Prokaryotic Expression And Immunogenic Analysis For The Tandem Arranged Multiple Epitope Gene Of IBDV-VP2

Infectious bursal disease virus (IBDV), the causative agent of infectious bursal disease (IBD), is one of the major infectious diseases against the world's poultry industry. It causes immunosuppression in chickens by destroying the precursors of B lymphocytes in the bursa of Fabricius and enhances any other pestilence susceptibility. 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.Epitope, or antigenic determinant, is the material basis of protein antigenicity, can stimulate the body to produce antibodies or sensitized lymphocytes and have the ability to combine with them. It plays a very important role in the structure and function of protein antigens. So precise epitope mapping may help design more reasonable vaccines and diagnostic reagents. Now the field of preventive medicine on the basis of epitopes, such as the epitope vaccine and diagnostic reagents and other research have become a hot spot. At the same time, a multi-epitope vaccine can provide protection against mutable virus because it may carry a broad spectrum of neutralizing epitopes. The subject of a multi-epitope vaccine as a starting point, were studied by the molecular design of the IBDV and the function of expression products in artificial protection test. The result shows that the research ideas of multi-epitope vaccine not only provide a new protective agent, but also will establish the foundation for the study of molecular immunology and genetically engineering vaccine.The research was divided into two parts: 1. Prokaryotic Expression and Immunogenic Analysis for the Tandem Arranged Multiple Epitope Gene of IBDVIn our laboratory, four mimic epitopes of infectious bursal disease virus have been identified from a 12-mer phage-displayed peptide library by 4 monoclonal antibodies in earlier research. 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 BL21(DE3)pLysS. The resultant protein of 4epis was called r4EPIS. The results from SDS-PAGE anlysis showed that the protein of the r4EPIS was procured expression and existed by inclusion body. After washing the inclusion body, purifying the protein, the purity of the r4EPIS was above 90%, and the molecular weight of r4EPIS was about 30kDa. The high titer anti-r4EPIS serum was also prepared in SPF chicken immunized with purified r4EPIS. ELISA results showed that the valence of antibody was above 1: 10240 and western blot analysis showed that the expressed protein r4EPIS reacted with monoclonal antibody. It explained that the protein r4EPIS was expressed and had satisfactory immunogenicity. It establish the foundation of immune mechanism and genetic engineering subunit vaccine of virulent IBDV for the study2. Artificial protection experiment for multi-epitope protein of IBDV-VP2In our research, one hundred of sixty SPF chickens were selected in 7 days old and divided 8 groups randomly. Average of the each group was 20. We tested according to the following groups: negative control, IBDV vaccine, FCA control, expression protein, VP2 antigen, 4EPIS+adjuvant (1:2), 4EPIS+adjuvant (1:1) and 4EPIS+adjuvant (2:1). The first immunization time was about 14 days age, 21-day in the second immunization, control group received salt solution. After 7 days, means 28 day-old of chicken, all groups were challenged with vvIBDV, then we got the samples (thymus, bursa, spleen, serum, etc) at the different challenge time: before and 3 days, 7 days, 14 days later. At the same time, we evaluated the index of immune organ, percentage of ANAE+ lymphocytes, activity of serum superoxide dismutase and the valence of antibody. The results showed: 200ELD50 virulent IBDV infected chicken from every group, the group of 4EPIS+adjuvant (2:1) gained the highest protection rate, above 90%. So we think that the multi-epitope vaccine, which combined the protein r4EPIS with the immune adjuvant, can promote the development of immune organs and prevent immune organ to damage of IBDV effectively. At the same time, the vaccine also can maintain high antibody levels, increase the percentage of ANAE+ lymphocytes and the SOD activity significantly, etc. At the end, we draw a conclusion that the r4EPIS can induce protection against IBDV infection and indicate the 4epis as a candidate gene of IBD multi-epitope vaccine.