Identification Of Infectious Bursal Disease Virus Variant Strain And Immunogenicity Analysis Of VP2 Protein Expressed In Different Expression Systems

Infectious bursal disease(IBD),also called as Gumboro disease,is a highly contagious epidemic disease of young chickens caused by infectious bursal disease virus(IBDV),which causes great economic loss for the poultry industry all over the world.It can cause immunosuppression because IBDV replicates in the bursa of Fabricius.Recently,a new variant strain occurred,leading to bursal atrophy and severe immunosuppression.Because IBDV is composed of two relatively independent segments,the recombination of different strains is greatly possibility.With the continuous prevalence of very virulent strains and the emergence of mutants,the recombinant viruses could be a great challenge to the prevention and control of infectious bursal disease.In this study,two novel IBDVs were isolated and identified.VP2 protein was expressed by prokaryotic expression system and baculovirus expression system respectively.SPF chickens were immunized with the expressed product roughly purified to verify its immune effects.1 Isolation and identification of infectious bursal disease virus variant strainIn this study,two strains of IBDV were isolated by inoculating chicken embryos with the clinical samples showed severe bursal atrophy.The virus could proliferate on chicken embryos.The VP1 and VP2 genes of a strain of IBDV were amplified and sequenced by using IBDV specific primers.Whole sequence of another virus was amplified and sequenced.The results showed that one of the two isolates was a novel recombinant virus and the other was a novel variant.Phylogenetic tree and amino acid sequence analysis by informatics software of the novel recombinant virus showed that VP2 gene belonged to the branch of very virulent strain,while VP1 gene belonged to the branch of attenuated strain.Amino acid sequence analysis showed that the amino acid encoded by VP2 gene of the isolate had the unique amino acid sequence of the very virulent strain,and had the highest homology with the very virulent strain(99%-99.4%).The amino acid encoded by VP1 gene had the unique amino acid sequence of the attenuated strain,and had the highest homology with the attenuated strain(99.3%-99.8%).The novel variant strain of isolations belonged to branch of novel variant,and had the highest homology with the novel variant strain(96.2%-98.2%).The amino acid of the isolated strain had the unique amino acid sequence of the novel variant.The isolation and identification of the recombinant virus and novel variant virus provide a reference for the epidemic trend and prevention and control of IBD.2 Expression of VP2 gene of IBDV in different expression systemsThe prevention and control of IBDV mainly depends on vaccine immunization.In this study,VP2 protein was expressed by prokaryotic expression system and baculovirus expression system,respectively.VP2 gene was cloned into prokaryotic expression vector pET-28a and baculovirus expression plasmid,and prokaryotic expression vector pET-28a-VP2 and recombinant baculovirus re-Bac-1-GP67-VP2 were constructed.VP2 protein was successfully expressed in both expression systems.Prokaryotic expression system was mainly expressed in inclusion body,and the expression amount accounted for about 33%of the total protein.Baculovirus expression system was secreted into the culture supernatant in soluble form,and the expression amount accounted for about 10%of the total protein.After optimizing the expression conditions,the expressed protein can react with monoclonal antibody to VP2.Our results provides the basis for the subsequent immunogenicity test.3 Comparison of immunogenicity of two VP2 recombinant proteinsThe vaccine was prepared by the expressed recombinant proteins which were crudely purified,and mixed with adjuvant.SPF chickens were immunized these vaccines.The serum antibody titers were measured and challenged at 21 days after immunization.The changes of bursa of Fabricius were observed by autopsy and histopathology.The shedding virus and viral load in tissues were titration in real time RT-PCR.The results showed that the immune effect of recombinant protein expressed by baculovirus was better than that expressed by Escherichia coli.The pathological degree of bursa of Fabricius was lower.The level of serological antibody was higher than that of prokaryotic expression product.The viral load and shedding were lower than that of prokaryotic expression product.Twice immunization is better than that of once immunization.These results provide reference data for the development of IBD subunit vaccine.