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Molecular cloning and characterization of a novel anti-TLR9 intrabody

Cellular & Molecular Biology Letters volume 18, pages 433–446 (2013)Cite this article

Abstract

Toll-like receptor 9 (TLR9) is a component of the innate immune system, which recognizes the DNA of both pathogens and hosts. Thus, it can drive autoimmune diseases. Intracellular antibodies expressed inside the ER block transitory protein functions by inhibiting the translocation of the protein from the ER to its subcellular destination. Here, we describe the construction and characterization of an anti-TLR9 ER intrabody (αT9ib). The respective single-chain Fv comprises the variable domains of the heavy and light chain of a monoclonal antibody (mAb; 5G5) towards human and murine TLR9. Co-expression of αT9ib and mouse TLR9 in HEK293 cells resulted in co-localization of both molecules with the ER marker calnexin. Co-immunoprecipitation of mouse TLR9 with αT9ib indicated that αT9ib interacts with its cognate antigen. The expression of αT9ib inhibited NF-κB-driven reporter gene activation upon CpG DNA challenge but not the activation of TLR3 or TLR4. Consequently, TLR9-driven TNFα production was inhibited in RAW264.7 macrophages upon transfection with the αT9ib expression plasmid. The αT9ib-encoding open reading frame was integrated into an adenoviral cosmid vector to produce the recombinant adenovirus (AdV)-αT9ib. Transduction with AdVαT9ib specifically inhibited TLR9-driven cellular TNFα release. These data strongly indicate that αT9ib is a very promising experimental tool to block TLR9 signaling.

Abbreviations

mAb:

monoclonal antibody

Intrabody:

intracellular antibody

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Authors and Affiliations

  1. Department of Gene Regulation and Differentiation, Helmholtz Centre for Infection Research, Inhoffenstraße 7, 38124, Braunschweig, Germany

    Elisa Reimer, Stefan Somplatzki, Diana Zegenhagen, Svenja Hänel, Alina Fels, Thorsten Bollhorst & Thomas Böldicke

  2. Department of Immunology, Eberhard-Karls-University, Auf der Morgenstelle 15, 72076, Tübingen, Germany

    Ludger Grosse Hovest

  3. Institut für Immunologie, Philipps-Universität Marburg, Hans-Meerwein Straße 2, 35043, Marburg, Germany

    Stefan Bauer

  4. Institut für Medizinische Mikrobiologie, Universitätsklinikum Essen, Hufelandstraße 55, 45122, Essen, Germany

    Carsten J. Kirschning

Authors
  1. Elisa Reimer
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  2. Stefan Somplatzki
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  3. Diana Zegenhagen
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  4. Svenja Hänel
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  9. Carsten J. Kirschning
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  10. Thomas Böldicke
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Corresponding author

Correspondence to Thomas Böldicke.

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These authors contributed equally to this work.

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Reimer, E., Somplatzki, S., Zegenhagen, D. et al. Molecular cloning and characterization of a novel anti-TLR9 intrabody. Cell Mol Biol Lett 18, 433–446 (2013). https://doi.org/10.2478/s11658-013-0098-8

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  • DOI: https://doi.org/10.2478/s11658-013-0098-8

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Cellular & Molecular Biology Letters

ISSN: 1689-1392

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