[Frontiers in Bioscience E3, 856-863, June 1, 2011]

Use of hr3 enhancer and P74 TM domain in baculovirus surface display

Yingying Liu1, Hui Feng1, Qian Feng1, Junlin Teng1, Zhiyin Wu1, Yiyu Lu3, Albert CH Yu4, Jianguo Chen1,2

1 The Key Laboratory of Cell Proliferation and Differentiation of Ministry of Education and The State Key Laboratory of Bio-membrane and Membrane Bio-engineering, College of Life Sciences, Peking University, Beijing 100871, P. R. China, 2The Center for Theoretical Biology, Peking University, Beijing 100871, P. R. China, 3Institute of Virology, Zhejiang Provincial Center For Disease Prevention and Control, Hangzhou, Zhejiang 310009, P. R. China, 4 Neuroscience Research Institute and Infectious Disease Center, Health Science Center, Peking University, Beijing 100191, P.R.China


1. Abstract
2. Introduction
3. Materials and methods
3.1. Materials
3.2. Cloning of Pgp64, Pvp39, hr3 and P74TM
3.3. Construction of the display vectors
3.4. Construction of HA gene displaying vector
3.5. Generation of the recombinant baculovirus
3.6. Purification of BVs
3.7. Purification of ODV viruses
3.8. Separation of the ODV components
3.9. Measurement of the recombinant baculovirus titer
3.10. Western blot
3.11. Luciferase assay
3.12. Immunization
3.13. Neutralization assays
4. Results
4.1. Comparison of baculovirus displaying efficiency
4.2. Surface display of HA protein
4.3. Immune effect of rBVs displaying HA protein
4.4. The application of P74TM in ODV display
5. Discussion
6. Acknowledgment
7. References


Baculovirus surface display technique provides a new platform for novel vaccine research and production. Unfortunately, the low display efficiency in current methods and the waste of occlusion-derived virion (ODV) products limited its application. We investigated the use of two motifs, BmNPV hr3 and transmembrane domain of P74 (P74TM), in display. Budding virus (BV) with hr3 showed a 14.2-time enhanced display efficiency than the current recombinant BV. Hemagglutinin (HA) protein of H5N1 influenza virus was displayed by using 3 different vectors to ensure the improvement of display efficiency and the characters of displayed protein in recombinant baculovirus. Immunoassay demonstrated that the recombinant BV with TM/CTD of vsvG protein, and hr3 could induce the highest level of neutralizing antibody against HA, suggesting that the optimized HA displaying BV could be a novel live virus-based vaccine candidate for influenza virus. In addition, GFP fused with the P74TM could be anchored to the ring zone in infected cell and the ODV envelope, which may luminate a new direction for ODV display and provide a promising strategy to use ODV products.