Development And Application Of Self-assembled IBDV Virus-like Particles

Infectious bursal disease virus(IBDV)infection by immature bursa tissue in B cells caused by acute death resistant chicken chicken;present immunosuppression,vaccination failure,increased susceptibility to other diseases,has brought huge economic losses to the poultry industry in the world.Currently,the most effective control of IBD is vaccination.However,live vaccines have the potential to be attenuated and to recombine with wild viruses,especially moderately virulent vaccines that cause direct damage to the central immune organs.Compared with the whole virus vaccine,virus-like particle vaccine does not contain viral nucleic acids,and has many advantages,such as safety and high efficiency.It is an important direction of vaccine development in the future.In this study,we obtained two recombinant IBDV virus-like particles by expressing IBDV-VP2 in yeast expression system(T=1 subunit virus-like particle,sVP & T=13 virus-like particle,VLP)?Through conditional optimization,a breakthrough in self assembling IBDV-sVP can be achieved without purification.A fermentation tank at a factory level produces a high level of expression of the target protein.The expression level can be up to 21 g/L and can be assembled into IBDV-sVP.The immunogenicity of two virus-like particles was evaluated and compared by animal immunity test.The results showed that the immunogenicity of IBDV-sVP was better than that of IBDV-VLP.Immunization experiments showed that the IBDV-sVP vaccine elicited high IBDV-neutralizing antibodies in each group,and all birds survived challenge with vvIBDV.Additionally,IBDV RNA was not detected,and sterile immunity was achieved,in the meanwhile.In the meantime,we explored the protective effect of oral immunization against IBDV-sVP yeasts.oral administration provides 90 % of the chickens survived,70 % of the bursal were protected,and IBDV RNA was not detected in the chickens that survived.This shows that the IBDV-sVP developed in this study has high vaccine value.Virus like particles are an effective tool for identification of viral cell receptors.The cellular receptor for IBDV has not yet been determined.In this study,the identification of vvIBDV cell receptor was carried out by using the above-mentioned IBDV virus-like particles.In this study,we obtained a large number of proteins which may interact with IBDV-Gx by VOPBA using IBDV-sVP and DT40 cell membrane protein and mass spectrometry analysis.After the screening by database KEGG and Ami GO 2,we designed siRNA to screen for 8 candidate proteins involved in IBDV infection.We finally chose epidermal growth factor receptor(EGFR)for follow-up study.Immunoprecipitation experiments showed that IBDV-Gx had a direct interaction with EGFR.Overexpression experiments showed that overexpression of EGFR on the surface of DT40 cells could promote IBDV-Gx replication,the titer of the virus has increased by 16 times.EGFR down regulation,anti EGFR antibody and soluble EGFR protein could significantly inhibit the replication of IBDV-Gx,and showed a dose-dependent manner.We also have shown that the DT40 cell surface EGFR protein was blocked by DT40 and the IBDV cells were significantly inhibited by invasion experiments.In summary,we determined that the EGFR protein is a receptor for IBDV-Gx.In summary,we determined that the EGFR protein is the cellular receptor component of vvIBDV-Gx in DT40 cells.In this study,an efficient and stable IBDV virus-like particle(IBDV-sVP)preparation process based on yeast fermentation system has been established,which is suitable for industrial production.The prepared IBDV-sVP has good immune activity,can resist 100 % vvIBDV attacks,and also has a lethal oral protection rate of 90 %.A new IBDV cell receptor component,EGFR,was also identified by using IBDV-sVP.This study is of great significance to the prevention and control of IBDV.