College Veterinary Medicine, Yangzhou University, Yangzhou 225009, China.
Joint International Research Laboratory of Agriculture and Agri-Product Safety, Yangzhou University, Yangzhou 225009, China.
Int J Mol Sci. 2023 Feb 18;24(4):4118. doi: 10.3390/ijms24044118.
The RIG-I-like receptors (RLRs) play critical roles in sensing and combating viral infections, particularly RNA virus infections. However, there is a dearth of research on livestock RLRs due to a lack of specific antibodies. In this study, we purified porcine RLR proteins and developed monoclonal antibodies (mAbs) against porcine RLR members RIG-I, MDA5 and LGP2, for which one, one and two hybridomas were obtained, respectively. The porcine RIG-I and MDA5 mAbs each targeted the regions beyond the N-terminal CARDs domains, whereas the two LGP2 mAbs were both directed to the N-terminal helicase ATP binding domain in the Western blotting. In addition, all of the porcine RLR mAbs recognized the corresponding cytoplasmic RLR proteins in the immunofluorescence and immunochemistry assays. Importantly, both RIG-I and MDA5 mAbs are porcine specific, without demonstrating any cross-reactions with the human counterparts. As for the two LGP2 mAbs, one is porcine specific, whereas another one reacts with both porcine and human LGP2. Thus, our study not only provides useful tools for porcine RLR antiviral signaling research, but also reveals the porcine species specificity, giving significant insights into porcine innate immunity and immune biology.
RIG-I 样受体 (RLRs) 在感知和抵御病毒感染方面发挥着关键作用，尤其是在 RNA 病毒感染方面。然而，由于缺乏特异性抗体，关于家畜 RLR 的研究很少。在这项研究中，我们纯化了猪的 RLR 蛋白，并针对猪的 RLR 成员 RIG-I、MDA5 和 LGP2 开发了单克隆抗体 (mAb)，分别获得了一个、一个和两个杂交瘤。猪 RIG-I 和 MDA5 mAb 分别针对 N 端 CARD 结构域以外的区域，而两个 LGP2 mAb 均针对 Western blot 中的 N 端解旋酶 ATP 结合域。此外，所有猪 RLR mAb 在免疫荧光和免疫化学测定中均识别相应的细胞质 RLR 蛋白。重要的是，RIG-I 和 MDA5 mAb 都是猪特异性的，与人类对应物没有任何交叉反应。对于两个 LGP2 mAb，一个是猪特异性的，另一个与猪和人 LGP2 都反应。因此，我们的研究不仅为猪 RLR 抗病毒信号研究提供了有用的工具，还揭示了猪种特异性，深入了解了猪的先天免疫和免疫生物学。
