Please use this identifier to cite or link to this item:
標題: Separation of pure and immunoreactive virus-like particles using gel filtration chromatography following immobilized metal ion affinity chromatography
作者: Cheng, Y.S.
Lee, M.S.
Lai, S.Y.
Doong, S.R.
Wang, M.Y.
關鍵字: infectious bursal disease;insect cells;escherichia-coli;genomic;segment;expression;protein;purification;chickens;pathogenicity;birnavirus
Project: Biotechnology Progress
期刊/報告no:: Biotechnology Progress, Volume 17, Issue 2, Page(s) 318-325.
A purification process was developed to obtain highly pure rVP2H particles, formed by a structural protein (VP2) of the infectious bursal disease virus (IBDV) with six additional histidine residues at its C-terminus. The ultimate 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 from the free rVP2H proteins or its degraded products. To separate the particulate form from the free form of rVP2H, an additional step was added, which used either gel filtration chromatography or CsCl density gradient ultracentrifugation. 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 process a larger sample volume than does the latter. By integrating IMAC and gel filtration chromatography, 1 mg of extremely pure rVP2H particles was routinely 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 respective 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 facilitates an understanding of higher order structure of the immunogenic particles and can therefore result in improved vaccines against the virus.
ISSN: 8756-7938
DOI: 10.1021/bp000155a
Appears in Collections:生物科技學研究所

Show full item record

Google ScholarTM




Items in DSpace are protected by copyright, with all rights reserved, unless otherwise indicated.