Results
Phylogenetic analysis
The phylogenetic analysis confirms the highly conserve of PDCoV M
protein. From the 139 sequences (Table 1), we found 111 sequences with
100% of similarity. The 28 rest of the sequences show similarities
> 94% and also, different branch length (Fig 1a). These 28
different sequences were mainly from Asia (China, Japan, Thailand, and
Vietnam) and one sequence from USA, the USA/Minnesota292/2014. The
sequence CHzm2019 from China, showed the longest branch in the tree with
ten different amino acid (Fig 1b). Therefore, a protein M consensus
sequence was used due to the high similarity between the sequences
r M-PDCoV protein structure
We made a comparison to evaluate if the protein M consensus sequence has
a significant difference at a structural level, (Fig 2). We compare the
protein structure from USA/Minnesota292/2014, CHzmd2019, and HKU15 (the
first PDCoV isolated reported) and the consensus sequence protein M
structure obtained (C-Score values up to -4.6). Also, we include the
protein M structure from two different SCoV: PED CV777 isolate and TGE
Miller M6 isolate (Fig 2a and 2b). According to TM-Score, the protein
structure for CHzmd2019, HKU15 and the consensus sequence protein M has
a TM-Score value > 0.8, (Table 2). Also, we found nine
potential antigenic sites along the sequence and correlate with
hydrophilic sites, indicating that the antigenic sites are in the
surface of the protein structure, available to be recognized by the
immune system (Fig 2c and 2d).
Expression analysis of r M-PDCoV in E. coli
A synthetic gene (654 bp) based on the consensus sequence using in this
study, was acquired from a commercial supplier. The integrity and weight
were evaluated (fig. 3a). The expression was conducted in the E.
coli BL21 (DE3), using the vector pET-SUMO with a His-tag. After the
purification with Ni-NTA agarose His-tag affinity column, the identity
of the r M-PDCoV was confirmed by Western blotting with a specific
antibody anti -His. The band size was observed at an estimated
size of 37.7 kDa (fig.3b). Also, the r M-PDCoV was observed in the
insoluble phase (fig.3b), suggesting that r M-PDCoV are found as
inclusion bodies. After solubilization of inclusion bodies and
purification, recombinant proteins were verified from the elutions using
SDS-PAGE and western blot. We found the purified r M-PDCoV in
elution 3 to 5 and the molecular weight was according to expected,
~37.7 kDa (fig. 3c).
Immunodetection of r M-PDCoV in E. coli.
The immune reactivity of the r M-PDCoV protein with specificanti -PDcoV-IgG antibodies in 17 serum samples from pigs infected
by PDCoV from “El Bajio” Mexico, was confirmed by Western blot
analysis. The Western blot detected a band of approximately 37.7 kDa
consistent with the molecular weight of the M protein. The results show
eight positive sera: 130, P-1, P-3, P-4 and P-10-P13 (Fig. 4). No bands,
of the corresponding molecular size were detected with serum from
control non- infected pigs. Negative control serum was from a sow
keeping in laboratory.
Antibody response of BALBc/mice to immunization with r M-PDCoV
protein.
The capability of r M-PDCoV to stimulate the immune system to
produce antibodies was determined using three mice groups. The
antibodies production was evaluated by iELISA (Fig. 5). The antibody
response increased after the first dose given from day 7 lasting to the
end of the experiment (day 28) being significantly higher (P <
0.0005) compared to the PBS control group. Also, the higher antibody
production was observed in Group-2: r M-PDCOV with
immunostimulating complex (Iscom®) being significantly higher (P
< 0.0005) compared to the PBS control group.