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Gram-negative bacterium Riemerella anatipestifer (RA) can cause septic infections in ducks, turkeys and other birdes, which belongs to Flavobacterium, without movement and spores. At present, various types of vaccines, including inactivated vaccine, attenuated vaccine and subunit vaccine, are used to control this disease. The results of these vaccine application show that some problems still exist, such as a relatively low level of immunity, a shorter duration of immunity, uneven immunity levels, and many other shortcomings. In addition, the serotype of RA are very complex, internationally recognized serotypes could be as many as 21 and no cross-protective immunity among them, so it is difficult to monitor antibodies in the immune prevention and diagnosis. In China, agar diffusion test, glass plate agglutination test and ELISA for the detection of seral antibodies against RA have been reported, but these methods can only detect individual serotypes and have low detection sensitivity, poor reproducibility and other shortcomings.The protein P25 was selected as a reaction protein from the RA1 genomic library using the RA1 antibodies of capsule membrane and outer membrane, which contains 229 amino acids, the gene encodes the ORF length of 687 bp. The BLAST analysis showed that P25 is included in GenBank sequence AF104936 and totally consistent to 1402～2088 regional nucleotide sequence. The cloning ORF sequences according to RA1-P25 gene specific primers from RA1, 2, 5, 8, 10 and other serotypes can be highly similar, encoding product with a high degree of amino acid sequence identity. The antigen epitopes and the epitope-based number were same among different serotypes through analysing the potential antigen of P25 using online tools.Logo sequence analysis showed that despite the inter-individual amino acids (AA) have some variation in the different serotypes, the antigenic determinant AA (Cys, Leu, Val) have no changed.These results showed that the P25 is co-expression protective antigen in different seral types, and can be used as different RA antigen in the diagnosis of infection, like other similar bacterial outer membrane protein.The ORF encoding P25 was recombined into E.coli Rosetta (pET-32a(+)-P25) and expressed in the induction of 1mmol/L IPTG at 37℃using conventional molecular biology methods. The soluble protein was highly purified by Ni-NTA Kit; the pure efficiency is more than 90% to meet the establishment of ELISA and other diagnostic antigen used in assays. Western blot analysis using recombinant product and ELISA results of various serotypes demonstrated that P25 is a common antigen shared by different serotype RA.An indirect ELISA was developed for detecting RA infection in ducks, based on P25 as the coated antigen.The optimal conditions for coated antigen concentration and antiseral dilution were 0.6μg/ml and 1:64, respectively, without cross-reaction against sera from E. coli and Pasteurella multocida infected ducks.In 1980s, the gold immunochromatographic assay (GICA) with low-cost, easy to operate, easy to detect a large number of samples, etc was developed. In order to overcome the shortage of above-mentioned methods for the diagnoses, a rapid and specific double antigen sandwich colloidal gold immunochromatographic assay was established to detect RA seral antibodies. In this study, recombinant protein P25 was used as capture reagent, and labeled with colloidal gold. Clinical samples were used to detect antibodies against RA using different methods,the results showed that the sensitivity of the test paper is lower than that of agglutination test.The optimal conditions need to be reseached in order to improve the sensitivity of the test paper and evaluate protective antibody levels.
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