| Duck Adenovirus(DAdV),belonging to the family Adenoviridae,are currently divided into duck adenovirus A(DAdV-A)and duck adenovirus B(DAdV-B).Duck adenovirus 1(DAdV-1),also named as egg drop syndrome virus(EDSV),is the only representative member of Duck adenovirus A(Atadenovirus genera).Duck adenovirus B belongs to the genera Aviadenovirus,including duck adenovirus 2(DAdV-2)and duck adenovirus 3(DAdV-3).DAdV-3 infection has been widespread in the central and southern China since the outbreak in 2014.The DAdV-3 infection,which mainly causes swelling and hemorrhage of liver and kidney in Muscovy ducks,has resulted in substantial economic losses to the domestic waterfowl industry.Penton,Hexon and Fiber are the main structural proteins of DAdV and the Fiber plays a vital role in mediating viral infection and inducing humoral immunity.In order to investigate the antigenic epitopes of DAdV-3 Fiber-1 and establish serological diagnostic method for the detection of antibodies against DAdV-3,the surface structural protein Fiber-1 of DAdV-3 was used as the target protein in this study.Based on the cloning of Fiber-1 gene and prokaryotic expression of Fiber-1 protein,polyclonal antibodies and monoclonal antibodies targeting Fiber-1 protein were prepared,and two indirect ELISA methods for the detection of antibodies against DAdV-3 were established based on purified recombinant protein.1.Prokaryotic expression of Duck adenovirus 3 Fiber-1 protein and preparation of polyclonal antibodiesTo obtain the recombinant Fiber-1 protein of DAdV-3,the Fiber-1 gene was amplified based on the genome of DAdV-3 and the Fiber-1 protein was expressed with the constructed plasmid pCold-Fl using the prokaryotic expression system in this study.Efficient expression of Fiber-1 protein was achieved by transforming the prokaryotic recombinant expression plasmid into BL21(DE3)with the induction of IPTG.The fusion protein,mainly expressed as inclusion bodies in the precipitation,was purified using the method specific to inclusion bodies.The purified His-Fiber-1 protein was used to immunize mice,and the serum against Fiber-1 protein was obtained and identified by indirect fluorescence assay(IFA)and western blot.The results showed that the prepared serum not only had high specificity with the exogenously over-expressed Fiber-1 protein,but also recognized DAdV-3 effectively.All the results laid the foundation for the development of murine-derived monoclonal antibodies against Fiber-1 protein.2.Preparation and characterization of monoclonal antibodies against Fiber-1 protein of Duck adenovirus 3To prepare monoclonal antibodies against Fiber-1 protein of DAdV-3,spleen cells from immunized mice were fused with mouse myeloma cells by cell fusion technique.Six novel monoclonal antibodies against Fiber-1 protein,designated as 1D8,1E6,3G6,4G1,4G2 and 6F10,were finally obtained after HAT screening,IFA identification and cell subcloning.The heavy and light chains of six monoclonal antibodies were identified using the monoclonal antibody subclass identification kit.The results showed that the subclass of monoclonal antibody 4G2 was IgG2b,and the subclasses of monoclonal antibodies 1D8,1E6,3G6,4G1 and 6F10 were IgG1.The specific reaction of the monoclonal antibodies was examined by IFA and western blot,indicating that monoclonal antibodies only recognized DAdV-3 specifically and did not react with other viruses.Five different epitopes of the Fiber-1 protein,which were highly conserved in different DAdV-3 isolates,were identified by constructed eukaryotic and prokaryotic recombinant plasmids of truncated Fiber-1 gene.In addition,monoclonal antibodies 3G6 and 6F10 could immunoprecipitate Fiber-1 protein effectively in LMH cells transfected with pcDNA3.1-F1 or infected with DAdV-3.Monoclonal antibodies 3G6 and 4G2 showed certain neutralizing activity against DAdV-3 infection in vitro neutralization assays.The generation of monoclonal antibodies against Fiber-1 protein of DAdV-3 and the identification of B cell epitopes provided important materials for the establishment of a rapid and efficient diagnostic method for DAdV-3 detection.3.Development of an indirect ELISA for detection of antibody against DAdV-3To establish a rapid and efficient serological diagnostic technique for detection of DAdV3,purified Fiber-1 and Fiber-2 recombinant proteins were used as coated antigens.By optimizing the reaction conditions,the concentration of coated His-Fiber-1 and His-Fiber-2 were determined to be 1μg/mL and 0.5 μg/mL,respectively.The optimal closure solution was a mixture of 5%skim milk and 1%calf serum,and the optimal incubation time was 120 min.The optimal solution,dilution and incubation time of primary antibody were the suspension of BL21 cell lysate and 1%skim milk,1:400 and 60 min,respectively.The optimal incubation time and dilution of secondary antibody were 90 min and 1:10000.The optimal substrate reaction time was 15 min.In this study,two indirect ELISA methods based on DAdV-3 Fiber proteins were established,which showed good specificity and reaction with positive duck sera.The ELISA methods also revealed good repeatability with the coefficients of variation(CV)less than 10%in the inter-and intra-assay.The above serological diagnostic method for DAdV3 detection based on Fiber protein provided technical support for epidemiologic surveillance and evaluation of vaccine immunogenicity. |
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