集胞藻6803藻胆体别藻蓝蛋白多克隆抗体的制备

Preparation of Polyclonal Antibody of Allophycocyanin Protein in the Cyanobacterium Synechocystis sp.Strain PCC 6803

藻胆体是蓝藻细胞主要的捕光天线色素超分子复合体,主要由核心体和外围的杆两部分组成,核心体主要由别藻蓝蛋白组装而成,参与光能向光合作用反应中心的传递.该研究通过PCR扩增出集胞藻6803别藻蓝蛋白α亚基(ApcA)编码基因apcA,构建表达质粒pET-32a(+)-apcA,并将其转入大肠杆菌BL21(DE3)pLysS菌株中;通过IPTG诱导表达重组蛋白,并利用组氨酸标签将可溶性目的蛋白进行亲和纯化后,免疫日本大耳白兔,从而获得多克隆抗体.间接ELISA法揭示ApcA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.表明该研究成功制备了集胞藻6803藻胆体别藻蓝蛋白多克隆抗体,为进一步研究藻胆体的核心体在光能传递过程中所承担的重要生理角色奠定了生化基础.

Phycobilisome is the major accessory light-harvesting supramolecular complexes,which is composed of core and peripheral rods.Furthermore,the core contains several cylindrical protein assemblies that mainly consist of allophycocyanin,and is involved in the transfer of light energy to the reaction centers of photosystems.In this study,the apcA gene was amplified from the unicellular cyanobacterium Synechocystis sp.strain PCC 6803.The expression plasmid pET-32a（＋）-apcA was constructed and transformed into BL21（DE3）pLysS,and the expression of ApcA protein was induced by IPTG.After purification by His-tag,the recombinant protein pET-ApcA was used to immunize Japanese White Rabbit to obtain the polyclonal antibody.The titer of the polyclonal 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.A polyclonal allophycocyanin antibody of Synechocystis 6803 was successfully obtained in this study,and it will further help in understanding the important roles of core of cyanobacterial phycobilisome during light energy transfer by using biochemical strategy.