Expressing Infectious bursal disease virus vaccine by coupling Bamboo mosaic virus vector and plant transgenesis

• Main Authors: Chin-Ya Yuan, 袁琴雅
• Other Authors: Yau-Heiu Hsu
• Format: Others
• Language: zh-TW
• Published: 2014
• Online Access: http://ndltd.ncl.edu.tw/handle/16764590416841798137

碩士 === 國立中興大學 === 生物科技學研究所 === 102 === Infectious bursal disease virus (IBDV) is an acute and highly contagious virus which is main cause of infectious bursal disease. It primarily occurrs in 3-6 weeks chickens and seriously affects the economy loss of poultry industry. Currently, inactivated and attenuated vaccines are two main strategies for chicken IBD prevention. In recent years, the safer and low-cost vaccines are produced by using plants as a platform. In this study, we constructed Bamboo mosaic virus (BaMV), Tobacco mosaic virus (TMV), Foxtail mosaic virus (FoMV), and Potato virus X (PVX) as plant viral vectors. The coat protein genes of viral vectors are replaced with IBDV VP2 gene and fused with His-tag in the C-terminal, to generate BaMVdC-VP2His, TMVdC-VP2His, FoMVdC-VP2His, and PVXdC-VP2His. To test the VP2 production, these four recombinant viruses were individually transfected into Nicotiana benthamiana leaves by agro-infiltration. The results showed that among four viral vectors, BaMV vector expressed the most high-level accumulation of VP2 protein. In order to stably express VP2 protein in plants, we generated BaMVdC-VP2His transgenic N. benthamiana plants by Agrobacterium- mediated transformation. Western blot analysis confirmed the VP2 protein could be expressed in homologous transgenic lines. The virus like particles (VLP) of VP2 approximately 20-30 nm in diameter were purified and observed by transmission electron microscopy. Taking into account the environmental safety and high expression of VP2His protein, different gene silencing suppressors replace BaMV TGB or co-expression with BaMVdC-VP2His. Furthermore, complementation coat protein function of BaMVdC-VP2His by co-expression of BaMV CP. The results indicate VP2 protein dose not significantly increase. To develop edible vaccine, the transgenic barley (Hordeum vulgare) were generated. The results showed that BaMVdC-VP2His transgenic barley accumulated low VP2 protein. However, we have successfully established tissue culture and plant regeneration system in barley. We will use VP2-VLP to immuning chicken and to test the neutralizing antibodies production. We hope in the future, to develop subunit vaccine against IBDV by the BaMV based vector system.