| Duck Tembusu virus disease is a new infectious disease caused by Tembusu Virus(TMUV).The disease has continued to harm Chinese waterfowl farming industry since 2010,causing serious economic losses.In November 2017,a group of ducks inoculated with attenuated duck Tembusu virus vaccine in Linyi area of Shandong Province developed a disease characterized by decreased egg production.Tissue suspension was prepared by treating brain,liver and other tissues.The 10-day-old duck embryos were inoculated and passaged four times with aseptic tissue suspension.After identification,a strain of duck Tambusu virus was obtained,and then animal regression test was carried out.The results showed that the isolated strain could cause embryo body hemorrhage and dwarfism.Anatomical examination of dead ducklings showed hemorrhagic edema of lungs and meninges,bleeding of glandular stomach,enlargement of spleen and bleeding spots on the surface,which were consistent with clinical manifestations.The genetic evolution analysis of the NS1 gene and prM gene of the strain showed that the isolate was located on the Chinese gene type II branch and had high homology with other reference strains.The homology of amino acid and nucleotide was 99.4% and 99.8% respectively.In conclusion,a wild strain of duck Tembusu virus was successfully isolated from immunized duck flocks.The prokaryotic expression vector pET-32a-prM was successfully constructed by amplifying prM gene from duck Tembusu virus.The vector was transformed into competent cells of E.coli BL21(DE3),and the prM protein of duck Tembusu virus was purified.The purified prM protein was used to immunize 3-month-old New Zealand white rabbits and obtain corresponding polyclonal antibodies.The titer of antibody was 1:51200 by indirect ELISA test.The results of Western-blot showed that the polyclonal antibody could specifically bind to the recombinant protein of pET-32a-prM,which proved that it has good reactivity.The indirect immunofluorescence assay showed that the polyclonal antibody of prM protein could specifically recognize the corresponding protein in TMUV infected cells.The purified prM protein was used as antigen to immunize BALB/c mice aged 6-8 weeks.After cell fusion and indirect ELISA screening,three hybridoma cell lines secretingmonoclonal antibodies against prM protein were obtained.All monoclonal antibodies were all IgG1 subtypes determined by antibody subclass kit.The indirect ELISA method was used to determine the titer.The titers of the cell supernatant and the induced ascites were higher than1:26 and 1:25600 respectively.Western-blot and indirect immunofluorescence assays showed that all three monoclonal antibodies could react specifically with prM protein.The preparation of the monoclonal antibody against the prM protein of Duck Tambusu virus lays a foundation for further study on the mechanism of TMUV ADE.Flavivirus is widely believed to have the action of antibody-dependent enhancement.TMUV is a member of the flavivirus genus,and the mechanism of ADE action has not been clear.The antibodies induced by prM protein are considered to be closely related to the ADE action of flavivirus.In this experiment,we combine different dilutions of antibodies with duck Tembusu virus to form a mixture of antibodies and viruses,and then infect HEK-293 cells.The proliferation of duck Tembusu virus in cells was detected by fluorescence quantitative PCR.The results showed that the HEK-293 cells infected with the antibody dilution at 1:800 had the highest viral load,and the infection effect was significantly enhanced,indicating that the intensity of antibody-dependent enhancement was related to the concentration of the antibody.In this study,the antibody-dependent enhancement of duck Tembusu virus was preliminarily explored by using the prepared monoclonal antibody against prM protein in vitro experiments,which laid a foundation for further study on the mechanism of ADE and development of a novel TMUV vaccine to attenuate specific prM antibody. |
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