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Construction and characterization of an enhanced GFP-tagged anti-BAFF scFv antibody

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Abstract

Elevated levels of B-cell-activating factor of the TNF family (BAFF) have been implicated in the pathogenesis of autoimmune diseases in human. In this study, an anti-BAFF single-chain antibody fragment (scFv) was genetically linked to the C terminus of the enhanced green fluorescent protein (EGFP) to generate an EGFP/scFv fusion protein. The EGFP/scFv fusion protein had an apparent molecular weight of 52 kDa and was identified by sodium dodecyl sulfate polyacrylamide gel electrophoresis and Western blot analysis. After being purified by immobilized metal affinity chromatography, the fusion protein exhibited similar fluorescence spectra with native EGFP. Enzyme-linked immunosorbent assay and fluorescence-activated cell sorting showed the EGFP/scFv could bind to human soluble BAFF and BAFF positive cell lines in vitro. The binding of EGFP/scFv can also be visualized under laser scanning confocal microscopy. Furthermore, the results of the competition assay indicated its antigen binding specificity. Therefore, the fusion protein EGFP/scFv has several characteristics including high sensitivity, stability and convenience for manipulation, and can be a powerful tool for the study of the underlying pathology of BAFF relevant to autoimmune diseases.

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Acknowledgments

This work was supported by the Grants of Nanjing Normal University and Jiangsu Province Graduate Innovation Project (No. 1612005024) and the International Cooperation of Jiangsu Province (No. BZ2007078). We are particularly grateful to Dr. Yuying Feng for the help and technical support in the experiments of fluorescence spectrum, to Dr. ChaoYing Zhang for the help in the experiment of LSCM, and to Dr Ling Wang in Nanjing Medical University for the help and technical support in the experiments of Flow Cytometry Analysis.

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Authors and Affiliations

  1. Jiangsu Province Key Laboratory for Molecular and Medical Biotechnology, Life Sciences College, Nanjing Normal University, Nanjing, Jiangsu, 210046, People’s Republic of China

    Meng Cao, Huaijiang Yan, Fang Ren, Wuguang Lu, Yunlong Hu & Shuangquan Zhang

  2. Laboratory of Molecular Medicine, Jiangsu Province Institute of Traditional Chinese Medicine, Nanjing, Jiangsu, 210028, People’s Republic of China

    Peng Cao

  3. Jiangsu Province Engineering Research Center for Biomedical Function Materials, Life Sciences College, Nanjing Normal University, Nanjing, Jiangsu, 210046, People’s Republic of China

    Shuangquan Zhang

  4. Department of Microbiology, Parasitology and Immunology, College of Basic Medical Science, Southeast University, Nanjing, Jiangsu, 210009, People’s Republic of China

    Meng Cao

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  1. Meng Cao
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Correspondence to Shuangquan Zhang.

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Cao, M., Cao, P., Yan, H. et al. Construction and characterization of an enhanced GFP-tagged anti-BAFF scFv antibody. Appl Microbiol Biotechnol 79, 423–431 (2008). https://doi.org/10.1007/s00253-008-1447-9

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  • DOI: https://doi.org/10.1007/s00253-008-1447-9

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