| Riemerella anatipestifer (RA) is a major cause of disease.Riemerella anatipestifer is the causative agent of septicaemic and exudative diseases in a variety of bird species. The organism is a gram-negative, nonmotile, nonsporulating rod.Economic loss to the duck industry from this disease is due to mortality, as well as weight loss and condemnations.Typically, ducklings of 1 to 8 weeks old are highly susceptible. Slide and tube agglutination tests with antisera differentiate 21 serotypes of R. anatipestifer,serotypes 1 and 2 are most prevalent in China.The main lesions are fibrinous serositis, caseous salpingitis and arthritis,but the acute disease is often rapidly fatal due to septicaemia while the chronic carrier state can be virtually asymptomatic and without distinctive lesions,economic loss in farm ducks throughout the world.Outer membrane proteins A of RA are generally very immunogenic.They play an important role in virulence of and immunity to bacterial diseases. The ompA gene is present in the R. anatipestifer type strain and in all serotype reference strains.It exhibits some minor genetic heterogeneity among different serotypes,which seems not to affect the strong antigenic characteristics of the protein. In this study, ompA genes of HLG1 strain were amplified by PCR. And recombinant expression plasmids pHtb-ompA were constructed. The sequence analysis showed the genes were right. The recombinant plasmids and pHtb-ompA were transformed into BL21(DE3) to express. SDS-PAGE analysis showed structure proteins expressed. The proteins were purified and quantified by SDS-PAGE, we report the cloning and expression of an immunogenic 55kDa outer membrane protein (OmpA) of R.anatipestifer.with an induction time of 4h and 1 mM IPTG.Antigenicity of proteins was analyzed by DNAStar and Western Blot.The recombinant protein was purified by His-Ni+affinity chromation with good purity.Using the purified recombinant protein, an indirect-ELISA was developed and its optimal reactions were determined. The optimized conditions were as following:concentration of antigen 2.09μg/mL, dilution of sera and conjugate were respectively 1:100 and 1:5000, the confining sulution was 5% skim milk.The threshold value of the ELISA was conformed according to the detection result of 133 negative sera. It was positive when the value of OD450 was more than 0.304, it was negative when the value was less than 0.260. The recombinant protein was highly specific because it had no cross reaction with positive sera of Escherichia coli, Pasteurella multocida,Reticuloendotheliosis virus,Duck plague virus,Duck hepatitis virus,Duck Influenza virus, but could block the reaction with the positive serum of RA. The recombinant antigen was also highly sensitive and could react with positive serum at 1:1204800.The coincidence rate between of ELISA and ELISA was 91.3%. The coefficient of variation of intro-batch and inter-batch duplicability tests were less than 15%. This value showed that the repetition of ELISA was good. Sera sa (?)ples collected in helongjiang province were tested by this ELISA and the negative rate was 80.2%.This study successfully expressed the OmpA protein. The antigenicity of those proteins analyzed by DNAStar and Western-Blot showed that the OmpA had the best antigenicity. Using OmpA protein, an indirect-ELISA with good specifity, sensitivity and repetition was developped.So the indirect ELISA assay would provide a simple and rapid means for Riemerella anatipestifer infection. it is necessary to detect RA antibodies of SPF ducks level and above.
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