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.