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A purification process was developed to obtain highly pure rVP2H particles, formed by a structural protein (VP2) of infectious bursal disease virus (IBDV) with additional six histidine residues at its C-terminus, for the study of its antigenicity and conformational structure and the development of an efficient subunit vaccine against IBDV infection. The expressed VLPs mimic th authentic structure of the critical epitopes in the native viruses and are capable of inducing a stronger immunological response than does the free form of rVP2H. Those particles in the inected High-FiveTM cell lysates were partially purifed employing immobilized metal-ion (Ni＋2) affinity chromatography (IMAC). This step recovered about 85 percent of immunogeric rVP2H proteins but did not separate the rVP2H particles from the free rVP2H proteins or the degraded products of rVP2H. A further separation of the particulate form of rVP2H from the free form of rVP2H was achieved using either gel filtration chromatography or CsCl density gradient ultracentrifugation. The latter method is suitable for the purification of small amount of rVP2H particles and the particle's buoyant density determine using this method was very close to 1.27 g/cm3. However, when large quantities of the rVP2H particles are needed, it is more efficient to purify the particles by using gel filtration chromatography than the conventional ultracentrifugation method. The separation of the particulate form from the free form of rVP2H proteins enables the evaluation of their own immunogenicity to induce the virus-meutralizing antibodies leading to the protection of chickens from IBDV infection. In addition, the abundant quantities of pure rVP2H particles coupled with the uniform dimensions of the particles will facilitate an examination of higher order structure of the immunogenic particles and, therefore, the design of better vaccines against the virus will evolve.
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