Expression Of Infectious Bursal Disease Virus VP2 Protein In Vitro And Evaluation Of Its Immunoprotective Effect

Objective:The eukaryotic expression system of Pchia pastoris was used to express the VP2 protein gene of infectious bursal disease virus(IBDV)in vitro.The immunogenicity and protective effect were evaluated by animal immunoprotection test.Methods:The gene sequence of infectious bursal disease virus VP2 protein was obtained from the NCBI gene database.Xho I and EcoR I enzyme cut sites and 6His tag gene sequences were added to the ends of the sequences,and the sequence of VP2 protein was optimized according to the preference of X33 Pichia pastoris codon.The recombinant expression vector pwPICZa-VP2 containing VP2 protein gene was constructed by gene recombination technique and introduced into Pichia pastoris X33 to get transgenic Pichia pastoris.VP2 proteins were expressed and identified by Coomassie brilliant blue staining and Western blot.In the animal immune protection test,the experimental chicken were divided into six groups and immunized by VP2 protein with the concentration of 10 ?g,40 ?g,160 ?g,10 ?g(adjuvan),40 ?g(adjuvan)and 160 ?g(adjuvan)respectively.The B87 vaccine was used as the positive control and the PBS as the negative control.The effects of VP2 protein on cellular immunity and humoral immunity were investigated by peripheral blood lymphocyte proliferation and virus antibody detection The protective effect of VP2 protein was evaluated by using BC6/85 virus strain to infect chickenResults:Coomassie brilliant blue staining,Western blot and deglycosylation analysis showed that VP2 protein was successfully expressed in vitro,and the proliferation of peripheral blood lymphocytes showed that VP2 protein had no effect on cellular immunity.The results of virus antibody detection showed that there was no significant difference between VP2 protein and PBS group.There was no significant difference in the antibody level between the 40 ?g(adjuvant)and the 160 ?g(adjuvant)group compared with the B87 vaccine group,and the antibody level in the 10 ?g(adjuvant)group was significantly lower than that in the B87 vaccine group(P<0.05).Pathological anatomy of bursa of Fabricius showed that there were lesions in bursa of Fabricius in VP2 protein test groupConclusion:The VP2 protein of infectious bursal disease virus was successfully expressed in vitro and was modified by N-glycosidic ligation.The chicken immunized with adjuvant(ISA206)VP2 protein produced a high level of IBDV antibody.The lesions of the bursa of Fabricius,showed that the IBDV antibody produced,failed to resist the attack of BC6/85 strain.In future,advance research design and follow-up of experiments are needed to find out a solution to this problem.