Department of Gene Regulation and Differentiation, Helmholtz Centre for Infection Research, Inhoffenstraße 7, 38124 Braunschweig, Germany.
Cell Mol Biol Lett. 2013 Sep;18(3):433-46. doi: 10.2478/s11658-013-0098-8. Epub 2013 Jul 26.
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.
Toll 样受体 9（TLR9）是先天免疫系统的一个组成部分，它可以识别病原体和宿主的 DNA。因此，它可以驱动自身免疫性疾病。细胞内抗体在 ER 内表达，通过抑制蛋白质从 ER 向其亚细胞靶位的易位来阻断瞬时蛋白质的功能。在这里，我们描述了抗 TLR9 ER 内抗体（αT9ib）的构建和表征。相应的单链 Fv 包含针对人源和鼠源 TLR9 的单克隆抗体（mAb；5G5）的重链和轻链的可变区。在 HEK293 细胞中共表达 αT9ib 和小鼠 TLR9 导致两种分子与 ER 标记物 calnexin 共定位。αT9ib 与小鼠 TLR9 的共免疫沉淀表明 αT9ib 与它的同源抗原相互作用。αT9ib 的表达抑制了 CpG DNA 挑战后 NF-κB 驱动的报告基因激活，但不激活 TLR3 或 TLR4。因此，在用 αT9ib 表达质粒转染 RAW264.7 巨噬细胞后，TLR9 驱动的 TNFα 产生被抑制。αT9ib 编码的开放阅读框被整合到腺病毒 cosmid 载体中，以产生重组腺病毒（AdV）-αT9ib。AdVαT9ib 的转导特异性抑制 TLR9 驱动的细胞 TNFα 释放。这些数据强烈表明，αT9ib 是一种非常有前途的实验工具，可以阻断 TLR9 信号。
