Characterization of Particles Formed by Infectious Bursal Disease Virus
Structural Precursor Protein, VPX, in Hi-5 Cells Using Immobilized
Metal-ion Affinity Chromatography and Size-Exclusion Chromatography
Meng-Shiou Lee1, Shyue-Ru Doong3, Su-yuan Lai2, and Min-Ying Wang1*,
1
2
Graduate Institute of Biotechnology, National Chung Hsing University, Taichung,
Taiwan 40227 ROC
Department of Food Science, Chungtai Institute of Health Sciences and Technology,
#11, Pu-tzu Lane, Pei-tun District, Taichung, Taiwan, 40605 ROC
3
Department of Chemical and Materials Engineering, National Central University,
No. 38, Wu-chuan Li, Chung-li, Tao-yuan, Taiwan 320, R.O.C.
*To whom correspondence should be addressed
Mailing Address: Graduate Institute of Biotechnology,
National Chung Hsing University, Taichung, 40227 Taiwan
Telephone: 886-4-2285-6697
Fax: 886-4-2285-6697, 886-4-2285-3527
E-mail address: mywang@dragon.nchu.edu.tw
Running title: Expression, Purification and Characterization of Infectious Bursal
Disease Virus Structural Precursor Protein, VPX
Abstract
The precursor protein (VPX) of infectious bursal disease virus (IBDV) host
immunogen VP2 protein was expressed in insect cells, including Sf9 and Hi-5 cells,
to examine its regenerated particle types and the immunogenicity induced by those
particles. When expressed in Hi-5 cells, although some degraded products appeared,
the recombinant protein expression level was much higher than that in Sf9 cells. Since
the expressed protein, rVPXH, was engineered a His-tag, consisting of six histidine
residues, at the C-terminal end, it was purified using immobilized metal-ion affinity
chromatography (IMAC). Following IMAC, particles produced in the infected Hi-5
cells were examined and unexpectedly at least three architectures of particles were
observed. The morphologies of particles included twisted tubular structure, 20-25 nm
particles and isometric particle structure. The IMAC partial purified particles were
further separated using a gel-filtration column fitted to an HPLC system to analyze
their composition. Results indicate that the expressed rVPXH protein formed
isometric particle structure and very few of twisted tubular structure, while
dodecahedral particles were formed by the degraded proteins of rVPXH protein.
Previously, when VPX was alone expressed in Sf9 cells, it has been shown that VPX
could either formed the isometric particle structure or tubular structure. In this study,
we further demonstrated that its partial degraded product could form small virus-like
particles.
Based on our result, we hypothes that VPX protein formed a loose
structure to interact with other proteins (VP3 or VP4) and VP1-RNA complex to
assemble into immature IBDV virion. Then, VPX in the immature IBDV was
processed to VP2 protein and complete the formation of the mature IBDV. However,
once it was processed (or degraded to VP2), the VP2 protein will self-assemble to
rigid small virus-like particles, which was difficult to interact with other proteins or
factor to form 60 nm of IBDV capsids. Finally, we also demonstrated that when
susceptible chickens were vaccinated with the IMAC-purified rVPXH protein (20
g/bird), virus-neutralizing antibodies were induced. This indicated that those
particles are highly immunogenic and might be an alternative vaccine candidate for
the development of IBDV subunit vaccine.
Key words: infectious bursal disease virus; virus-like particle; baculovirus expression
system;
immobilized
chromatography.
metal-ion
affinity
chromatography;
gel-filtration