| Rabbit hemorrhagic disease (RHD) is a highly contagious and fatal disease by Rabbit hemorrhagic disease virus (RHDV). It is associated with necrosis in hepatic cells, congestion and bleeding in parenchymatous organs and high mortality, which caused serious economical losses in rabbitry. The phage display technology is a technology for screening specific ligand from phage display random peptide library though the Specific bingding between ligand and acceptor. It is widely used to select antigen epitopes, detect diseases, study vaccines and select polypeptide drugs. The biopanning is mainly used in the research of selecting antigen epitopes. The specific phage clones are enriched effectively after three to four rounds of biopanning, and antigen epitopes are got.In order to get the epitope of RHDV, the McAb A3c against RHDV developed by the laboratory had been used as solid-phase selective molecule to screen by phage display technology. In order to get a new method to detect RHDV, the indirect ELISA to detect the antibodies against RHDV had been developed with phage clone got from biopanning at the basis of result of biopanning. The study of antigen epitope will provide theoretical basis for studying the molecular biology of RHDV and new vaccines. The development of indirect ELISA will supply a favorable tool to evaluate immunity effect in rabbitry and survey epidemiology.The details of content and result were as following:1. Screening of antigen mimotopes of RHDV. McAb A3c hybridoma cell strain against RHDV in the laboratory was recovered and the ascites antibody was prepared. The purity, concentration, titer and the affinity constant of the antibody were determined after purifying. Using highly pure and active McAb as solid-phase selective molecule, the biopanning had been done by phage display technology. The phage clones selected were determined and sequenced to get highly homogenous sequence comparing to antigen, which is the antigen epitope. The results showed that the purity of McAb A3c was more than80%, the ELISA titer was1:81920and the affinity constant was3.5×107after purifying. The purified McAb can be used as solid-phase selective molecule. After three rounds of biopanning, the specific phages were enriched effectively. The sandwich ELISA showed that19out of25phage clones were positive and the competitive ELISA further suggested that the positive phage clones can specifically bind to the monoclonal antibody. The sequence analysis of the positive clones showed that highly homogenous sequence (GTDDMDPGTTAA) comparing to antigen was got, which is the antigen mimotopes of RHDV. The sequence DXXDP was the core amino acids in epitope.2. The indirect enzyme-linked immunosorbent assay (iELISA) method was developed to detect rabbit antibody against RHDV, using the phage clone Kl got from biopanning as antigen. The reaction conditions of ELISA were optimized; the specificity, sensitivity, repetitiveness and retention period of ELISA plate coated were detected; and100serums of rabbit were detected by the method. The result showed that the best concentration of phage was1.56×107pfu/ml and the dilution of the serum sample was1:200;5%BSA was the best confining liquid; the dilution of HRP-goat anti-rabbit IgG was1:5000; the reaction time of confining liquid, antigen and antibody, HRP-goat anti-rabbit IgG and substrate were90min,30min,30min and10min separately. Antibodies against RHDV in100serums of rabbit were detected by the method and the positive samples were86(86%), which were73(73%) by the method of RHDV VP60-indirect ELISA developed by the laboratory. The concordant rate between the two methods was85%. Thus, indirect ELISA developed in the study was specific, sensitive, reproducible and easily operate in detection, which indicated that this method was appropriate for application in clinical diagnose. |
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