Molecular cloning and characterization of a novel anti-TLR9 intrabody

• Autorzy: Reimer E. , Somplatzki S. , Zegenhagen D. , Hanel S. , Fels A. , Bollhorst T. , Grosse Hovest L. , Bauer S. , Kirschning C. J. , Boldicke T.
• Języki publikacji: EN

Abstrakty

EN

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.

• Wydawca: -
• Czasopismo: Cellular and Molecular Biology Letters
• Rocznik: 2013
• Tom: 18
• Numer: 3
• Opis fizyczny: p.433-446,fig.,ref.

Twórcy

autor

Reimer E.

• Department of Gene Regulation and Differentation, Helmholtz Centre for Infection Research, Inhoffenstrase 7, 31124 Braunschweig, Germany

autor

Somplatzki S.

• Department of Gene Regulation and Differentation, Helmholtz Centre for Infection Research, Inhoffenstrase 7, 31124 Braunschweig, Germany

autor

Zegenhagen D.

• Department of Gene Regulation and Differentation, Helmholtz Centre for Infection Research, Inhoffenstrase 7, 31124 Braunschweig, Germany

autor

Hanel S.

• Department of Gene Regulation and Differentation, Helmholtz Centre for Infection Research, Inhoffenstrase 7, 31124 Braunschweig, Germany

autor

Fels A.

• Department of Gene Regulation and Differentation, Helmholtz Centre for Infection Research, Inhoffenstrase 7, 31124 Braunschweig, Germany

autor

Bollhorst T.

• Department of Gene Regulation and Differentation, Helmholtz Centre for Infection Research, Inhoffenstrase 7, 31124 Braunschweig, Germany

autor

Grosse Hovest L.

• Department of Immunology, Eberhard-Karls-University, Auf der Morgenstelle 15, 72076 Tubingen, Germany

autor

Bauer S.

• Institut fur Immunologie, Philipps-Universitat Marburg, Hans-Meerwein Strase 2, 35043 Marburg, Germany

autor

Kirschning C. J.

• Institut fur Medizinische Mikrobiologie, Universitatsklinikum Essen, Hufelandstrase 55, 45122 Essen, Germany

autor

Boldicke T.

• Department of Gene Regulation and Differentation, Helmholtz Centre for Infection Research, Inhoffenstrase 7, 31124 Braunschweig, Germany

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