Characterization of Goatpox virus major intracellular mature virion proteins A27L and P32 expressed by baculovirus system: Superior to Escherichia coli

Baculovirus expression system has become the most widely used eukaryotic system for recombinant protein production due to its ability to perform post-translational modifications and produce proteins with native-like conformation and antigenicity. In this study, two major immunodominant intracellular mature virion genes of Goatpox virus were amplified into three constructs: A27L, full-length P32, and truncated P32 (tr-P32), respectively. The constructs were cloned into the pFastBac HTA vector and expressed in Trichoplusiani (TN5) insect cells using the baculovirus expression system. Recombinant A27L was purified under native conditions, however full-length P32 and tr-P32 required denaturing conditions. tr-P32 showed markedly higher expression and solubility than full-length P32. All recombinant proteins were immunoreactive with hyperimmune GTPV sera, as confirmed by western blotting. Native PAGE analysis of rA27L revealed concentration-dependent oligomerization into dimers, trimers and tetramers. Furthermore, in indirect ELISA, both A27L and P32 exhibited strong reactivity with sera from GTPV and SPPV infected or vaccinated animals at 1:50 dilution and did not show any cross-reactivity with related viruses. These results quantitatively demonstrated that baculovirus-expressed A27L and P32 proteins are superior diagnostic antigens, providing higher sensitivity and specificity for Capripoxviruses serodiagnosis and offering potential as improved candidates for both serological and prophylactic applications.