Abstract
Porcine circovirus associated disease (PCVAD) is a prevalent porcine disease. Porcine circovirus type II (PCV2) is the major etiologic pathogen and causes tremendous economic loss. Although the capsid (Cap) protein-based subunit vaccines have been commercialized, the high price of vaccination entails the need for effective and inexpensive vaccines. We have produced PCV2 Cap protein as the vaccine antigen using baculovirus/insect cell expression system and proved the antigen could elicit protective immune response against PCV2 infection in swine. Here we aimed to develop a cost-effective process to extract and purify PCV2 Cap-based vaccine. First, we attempted to use various detergents and salt solutions as extraction buffers, which however resulted in poor extraction efficiency. Therefore, we employed physical methods including sonication and high pressure homogenization in the process and elevated the extraction efficiency significantly. The optimal extraction process involved the use of phosphate-buffered saline and 3 cycles of freeze and thaw, followed by extraction of the Cap protein into 20 mM sodium phosphate with high pressure homogenizer. This process enabled the extraction of Cap protein at an efficiency exceeding 70% and a purity of 38%. Immunization of porcine with the extracted Cap proteins were able to elicit significant immune effects. We further developed a purification process based on cation exchange chromatography. By changing the salt concentrations and pH values of binding and eluting solutions, the purity of Cap protein was increased to higher than 90%. The purified Cap protein self-assembled into virus-like particles, as confirmed by electron microscopy. In summary, we developed an efficient and cost-effective process for the extraction and purification of Cap proteins as the potential PCV2 vaccine.