The SP2/0 myeloma cell line, HEK293T cell line, and immortalized goat endometrial epithelial cells (iGEEC) were maintained in our laboratory and cultured at 37°C in a humidified incubator with 5% CO₂. The basal medium for the SP2/0 myeloma cell line is RPMI-1640 (Share-bio, China). The basal medium for HEK293T and iGEEC is Dulbecco’s modified Eagle medium (Invitrogen, USA). The cells in this study were cultured in basal medium supplemented with 10% FBS (Gibco, USA), 100 IU/ml penicillin, and 100 μg/ml streptomycin. The ORFV HCE isolate is an attenuated vaccine strain purchased from Shandong Huahong Biotechnology Company Limited. The prokaryotic expression vector pET-28a, the eukaryotic expression vector pEGFP-C1, and pCMV-HA were kept in our laboratory.

The characteristics of the ORFV F1L protein were analyzed using Lasergene v7.0 software (Lasergene, USA). The molecular weight (MW) and amino acid (aa) sequence of the cloned ORF059 (F1L) gene were analyzed by the EditSeq program of the Lasergene software. The secondary structure of the F1L protein was analyzed using the Protean program of the Lasergene software. The analysis focused on two aspects: antigenicity and hydrophilicity, which are important for the exogenous expression of the target proteins in Escherichia coli. The potential transmembrane domains of the F1L protein were predicted using the online program TMHMM.¹

The prokaryotic expression system was used to express a truncated form of the F1L protein, designated as tF1L, covering amino acids 1–281. The F1L gene was amplified from a sample of orf collected in the field. The F1L gene has been deposited in GenBank with the accession number OQ686990. The tF1L gene was amplified using a pair of primers (tF1L-F and tF1L-R) (Supplementary Table 1) carrying the restriction enzyme sites HindIII or XhoI. The amplified gene was then digested with the appropriate restriction enzymes and ligated to the plasmid pET-28a between the HindIII and XhoI cleavage sites. The recombinant plasmid pET-28a-tF1L was confirmed by sequencing and subsequently transformed into competent BL21(DE3) cells (Tolobio, China). The single positive colony of pET-28a-tF1L transformed BL21(DE3) was induced to express the recombinant protein His-tF1L. The expression of His-tF1L was confirmed by Coomassie Brilliant Blue staining. The SDS-PAGE coupled gel-cutting method was used to purify His-tF1L from the inclusion bodies of the induced E. coli lysates, following the procedure described by Han et al. (2019). Briefly, after separation by SDS-PAGE, the protein samples were stained with 0.25 M KCl for approximately 5 min. The proteins with different MWs appeared as white bands in the polyacrylamide gels. The protein bands corresponding to the position of His-tF1L were excised, and homogenized, and an appropriate amount of PBS was added to the gels. After several cycles of freezing and thawing, a portion of the target proteins migrated from the gels into the PBS. The PBS was then separated by centrifugation and aspirated. The purified His-tF1L was identified by staining with Coomassie Brilliant Blue and Western blot analysis using an anti-His mAb (Biodragon, China).

Enzyme-linked immunosorbent assay (ELISA) plates were coated with the purified His-tF1L protein (1 μg/ml, 100 μl/well) and incubated at 4°C overnight. The plates were then blocked with 5% skim milk in PBS at 37°C for 1 h, washed three times with PBS containing 0.05% Tween-20 (PBST), and incubated with hybridoma culture supernatant at 37°C for 1 h. After three washes with PBST, HRP-labeled goat anti-mouse IgG (Beyotime, China) was added to each well at a dilution of 1:3,000 and incubated at 37°C for 1 h. After washing with PBST, 100 μl/well of TMB substrate (Beyotime, China) was added and the color was allowed to develop for 5 min at 37°C. To stop the reaction, 50 μl of 2 M H₂SO₄ was added to each well. Absorbance was measured at 450 nm using Multiskan FC (Thermo, USA).

Monoclonal antibodies against the recombinant F1L protein of ORFV were generated according to a previously described procedure (Zhang, 2012). Three female BALB/c mice, aged 7 weeks, were immunized three times with 100 μg of purified His-tF1L via subcutaneous injection at 21-day intervals. The antigen was emulsified with complete Freund’s adjuvant for the first dose and incomplete Freund’s adjuvant for the subsequent booster immunizations (Sigma, USA). The antibody levels of the immunized mice were assessed through indirect ELISA after three immunizations, using purified His-tF1L as the coat antigen. A booster dose of 100 μg His-tF1L protein without adjuvant was administered intraperitoneally to the mouse with the highest antibody level 3 days before cell fusion. The mouse selected for the experiment was euthanized and its splenocytes were fused with SP2/0 myeloma cells according to standard procedures (Galfre and Milstein, 1981). The resulting hybridoma cell supernatants were tested for His-tF1L antibodies by indirect ELISA. The hybridoma cells were subcloned three times by limiting dilution and verified as His-tF1L specific antibody positive by ELISA. The purified hybridoma cells were then injected into the peritoneal cavity of BALB/c mice to generate ascites fluid against the F1L protein. The isotype of the F1L mAb was determined using a Mouse Monoclonal Antibody Ig Class/subclass Identification ELISA Kit (Biodragon, China) and an IsoQuick Strips and Kits for Mouse Monoclonal Isotyping (Sigma-Aldrich, MO, USA) according to the manufacturer’s protocol. The reactivity of the F1L mAb with ORFV were evaluated using Western blot analysis and immunofluorescence assay (IFA).

To determine the minimal epitope recognized by the F1L mAb, the amino acids at both ends of the tF1L protein were gradually deleted until the core epitope was identified. A series of truncated F1L proteins that were used in the study are shown in Figures 4A, 5A. The genes encoding the truncated forms of the F1L protein were inserted into the pEGFP-C1 plasmid via HindIII and KpnI restriction sites. The HEK293T cells were transfected with the recombinant plasmids pEGFP-C1-tF1L or its derivatives to express the F1L protein and its various truncations. Simply, when the cell culture reached 70%–80% confluence, 2.5 μg of each recombinant plasmid was transfected into HEK293T cells in a six-well plate. Cells were harvested at 48 h post-transfection, and the expression of each GFP-fused protein was confirmed through Western blot analysis using both GFP mAb and F1L mAb.

For pCMV-HA-F1L construction, the intact ORF059 gene was amplified using the primers HA-F1L-F and HA-F1L-R (Supplementary Table 1), containing EcoRI and XhoI restriction sites, respectively. The resultant amplicon was inserted into the eukaryotic expression plasmid pCMV-HA via EcoRI/XhoI restriction sites to generate the recombinant plasmid pCMV-HA-F1L. For in vitro expression of HA-F1L, HEK293T cells were transfected with recombinant plasmid pCMV-HA-F1L as described above.

The expression of HA-F1L protein in HEK293T cells was assessed through IFA 36 h after transfection. The native F1L protein was obtained by infecting iGEEC with ORFV HCE strain at an MOI of 0.5. Briefly, iGEEC seeded in glass-bottomed culture dishes at approximately 90% confluence were inoculated with ORFV. When there were obvious cytopathic effects after infection, cells were fixed with 4% paraformaldehyde (Biosharp, China) in PBS for 30 min at room temperature, and rinsed three times with PBS for 5 min each time. The fixed cells were then permeabilized with 0.1% Triton X-100 (Macklin, China) in PBS/2% BSA (Saiguo, China) for 10 min. The liquid was aspirated and the cells were blocked with PBS/2% BSA for 30 min at 37°C. After washing with PBS, the cells were incubated with mAb Ba-F1L (1:500 dilution) for 1 h at 37°C. After washing, cells were incubated with donkey anti-mouse IgG antibody (1:1,000) conjugated to FITC (Share-bio, China) for 1 h at 37°C. Cells were washed thrice with PBS and stained with DAPI (Sigma, USA) for 10 min at room temperature before final washing with PBS. Images were captured by fluorescence microscopy (Nikon, Japan).

Western blot was used to analyze the reactivity of mAb Ba-F1L with viral native F1L and GFP-fused F1L protein for the fine determination of the core epitope. Culture lysates containing native F1L or GFP-fused F1L polypeptides were subjected to 10% SDS-PAGE followed by transfer to polyvinylidene fluoride (PVDF) membrane. After blocking with 5% skim milk in PBS at 37°C for 1 h, PVDF membranes were incubated with mAb Ba-F1L (1:500 dilution) or GFP (1:1,000 dilution, Share-bio, China) at 4°C overnight. After three washes with PBS containing 0.05% Tween-20 (PBST), the membranes were incubated with HRP-conjugated goat anti-mouse IgG (1:3,000 dilution, Share-bio, China) at room temperature for 1 h. The membranes were washed with PBST and developed using an ECL imaging system (Amersham Imager 600, General Electric, USA).

To analyze whether the epitope identified in the present study is conservative among different ORFV strains, the epitope sequences and their flanking sequences of the F1L proteins were aligned among 17 ORFV reference strains using BioEdit software V7.2.5.

In order to ascertain the antigenicity, hydrophilicity, MW, and transmembrane region of the F1L protein, we initially conducted a targeted analysis using bioinformatics software. The results are depicted in Figures 1A, B. It was revealed that the net MW of tF1L is approximately 31 kDa. In comparison to the full-length F1L protein, the N-terminal 281 amino acids (tF1L) exhibit higher immunogenicity and hydrophilicity. The F1L protein has multiple transmembrane regions at its C-terminus. Consequently, we chose to express the N-terminal 281 amino acids (tF1L) of the F1L protein instead of the full form F1L.

The prokaryotic expression vector pET-28a was used as the vehicle to express the F1L protein. In the present study, tF1L was expressed as a fusion protein in the form of hexahistidine-tF1L-hexahistidine. The MW of the recombinant protein His-tF1L, as estimated by the software lasergene, is approximately 37 kDa. As shown in Figure 2A, the His-tF1L protein was successfully expressed and almost all the target proteins were present in an insoluble fraction. However, the actual MW of the prokaryotic form of His-tF1L is a slightly larger than the deduced MW. We purified the insoluble His-tF1L by a method called “SDS-PAGE and gel-cutting” as described previously (Han et al., 2019). The purity of purified His-tF1L was relatively high as shown by SDS-PAGE (Figure 2B). Western blot analysis showed that the purified His-tF1L could be recognized by the anti-His mAb (Figure 2C).

The purified His-tF1L protein was used both as an immunogen and as a screening antigen for the development of hybridomas. Positive clones were screened from hybridoma supernatants using purified His-tF1L protein as the coating antigen in an indirect ELISA. After three rounds of screening and subcloning, a hybridoma cell line that secretes F1L protein-specific antibodies was selected and designated Ba-F1L. Ascites fluid was produced by infecting mice with the Ba-F1L hybridoma cell line. The isotype of the mAb Ba-F1L light chain was determined using an IsoQuick Strips and Kits for Mouse Monoclonal Isotyping. The right strip in Figure 3A has been extracted from the instructions accompanying the Strips and Kits, which provide guidance on how to judge the results. As shown in Figure 3A, the left strip developed a test line and a kappa line, which indicated that the light chain of mAb Ba-F1L was κ. The heavy chain subclasses were identified by another commercial kit. The results clearly indicated that the mAb Ba-F1L heavy chain was IgG1, as demonstrated by testing at a dilution of 1:100,000 of the ascites (Figure 3B).

To assess the reactivity of the mAb Ba-F1L, we used Western blot and IFA to test its ability to recognize the F1L protein. As shown in Figure 3C, a specific band at the position of the expected MW of F1L (approximate 39 kDa) was present in ORFV-infected iGEEC but not in mock-infected cells. Green fluorescence signals were observed in both ORFV-infected iGEEC and HEK293T cells transfected with the eukaryotic plasmid pCMV-HA-F1L when using the mAb Ba-F1L as the primary antibody in IFA. However, no fluorescence signals were present in mock-infected iGEEC (Figure 3D). The fluorescence signals of HEK293T cells transfected with recombinant plasmids are stronger than those of iGEEC infected with ORFV. This may be due to the higher expression level of exogenous F1L in HEK293T compared to the viral native F1L protein in iGEEC.

The basic strategy for identifying the epitope-containing domain in F1L involves progressively splitting and truncating the positive proteins/polypeptides that react with mAb Ba-F1L. In this section, a series of four successive rounds of overlapping GFP-tagged proteins or polypeptides were constructed and expressed in HEK293T cells through transfection of the corresponding recombinant plasmids (Figure 4A). The tF1L protein is 281 aa in length. For the first round, the tF1L protein was divided into two parts, fragment A (150 aa) and fragment B (151 aa), with an overlap portion of 20 aa. The anti-GFP mAb recognized the tF1L protein, fragments A, and B, all of which bear a GFP tag (Figure 4B). However, only the tF1L protein and fragment A exhibited reactivity with the mAb, thereby signifying that the specific epitope recognized by mAb Ba-F1L was situated within fragment A (Figure 4B). Subsequently, over the course of the three rounds of epitope mapping, fragments A2, A3, and A7 were successively identified as the epitope-containing domains (Figures 4C–E). Consequently, it can be concluded that the epitope-containing region of the F1L protein is located between amino acids 96 and 115.

For fine mapping of the core epitope, a stepwise deletion of amino acids was carried out from either the N-terminal or C-terminal of fragment A7 (96–115 aa), resulting in a total of 14 polypeptides derived from A7 (Figure 5A). These polypeptides were expressed as GFP-fused proteins through the transient transfection of the corresponding plasmids into HEK293T cells. Subsequently, Western blot analysis was conducted on lysates from cells transfected with each recombinant plasmid to assess their reactivity with either the GFP mAb, F1L mAb, or both. As depicted in Figures 5B, C, all polypeptides exhibited reactivity with the GFP mAb, indicating the successful expression of each recombinant protein. Notably, the polypeptide A7-7 failed to be recognized by the F1L mAb, implying the essential nature of the cysteine at position 103 in F1L for the mAb-specific epitope (Figure 5B). Polypeptides A7-8 to A7-14 share the same amino acid ¹⁰³C in their N-terminus, differing solely in their C-terminus due to the progressive deletion of one amino acid at a time (Figure 5A). In Figure 5C, it is evident that polypeptides A7-8 and A7-9 exhibited a strong reaction with the F1L mAb, whereas A7-10 only yielded a weak signal. The remaining polypeptides, A7-11 to A7-14, showed no reactivity with the F1L mAb (Figure 5C). The polypeptide ¹⁰³CKSTCPKE¹¹⁰ displayed weak reactivity with the mAb Ba-F1L. However, the addition of the amino acid M¹¹¹ to the polypeptide ¹⁰³CKSTCPKE¹¹⁰ resulted in a strong reactivity with the F1L mAb, as observed in sample A7-9 in Figure 5C. These findings indicate that the core epitope recognized by the mAb Ba-F1L is ¹⁰³CKSTCPKEM¹¹¹ within the F1L protein.

Sequence alignment was performed to determine the conservation level of the epitope recognized by mAb Ba-F1L among 17 representative ORFV strains from China and other countries (Table 1). The results showed that the epitope ¹⁰³CKSTCPKEM¹¹¹ exhibits complete conservation across all ORFV reference strains, both domestic and abroad (Figure 6). The polypeptide ¹⁰³CKSTCPKEM¹¹¹ represents a highly conserved epitope on the F1L protein of ORFV, indicating the mAb Ba-F1L has the potential to react with any ORFV strain globally.