The expression of the capsid protein VP2 from human bocavirus identified in Beijing and the formation of virus-like particles (VLPs) in insect cells.
- Author:
Lin-Qing ZHAO
1
;
Yuan QIAN
;
Ya-Xin DING
;
Ru-Nan ZHU
;
Jie DENG
;
Fang WANG
;
Yu SUN
;
Yan LI
Author Information
1. Laboratory of Virology, Capital Institute of Pediatrics, Beijing 100020, China.
- Publication Type:Journal Article
- MeSH:
Animals;
Blotting, Western;
Capsid Proteins;
genetics;
metabolism;
Cell Line;
Electrophoresis, Polyacrylamide Gel;
Fluorescent Antibody Technique, Indirect;
Human bocavirus;
genetics;
metabolism;
Microscopy, Electron, Transmission;
Polymerase Chain Reaction;
Spodoptera;
Virion;
genetics;
metabolism;
ultrastructure
- From:
Chinese Journal of Virology
2009;25(5):333-338
- CountryChina
- Language:Chinese
-
Abstract:
The aim of this study was to obtain the capsid protein VP2 of human bocavirus (HBoV) identified in Beijing recently and construct virus-like particles (VLPs) in insect cells for further study of this virus. The full-length VP2 gene of HBoV from BJ3722 was inserted into the baculovirus expression transfer vector (pFastBac 1) to obtain the recombinant Bacmid, and generation of recombinant baculoviruses was followed by transfection of the recombinant Bacmid into insect cells. Then the recombinant VP2 protein was recognized by SDS-PAGE using Coomassie-blue staining and Western blot using hyper-immune serum against VP2 of HBoV from rabbit. The recombinant baculoviruses were harvested and amplified to gain large amounts of viruses with high titers to infect insect cells at a multiplicity of infection (MOI) of 0. 5. After 7-10 days or 4-5 days of the infection, the supernatants of culture or the cell lysates treated with lysing solution were harvested, and ultracentrifuged twice through 40% sucrose cushion to obtain purified VLPs, which were followed by Western blot and IFA for VLPs' composition and specificity analysis, by electron microscopy for VLPs' morphologic structure. The recombinant VP2 protein with molecular weight of approximately 61 kD expressed in recombinant baculoviruses was recognized by SDS-PAGE using Coomassie-blue staining and Western blot. The presence of VP2 on VLPs was demonstrated by Western blot and IFA from samples collected during the purification of VLPs from the supernatants of culture or the cell lysates, and the expression of VP2 in insect cells led to the formation of VLPs which formed the typical icosahedral appearance of parvoviruses with a diameter of approximately 20 nm. In conclusion, the recombinant baculoviruses were constructed, the HBoV VP2 protein was expressed in insect cells with high specific antigenicity and VLPs was formed successfully.
