndhO,构建表达质粒pET32a(+)-ndhO,转化大肠杆菌BL21(DE3)pLysS,用IPTG诱导表达、经His-tag纯化后免疫日本大耳白兔获得多克隆抗体。间接ELISA法揭示NdhO抗体效价可高达1∶1 025 000;蛋白免疫印迹确定该抗体具有高度特异性。应用该抗体进行蛋白免疫印迹检测,结果发现NdhO亚基仅存在于NDH-1活性复合体中。因此,NdhO多克隆抗体的获得将为进一步阐明该亚基所担任的功能角色奠定了生化基础。

Cyanobacterial NADPH dehydrogenase （NDH-1） is an important photosynthetic membrane protein complex, and is essential to CO2 uptake, cyclic electron transport around photosystem I and cellular respiration. This enzyme accepts electrons from NADPH and consists of at least 17 subunits, i. e. , NdhA to NdhQ. Recently, an ndhO gene inactivation mutant, AndhO, has also successfully been obtained. However, little is known regarding the functional roles of NdhO subunit in cyanobacteria. Therefore, the encoding gene, ndhO, was PCR amplified from the unicellular cyanobaeterium Synechocystis sp. strain PCC 6803, the expression plasmid pET32a（ ＋ ）-rtdhO was constructed and transformed into BL21 （DE3）pLysS, and the expression of NdhO protein was induced by IPTG. After purification, the fusion protein pET-NdhO was used to immunize Japanese white rabbit to obtain the polyelonal antibody. The titer of the polyelonal antibody was detected by ELISA and its specificity was analyzed by immunoblotting. The titer of polyclonal antibody was found to be up to 1：1 025 000, and thus possessed a high specificity. Further, immunoblotting results using the polyclonal antibody showed the presence of NdhO in active NDH-1 mediumeomplex, and not active NDH-1 supercomplex. Therefore, the antibody of NdhO obtained will further help us to reveal the functional roles of cyanobacterial NdhO subunit.