Generation of Three Recombinant D78 Infectious Bursal Disease Viruses and Purification with Immobilized Metal-ion Affinity Chromatography

• Main Authors: Sheng-Tse Liu, 劉聖哲
• Other Authors: Min-Ying Wang
• Format: Others
• Language: zh-TW
• Published: 2018
• Online Access: http://ndltd.ncl.edu.tw/handle/6hkms5

碩士 === 國立中興大學 === 生物科技學研究所 === 106 === Infectious bursal disease virus (IBDV) causes severe immunosuppression in 3-7 week-old young chickens by infecting B cells in the bursa of Fabricius and thus causes severe damages to their immune systems. The immunosuppressive diseases will enable the IBDV-infected chickens more susceptible to other infectious agents leading to death. The IBDV infection usually results in tremendous economic loss in poultry industry. Previous study reveals that the capsid protein VP2 of IBDV contains neutralizing epitopes of IBDV and plays an important role in virulence, as well as antigenic variation. Recently, we concluded that the exposed residue His253 of a VP2-formed subviral particle (SVP) is crucial for the binding affinity of SVP to Ni-NTA. In order to develop a method for the purification of IBDV using immobilized metal-ion affinity chromatography (IMAC), the recombinant D78 (rD78) IBDV with site-directed mutagenesis rD78-Q221H, -P222H, and -Q324H were generated and characterized. The mutant sites were calculated and predicted to be exposed at the surface of virions. Initially we compared the efficiency of IBDV replication levels in DF-1 cells and Vero cells by infecting IBDV at the multiplicities of infection (MOI) of 1, 0.1 and 0.01. Our results indicated that at 0.01 MOI the virus could be amplified up to 106 pfu/mL at 3 days post-infection (dpi) in DF-1 cells, and 105 pfu/mL at 6 dpi in Vero cells. Therefore, following we propagated recombinant viruses by infecting DF-1 cells with 0.01 MOI while the rD78 mutants couldn’t amplify as much as rD78-WT. The virus solutions were further purified by IMAC. The recombinant virus VP2 and VP3 proteins could be purified by Ni-NTA column, among all of them rD78-P222H is mostly centralized eluted at pH 4.0.