Simple Summary Duck astrovirus type 1 (DAstV-1) disease constitutes a reason behind viral hepatitis in ducklings and small is known on the subject of the B-cell epitope of DAstV-1. conserved linear B-cell epitopes of 454STTESA459 in DAstV-1 ORF2 proteins. Sequence evaluation, dot blot assay, and cross-reactivity check indicated how the epitope peptide was extremely conserved in DAstV-1 series and mAb 3D2 had no cross-reactivity with other DAstV serotypes. To the best GDC0853 of our knowledge, this is the first report about identification of the specific conserved linear B-cell epitope of DAstV-1, which will facilitate the serologic diagnosis of DAstV-1 infection. family includes two genera of (MAstV) and (AAstV), causing infection in GDC0853 mammalian and avian species, respectively [2]. Although MAstVs have been considered as enteric pathogens usually with mild and self-limiting characteristics in mammals [3,4,5,6], it had also been reported Rabbit polyclonal to ZCCHC12 that MAstV could cause serious disease such as encephalitis in different species [7,8,9]. In terms of AAstV, it could induce severe disease to poultry, such as poultry mortality syndrome and enteritis in turkeys [10,11], acute nephritis in chickens [12], fatal hepatitis in young ducklings [13], and fatal visceral gout in goslings [14]. Duck astrovirus (DAstV) was divided into four serotypes: DAstV-1 [13], DAstV-2 [15], and the newly found DAstV-3 [16] and DAstV-4 [17]. DAstV-1 disease has spread worldwide and continued to threaten the duck industry because of the symptom of fatal hepatitis in young ducklings [13,18]. The genome of DAstV-1 is 6.4C7.9 kb in length, comprising of three open reading frames (ORF1a, 1b, and 2), 5 and 3 untranslated region (UTR), and a poly A tail [19]. Both the ORF1a and ORF1b encode the nonstructural proteins (NSPs), containing enzymes and participating in viral replication, whereas the ORF2 encodes the viral capsid polyprotein [20,21]. ORF2, containing antigenic determinant, can induce the production of neutralizing antibody interacting with the host [22,23,24]. It is recognized that monoclonal antibodies (mAbs) consisting of one specific antibody molecule are superior to their polyclonal antisera in many facets of immunology [25,26,27]. Their characteristics of sensitive and specificity make hybridoma-derived antibodies the effective immunological reagents in immunoassays, immunotherapy, immunoaffinity chromatography and immune diagnosis. Until now, the application of mAb in DAstV diagnosis has not been reported. In this study, taking the prokaryotic-expressed ORF2 protein as the immunogen, a DAstV-1 ORF2-specific mAb 3D2 was generated using cell hybridization technique, and a highly conserved B-cell epitope in DAstV-1 ORF2 protein was identified with the mAb. These findings shall be valuable for developing epitope-based diagnostic package for DAstV-1 infections. 2. Methods and Materials 2.1. Infections, Cells, and Antibodies DAstV-1 virulent stress D51 (GenBank accession no. “type”:”entrez-nucleotide”,”attrs”:”text message”:”MH712856″,”term_id”:”1603711543″,”term_text message”:”MH712856″MH712856) was isolated through the liver of ill cherry valley ducks in the Shandong province of China in 2012 [28]. The gene of DAstV-1 D51 stress was cloned in to the prokaryotic manifestation vectors pET-32a (+) (Novagen, Darmstadt, Germany) and pGEX-6p-1 (GE Health care, Amersham, UK) to create recombinant histidines tagged ORF2 (His-ORF2) and glutathione S-transferase tagged ORF2 (GST-ORF2). The purified His-ORF2 proteins was utilized to immunize BALB/c mice. The hybridoma cell range creating mAb 3D2 was made by fusion of B-lymphocytes from immunized mice with mouse myeloma cells. Subtype recognition exposed that mAb 3D2 was from the IgG2b/kappa type. Horseradish peroxidase (HRP) tagged GDC0853 goat anti-mouse antibody and fluorescein isothiocyanate (FITC) tagged goat anti-mouse antibody had been bought from KPL (Gaithersburg, MD, USA). The positive anti-DAstV-1 serum was from five mice immunized with purified His-ORF2 proteins and kept in the veterinary molecular etiology lab of Shandong Agricultural College or university. The infant hamster kidney (BHK-21) cells and duck embryo fibroblasts (DEF) cells had been cultured in Dulbeccos customized Eagles moderate (DMEM) including 10% fetal bovine serum (FBS) at 37 C inside a 5% CO2 atmosphere. A DNA-launched infectious clone of DAstV-1 D51 stress, named pABX-D51,.