Materials and Methods
In Silico Analysis of M-PDCoV
A total of 138 PDCoV M protein sequence available in GenBank were obtained. The 138 sequences were from China, Laos, Japan, Vietnam, Thailand, and USA dated from 2012 to 2019 (Table 1). To determine phylogenetic relationships among the 138 sequences, a maximum likelihood phylogenetic tree using RaxML v8.0 was made (Stamatakis, 2014). The sequences were aligned using Clustal Omega online server (Sievers et al., 2011). RaxML was running using a HIVb amino acid substitution model (Nickle et al., 2007) calculated by ProtTest software (Abascal et al., 2005). 1,000 bootstraps were evaluated to assess support values. Also, from the 138 PDCoV M protein sequences available in GenBank we obtained a consensus sequence (217 a.a.) using Jalview software (Clamp et al., 2004). The potential antigenicity sites were predicted whit Jameson‐Wolf method using Protean, DNAStar v17.2 (Plasterer, 2000). The hydrophobic and hydrophilic sites were predicted with Kyte and Doolittle and Emini et al. methods using Protean, DNAStar v17.2 (Emini et al., 1985; Kyte & Doolittle, 1982). Additionally, predicted tertiary structure for the consensus sequence, HKU15 (GenBank: JQ065042) and CHzmd2019 (GenBank: MN781985) were determined using I-TASSER (Roy et al., 2010). Also, we include the M protein predicted tertiary structure of the PEDV CV777 (GenBank: AAK38659) and TGEV Miller M6 (GenBank: ABG89300) for comparison. The predicted tertiary structures were selected according to a suitable C-Score value (range of -5 to 2) where higher value signifies a model with a high confidence (Yang et al., 2015; Zhang, 2008). The selected tertiary structures were visualized using PyMOL Molecular Graphics System, Version 2.0 Schrödinger, LLC software. To assess the topological similarities, we determinate the template modeling score (TM-Score). TM-Score ranged from 0 to1, where 1 indicates a perfect match between two structures (Zhang & Skolnick, 2004) (Table 2). Finally, the amino acid identity was determined using the Sequence Manipulation Suite (SMS) on line (Stothard, 2000).
Molecular cloning of M-PDCoV in pETSUMO vector.
Since PDCoV is an emergent disease, it was not possible to collect a PDCoV sample from a pig farm. Therefore, a synthetic gene (654 bp) from the consensus amino acid sequence was obtained from a commercial supplier and was verified with the following primer sequences: Fw 5’-GACGCAGAAGAGTGGCAAATTATT-3’ and Rv 5’-GCGCTACTACATATACTTATACAGGCG-3. The synthetic gene was cloned in pETSUMO vector and positive recombinant plasmids were transformed into E. coli BL21 (DE3). To perform a competent cell, MgCl2 and CaCl2 protocol was used. Transformants were selected in LB agar plates supplemented with kanamycin, 50 µg/ml. The positive colonies were propagated and used to purify the plasmid DNA, using Wizard® Plus SV Miniprepr kit (Promega). The clones were confirmed by PCR using the primers sequences mentioned above. Also, a PCR using the above forward primer sequence a the reverse T7 sequence primer 5’-TAGTTATTGCTCAGCGGTGG-3’ was made to confirm the right direction of the inserted synthetic gene with the vector expression promoter.
Expression of the recombinant M-PDCoV in E. coli BL21 (DE3)
A transformant colony was inoculated into 3 ml of LB supplemented with kanamycin (50 µg/ml) medium at 37 °C, 250 rpm, 12 h as a pre-culture. Then, 100 ml of LB medium was inoculated starting 0.1 OD600 nm. The medium was incubated at 37 °C, 250 rpm until the culture reached the 0.5 OD600 nm. Then, the expression of recombinant M-PDCoV (r M-PDCoV) protein was induced by addition of Isopropyl β-D-1-thiogalactopyranoside, (IPTG) (Merck, Germany) at a final concentration of 1.5 mM followed by incubation over night at 37°C.
r M-PDCoV purification.
The induced medium was centrifuged (12,000 g for 10 min), the pellets were resuspended 400 ml of 0.1 M Tris-HCl pH 8.0 buffer. The cells were disrupted mechanically with a cell disrupter (GAULIN) at 550 kg/cm2, 15 min. The insoluble phase was separated by centrifugation at 15,000 g, 20 min, 4 °C. Then, the inclusion bodies from the insoluble phase, were solubilized into union buffer (7% N-Lauroylsarcosine sodium salt, Tris-HCl 50 mM pH 8) with agitation 250 rpm, 12 h, 25°C. After the inclusion bodies solubilization, the recombinant proteins were purified using Ni-NTA agarose column with His-tag affinity (Amersham), 1.5 ml of resin packed into a vertical column. The column was equilibrated with 5 CV of binding buffer (Tris-HCl 50 mM pH 8). Then, 2 CV of solubilized inclusion bodies (2 mg/ml) were loaded. Other proteins will be eliminated with 5 CV of wash buffer (Tris-HCl 50 mM pH 8, 30 mM imidazole) and the recombinant proteins were recovered using 4 CV of elution buffer (Tris-HCl 50 mM pH 8, 500 mM imidazole).
SDS-PAGE, Western blot analysis
The purified recombinant protein was separated by 12% SDS-PAGE and confirmed by Western blot. The recombinants proteins were transferred in to polyvinylidene fluoride membrane (PVDF) and blocked with 5% non-fat milk in PBS-Tween buffer (20 mM Tris-HCl, pH 8, 0.15 M NaCl, 0.05% Tween 20) at 4°C for 16 h with moderate agitation. Membrane was washed with PBS-Tween buffer and incubated with primary antibodies. To identify the r M-PDCoV an anti-his conjugated by horseradish peroxidase (HRP, Invitrogen) diluted 1:5000 was used and anti-pig IgG+HRP as secondary antibody diluted 1:5000. To visualize the protein bands in western blot, 10 ml development solution (PBS, 12 mg of 3,3’-Diaminobenzidine Tetrahydrochloride, 300 µl 3.4% H2O2) was used. Finally, the protein concentration will determinate according to Bradford (Bradford, 1976) using bovine albumin as a standard.
Antigenicity assay of r M-PDCoV in swine serum samples
Western blot assay to determinate the recognition by immunodetection ofr M-PDCoV with 17 swine sera from “El Bajio”, Mexico, pig farm. The sera came from pigs affected by lethargy, watery diarrhea, vomiting and dehydration. The Sera were randomly named:
102, 105, 130, 136, and P1-P13 and were diluted 1:1000, the methodology used was the previously mentioned. Negative control serum was from a sow keeping in laboratory
BALBc mice Immunization
The evaluation of antibodies anti-r M-PDCoV production was made using three experimental groups. Each group was performed by eight BALBc mice 28 days old. The immunization of Group-1 consisted in 5 µg/mouse ofr M-PDCOV protein diluted in PBS buffer, 1X pH 7.4. Group-2, 5 µg/mouse of r M-PDCOV protein and 5 µg/mouse of using immunostimulating complex, Iscom® (ISCONOVA AB, MATRIX Q, Uppsala, Sweden) as an adjuvant. Group-3, 200 µL de PBS buffer as a negative control. The final volume dose was 200 µL, and two doses were applied to each mouse subcutaneously at day one and at day 14, respectively. The blood sample were collected through the caudal vein at days 7, 14, 21, and 28. The IgG antibodies production were evaluated by indirect ELISA (iELISA). The mice were euthanized using a CO2 chamber. The IgG antibodies production were evaluated by indirect ELISA. The animals were handled at the house facility at National Microbiology Research Centre (CENID-SAI), Instituto Nacional de Investigaciones Forestales, Agrícolas y Pecuarias (INIFAP), (NOM-062-ZOO-1999; SAGARPA).
iELISA for antibody detection
An indirect enzyme-linked immunoassay (iELISA) was performed to determine the presence on IgG in mice sera. The 96 wells plates (microwell plate Batch 011606) were coated with 50 ng of r M-PDCoV in 0.05 mol/l carbonates buffer pH 9.6 (Sigma-Aldrich, USA) at 4°C and blocked with 5% skimmed milk at 37°C for 1 hr. After washing four times with PBS buffer, pH 7.4, Tween 0.1%, 100 µl of serum and negative control (Group-3), diluted 1:150, was added to the wells in triplicate and incubated at 37°C for 2 hr. The plates were washed four times and incubated with 100 µl of HRP-conjugated rabbit anti-mouse IgG diluted (Merck KGaA, Darmstadt, Germany) 1:7000 in PBS pH 7.4, Tween 0.1%, with 5% skimmed milk at 37°C for 2 hr. After adding 100 µl of a 3,3′,5,5′-Tetramethylbenzidine (TMB) substrate solution an incubating at room temperature for 10 min, the reaction was stopped by adding 100 µl of 2N H2SO4, and the absorbance at 450 nm was measured. The result was graphed using SigmaPlot software.