Screenping Of The Propective Antigen And Developing A Differentiante ELISA For Antibody Of Ery

Our research selected 51 metal transport associated protein, surface protein, membrane protein and lipoprotein of Erysipelothrix Rosenbach based on whole genome analysis, after trans-membrane domain, signal peptide analysis we designed primers to clone these genes to pET-28a(+). Recombinant plasmid identification results showed that all of the plasmids were constructed successfully. Host cells BL21 (DE3) containing recombinant plasmid were induced to express recombinant proteins and 14 soluble proteins as well as 9 insoluble proteins were verified by SDS-PAGE analysis. Subunit vaccines containing each recombinant protein immunized mice before challenge showed that Su-1(SpaA) as well as comercial available vaccines confered fully protection, at the same time, three other proteins named SpaA truncated Su-2-2, Su-2-4 and Lp-2(lipoprotein) protected 17% of mice.We further tested the protective efficacy of the N-terminal protective domain of SpaA(SpaA-N) absorbed with Al(OH)3, the results also showed that the vaccine confered completely protection to mice and pigs. These results demonstrated that SpaA-N is a potential subunit vaccine candidate.In contrast natural infections, subunit vaccine induced humoral responses were less complex, this basement rised the possibility to establish ELISA methods which can be used to differntiate natural infections and vaccination. Therefore, we selected 10 soluble proteins as candidate antigens to coat on ELISA plates to detect sera which sampled from infected pigs. The results showed that Fe-2-coated plates was able to detect antibodies in pigs 2 weeks after infection. Importantly, Fe-2-coated plates have no cross-reaction with hyper-immune sera prepared by infection of E. coli, staphylococcus, streptococcus, Haemophilus parasuis, Pasteurella, Salmonella. Based on these results, we optimized the ELISA method as a diagnosis method to discremate natural infections and SpaA-N induced antibody responses. The optimized ELISA procedures were showed as follows:coating 100 ng/well SpaA-N on plates, dilluting the sample sera 100 times, incubation both sample and secondary antibodies with 30 min, and incubation substrates with 10 min.