Female BALB/c mice were purchased from the laboratory animal center of the Air Force Medical University, Xi’an city, Shaanxi Province, China. All mice were bred in the animal center of NWAFU in specific pathogen-free facilities and treated in accordance with the guidelines of the Care and Use of Laboratory Animals of the Ministry of Health, China. The mice were euthanized by spine dislocation during mAb collection. Eight healthy 2-month-old goats were purchased from a farm in Yangling city. During sample collection, goats were anesthetized with sodium pentobarbital (Sigma-Aldrich, St. Louis, Missouri, USA) at a dose of 20 mg/kg intravenously. After the study, all goats were fully recovered and normally fed in the animal center of NWAFU.
Analysis of the expression of IFN-γ from goat PBMCs infected with Orf virus using real-time PCR
Goat blood was collected on 0, 10th, and 20th day after infection with Orf virus and lymphocytes were separated by lymphocyte separation solution (Haoyang, Tianjin, China). RNAiso plus was added (Takara Bio, Beijing, China) to save at − 80 °C. Total RNA extraction was done by using the following methodology: First, take above samples out of − 80 °C and add 1/5 volume of chloroform after they melt. To mix the solution properly, vigorously shake for 15 s. Then centrifuge at 12000 rpm for 15 min at 4 °C and pipette the upper aqueous phase into a new Eppendorf tube. Second, add the equal volume of isopropanol, mix and leave it at room temperature for 10 min. Then centrifuge at 12000 rpm for 10 min at 4 °C and discard the supernatant. Third, add 1 ml of 75% DEPC ethanol to wash RNA and centrifuge at 12000 rpm for 10 min at 4 °C. After the centrifugation discard the supernatant. Finally, add 10 to 100 μL of RNase-free double distilled water to dissolve the RNA for reverse transcription. The RNA reverse-transcription was done with the help of a Fastking RT kit (Tiangen, Beijing, China). Real-time PCR was performed with a SYBR Green Real-time PCR kit (Tiangen) and the mRNA expression of IFN-γ was normalized to that of β-actin. The sequence of caprine IFN-γ was obtained from GenBank (NM_001285682.1) and the primers was designed with Primer Permier 5.0 software. The primer sequences are AGATCCAGCGCAAAGCCATA (forward) and TCTCCGGCCTCGA AAGAGAT (reverse). Each test set up 3 technical repeats.
Preparation of natural and recombinant caprine IFN-γ
Natural caprine IFN-γ could be produced by spleen cells and PBMCs . In the present study, the coding sequence region of caprine IFN-γ (signal peptide sequence removed) was cloned from Con A (Sigma-Aldrich) activated PBMCs and then inserted into the expression vector PET 32a, and the recombinant vector was transformed into the host cell BL21 (DE3) to construct a recombinant expression bacteria named “cIFN-γ-PET 32a-BL21 (DE3)”. The recombinant expression bacteria “cIFN-γ-PET 32a-BL21 (DE3)” was cultured in ampicillin LB medium to logarithmic phase at 37 °C. After that, 0.03 mmol/L isopropyl-D thiogalactopyranoside (IPTG) (Sigma-Aldrich) was added to the bacterium solution to induce IFN-γ expression for 6 h at 30 °C. Bacteria were harvested by centrifugation at 6000 g for 30 min at 4 °C and pellets were resuspended in 100 ml of phosphate buffer (PBS). Then, bacteria were lysed by sonication on ice and the supernatant was collected by centrifuging at 12000 rpm for 30 min. Expression levels of IFN-γ proteins in both supernatant and precipitates were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE).
The prokaryotically expressed recombinant fusion protein caprine IFN-γ (rIFN-γ) protein was purified from the supernatant under native conditions. The Ni-NTA agarose (Solarbio, Beijing, China) was used to purify recombinant caprine IFN-γ protein according to the manufacturer’s instructions. In brief, the supernatant was added to the Ni-NTA agarose and incubated for 3 h at 4 °C. Then, the heterologous proteins were eluted with 40 mmol/L imidazole (Solarbio) and the target proteins were eluted with 400 mmol/L imidazole. The concentration of purified protein was determined by using the BCA protein concentration assay kit (Biosharp, Shanghai China) according to the manufacturer’s instructions.
Animal immunization and hybridoma lines production
Animal immunization was conducted as previously reported . Briefly, six-to-eight-week-old female BALB/c mice were immunized intraperitoneally with 100 μg of purified rIFN-γ protein for three times with an interval of 21 days. The first injection was accompanied with complete Freund’s adjuvant (Sigma-Aldrich), the second and third injections were accompanied with incomplete Freund’s adjuvant (Sigma-Aldrich). For the fourth immunization, 100 μg of rIFN-γ protein solution (pH = 7.4) was injected through a caudal vein. Three days after the last boost injection, splenocytes were harvested from the mice and fused with SP2/0 myeloma cells (10:1 ratio) to establish hybridoma lines, using polyethylene glycol 1500 (Roche, Basel, Schweizer) as fusogen. The fused cells were resuspended in RPMI 1640 medium (Thermo Fisher, Shanghai, China) supplemented with 10% FBS (ExCell Bio, Shanghai, China), 100 units/ml penicillin and streptomycin (Hayao, Haerbin, China), and HAT media supplement (Sigma-Aldrich). They were then plated into 96-well tissue culture plates and cultured at 37 °C with 5% CO2. After incubation for 7 to 10 days, the culture medium in each well was analyzed by indirect ELISA to detect the presence of mAb against rIFN-γ. Positive hybridoma cells were subjected to limiting dilution three times. Finally, one hybridoma line 2C was selected for further investigation.
Indirect enzyme-linked immunosorbent assay
The mAb screened by indirect ELISA was performed as follows: 96-well polystyrene microtiter plates (Corning, New York, USA) were coated with purified rIFN-γ protein (100 μL/well, 10 mg/ml) and incubated overnight at 4 °C. The plates were washed three times by PBST (PBS containing 0.05% tween-20) and blocked with 0.1 M carbonate buffer containing 5% skimmed milk powder. After washed as above, cultured supernatant from the hybridomas was added to each well and incubated for 1 h at 37 °C. Followed by previous step, 100 μL of HRP-conjugated goat-anti-mouse IgG (Bioss, Beijing, China) diluted in 1:5000 with PBST was added as secondary antibody and incubated for 1 h at 37 °C. After washing, 100 μL of 3,3′,5,5′-tetramethylbenzidine (Tiangen) was added and incubated for 18 min at 37 °C. After chromogenic termination by 0.2 M H2SO4, absorbencies were measured with an automatic ELISA reader (Bio-Rad, California, USA) at 450 nm.
Preparation of mice ascites
For this, 10-week-old female BALB/c mice were selected and received sterile liquid paraffin with 0.5 ml by intraperitoneal injection. After 7 to 10 days, the cultured hybridoma cells were collected by centrifugation at 800 rpm for 10 min and 106 cells in 0.5 mL were injected into mice intraperitoneally. After 10 days, the abdominal circumference of the mice increased significantly and the ascites were collected. The fluid from the mouse ascites was centrifuged and stored at − 20 °C.
SDS-PAGE and Western blot
To identify the specificity of mAb, SDS-PAGE and western blot techniques were applied. For western blot assay, rIFN-γ and some unrelated proteins were respectively mixed with SDS (Sigma-Aldrich) sample buffer supplied with β-mercaptoethanol (Sigma-Aldrich) and denatured for 10 min at 100 °C. First, the mixtures were separated by SDS-PAGE and then transferred to polyvinylidene difluoride membranes (TianGen) under 60 V for 50 min using transfer buffer (58 mM Glycine, 71.8 mM Tris base, 1.9 mM SDS). After the membranes were blocked with 3.5% fish gelatin in PBST at 37 °C for 2.5 h and washed three times with TBST, hybridoma culture supernatant was served as the primary antibody and incubated overnight at 4 °C. Followed by previous step, HRP-conjugated goat-anti-mouse IgG (Sigma-Aldrich) was served as the secondary antibody incubated at 25 °C for 1 h. Finally, the signal was visualized with enhanced chemiluminescence reagent ECL and ChemiDocTMMP Imaging System (Bio-Rad).
To identify whether mAb recognize IFN-γ secreted by histopathological sites, frozen sections of lip tissues of goat infected with Orf virus were used for immunofluorescence analysis. First, frozen sections were rewarmed with an antigen-repairing solution and incubated at room temperature (RT) for 15 min. After being washed six times with PBS (pH = 7.4), 5% Triton-100 was added and incubated at RT for 15 min to permeabilize the cells. Followed by washing, frozen sections were blocked with PBS containing 5% donkey serum for 2 h at RT. Then, hybridoma culture supernatant was served as primary antibody and incubated for 1 h at 37 °C. Followed by previous step, fluorescein isothiocyanate (FITC)-conjugated goat-anti-mouse IgG (Proteintech Group, Chicago, USA) was diluted 400 times with PBS as secondary antibody at RT for 2 h in the dark. Nuclei were stained with DAPI (BioFROXX, Guangzhou, China) for 30 min at RT in dark. Corresponding tissue section of healthy goat was served as negative control. Finally, sections were analyzed using an inverted fluorescence microscope (Leica, Wetzlar, Germany).
The statistical analysis was performed using the two-tailed Students’ t test. Differences were considered statistically significant if p < 0.05 (* p < 0.05, ** p < 0.01, *** p < 0.001).