Figure legends
Figure 1. (a) Maximum likelihood phylogenetic tree displaying the 138 PDCoV protein M sequences available in Gen Bank. In blue, the consensus sequences used in this study. In red, 18 sequences showed differences in branch length. (b) Comparison of 18 sequences showed differences at amino acid level.
Figure 2 . Comparison of five models predicted by I-TASSER, antigenic sites and surface properties. (a) Predicted models for Consensus sequence, Chzmd2019, HKU15, M-PED CV777 and M-TGE Miller M6. (b) Amino acid sequence for each model indicated the predicted secondary structure, red: helix, blue: strands, black: coil.(c) Nine Antigenic sites predicted and their positions along the consensus sequence. (d) Consensus sequence surface properties indicating the hydrophobic and hydrophilic sites.
Figure 3 . (a) Integrity and weight verification of PDCoV-M 654 bp synthetic gene (b) Western blot and SDS-PAGE to verified r M-PDCoV in the expression essay. Lines 1 to 3 are an aliquot of the culture media before cell disruption. Line 4 is an aliquot after cell disruption. Line 5 and 6 are an aliquot after centrifuging the cultures, soluble and insoluble phase, respectively. The r M-PDCoV was found in insoluble phase as inclusion bodies.(c) Confirmation of r M-PDCoV after purification with Ni-NTA agarose column with His-tag affinity. The purified recombinant protein was observed in elutions 5 to 6.
Figure 4 . Western blot to determinate the capability ofr M-PDCoV to recognized antibodies in 17 pig blood sera from “El Bajio” pig farm. Eight positives sera were determined and are indicate in the image. Negative control serum was from a sow keeping in laboratory
Figure 5. anti -PDCoV IgG distribution in sera samples obtained from three groups of eight mice each. Black dots line indicates PBS control group. White dots line indicates r M-PDCoV protein group. Triangle line indicates rM-PDCoV + Iscom as an adjuvant to enhance the immune response. Dots and triangle indicated the days of collection sera: 7, 14, 21, and 28 Bars indicated the standard error. The statistical significance P < 0.01 (**) and P < 0.001 (***).