Cloning, expression, and characterization of potential immunogenic recombinant porcine deltacoronavirus (PDCoV) membrane protein from a synthetic gene.

Abstract

Porcine Deltacoronavirus (PDCoV) is an emergent swine coronavirus that infects epithelia cells from the small intestine, and inducing watery diarrhea, vomiting and dehydration. Clinical signs are more aggressive in piglets causing high mortality rates (>40%) representing serious economic losses. Despite the importance of PDCoV as an emerging coronavirus, little is known about the currently prevalence in México. We select from GenBank a group of 138 sequences and obtained a consensus PDCoV membrane protein (M-PDCoV) sequence of 216 a.a. A Maximum Likelihood phylogenetic tree was constructed and evaluate the relationship between the 138 sequences. Also, a protein tertiary structure analysis was performed to analyze and compare the topological differences. The phylogeny and the tertiary structure analysis showed that M-PDCoV is highly conserved and therefore suitable to use as an antigen in a diagnostic system. Hence, an expression system performed in E. coli BL21 (DE3) using the vector pET-SUMO with a His-tag was prepared, resulting in a synthetic M gene of 654 pb to produce a recombinant M-PDCoV protein ( rM-PDCoV). Western blot test confirmed the rM-PDCoV immune detection in 8 of 17 sera samples. The rM-PDCoV was able to successfully stimulate immune response in immunized mice to produce antibodies after day 7 of immunization (P < 0.001). Our results show that rM-PDCoV is suitable to use in diagnostic systems like an ELISA.