Epitope Identification Of VvIBDV By Phage Display And Expression For Multi-epitope Simulated Albumin

Epitope, or antigenic determinant, is an antigen domain with affinity to antibody in Ag-Ab complex. Epitopes play a very important role in the structure and function of protein antigens. Precise epitope mapping may help design more reasonable vaccines and diagnostic reagents. A multi-epitope vaccine can provide protection against mutable virus because it may carry a broad spectrum of neutralizing epitopes. In addition, epitope-based vaccines can be easily distinguished from wild-type viruses, which facilitate the eradication of virial diseases. Phage-displayed peptide libraries technology provides a new approach to disclose protein structure and function, and has been used to map large number of epitopes.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 bilght susceptibility, increased feeding cost, drug residue, threated human health, causing economic loss. Recent years, molecular biology research of infectious bursal disease virus(IBDV) achieved great advance, but the study of immune and pathogenic mechanism,molecular structure and function of infectious bursal disease virus(IBDV) need further research. Thus, we had identified the epitope of infectious bursal disease virus(IBDV) which is valuable for further research such as the interaction between infectious bursal disease virus(IBDV) and its native host.The research divide two parts:1. Identification and Sequencing of Epitopes of Four IBDV-VP2 Monoclonal AntibodiesIn order to study mechanism of pathogenesis and immune, the epitope of VP2 of infectious bursal disease virus was screened by phage display. Four monoclonal antibodies to protein VP2 of infectious bursal disease virus (IBDV), 1B5,5D1,2H11 and I-4-4-3 were used to screen for binding peptides from peptide 12-mer phage display library. After three rounds of panning (absorption-elution-amplification), sixty positive monoclonal phages (fifteen for each monoclonal antibody) were selected and the phage displayed 12-peptides were detected and identified with indirect ELISA (A405 greater than negative control second times, A value>1.00) and competitive inhibition ELISA (inhibition rate>40%). The results indicated that 12-peptides contained epitopes of IBDV. Twenty positive monoclonal phages were sequenced, and these quences of nucleotides and amino acids of the four different epitopes on IBDV were determined and analyzed. Comparison with sequences of IBDV registered in GenBank, 1B5,5D1,2H11 and I-4-4-3 selected peptides had no more than three continuous amino acid residues similarity with the sequences of IBDV. The results indicated that the epitope 1B5,5D1,2H11 and I-4-4-3 were conformation-dependent.2. Expression and immunogenic analysis for the tandem arranged multiple mimic epitope gene of Infectious Bursal Disease VirusIn ordor to develop an effective caccine, four mimic epitopes of infectious bursal disease virus have been identified from a 12-mer phage-displayed peptide library by 4 monoclonal antibodies. Based on the sequences of the four epitopes, multiple epitope gene 4epis was constructed by the four epitopes being tandemly arranged and linked with 4-peptide GGGS. The expression plasmid pET-4epis was constructed and successfully expressed in E.coli. The resultant protein of 4epis was called r4EPIS. The results from SDS-PAGE anlysis showed that the proportion of r4EPIS was 20% of the total bacterial proteins and the molecular weight of r4EPIS was 30kDa. By use of parallel immunoblotting test with corresponding monoclonal and polyclonal antibodies, the immunological specificity and reactivity of r4EPIS against IBDV have been verified. Rabbits were subcutaneously injected with r4EPIS, twice with 7 days internal. The titers of the IBDV specific antibody measured by indirect ELISA were up to 1:212 at the 7th day after first immunization and 1:218 at the 14th day after the second immunization. To determine the protective ability of r4EPIS to the challenge of IBDV, chickens were injected intramuscularly with r4EPIS in adjuvant twice with 7 days internal and the resultant antibody titer was up to 1:214 at the 7th day after the second immunonization.After challenge with 200ELD50 of virulent IBDV GX8/99 strain, all the chicken in r4EPIS- immunized group were survived in contrast to the moutality of 80%(12/15) in adjuvant control group, suggesting that r4EPIS had a potent ability to generate protective immune response and it implied that the constructed gene 4epis is a prospective candidate for the development of epitope-based IBD vaccine.