Expression and immunogenic analysis for the tandem-arranged multiple mimic epitope gene of Infectious Bursal Disease Virus

Abstract:

Five mimic epitopes of Infectious bursal disease virus (IBDV) have been identified from a 12-mer phage-displayed peptide library by 5 monoclonal antibodies. Based on the sequences of the five epitopes, multiple epitope gene 5epis was constructed by the five epitopes being tandemly arranged and linked with 4-peptide GGGS. The expression plasmid pET-5epis was constructed and successfully expressed in E. coli. The resultant protein of 5epis was called r5EPIS.The results from SDS-PAGE analysis showed that the proportion of r5EPIS was 15% of the total bacterial proteins and the molecular weight of r5EPIS was 10kDa. By use of parallel immunoblotting test with corresponding monoclonal and polyclonal antibodies, the immunological specificity and reactivity of r5EPIS against IBDV have been verified. Rabbits were subcutaneously injected with r5EPIS (400μg per injection), twice with 7 days interval. The titers of the IBDV-specific antibody measured by indirect ELISA were up to 1∶4000 at the 7th day after first immunization and 1∶256000 at the 14th day after the second immunization. To determine the protective ability of r5EPIS to the challenge of IBDV, chickens were injected intramuscularly with r5EPIS in adjuvant twice with 7 days interval (50μg per injection) and the resultant antibody titer was up to 1∶12800 at the 7^th day after the second immunonization. After challenge with 200ELD₅₀ of virulent IBDV GX8/99 strain, all the chicken in r5EPIS-immunized group were survived in contrast to the mortality of 86.7%(13/15) in adjuvant control group, suggesting that r5EPIS had a potent ability to generate protective immune response and it implied that the constructed gene 5epis is a prospective candidate for the development of epitope-based IBD vaccine.

Key words:Infectious bursal disease virus （IBDV）; Epitope; Expression; Immunogenicity