蛋白A-藻蓝蛋白β亚基融合蛋白的性质及应用

蛋白A-藻蓝蛋白β亚基融合蛋白的性质及应用 Properties of Protein A-β Subunit of Phycocyanin（SPA-CpcB） and Its Application 孙雅芳 SUN Yafang1 1 first-author 谢冰清 XIE Bingqing2 2 夏坤 XIA Kun2 2 陈思礼 CHEN Sili1 1 周明 ZHOU Ming3 3 4 中南民族大学生命科学学院 中南民族大学生命科学学院 College of Life Science,South-Central University for Nationalities College of Life Science,South-Central University for Nationalities 华中科技大学环境科学与工程学院 华中科技大学环境科学与工程学院 School of Enviromental Science and Engineering,Huazhong University of Science and Technology School of Enviromental Science and Engineering,Huazhong University of Science and Technology 华中农业大学农业微生物国家重点实验室 华中农业大学农业微生物国家重点实验室 State Key Laboratory of Agricultural Microbiology,Huazhong Agricultural University State Key Laboratory of Agricultural Microbiology,Huazhong Agricultural University 华中农业大学生物质与生物能源研究中心 华中农业大学生物质与生物能源研究中心 Biomass and Bioenergy Research Centre,Huazhong Agricultural University Biomass and Bioenergy Research Centre,Huazhong Agricultural University PCR扩增金黄色葡萄球菌蛋白A基因spa,通过酶切位点的转换构建重组质粒pET-spa,进一步成功构建能表达融合蛋白SPA-CpcB的质粒pET-spa-cpcB.将重组质粒pET-spa-cpcB与在大肠杆菌内生成藻胆色素必需的质粒,以及藻胆蛋白裂合酶质粒,共同转入大肠杆菌BL21（DE3）内,进行体内重组,生物合成融合色素蛋白.蛋白质电泳以及锌染色结果表明,体内重组分别获得了融合色素蛋白SPA-CpcB-PCB或SPA-CpcB-PEB.吸收光谱与荧光光谱结果表明,融合色素蛋白SPA-CpcB-PCB或SPA-CpcB-PEB具有与CpcB-PCB或CpcB-PEB相同的光谱特征.将SPA-CpcB-PEB与生物素标记的兔抗体进行荧光免疫反应,通过多功能微孔板荧光检测仪检测,结果表明融合色素蛋白SPA-CpcB-PEB具有SPA能与多种哺乳动物血清中的IgG的Fc段结合的特点.利用SPA-CpcB-PEB来检测包被于聚苯乙烯微量反应板的基因编码为all5292的蛋白,证实了该融合色素蛋白可用于免疫检测. In this research,the spa gene was amplified by PCR from Staphylococcus aureus genome and cloned.Molecular biology technique was used to construct recombinant plasmid pET-spa-cpcB,and in this way we constructed a fusion protein consisting of SPA and CpcB.By reconstituting CpcB with phycobilin,the fluorescence can be detected by observing the fluorescence emission.Protein A has the property to selectively bind to immunoglobulins G（IgG） of many mammalian.The fusion protein could be applied to immunoassay as a new alternative reagent.The fluorescence activity of the fusion protein was tested by reconstitution in vivo and the immunocompetence was tested by immunoreaction.The results showed that the fusion protein retained the original activities of the two proteins.Then by using the bifunctional engineered fluorescence proteins as a second antibody,and detecting its fluorescence,it was indicated that SPA-CpcB-PEB can be used for fluoroimmunoassay. 融和蛋白 金黄色葡萄球菌蛋白A 藻蓝蛋白 fusion protein Staphylococcus aureus protein A phycocyanin 10.14188/j.1671-8836.2010.03.004 Q51 国家自然科学基金资助项目（30870519;30870541） 2010-03-01 2021-04-01 3 56