2. Materials and methods

2.1 Strains, reagents, cell lines

The SP2/0 myeloma cells were kept by the Henan Provincial Key Laboratory of
Animal Immunology (Zhengzhou, China), and SP2/0 cells were cultured in Roswell Park Memorial Institute 1640 (RPMI 1640, Gibco) medium supplemented with 10% fetal bovine serum (FBS, Gibco, USA). ASFV-positive sera (Convalescent sera from surviving pigs naturally infected with ASFV) were a kind gift from Professor Hu of Institute of Military Veterinary Medicine, Academy of Military Medical Sciences, Changchun, China. Human embryonic kidney 293T (HEK293T) cells were obtained from ATCC (Manassas, VA, USA) and stored in DMEM medium (Solarbio, Beijing, China) supplemented with 10% (v/v)fetal bovine serum (FBS, Gibco, USA).Escherichia coil BL21 (DE3) competent cells were purchased from Takara Biomedical Technology (Beijing China). Animals care and study procedure followed the guideline of the Animal Research Ethics Board of Zhengzhou University.

2.2. Expression and purification of the ASFV p30 recombinant protein

In response to efficient expression, we constructed three co-expression system with chaperone plasmids. The chaperone plasmids include pKJE7, pGro7 and pTf16. And then chosen the best one. Eventually we found that 0.1 mmol/L of IPTG, 12 h of expression time, and 18 ℃ of the temperature, 0.4 mg/mL of L-arabinose are the best condition to obtain the highest yield of soluble p30 protein. The soluble recombinant protein was purified by His Trap FF and His Trap Q HP affinity column.
The purified p30 protein was mixed with 5 × loading buffer, the mixture was boiled for 10 min. Following separation by sodium dodecyl sulfate-poly-acrylamide gel electrophoresis (SDS-PAGE), proteins were transferred onto a polyvinylidene fluoride membrane. The membrane was blocked overnight with 5% skim milk at 4 ℃ and incubated with anti-His tag mAb (Solarbio, Beijing, China) 1:2000 for 1 h. After washing three times with PBST, and then probed with a 1:2000 HRP-conjugated goat anti-mouse IgG antibody at 37 ℃ for 1 h. The reactivity was visualized using AEC reagents.

2.3. Construction of pcDNA3.1-p30

According to the genome information of strain BA71V (GenBankYP_009704045) Georgia 2007/1, the full-length ASFV p30 protein gene with Bam HⅠ and Xho Ⅰ restriction sites was synthesized and subcloned into the pcDNA3.1 vector (Invitrogen, Shanghai, China). The recombinant construct was named pcDNA3.1-p30 and confirmed by PCR and sequencing.

2.4. Monoclonal antibody generation and characterization

Monoclonal antibodies against p30 were produced as described below. Briefly, female, 6 weeks old, BALB/c mice were immunized subcutaneously on week 0 with 50 μg purified p30 mixed with the same amount of complete Freund adjuvant. Two booster immunizations with p30 emulsified in incomplete Freund adjuvant were administrated on weeks 2 and 4. The mice were administered 100 µg of p30 protein without adjuvant intraperitoneally. Mice were euthanized 3 days later after the last administration, and harvested spleen cells were fused with SP2/0 cells using polyethylene glycol (PEG 1500). After HAT/HT medium selection, positive hybridomas cells were screened by ELISA. Then, the supernatant was collected to identify p30-specific antibodies with ELISA assay. Positive clones were sub-cultured three times. The selected clones were cultured in the peritoneal cavities of BALB/c mice primed with paraffin to obtain ascites fluid.

2.5. Indirect enzyme-linked immunosorbent assay

Ninety-six-well plates were coated overnight at 4 ℃ with the purified p30 protein (4 µg/mL, 100 µL/well) in 0.05 M carbonate-bicarbonate buffer (CBS, pH 9.6). After washing with PBST (1×PBS with 0.05% Tween 20, pH 7.4), the plates were incubated with 5% skim milk at 37 ℃ for 2 h. Then, the first antibodies diluted with PBST were incubated at 37 ℃ for 30 min. After the incubation, 100 µL/well of HRP-conjugated goat anti-mouse IgG antibody was added at a dilution of 1:5,000, and the sample was incubated at 37 ℃ for 30 min, followed by washing three times. Tetramethylbenzidine (TMB) was added and the signal was collected at 450 nm.

2.6. Immunofluorescence assay (IFA)

HEK293T cells were seeded into 96-well cell culture plates at a density of 4×104 cells/well and incubated overnight at 37 ℃ with 5% CO2. When the cells were 70-80% confluent, the recombinant plasmid pcDNA3.1-p30 was transfected into the cells using Lipofectamine® 2000 and cultured at 37 ℃ for 48 h. After cultured, the plates were fixed with methanol containing 3% paraformaldehyde and permeabilized with 0.3% Triton X-100 for 15 min at room temperature (RT). Next, the plates were blocking with 5% skim milk at 37 ℃ for 2 h. After three times washes, anti-p30 mAbs were incubated at 37 ℃ for 30 min, the plates were washed three times with PBS. Subsequently, the plates incubated with FITC labeled goat anti-mouse IgG (1:100 in PBS) for 30 min at 37 ℃. Cells were simultaneously stained by 4,6-diamidino-2-phenylindole (DAPI, Solarbio, Beijing, China). After the final wash, the fluorescence signals were visualized by fluorescence microscopy. Mice immune and pre-immune sera were used as positive and negative controls.

2.7. Peptide design and synthesis

Bioinformatics tools were applied to analyze the entire sequence of p30 protein (201 aa). The secondary structure prediction was performed on Ramachandran server
(http://www.ebi.ac.uk/thornton-srv/databases/pdbsum/Generate.html). The B cell epitope regions were predicted using the online tool BepiPred (BepiPred-2.0 (dtu.dk), and the epitope threshold was set up at 0.5 as previously reported(Petrovan et al., 2019). The cleavage sites were selected in the rigid regions to conduct overlapping peptides of p30 protein without destroying the protein flexible structure. Based on a comprehensive consideration of bioinformatics prediction, 13 overlapping peptides (with an offset of 5 amino acids) covering the entire p30 protein and the further truncated peptides were synthesized by GL Biochem (Shanghai, China).

2.8. Dot-blot assay and peptide ELISA

Dot-blot hybridization for identification of linear epitopes was based on the method described by previously described method(Chen, Wu, Huang, Cheng, & Chang, 2015). Dot immunoblotting assay was employed to determine the epitopes recognized by mAbs. Polypeptide were prepared dissolving 1mg of solid in 100 µL of DMF (HPLC grade), 2.5 mg of bovine serum albumin (BSA) was mixed with 1.5mg of EDC in 1.25 mL of PBS. And then, the above solution was mixed slowly and after 10 min, 0.5mg of EDC was added, and stired at 4 ℃ for 18 h. The reacted mixture was dialyzed for 3 days. Centrifuged at 3500 r/min for 10 min, the supernatant was kept at -20 ℃. ASFV p30 protein peptides were spotted onto nitrocellulose membranes (Pierce, USA), and blocked with 5% skim milk in PBST at 4 ℃ overnight. After washing 5 times with PBST, the membranes were incubated with anti-p30 antibody at 37 ℃ for 1 h. HRP-conjugated goat anti-mice IgG antibody was used as the secondary antibody, and the results were observed by incubating with AEC reagent.
In peptide ELISA, 96-well plates were coated with 6 µg/mL peptide-BSA conjugates (100 µL/well) in 0.05 M CBS buffer (pH 9.6) at 37 ℃ 2 h. Then, the plates were blocked with 5% skim milk in PBST. Next, mAbs anti-p30 were used as the primary antibodies. BSA was used as negative control. HRP-conjugated goat anti-mouse IgG was used as the secondary antibody. The reactions were developed using TMB. The OD values of each well were measured at 450 nm using an ELISA microplate reader.

2.9. Reactivity of the linear B cell epitopes with ASFV-positive serum

To assess whether the linear B cell epitopes could be recognized by ASFV-positive serum, the BSA and peptides were then conjugated using the EDC reaction. The reactivity of the synthesized peptides with ASFV-positive swine serum was determined by the above-mentioned dot-blot.