ESTABLISHMENT OF A BACULOVIRUS EXPRESSION SYSTEM FOR PRODUCTION OF THE PORCINE CIRCOVIRUS TYPE 3 (PCV3) VIRUS-LIKE PARTICLES

Abstract

Intro: Porcine circovirus type 3 (PCV3) is a newly emerging porcine circovirus that has been reported to be associated with porcine dermatitis and nephropathy syndrome (PDNS) and has affected pig populations worldwide. Porcine circoviruses contain two major open reading frames (ORFs) and the ORF2 encodes the sole viral capsid protein (Cap) which is considered as the most antigenic structure protein to be a potential candidate for diagnostic reagent and vaccine development. In addition, the PCV3 cap protein with the morphological structure of virus-like particles (VLPs) is similar to that of natural virus particles, does not contain viral genomes, and has strong immunogenicity and biological activity. The proteins produced by the E. coli expression system are often misfolded, whereas the baculovirus expression system usually produces proteins with properly folded VLP structures. Methods: 1. Immunofluorescence assay was for localizing recombinant proteins. 2. High-performance liquid chromatography was for purifying recombinant proteins. 3. Transmission electron microscope was for observing the structure of VLP. 4. Immunogold staining was for confirming the antigenicity of VLP. Findings: In this study, we produce the original sequence PCV3 Cap (ori- PCV3Cap) and the codon-optimized sequence of the PCV3 Cap (opti-PCV3Cap), respectively. Both recombinant proteins can be successfully produced in the baculovirus expression system, but the yield of opti-PCV3Cap was slightly higher than that of ori-PCV3Cap. Both expressed proteins were further observed to assemble into 17-20 nm particles under transmission electron microscopy and could be recognized by the monoclonal antibody specific to PCV3 Cap in immunogold staining. Furthermore, mutation of the PCV3 Cap within the putative nucleolar localization sequence (NoLS) resulted in reducing protein accumulation in the nucleus and increasing protein extraction efficiency. Conclusion: All the results indicate that opti-PCV3Cap with the putative NoLS mutation had higher protein yield in the baculovirus expression system and formed a VLP structure as a candidate for PCV3 vaccine.