Separation of pure and immunoreactive virus-like particles using gel filtration chromatography following immobilized metal ion affinity chromatography

A purification process was developed to obtain highly pure rVP2H particles, formed by a structural protein (VP2) of the infectious bursal disease viru s (IBDV) with six additional histidine residues at its C-terminus. The ulti mate goal was the development of an efficient subunit vaccine against IBDV infection. The particles within the infected High-Five (Hi-5) cell lysates were partially purified by employing immobilized metal ion (Ni2+) affinity chromatography (IMAC). The initial step could recover approximately 85% of immunoreactive rVP2H proteins but failed to separate the rVP2H particles fr om the free rVP2H proteins or its degraded products. To separate the partic ulate form from the free form of rVP2H, an additional step was added, which used either gel filtration chromatography or CsCl density gradient ultrace ntrifugation. Both were able to produce extremely pure rVP2H particles with a buoyant density close to 1.27 g/cm(3). However, the former method can pr ocess a larger sample volume than does the latter. By integrating IMAC and gel filtration chromatography, 1 mg of extremely pure rVP2H particles was r outinely obtained from a 500 mt Hi-5 cell culture broth. The separation of the particulate form from the free form of rVP2H proteins exposes their res pective immunogenicity to induce the virus-neutralizing antibodies and the ability to protect chickens from IBDV infection. Additionally, the abundant quantities of pure rVP2H particles coupled with their uniform dimensions f acilitates an understanding of higher order structure of the immunogenic pa rticles and can therefore result in improved vaccines against the virus.