First-strand cDNA was synthesized from purified RNAs of PAMs and 3D4/21 cells using a First-Strand Synthesis System (Transgen, Beijing) according to the manufacturer's instructions. The FcεRI gene was subsequently amplified using primers FcεRI-F and FcεRI-R, based on known sequences of FcεRI genes, as shown in Supplementary Table S1, and the amplified fragments were cloned into pGEM®-T Easy Vector (Transgen). The vector containing the FcεRI gene was sent to Genewiz (Beijing, China) for sequencing. Enzymes used for cloning procedures were purchased from TaKaRa (Dalian, China). A DNA thermal cycler (Biometra Tgradient, Germany) was used to perform the reaction. The FcεRI gene sequence was deposited at NCBI (GenBank Number: XM_001929139).
Table Table S1. The primers used for PCR amplification
The pcDNA3.1-FcεRI eukaryotic expression vector was constructed. Primers pcDNA3.1-FcεRI-F and pcDNA3.1- FcεRI-R (Supplementary Table S1), harboring common sequence with the vector (underlined), were used to amplify the FcεRI gene from the FcεRI clone plasmid. Through the common sequence, the PCR product was ligated with pcDNA3.1 vector by using a one-step cloning kit (Vazyme, China). The prokaryotic expression plasmid pGEX-6p-1 was constructed using primers pGEX-6pFcεRI-F and pGEX-6p-FcεRI-R, to express the recombinant protein GST-FcεRI according to the aforementioned protocol.
PAMs and PBMCs were collected from PRRSV-negative piglets from Tianjin Ninghe original pig farm according to a protocol described previously (Arce et al. 2010; Sinha et al. 2012; Uddin et al. 2012). The intestinal porcine epithelial cell line IPEC-J2 and the porcine kidney cell line PK-15 were purchased from the Chinese Veterinary Drugs Inspection Institute (Beijing, China). All applicable international, national and/or institutional guidelines for the care and use of animals were followed. Pigs were maintained in individually ventilated cages at the Tianjin Laboratory Animals Center.
PRRSV-permissive derivative 3D4/21 cells (PAMpCD163, obtained by transfecting the viral receptor CD163 into CRL2843 AKA 3D4/21 cells), were kindly contributed by Han Jun, professor of China Agricultural University (Lee et al. 2010). 3D4/21 cells were cultured in RPMI 1640 medium (Gibco, USA) supplemented with 10% (V/V) fetal bovine serum (FBS) plus 100 μg/mL penicillin and streptomycin, were used to inoculate and propagate PRRSV. Human embryonic kidney (HEK) 293T cells, IPEC-J2 and PK-15 were maintained in Dulbecco's modified eagle's medium (DMEM, Gibco) supplemented with 10% (V/V) FBS plus 100 μg/mL penicillin and streptomycin. All cells were cultured in a humidified incubator with 5% CO2 at 37 ℃.
PRRSV (JXwn06) were contributed from China Agricultural University. PRRSV was propagated in 3D4/21 cells, and the PRRSV titer was determined to be 106.54 TCID50/0.1 mL using the Reed-Muench method (Reed and Muench 1938). Viruses were frozen at - 80 ℃ before use.
PRRSV-specific IgG (IgG+) and specific-pathogen-free pig serum (PRRSV-negative IgG, marked as IgG-) were identified using an IDEXX HerdChek PRRS X3 Ab enzyme-linked immunosorbent assay (ELISA) (Westbrook, MA), and purified antibodies were further precipitated by using saturated ammonium sulfate to yield a protein content of 8.160 and 7.92 mg/mL, respectively. Anti-b-actin mouse monoclonal antibody and goat anti-mouse IgG (H+L) horseradish peroxidase conjugate were purchased from TIANGEN (Beijing, China).
The polyclonal antibody against FcεRI was derived from serum of rabbit immunized with extracellular recombinant GST-FcεRI proteins coupled with mineral oil adjuvant as previously described (Zhang et al. 2016).
The ELISA plate (Bio-Rad, USA) was coated with the recombinant FcεRI protein at 4 ℃ overnight, and then blocked with 5% horse serum (Gibco, USA). After washing three times with PBS, the diluted FcεRI antiserum (with the dilution ratios of 1:50, 1:100, 1:200, 1:400, 1:800, 1:1600, 1:3200, 1:6400, 1:12, 800 and 1:25, 600) was added to the corresponding well and incubated at 37 ℃ for 1 h. Subsequently, the plates were washed with PBS and incubated with enzyme-labeled secondary antibody Goat-Anti-Rabbit-HRP (CWBIO, Beijing) diluted 1:5000. Then add freshly prepared OPD substrate coloring solution and stop solution to the corresponding well. Absorbance was measured at 490 nm using a microplate reader (Thermo Fisher, USA) within 15 min after termination.
The immunofluorescence assay was used to detect FcεRI expression. HEK 293T Cells were transiently transfected with 1 μg plasmid of pcDNA3.1-FcεRI or pcDNA3.1 for 24 h, The transfected cells were fixed and the rabbit anti-FcεRI polyclonal antibody or rabbit FcεRInegative antibody (from PBS-immunized rabbit) were added to react at 37 ℃ for 30 min, and then the goat antirabbit FITC conjugate (Abcam, UK) was added at 1000-fold dilution for 30 min, and the nucleus was stained with 40, 6-Diamidino-2-Phenylindole (DAPI). The localization of FcεRI were observed under an inverted confocal microscope (PerkingElmer, UltraView Vox, USA). Images were taken at a ×100 magnification.
The purified IgG+ or IgG- with the concentration of 6.25 μg/mL was mixed with 1 MOI of PRRSV, and incubated on ice for 1 h to promote the formation of virusantibody immune complexes.
The 3D4/21 cells were seeded into 6-well plates and inoculated with 1 MOI of PRRSV, 1 MOI of PRRSV and 200 μL negative IgG (PRRSV + IgG-), 1 MOI of PRRSV and 200 μL positive IgG (PRRSV + IgG+) or phosphatebuffered saline (PBS) respectively at 4 ℃ for 1 h. After 24 h, cells were harvested and the FcRs mRNA expression level were analyzed by quantitative reverse transcription PCR (RT-qPCR).
The 3D4/21 cells or HEK 293T were transfected with pcDNA3.1-FcεRI or pcDNA3.1 (1 μg) with 5 μL PEI (Head, Beijing) respectively following the manufacturer's instructions. After 24 h, the layer cells were respectively inoculated with PRRSV, PRRSV + IgG-, PRRSV + IgG+ or PBS at the previous dose. After 24 h, the cells were harvested and subjected to analyze the PRRSV RNA load level and the expression level of inflammatory- related genes by RT-qPCR.
Total cellular RNA was extracted from cells using TRIzol, and complementary DNAs (cDNAs) were synthesized using a First-Strand Synthesis System (Transgen, Beijing) according to the manufacturer's instructions.
The amplification reaction contained 2 × Multiplex PCR Mix (10 μL), 10 mmol/L forward and reverse primers, 2 μL of the cDNA template, and sterile double-distilled water to a final volume of 20 μL. All RT-qPCRs were performed under the following conditions: 95 ℃ for 5 min followed by 40 cycles of 95 ℃ for 30 s and 56 ℃ for 30 s. SYBR Green Master Mix (Vazyme) was used according to the manufacturer's instructions, and the real-time PCR was performed on a 7500 Real-time PCR system (Applied Biosystems, Foster city, CA, USA). All data presented were quantified relative to the mRNA level of the endogenous gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and analyzed using GraphPad Prism 6.0 software (La Jolla, CA 92037 USA). The primer pairs used for RT-qPCR are shown in Supplementary Table S2.
Table Table S2. The primers used for RT-qPCR detection
HEK 293T cells transfected with pcDNA3.1-FcεRI or pcDNA3.1 (1 μg) with 5 μL PEI (Head, Beijing) respectively following the manufacturer's instructions. Twentyfour hours after transfection, cells were washed twice with PBS and harvested in RIPA buffer. Cell lysates were heated in buffer for 10 min and separated by SDS-PAGE. The separated proteins were transferred onto a nitrocellulose fitter membrane (NC) (PALL, USA), and then membranes were blocked with PBST (1 × PBS in 0.05% Tween 20) and 5% skim milk for 1 h at room temperature. The membranes were then stained with primary antibodies at 4 ℃ overnight. The membranes were washed thrice with PBST for 3–5 min in each interval in order to remove excessive antibodies, and then incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies at room temperature for 2 h. To detect the bioactivity of FcεRI polyclonal antibody, the primary antibodies are GST monoclonal and FcεRI polyclonal antibody. To detect the FcεRI expression in transfected HEK 293T cells, the primary antibodies are the Myc antibody (TransGen, Beijing) (1:5000) and β-actin antibody (TransGen, Beijing) (1:5000).
Signals were visualized using Pierce ECL Western Bloting Substrate (Thermo Scientific), and images were obtained using a chemiluminescence apparatus (Gel Doc XR + imaging system; Bio-Rad, USA).
One million 3D4/21 monolayer cells were inoculated respectively with 1 MOI of PRRSV, 1 MOI of PRRSV and 200 μL negative IgG (PRRSV + IgG-), 1 MOI of PRRSV and 200 μL positive IgG (PRRSV + IgG+) or PBS. The cells were collected and washed with PBS after 24 h of inoculation, resuspended in 1 mL TRIzol LS (Invitrogen, USA) and transferred to cryopreservation tubes. Transcript mRNA was obtained by using oligo (dT)18 enrichment mRNA that was fragmented, purified, and subjected to mass detection. A terminal sequence was ligated, and the cDNA was amplified by reverse transcription before using a machine for high-throughput sequencing. All samples were sent to Guangzhou GENE DENOVO (Guangzhou, China) for transcriptome sequencing.
The above experiments were carried out with three independent replicates, and the data were analyzed using the 2-ΔΔCT method. Data were expressed as mean ± SD. Pairwise multiple comparisons were conducted to determine differences between groups by two-way analysis of variance (ANOVA) followed by Bonferroni post-tests using GraphPad Prism 6.0 (GraphPad Software Inc.). P-values < 0.05 were considered statistically significant.