Establishment Of Latex Agglutination Test And Preparation Monoclonal Antibody Based On Surface Antigen D15 Of Riemerella Anatipestifer Feng Bin (Preventive Veterinary Medicine)

So far, limited further studies on tDl5 protein of Riemerella anatipestifer has been reported yet. In the present experiment, bioinformatics analysis, clone and prokaryotic expression, recombinant protein purification and polyclonal antibodies preparation of the RA D15 gene (accession no. CP003787) which was registered to NCBI Gene Bank by our lab, development of monoclonal antibodies against recombinant tD15 of RA and establishment of latex agglutination test using the tD15 antigen to detect antibody of Riemerella anatipestifer. The results are as follows:1. Bioinformatics analysis of D15 gene sequence from Riemerella anatipestifer. The structure, antigenicity, motif and rare codon of D15 gene and its encoding protein were comprehensively analysed by bioinformatics. The results showed its encoding protein D15 had two conserved domains. Moreover, the nucleotide sequence and amino acid sequence of RA D15 had higher homology with related homologous protein of Flavobacterium than others. RA D15 contained one signal peptide and one transmembrane domain and can target to outer membrane of cell.It had abundant epitopes and most of them located in N terminus of the polypeptide. In addition, D15 gene had no more contiguous rare codon.2. Prokaryotic expression and polyclonal antibody preparation of RA D15 N-terminal gene. On this part, the gene was cloned by PCR, and the pET-28a(+) prokaryotic expression system, which was used to express the protein encoded by the partial ORF. The recombinant proteins about 34 kDa were produced after induction.Western blot analysis proved that fusion proteins could react with anti-RA polyclonal antibodies. The fusion proteins pET28-tD15 were purified and used to immunize the rabbits for preparing polyclonal antibodies. The antibodies titer was 1:16. And the result of western blot proved that the antibodies could react with the native protein of RA.3. Establishment of latex agglutination test to detect antibody of Riemerella anatipestifer. There are very limited studies on the establishment of latex agglutination test to detect antibody of Riemerella anatipestifer. To establish a rapid detection of Riemerella anatipestifer (RA) in latex agglutination test assay, the protein pET28a-tD15 was purified by Ni-NTA His. Bind balsam. The carboxylation of latex acted as a carrier, using the method of chemical crosslinking latex allergens to test the quality after sensitization optimization.The result showed that when the pH of PBS was 7.4, the concentration of latex particles and protein was respectively 1% and 0.225mg/mL. the latex agglutination reaction was optimal after 6h coupling, and no self-curing phenomenon of sensitization latex antigen. No agglutination was detected with sera from ducks inoculated with other pathogens, the duplicate test both of end-batch and inter-bacth were steady and the positive serum sensitivity was up to 1:256. The coincidence rate compared LAT with ELISA which was established by our lab, was 80% in the comparison test, the result was match to a certain extent. This study has shown that LAT using the tD15 antigen is a simple, quick, sensitive, specific, cheap and useful assay for detecting antibodies against Riemerella analipestifer for the basic veterinary units.4. Generation and preliminary identification of monoclonal antidodies against recombinant tD15 protein of Riemerella anatipestifer. To prepare monoclonal antibody (McAb) against RA tD15 protein and characterize its biological features, female Balb/c mice were immunized with RA tD15 protein which purified and refolded. Then splenocytes from the immunized micewere fused with SP2/0-Ag-14, the hybridoma cell colonies were harvested by limited dilution; and induced ascites methods was used to prepared the McAb of RA tD15 protein, saturated ammonium sulfate was used to depurate the ascites; we employed indirect ELISA to screen the hybridoma cells by determining the specificities, titers and relative affinities of the produced McAbs. And the subtypes of IgG were identified using a monoclonal antibody subclass determination kit. We got two strains hybridoma cell lines steadily secreting RA tDl5 protein specific mono-clonal antibodies, they were named 4# and 16#, monoclones were frozen and resuscitated timely. The subtype of heavy and light chains of the 4# was IgG1 and κ, and 16# was 1gG2b and κ, respectively; the indirect ELISA titers of these in culture supernatant were 1:3200 and 1:6400, McAbs ascites were 1:12800 and 1:204800 respectively; western blot analysis revealed that the reacted band was around 34 kDa which is the predicted size of tD15 of RA; The purified McAbs showed strong reactivity only with the RA, confirmed that the newly developed McAbs does not show any cross-reactivity, and affinity constant of monoclonal antibody from 4# was 1.52×109 L/mol,16# was 1.0×1010 L/mol; two different antigenic epitopes might be recognized by the McAbs. The development of McAbs against tD15 protein provided a useful tool for the study on further identification of the B cell epitopes, may have important application value in further studies on biological character of D15 protein and diagnosis of Rimerella anatipestifer infection.