Screening Mimic Epitopes Of McAb Against IBDV By Phage Display12Peptide Library

Infectious bursal disease virus (IBDV) is the causative agent of infectious bursaldisease(IBD) and results in a highly contagious and immunosuppressive disease.IBDV mainly do damage to the young chicken about3-12weeks, it also can lead tothe death of the infections chicken. Due to the systemic immunosuppression of thesurvival of chicken, they have a weakened immune system and easy to infected withother disease. IBDV is one of the three epidemics against the poultry industry anddistribution worldwide. There is no magic drug to treat the disease for the present, it isinstant to explore the IBDV efficient vaccine. Establishing the epitopes of the IBDVwill contribute to the epitope vaccine. IBDV contains a double-stranded RNA genomeincluding two segments(A and B), A fragment encoding poly protein of VP2-4-3andVP5. VP2and VP3are the major structural protein; VP4is the protease of virus; VP5can only be detected in IBDV-infected cells. B fragment encoding VP1protein, it isthe RNA-dependent polymerase. So far, multi-epitope sequence on VP2and VP3isfound.In this study, we carried out bio-panning of peptides targeted by threemonoclonal antibodies (H53、 V1and V38) to IBDV by phage display peptide library,which were saved in our laboratory, picked out the phage strains specially bindingwith the monoclonal antibodies. Then screened out the phage strains competitive withthe monoclonal antibodies. Finally the sequences of the phage strains were sequencedand analyzed. The specific operation is as follows:First,3strain cells (H53、V1and V38) which were saved in the laboratory wereresuscitated and cultured. The titers in the supernatant of cells, H53、V1and V38, inELISA were1:256,1:64and1:128respectively.8-weeks Balb/c mice were injectedwith the cells to preparate ascites. The results of analysis revealed that the titers ofMcAbs, H53, V1and V38in ascites by ELISA were1:2×10~5,1:3×10~4and1:1×10~5respectively. The sub-class of H53、V1and V38were identificated as IgG2b. Themonoclonal antibodies were purificated with a saturated ammonium sulfate method.A light chain and a heavy chain were detected in the picture of SDS-PAGE. After thegel analysis software to scan the protein band, the purity of the three monoclonalantibodies were99.5%,98.6%and99.1%respectively. The titers of indirect ELISAreached up to1.6×10~4, which indicated that the purificated monoclonal antibodieshad high purity and activity. It was ensure that the pro-screening process and the specificity of phage binding. Secondly, the screening of mimic epitopes for threestrains of monoclonal antibody by phage display12peptide library was carried out, inwhich the bingding by reducing the packet concentration and increasing the elutiontime was improved. Thirty clones were randomly picked and identified by indirectELISA; in terms of these clones, there were twenty two clones binding antibodieswith higher affinity. Fourteen phage clones were obtained from the indirectcompetitive ELISA, and their inhibittion rates reached up to50%. Finally, sequenceanalysis of these positive phages by DNAStar and the common sequences weredetermined.In conclusion, we screened three mimic epitopes by phage display12peptidelibrary successfully in this shudy. The mimic epitope identify H53wasXILRXXXXXXHM, V1could recognize the mimic epitope of RXLRLXXPIXQX,and XRXXRXXRRPRP was the mimic epitope of V38(X was any amino acid). Thesemimic epitopes provide bases for research of IBDV pathological mechanism and theeffective epitope vaccine, as well as an important tool for the prevention and controlof IBD.