Abstract
Porcine circovirus type 2 (PCV2) is the primary causative agent of porcine circovirus-associated disease (PCVAD). Many of the PCVAD syndromes result from the coinfection of PCV2 virus with other agents including mycoplasma. PCV2 infection is present in every major swine-producing country in the world, and the number of identified cases of PCVAD is rapidly increasing. The tremendous economic loss underscores the importance of developing cost-effective vaccines. The PCV2 ORF2 encodes the major capsid protein (Cap), which is also the main antigenic determinant of the virus. Therefore the primary objective of this study was to improve the quality and quantity of Cap protein produced from the baculovirus/insect cells system. We constructed multiple Cap-expressing recombinant baculoviruses using different vector systems: Bac-ORF2 using Bac-to-Bac® system, BacF-ORF2 using the flashBACTM GOLD system and BacU-ORF2 using the flashBACTM ULTRA system. For all 3 viruses, High-5 cells resulted in a significantly higher Cap yield than Sf-9 cells in intracellular space. The Cap protein yield in High-5 cells infected with BacU-ORF2 (123.2 mg/L) was 5-fold higher than that in the cells infected with Bac-ORF2 (25.6 mg/L). In addition, High-5 cells infected with BacU-ORF2 resulted in significantly alleviated protein degradation. The Cap protein was further purified by Cytoplasmic/nuclear fraction protein extraction and sucrose gradient ultracentrifugation, and the purity of the sample was greater than 90%. The formation of virus-like particles(VLP) was visualized by transmission electron microscopy(TEM), the size and morphology of VLPs appeared indistinguishable from those of PCV2 virus. These data implicate the potential of BacU-ORF2 for future Cap protein production, purification and application as a vaccine.