Research In Comparative Proteomics Of Riemerella Anatipestifer Serotype1and Development Of An Antibody Detection For Riemerella Anatipestifer

Riemerella anatipestifer is a gram-negative bacterium, which is sensitive to water birds and turkey. It is the pathogen of Riemerella anatipestifer disease. The disease is characterized by fibrinous pericarditis, perihepatitis, and airsacculitis. It was a threaten to the aquaculture industry. So the research in vaccine is very important. And establishing a method for detecting antibody of Riemerella anatipestifer is of guiding significance. In this research, the secreted proteins of Riemerella anatipestifer which was cultured both in iron restriction environment and normal environment were compared to identify the different expression proteins. What’s more, the ompA protein of Riemerella anatipestifer was coated antigen, and the diagnosis method for RA antibody was established. And the work below has been finished:In this study, according to the iron limition in animal body, RA was cultured in this experimental model in vitro. By comparative proteomics, using two-dimensional gel electrophoresis and mass spectrometry identification technology,23proteins were identitied, which increased expressed in the iron limited environment.12protein points were selected to do MS analysis. They represented8kinds of proteins, included fibronectin type III domain protein, secreted subtilase family protein, phosphoglycerate kinase, leucine-rich repeat-containing protein,2kinds of hypothetical proteins, Galactose-binding domain-like protein and translation elongation factor1a （ef-1a/ef-tu）.Two proteins named Fe10and Fe19（hypothetical protein Riean₁750and hypothetical protein Riean1752） were selected according to the regulation that the theoretical isoelectric point, molecular weight similar with the actual isoelectric point, molecular weight respectively and expressed in vitro. Recombinant plasmid of pET-28a（+）-Fe10and pET-28a（+）-Fe19were constructed. Induced expression technology was used to express proteins Fe10and Fe19. After affinity purification, Fe10and Fel9were got in high purity.On the basis of analysising the result of the Western-blotting and immune challenge test, it was conluded that Fe10and Fe19could induce the immunity protection in ducks. Additionally, combined these two kinds of proteins together, the protection effect was better than one single protein.A recombinant plasmid of pET-28a（+）-ompA was constructed, and the ompA protein was expressed in vitro. The coated material was recombinant protein ompA after purified. The concentration of antigen was1.5μg/mL. Test serum was used after dilution1:100. An indirect ELISA was erected in this research, which was more sensitive16-128times higher compared with common micro-agglutination test. There were no cross-reaction with E.coli, P.multocidam, Steptococcus, Reovirus, Duck hepatitis virus type â…  and Duck plague virus from ducks, which mean the method was specific. The panels were stored at-20℃for3months. After the repeat test, the result indicated that coefficient variation （CV） of repeated batch was less than5%（1.8%-4.6%） and the CV of different panels was less than9%（1.2%-8.8%）. That mean the repeat of this method was reliable.With this method, we test the varition of antibody in ducks after injection of inactivated oil emulsion vaccine of RA. The result indicated that the antibody in ducks increased after immuned by vaccine. And the concentration of antibody reached the peak at the35th day. Then it decreased gradually, until the77th day the concentration of antibody was similar with the14th day.