| The infection of reovirus could cause contagious disease that was characterized by swelling nape, eye conjunctival congestion or bleeding and extensive hemorrhage of infected ducks. Since2010, a virulence contagious disease emerged in succession on egg laying ducks in the southern part of our country, and it was characterized by egg laying drop. Reovirus was isolated and identified from the illed ducks by my laboratory. In order to diagnose and control duck reovirus (DRV) that could cause duck egg laying drop, methods for detecting antibody or antigen of DRV were established. This study included two aspects:one was using the aB protein of DRV expressed by escherichia coli as coating antigen to eatablish an indirect ELISA method for detecting antibody of DRV; two was using monoclonal antibody (McAbs) against DRV to eatablish a double antibody sandwich ELISA (DAS-ELISA) method for detecting antigen of DRV.The σB gene of DRV ZY/AH/1/2011strain was amplified by RT-PCR, then the aB gene was cloned into prokaryotic expression vector pET-32a(+) and was reconversed into Transetta (DE3). The55ku recombination aB protein was obtained in the form of inclusion body with IPTG induction. Western-blotting analysis showed that the recombination aB protein in the form of inclusion body could be recognized by polyclonal antibodies of rabbit against DRV. The recombinant aB protein was purified with High-Affinity Ni-NTA Resin under denaturing and renatured by gradient urea. By using the purified recombination σB protein, the indirect ELISA assay for the detection of DRV antibody in duck serum was applied. By detecting serums from infected ducks and specific pathogen-free ducks, the agreement ratio reached at100%between the indirect ELISA and neutralization test. The indirect ELISA showed no cross reaction with the positive serum of duck plague virus, duck virus hepatitis and avian influenza virus. The intra-assay variability was less than11%and the inter-assay variability was less than14%. DRV ZY/AH/1/2011was purified by differential centrifugation and sucrose density gradient ultracentrifugation, and eight-week-old BALB/c mice was immunized with the purified virus as antigen, then the spleen cells of the mice was fused with SP2/0. Positive clones were screened with purified recombination σB protein as detection antigen, and six hybridoma cells named2D7,2D10,2B11,6B2,4D7,7H9that could stably secrete McAbs against DRV were produced after subcloning three or four times by limiting-dilution. The six McAbs had an titer ranged from1:1600to1:51200; McAbs2D10was IgG1, and the other five McAbs were IgG3; indirect fluorescence assay showed that the six McAbs could react with Vero cells infected with DRV specifically; Western-blotting analysis showed that the six McAbs could be recognized by recombination aB protein; in these McAbs2D10might recognize different antigenic determinants with6B2and4D7. Rabbit-anti-DRV polyclonal antibody and McAbs2D10that prepared in large-scale were puried by Protein G resin. Then a DAS-ELISA method for the detection of DRV antigen was established based on puried rabbit-anti-DRV polyclonal antibody as capture antibody and puried McAbs as detection antibody. The sensitivity of this method reached0.522μg/mL. The DAS-ELISA showed no cross reaction with the positive viruses of duck plague virus, duck virus hepatitis and avian influenza virus, and the results of repetition test displayed that intra-assay variability and inter-assay variability both less than10%.The results showed above that the two ELISA methods eastablished for detecting antibody or antigen of DRV that causing duck egg laying in this study both had good sensitivity、 specificity and repetitiveness. |
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