S.W., X.R., W.X., H.H., G.W. synthetic peptides analysis, we finely mapped the neutralizing epitopes to two 10-amino acid (aa) peptides within the CAdV Hex: 634RIKQRETPAL643 located on the surface region; and 736PESYKDRMYS745 located in the inner region of the expected 3D structure of trimeric Hex. Importantly, the two epitopes are highly conserved among all CAdV isolates by sequence alignment analysis. Thus, these results provide insights into the interaction between virus and mAbs at the aa level and may have potential applications in the development of novel therapeutic or epitope-based vaccines, antibody therapeutics and a diagnostic method suitable for the rapid 25,26-Dihydroxyvitamin D3 detection of all CAdVs. of the family [6]. Mouse monoclonal to KARS The genome of CAdV includes 30 open reading frames (ORFs) flanked by two identical 161 bp inverted terminal repeats [7]. The major coat protein of CAdV is hexon (Hex), which plays a major role in viral tropism and neutralization [8]. The N-terminal domain (with a core size ~484 amino acids [aa]) and C-terminal domain (core size ~221 aa) adopt the same PNGase F-like fold, although they are significantly different in length. The CAdV capsid structure is relatively well conserved, and is primarily composed of homo-trimers of Hex; 240 trimers form the icosahedrons 20 facets [9,10]. There are 12 vertex penton capsomers, each with a fiber protruding from the surface [10,11,12]. The trimeric Hex has a pseudohexagonal base with three towers extending upwards [10,11]. Apart from these, the capsid is also stabilized by other Hex-associated proteins [13]. It is estimated that most of the adenoviral neutralizing antibodies are generated against Hex [14]. In this study, we characterized the potent neutralizing monoclonal antibody (mAb) 2C1, and a partially neutralizing mAb 7D7, raised against CAdV-2. We expressed and purified a truncated His-fused Hex (552C850 aa) containing most of C-terminal domain; then tested the neutralizing mAbs for recognition in Western blot (WB) assays. Using a phage display approach, we finely mapped the recognized epitopes to 634RIKQRETPAL643 and 736PESYKDRMYS745 on the CAdV-2 Hex. Since the two linear epitopes are highly conserved among CAdV-1 and CAdV-2 strains, these research results may assist in the design of structure-based novel epitopes vaccines or therapeutic vaccines and antibody therapeutics, and the development of rapid detection techniques for CAdV antigens or antibodies. 2. Materials and Methods 2.1. Virus Strains, Hybridoma Lines, Plasmids and Phage Peptide Library The CAdV-2 strain used in this study was stored at the Harbin Veterinary Research Institute of the Chinese Academy of Agricultural Science. Hybridoma lines secreting CAdV-2-specific mAbs were generated in our lab previously [15]. The pET-30a (+) vector was purchased from Tiangen, China. A commercially available Ph.D.-12TM Phage Display Peptide Library Kit was purchased from New England BioLabs Inc. CAdV-2 was cultured in MadinCDarby canine kidney (MDCK) cells in Dulbeccos modified eagles medium with 10% fetal calf serum (Gibco, USA) at 25,26-Dihydroxyvitamin D3 37 C, and ER2738 and Rosetta (DE3) were cultured in LuriaCBertani (LB) medium. 2.2. Characterization of mAbs against CAdV-2 Hybridoma lines secreting CAdV-2-specific mAbs were cultured, and the isotype was determined using the SBA ClonotypingTM System/HRP Kit (Southern Biotech, USA). Specificity characterization of CAdV-2-specific mAbs 2C1 and 7D7 was carried out by indirect immunofluorescence assay (IFA) and WB according to standard procedures [15], but WB using denaturing SDS-PAGE and native PAGE, respectively. Denaturing SDS-PAGE was carried out according to standard procedures. In brief, 15 L of virus sample was mixed with 5 L 4X SDS-PAGE loading buffer and boiled for 10 min. Samples were then loaded into 12% SDS-PAGE gel (Invitrogen), and electrophoresis was performed at room temperature using a constant voltage (120 V). In NSDS-PAGE, 15 L of virus sample was mixed with 5 L of 4X NSDS sample buffer (150 mM Tris base, 100 mM Tris HCl, 10% (v/v) glycerol, 0.0185% (w/v) Coomassie G-250, 0.00625% 25,26-Dihydroxyvitamin D3 (BL21 competent cells (Tiangen); the truncated Hex protein was produced via isopropyl–D-thiogalactopyranoside (IPTG, GE Healthcare, Chicago, IL, USA) induction using pET30a- Hex at 37 C. The truncated Hex protein was detected by SDS-PAGE and Western blotting (WB) analysis by anti-His mAb. Immunoreactive bands were verified by an enhanced chemiluminescence system (ECL; PerkinElmer Life Sciences, Fremont, CA, USA) [15]. The three-dimensional (3D) structure of the expressed protein was predicted with PyMOL software based on data from the SWISS-MODEL online server. 2.6. Biopanning of Phage Random Peptide Library against Anti-CAdV-2 mAbs A commercially Phage Display Peptide Library Kit (New England BioLabs Inc., Ipswich, MA, USA) was used to carry out four rounds of biopanning according to the manufacturers manual. Briefly, 10 g purified mAb 2C1.