| Duck tembusu virus (DTMUV), a member of the Flavivirus genus in family Flaviviridae, consists of single-stranded positive-sense viral RNA encoding three structural proteins (C, prM/M, and E) and seven nonstructural proteinsCNS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5). DTMUV is a causative agent, which caused a substantial drop in egg production in infected ducks in China. The disease was devastating, eliminating egg production and causing serious economic losses. Envelope protein is the main structural protein of the virion, contains multiple antigenic determinants that can induce neutralizing antibodies causing the host to produce protective immune response.To better understand the properties of this causative pathogen, to expand the available flavivirus sequences in public databases, and to establish phylogenetic relationships between the duck flavivirus and others, the full genomic sequence of the HN strain was determined. Features of the HN strain genome, polyprotein cleavage sites,3'UTR secondary structure, and3'-sequence analysis were also characterized.The E-encoding gene was obtained by RT-PCR and cloned into pET30a, named pET-E. The recombinant E protein was purified by the SDS-PAGE gel extraction. BALB/c mice of6week-old were immunized with purified E protein and the spleen cells from the immunized mice were fused with mouse myeloma cells SP2/0. Six hybridoma cell lines stably secreting MAbs (2A5,1F3,1G2,1B11,3B6, and4F9) were obtained after three round cloning. Dot-ELISA and Western blot analysis confirmed that six MAbs could recognize E protein specifically. IFA analysis showed that the six MAbs reacted with DEF infected with DTMUV and fluorescence is mainly located in the cytoplasm.The MAbs would be useful for development rapid and accurate diagnosis for DTMUV infection. MAbs would also be helpful in structural and functional study for E protein, which may establish a good foundation for vaccine development and molecular pathogenesis study of DTMUV. |
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