Huang Qi-Lai, Chen Cheng, Chen Yun-Zi, Gong Chen-Guang, Wang Jin, Hua Zi-Chun
The State Key Laboratory of Pharmaceutical Biotechnology and Department of Biochemistry, College of Life Sciences, Nanjing University, Nanjing 210093, People's Republic of China.
Biotechnol Appl Biochem. 2006 Feb;43(Pt 2):121-7. doi: 10.1042/BA20050136.
In the present study, a red fluorescent protein (DsRed) from the coral Discosoma was fused to the C-terminus of protein ZZ, a synthetic artificial IgG-Fc-fragment-binding protein derived from the B-domain of staphylococcal Protein A. The chimaeric protein, tagged with six histidine residues at the N-terminus, was expressed in Escherichia coli and easily purified by one-step Ni2+-chelating affinity chromatography. Its fluorescence and IgG-binding activities were validated using fluorescence-spectrum analysis, ELISA and dot-blot analysis. Furthermore, in subsequent dot-blotting immunoanalysis of glutathione S-transferase and tumour necrosis factor-alpha, and immunofluorescent microscopy assay of interferon regulatory factor 3, the chimaeric protein enabled effective detection of target molecules. Compared with fluorescence-conjugated antibodies, ZZ-DsRed is less susceptible to photobleaching and easy to produce. In addition, unlike HRP (horseradish peroxidase)-conjugated antibodies, using ZZ-DsRed needs no addition of a chromogenic reagent. Our results indicate that ZZ-DsRed shows a wide and promising application potential in immunological detection as a substitute for fluorescent or HRP-conjugated anti-IgGs.
在本研究中，来自珊瑚盘碟珊瑚的红色荧光蛋白（DsRed）与蛋白ZZ的C末端融合，蛋白ZZ是一种源自葡萄球菌蛋白A的B结构域的合成人工IgG-Fc片段结合蛋白。该嵌合蛋白在N末端带有六个组氨酸残基标签，在大肠杆菌中表达，并通过一步Ni2+螯合亲和层析轻松纯化。使用荧光光谱分析、酶联免疫吸附测定（ELISA）和斑点印迹分析验证了其荧光和IgG结合活性。此外，在随后对谷胱甘肽S-转移酶和肿瘤坏死因子-α的斑点印迹免疫分析以及对干扰素调节因子3的免疫荧光显微镜检测中，该嵌合蛋白能够有效检测靶分子。与荧光偶联抗体相比，ZZ-DsRed不易发生光漂白且易于制备。此外，与辣根过氧化物酶（HRP）偶联抗体不同，使用ZZ-DsRed无需添加显色剂。我们的结果表明，作为荧光或HRP偶联抗IgG的替代品，ZZ-DsRed在免疫检测中显示出广泛且有前景的应用潜力。
