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.