Development of Specific Monoclonal Antibodies against Porcine RIG-I-like Receptors Revealed the Species Specificity

Abstract

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.

Keywords: RLRs; human; monoclonal antibody; porcine; species specificity.

Figure 1

The porcine RLR purified proteins and the isotypes of four monoclonal antibodies against RLRs. (A) The purified proteins of porcine RLRs, RIG-I, MDA5 and LGP2, together with control BSA protein were subjected to SDS-PAGE and stained with Coomassie brilliant blue. The arrow head indicates the full-length MDA5. (B) The pRIG-I, pMDA5 and pLGP2 proteins were subjected to SDS-PAGE, transferred to PVDF membrane and detected by immunoblotting with anti-HA mAb. The protein markers are labeled on the left. (C) With the mouse monoclonal antibody Ig class/subclass/subtype identification kit, 4 strains of porcine RLR monoclonal antibodies derived from ascites all belong to IgG2b.

Figure 2

The reactivity of RLR mAbs with exogenous and endogenous porcine RLR proteins in Western blotting. 293T cells grown in 24-well plates (3 × 10⁵ cells/well) were transfected with HA tagged pLGP2, pRIG-I, pMDA5 or pcDNA (0.5 μg each) using Lipofectamine 2000 for 24 h. PAM cells and PK15 cells were plated on 24-well plates (2 × 10⁵ cells/well) and stimulated with human IFN-β (500 IU/mL, 1000 IU/mL) for 8–12 h. The above cell lysates were analyzed by Western blotting using the pRIG-I, pMDA5 and pLGP2 ascite mAbs, ISG56 pAb, HA mAb and Actin mAb (1:1000 dilution each). The protein markers are labeled on the left.

Figure 3

The cellular localizations of porcine RLRs identified by RLR mAbs in immunofluorescence assay (IFA). The PAM cells in 15 mm glass bottom cell culture dishes were grown to 70–80% and stimulated with 500 IU/mL human IFN-β for 8-12 h (A) or without stimulation (B). The cells were fixed, permeabilized and incubated with pRIG-I, pMDA5 and pLGP2 ascite mAbs (1:200 each) at 4°C overnight. Then, the cells were stained with secondary antibody Alex Fluor^TM 488 Donkey anti-Mouse Antibody (1:800) and counterstained with DAPI. The cellular localizations of pRIG-I, pMDA5 and pLGP2 in the PAM cells were examined under a fluorescence microscope. The boxed areas are magnified and shown on the upper right corners of each panels in (A). There was no reaction of non-stimulated PAM cells with RLR mAbs (B).

Figure 4

The identification of regions on three porcine RLR proteins recognized by four RLR mAbs. We transfected 293T cells (2 × 10⁵ cells/well) in 24-well cell culture plates with HA-tagged expression plasmids for pRIG-I-CARDs, pRIG-I-ΔCARDs, pMDA5-CARDs, pMDA5-ΔCARDs, pLGP2-L1, pLGP2-L2 and pLGP2-L3 (0.5 μg each) for 48 h, and the harvested cell lysates were subjected to Western blotting with HA mAb and the indicated porcine RLR ascite mAbs. The protein markers are labeled on the left.

Figure 5

Examination of the cross-reactivity with human RLR proteins by the four porcine RLR mAbs. We transfected 293T cells (2 × 10⁵ cells/well) in 24-well cell culture plates with expression plasmids including pcDNA-pRIG-I-2HA, pcDNA-pMDA5-2HA, pcDNA-pLGP2-2HA and pcDNA-hRIG-I-3FLAG, pcDNA-hMDA5-3FLAG and pcDNA-hLGP2-3FLAG (1 μg each) for 24 h. The cell lysates were analyzed by Western blotting. The porcine RLR protein expressions were confirmed by HA mAb, whereas human RLR protein expressions were confirmed by FLAG mAb. The protein markers are labeled on the left.

Figure 6

The expressions and distributions of RLR gene transcripts in various pig tissues in RT-qPCR. The heart, liver, spleen, lung, kidney and lymph node tissues were collected from pigs infected with HP-PRRSV (+) and non-infected control pigs (-), and the total RNA in each tissue was extracted to synthesize cDNA by reverse transcription. The RLRs, RIG-I, MDA5 and LGP2, gene expressions in these tissues were analyzed by qPCR. The three RLR genes exhibited similar patterns. * p < 0.05, ** p < 0.01 vs. normal tissues.

Figure 7

Immunochemistry analysis of RLR expressions in various tissues from PRRSV-infected pigs. Tissues from PRRSV-infected pigs including heart, liver, spleen, lung, kidney and lymph nodes were collected for tissue sectioning. Tissue sections were stained with pRIG-I mAb, pMDA5 mAb and pLGP2 (B9-2) mAb, respectively, followed by hematoxylin counterstaining. The staining results were observed under a microscope at a magnification of 200×, and the boxed areas have been enlarged and are shown on the right of each images. There was no reaction of normal pig tissues with RLR mAbs (not shown).