Development And Application Of The Method For IBDV Concentration And Purification With ELP-Hsp90α Fusion Protein

Infectious bursal disease (IBD) is a highly contagious disease in chickens of 3-6 week old which causes severe immunosuppression, vaccination failure, increased susceptibility to other pathogens and heavy losses to poultry industry worldwide. Infectious bursal disease virus (IBDV) is classified within the family of Birnaviridae and the viral particles is stable to heat, pH3-9 and chloroform treatment due to the lack of envelope. IBDV can be concentrated with chloroform and PEG, and purified by sucrose or cesium chloride density gradient centrifugation and gel filtration, which are time-consuming and expensive with low recovery. By using the specific interaction between viruses and the receptors, virus receptor-binding capture technique can capture viruses from tissue and environmental samples with receptor-conjugated solid phases, which is simple, quick and economical. In addition, the technique has been used successfully to monitor virus contamination in environment. By replacing the solid phase with elastin-like polypeptide (ELP), our lab has established more simple and economical method for concentration and purification adenoviruses. Previous study has shown that heat shock protein 90a (Hsp90a) is the component of IBDV receptor and its C-terminal 444 amino acid region (C444) contained the binding domain of IBDV. In this study, we defined further IBDV-binding region of C444 and established a quick method for IBDV purification using the ELP fusion protein.The C444 region of Hsp90α was divided into three overlapping segments of 283-449aa, 356-606aa and 597-728aa. Each of the three coding sequences was amplified by PCR and fused in frame with ELP sequence in prokaryotic expression vector. The resultant vectors pELP-283-449, pELP-356-606 and pELP-597-728 were transformed individually into BLR (DE3) E. coli and the expression of fusion proteins was induced at 20℃ with IPTG, which were purified to high purities by ELP-mediated inverse transition cycling (ITC). Each of three fusion proteins was incubated with IBDV and the virus-fusion protein complexes were recovered by ITC. RT-PCR analysis showed that ELP-283-449 protein, but not ELP-356-606 and ELP-597-728 proteins, was able to bind IBDV. IBDV was incubated with different concentrations of ELP-283-449 protein for different times at different temperatures and pH, and the virus-protein complex was recovered by ITC. Viral titration assay showed the best conditions for IBDV recovery were incubation with 160μg/mL of ELP-283-449 protein for 1 h at 16 ℃, pH7.0. The fusion protein-bound IBDV was eluted for different times at different temperatures and pH. Viral titration assay showed the best conditions for IBDV elution were incubation for 30 min at 16℃ and pH9.0. IBDV was recovered from the culture medium of infected cells and cell lysate under the optimized conditions with recoveries of 75.4% and 71.7% after ITC or 39.3% and 42.5% after elution, respectively. Different doses of IBDV were seeded into tap water or PBS and the virus was recovered successfully with a recovery of 73% or 68%. These experimental data suggest that the IBDV binding domain of Hsp90a is located within the middle region of 283-449 amino acids and the ELP fusion protein can been used to concentrate and purify IBDV from tissue and environmental samples.