Generation Of Monoclonal Antibody Against IBDV Nucleocapsid Protein VP3 And Establishment Of A Stable Cell Line Expressing Shrna Targeting VP3

Infectious bursal disease(IBD)is a highly contagious,acute,immunosuppressive disease of chicken,which is caused by the infectious bursal disease virus(IBDV).Recent years,vaccination is the only effective way to prevent this disease.Because of the appearance of new very virulent and gene variant strains of IBDV,the effect of the vaccination was greatly reduced,resulting in considerable economic losses to the poultry industry.VP3 is the structural nucleocapsid protein of IBDV,and plays an important role in virus genome replication,assembly of viral particles.And the inhibition of the innate immunity.In order to provide tools and the cell model for the studies on the role of VP3 in the replication of IBDV,the prokaryotic expression vector expressing His-tagged VP3 was constructed and the VP3 was then expressed in E.coli.After purification via His affinity column,the purified His-VP3 was used to immunize BALB/c mice.After a series of rounds of selections and ELISA-based screening,two hybridoma strains producing monoclonal antibodies against VP3 were successfully obtained.The secreted monoclonal antibodies were named mab-VP3-1 and mab-VP3-2,both of which reacted specifically with VP3 of IBDV.The ascites titers were 1: 32000 and 1: 64000,respectively.A number of shRNAs targeting various regions of VP3 gene were designed and synthesized by using bioinformatics software according to the conserved VP3 gene coding region sequence of different IBDV strains.The effect of each shRNA on silencing of VP3 expression in 293 T cells was evaluated via mab-VP3-1 mediated Western blot analysis,and the most effective shRNA in silencing of VP3 was cloned into the pLVX-IRES-Puro lentiviral vectors.Then the recombinant lentiviral vectors and viral packaging helper plasmids were co-transfected into HEK293 T cells for lentiviral packaging.The DF-1cells were infected with packaged lentiviral.After puromycin selection for 14 days,a DF-1 cell line stably expressing shRNA was obtained.Western blot assay and virus plaque neutralization test showed IBDV susceptibility to this stable cell line expressing shRNA against VP3 was significantly decreased.Results:(1)Two strains of mouse monoclonal antibodies against VP3 protein were obtained.(2)VP3 gene's RNA interference lentiviral expression vector was constructed.(3)The DF-1 cell line with stable interference of VP3 gene was obtained.(4)The stable cell lines had more significant resistance toviral protein expression and virus replication.Conclusion: Two hybridoma strains secreting monoclonal antibodies against IBDV VP3 were generated and a stable VP3 knockdown DF-1 cell line was established using lentiviral vector-mediated RNA interference.The will provide as a useful tool and a cell model for exploring the new insights into IBDV-mediated disease mechanisms.