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.