Viruses, cells and plasmids
DAdV-1, DAdV-3, serotype 4 fowl adenovirus 4 (FAdV-4), H9N2 avian influenza virus, Tembusu virus (TMUV), and goose astrovirus type 1 (GAstV-1) were maintained in our laboratory. duck hepatitis virus type 1 (DHV-1), duck hepatitis virus type 3 (DHV-3), duck reovirus (DRV), and avian paramyxovirus type 4 (APMV-4) were provided kindly by Prof. Zongyan Chen (Shanghai Veterinary Research Institute). LMH cells from ATCC were cultured in DMEM/F12 (Gibco, NY, USA) supplemented with 10% FBS (Lonsera, Shanghai, China) at 37°C with 5% CO2. Plasmids pcDNA3.1 and pCold-1 were stored in our laboratory. pcDNA3.1-Fiber-2 carrying with the Fiber-2 of DAdV-3 was generated and stored in our laboratory.
Expression and purification of recombinant Fiber-2
The recombinant plasmid pCold-1-Fiber-2 was constructed by full-length Fiber-2 gene (GenBank accession number: ON995379) and plasmid pCold-1 digested with EcoR I and Xho I restriction enzyme. The corresponding primers were listed in Table 1. The recombinant plasmid pCold-1-Fiber-2, confirmed by sequencing, was transformed into E. coli BL21 (DE3) cells. A single positive E. coli BL21 colony was cultured overnight and the culture was inoculated into 1L LB medium with a ratio of 1:100 at 37°C. When the OD value reached 0.6, isopropyl β-D-thiogalactoside (IPTG) was added with a final concentration of 1mM for overnight induction at 16°C. The E. coli BL21 cells transformed with pCold-1-Fiber-2 were collected, resuspended in binding buffer (10mM Na2HPO4, 10mM NaH2PO4, 500mM NaCl, 20mM imidazole, pH 7.4), ultrasonicated, and then centrifuged at 13,000g at 4°C for 20min. The Ni-NTA agarose column filled with the supernatant was incubated on a four-dimensional rotating mixer at 4°C for 2h, then washed with binding buffer, and eluted with elution buffer (10mM Na2HPO4, 10mM NaH2PO4, 500mM NaCl, 600mM imidazole, pH 7.4).
Generation of mAbs against Fiber-2 protein of DAdV-3
Purified His-fused Fiber-2 protein mixed with Freund’s complete adjuvant (Sigma-Aldrich, Missoula, MO, USA) was injected into 6-week-old BALB/c mice for the first immunization. For subsequent immunizations, the Freund’s complete adjuvant was instead by Freund’s incomplete adjuvant (Sigma-Aldrich). There is a two-week interval between the injection of Fiber-2 protein. During the interval, the antibody titers of the sera from the immunized mice were detected. When the antibody titers of the sera were high enough, the Fiber-2 protein without adjuvant was injected for the last time. After three days, the prepared SP2/0 cells were fused with the spleen cells of immunized mice by PEG1500 (Roche, Mannheim, Germany) as previously described (Wang et al. 2018). On the tenth day after the cell fusion, the supernatant of the hybridoma cells were detected using LMH cells infected with DAdV-3 through IFA. The positive hybridoma clones were subcloned, and the isotypes of the generated mAbs were determined with a mouse mAb isotyping kit (Thermo Scientific, Massachusetts, USA) according to the manufacturer’s instructions.
Indirect immunofluorescence assay
LMH cells infected with DAdV-3 or transfected with pcDNA3.1-Fiber-2 and its serial truncations were fixed with the fixative solution (The prechilled acetone and ethanol with the ratio of 3:2) for 5min. Then, the prepared corresponding mAbs diluted were incubated with the fixed LMH cells for 45min at 37°C. After three washes with PBS, the LMH cells were incubated with FITC-conjugated goat anti-mouse IgG (Sigma-Aldrich, USA) for another 45min. After three washes with PBS, the cells were observed using an inverted fluorescence microscope.
Western blot
LMH cells infected with DAdV-3 or transfected with pcDNA3.1-Fiber-2 were washed and lysed with RIPA buffer (CWbio, Beijing, China) containing phosphatase inhibitors and protease (CST, MA, USA). The lysates mixed with loading buffer were boiled for 10min and centrifuged for 3min. After SDS-PAGE, the denatured proteins were transferred onto nitrocellulose membrane (GE Healthcare Life sciences, Freiburg, Germany). After blocked with 5% skim milk in PBST for 2h at room temperature (RT), the membrane was incubated with the corresponding mAbs diluted with 5% skim milk in PBST for 2h at RT. After three washes with PBST, the membrane was incubated with HRP-conjugated goat anti-mouse IgG diluted with 5% skim milk in PBST for 1h. After another three washes, the membrane was developed with chemiluminescent reagents and imaged with an automatic imaging system (Tanon 5200).
Immunoprecipitation
The Thermo Scientific™ Pierce™ Crosslink Magnetic IP/Co-IP Kit was used for immunoprecipitation according to the manufacturer’s instructions. Firstly, the purified mAb was coupled to the protein A/G magnetic beads, and they were covalently crosslinked with disuccinimidyl suberate (DSS). The mAb-crosslinked beads were washed to remove mAb that was not covalently bound. After incubated with the lysate of LMH cells transfected with pcDNA3.1-Fiber-2 or infected with DAdV-3 for 1h, the prepared beads were washed to remove non-bound material and eluted in a low-pH elution buffer that dissociated the bound Fiber-2 protein from the antibody-crosslinked beads. The eluted low-pH sample, neutralized by neutralization buffer, was applied to Western blot analysis.
B cell epitope mapping
The recombinant plasmid pcDNA3.1-Fiber-2 and its serial truncations were constructed based on the linearized plasmid pcDNA3.1 and the corresponding fragments using ClonExpress II One Step Cloning kit (Vazyme, Nanjing, China). The corresponding primers were listed in Table 1. After sequenced to confirm the correctness, the plasmids with different truncations of Fiber-2 were transfected into LMH cells respectively. Then the transfected cells were fixed with fixative solution and incubated with corresponding mAbs and second antibody. The epitopes were finally identified by IFA.
Alignment of epitope sequences in Fiber-2 protein
To analyze the conservation of the four epitopes identified, the sequence alignment was performed using Clustal W program based on the software DNAStar (DNAStar Inc., Madison, WI, USA). The Fiber-2 protein sequences of DAdV-3, DAdV-4 and Fiber protein sequences of DAdV-1, DAdV-2 were downloaded from GenBank (http://www.ncbi.nlm.nih.gov). All of the downloaded sequences were put into DNAStar together for alignment.
mAb-based sandwich ELISA
To develop sandwich ELISA for detection of DAdV-3, mAb 3D9 was purified and HRP-conjugated as previously described (Wang et al. 2018). In the sandwich ELISA, the purified mAb 3D9 diluted in 0.1M carbonate buffer with the final concentration of 1.25μg/mL was coated into a 96-well ELISA plate (100μL/well) at 4°C overnight. The following morning, the 96-well ELISA plate was blocked with 360μL of 5% skim milk in PBST for 2h at 37°C. After the blocking and washes, the virus supernatants or samples diluted were added into the 96-well ELISA plate for 1h at 37°C and the 96-well ELISA plate was washed with PBST for three times. Then, the HRP-conjugated mAb 3D9, diluted by PBST with a final concentration of 0.625μg/mL, was added into the 96-well ELISA plate for 30min at 37°C. After washed with PBST for three time, the 96-well ELISA plate was added with 100μL TMB single-component substrate solution (Solarbio) per well and reacted for 15min at 37°C. Finally, 50μL of 2M H2SO4 per well was used to stop the chromogenic reaction and an ELISA reader was used to measure the absorbance values at 450nm.