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

Anand S. Kushwaha,Poulinlu Golmei,Amit Kumar,B. Mondal,G. Venkatesan

Published 2025 in Journal of Virological Methods

ABSTRACT

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.

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