Prokaryotic expression, purification and evaluation of goatpox virus ORF117 protein as a diagnostic antigen in indirect ELISA to detect goatpox

摘要

Goatpox is an economically significant transboundary viral disease of goats that is caused by goatpox virus (GTPV). This study describes the prokaryotic expression of the GTPV ORF117 protein, a homologue of vaccinia virus A27L, and evaluation of its diagnostic potential in ELISA. The GTPV ORF117 gene was cloned into the pET32a vector to express recombinant ORF117 protein (rA27L) in E. coli BL21-CodonPlus (DE3)-RIPL. The bacterial expression of the protein was confirmed by western blot analysis using anti-GTPV polyclonal antibodies that detected rA27L, which is similar to 35 kDa in size. rA27L was affinity purified under native conditions and used to assess the antibody response in an optimized indirect ELISA. The purified antigen specifically reacted with anti-GTPV and anti-SPPV serum in ELISA. A preliminary screening of random and purposive serum samples (n = 520) from sheep and goats using this optimized ELISA gave a positivity rate of 19.4 % with a diagnostic specificity of 88.7% and diagnostic sensitivity of 98.5% when compared to the gold standard serum neutralization test. Our results suggest that the indirect ELISA based on the rA27L protein has potential for serosurveillance and seromonitoring of GTPV in goats.