Isolation of feline-derived scFvs against the VP1-CDE region of feline calicivirus from a phage display library and characterization of their antigen-binding and antiviral potential in vitro

Fig. 1. Prokaryotic expression and purification of the recombinant VP1-CDE protein. A PCR amplification of the FCV VP1-CDE gene fragment. Lane A1: VP1-CDE amplicon (~ 430 bp); lane A2: negative control (no template). M, DNA molecular weight marker. B Schematic diagram of the pET-28a-VP1-CDE expression construct. C SDS‒PAGE analysis of the Ni-affinity purification of VP1-CDE. Lanes C1–C4: eluted fractions containing the purified VP1-CDE protein (~ 18 kDa). D Western blot analysis using anti-FCV feline serum as the primary antibody. Lane D1: purified VP1-CDE protein (~ 18 kDa); lane D2: FCV viral lysate showing the VP1 capsid protein (~ 65 kDa)

Fig. 2. Longitudinal monitoring of VP1-CDE-specific serum antibody responses in immunized cats. Serum samples were collected from two cats (Cat A and Cat B) prior to immunization (negative control) and 14 days after each of the five consecutive immunizations. Antibody levels were assessed by indirect ELISA using VP1-CDE as the coating antigen (100 ng/well) and a fixed serum dilution of 1:16,000. The data are presented as OD450 values. The corresponding raw OD450 values and negative control readings are provided in Supplementary Table S1

Fig. 3. Amplification of the VH and Vλ genes and assembly of the scFv fragment by overlapping PCR. A Amplification of the VH gene via the primer pair FeIGHV-F/FeIGHV-R at annealing temperatures of 56 °C (lane A1), 59 °C (lane A2), and 62 °C (lane A3). B Amplification of the Vλ gene via the primer pair FeIGLV-F/FeIGLV-R at annealing temperatures of 56 °C (lane B1), 59 °C (lane B2), and 62 °C (lane B3). C Assembly of the full-length scFv fragment by overlapping PCR via the primers FeIGHV-F/FeIGLV-R at annealing temperatures of 53 °C (lane C1), 56 °C (lane C2), and 59 °C (lane C3). M, DNA molecular weight marker

Fig. 4. Construction and sequence diversity of the feline scFv phage display library. A Schematic representation of the pComb3XSS-scFv expression cassette, depicting key functional elements, including the lac promoter, a common primer binding site, the scFv gene (VH-linker-Vλ), and unique restriction enzyme sites. B Multiple sequence alignment of the deduced amino acid sequences of 24 randomly selected scFv clones. Framework regions (FRs), complementarity-determining regions (CDRs), and the (Gly4Ser)3 linker peptide are indicated, highlighting the primary sequence diversity of the constructed library

Fig. 5. Phage ELISA screening of enriched clones and sequence alignment of unique scFv variants. A Phage ELISA analysis of 92 randomly selected clones after the third round of panning. Clones with a sample-to-negative (S/N) ratio of OD450 >10 were identified as high-affinity binders. Wells 1–92 correspond to supernatants containing monoclonal phage particles, whereas wells 93–96 serve as negative controls and contain 2YT medium supplemented with M13KO7 helper phage alone. B Multiple sequence alignment of the deduced amino acid sequences of five representative scFv clones (3A5, 3B8, 3H5, 2H5, and 2I5). Framework regions (FRs) and complementarity-determining regions (CDRs) are indicated. The VH domains are highly conserved among the five clones, whereas the Vλ domains exhibit substantial diversity, particularly within the CDR3 region

Fig. 6. SDS‒PAGE and Western blot analysis of eukaryote-expressed scFv‒Fc fusion proteins. A Reducing SDS‒PAGE analysis of protein A-purified scFv-Fc proteins. Lanes 1–5: scFv-Fc-3A5, scFv-Fc-3B8, scFv-Fc-3H5, scFv-Fc-2H5, and scFv-Fc-2I5, respectively. The single band at approximately 55 kDa corresponds to the monomeric scFv-Fc polypeptide. B-F Western blot analysis of the binding specificity of each scFv-Fc clone. Lane 1: recombinant VP1-CDE protein (~ 18 kDa); lane 2: FCV viral lysate (VP1 capsid protein, ~ 65 kDa); lane 3: FPV VP2 protein (negative control). The scFv-Fc proteins were used as primary antibodies, followed by an HRP-conjugated anti-human IgG-Fc secondary antibody. All five clones specifically recognized both the recombinant VP1-CDE antigen and native FCV VP1 but showed no reactivity toward FPV VP2

Fig. 7. Biolayer interferometry (BLI) analysis of the binding affinity between VP1-CDE and five scFv-Fc fusion proteins. Each scFv-Fc was captured onto protein A biosensors and exposed to a twofold dilution series of the VP1-CDE analyte (3.125–200 nM). Sensorgrams are shown together with global fits to a 1:1 Langmuir binding model

Fig. 8. IFA detection of scFv-Fc binding to FCV-infected F81 cells. F81 cells were infected with FCV (MOI = 5) for 3 h and fixed with 80% acetone. ScFv-Fc fusion proteins (20 µg/mL) were used as primary antibodies, followed by Cy3-conjugated anti-human IgG-Fc secondary antibodies (red). Nuclei were counterstained with Hoechst 33,342 (blue). A scFv-Fc-3A5; (B) scFv-Fc-3B8; (C) scFv-Fc-3H5; (D) scFv-Fc-2H5; (E) scFv-Fc-2I5; (F) Negative control (uninfected F81 cells). Scale bars, 400 μm

Fig. 9. Dose‒response neutralization curves of scFv‒Fc fusion proteins against FCV. F81 cells were infected with FCV (100 TCID50) preincubated with serial fourfold dilutions of each scFv-Fc protein (initial concentration of 75 µg/mL). Cell viability was measured at 48 h post infection via the CCK-8 assay and normalized to that of the uninfected (100%) and virus-only (0%) controls. The data points represent the means ± SDs from three independent experiments, each performed in quadruplicate