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

[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 Chen^1,2

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

TABLE OF CONTENTS

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

1. ABSTRACT

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.