Expression Of IBDV Structural Protein VP2 Gene In Bombyx Mori And Its Immune Effect As Subunit Vaccine

Infectious bursal disease virus(IBDV),the causative agent of infectious bursal disease(IBD),causes immunosuppression in young chickens by destroying the precursors of B lympHocytes in the bursa of Fabricius.And enhanced any other blight susceptibility. The IBDV has beeen the cause of significant economic losses in poultry industry for a long time. Classic method of inactive vaccine and low-virulence vaccine have been confronted with the threat of variant strains and very virulent strains of IBDV isolated since 1980,s.The scale of Poultry industry in China is enlarging increasingly,however,the epidemic profile of IBDV is very complicated in China and only a few IBDV vaccines are acquirable.As an access to the contradietion,we made the program of expressing IBDV viral proteins with bombyx mori expression system.Bombyx mori industry is one superior industry in our country, the study should help us to make sure that whether silkworm is fit for expressing IBDV proteins as bioreactor and whether the VP2 protein is a more suitable antigen for subunit vaccine against IBDV.The major structure protein of infectious bursal disease virus,VP2 is the major protective of the virus and point mutations in the variant a rea located in VP2 always lead to antigen shift and sequenced circumvent of vaccination.According to published IBDV sequence in Genbank, a pair of primers was designed and synthesized, amplify VP2 gene and its outer region by RT-PCR.In this study a VP2 gene of IBDV was inserted into a Bombyx mori baculovirus transfer vector.PBKblue and cotransfected with linear genomic DNA of baculovirus Bm-Ex into BmN cells.Recombinant baculovirus(rBV) vBacPAK-IVP2 was then acquired after several rounds of purification and was used to infect BmN cells and the fifth-instars silkworm. DNA extracted from the infected cells and the infected larvae's haemolymph were examined with Agar diffusion method and Westernblotting ,respectively.It suggested that the expression product remains the characteristics of immunoreactivity and the expression achieved the highest level 6-day post inueolation.Expression amount of the protein in larvae was obviously higher than that in BmN cells,which indicated that silkworm larvae is a more efficient bioreactor in the case of target protein expression.TheVP2-contained silkworm pupa haemolymph harvested in 5-day and normal haemolymph were emulsionized respectively with mineral oil as vaccines.Chiekens were inoeulated subeutaneously with 0.6ml of VP2 protein vaccine for the examination of vaccine safety.There's no reaction among the chickens since the inoculation and no abnormality in the autopsy 5 days post inoculation.Chiekens were divided into 6 groups randomly for inoculation with emulsions prepared with VP2 respectively,as the primary at 28-day old,and the rest two groups were set for challenge control and normal control.Blood of the immunized chickens was collected periodieally for antibody titration until the chickens were challenged with virulent IBDV BC6/85 21 days post the boost.Chickens were killed 4 days post challenge and autopsied.Results indicated that the VP2 protein vaccine can resist the challenge of virulent IBDV(BC6/85strain) completely.The results showed that the VP2 protein containing IBDV haemolymph preparation of vaccine safety and good immunogenicity, and this study lay on foundation for the development of the new IBDV subunit vaccine.