Production Of Porcine Circovirus Type 2 Capsid Protein In Different Expression Systems

Porcine circovirus type 2 (PCV2) is an invasive pathogen, which causes a series of related diseases in pigs (PCVAD) and significant economic losses to the worldwide swine industry. PCV2 capsid protein (Cap) is an important protective antigen, and its N terminal sequence including 41 amino acids is dentified to be the nuclear localization signal sequence (NLS). Studies have shown that the truncated Cap (tCap) with the deletion of its NLS sequence has the same immunogenicity to Cap, thus has the potential applications in developing a PCV2 subunit vaccine. In this work, we used three expression systems, including Escherichia coli, Pichia pastoris and baculovirus to produce tCap and further investigated the immunogenicity of produced tCap. Our work provided aternaltive strategies to produce the protective antigen tCap, which faciliates to develop PCV2 subunit vaccines.First, tCap was produced via E. coli-based expression system. Codon optimized opti-tcap and tcap were inserted into pET28a(+) expression vector before transformating into E. coli expression strains BL21. Codon optimization significantly increased the soluble expression of active tCap. And the purified tCap showed good immunogenicity with the title of 1:8 via double agar diffusion assay.Secondly, tCap was produced via Pichia-based expression system. Codon optimized opti-tcapl and tcap were respectively inserted into pPIC9K expression vector. Recombinant strains with the expression cassate integrated into the GS115 in high-copy number were selected and fermented in 5 L bioreactor, the production of tCap in supernatant reached 0.25 g/L with the titer of 1:1 via in vitro assay. Serum antibody tests demonstrated that tCap induced significant increase of specific PCV2-Cap antibody over time in mice and even similar antibody level in piglets compared with a commercial Cap-based subunit vaccine.Finally, tCap was produced via baculovirus expression system, tcap was inserted into the transfer vector of pFastBacHTB, and then switched into bacmid by transposition. The recombinant baculovirus vector bacmid-tcap was transfected into Sf9 cells to harvest the virus, and the P3 virus were used to infect cells to produce tCap. The harvested tCap demonstrated good immunogenicity with the titer of 1:1 via in vitro assay.