Subsequently, HRP-labeled goat anti-pig IgG (KPL Inc., Gaithersburg, MD, USA) was incubated in wells for 15?min at 37?C, after which wells were washed five occasions with PBST and 50?L of tetramethyl-benzidine (TMB, Sera Care Life Sciences, Inc., Milford, MA, USA) was incubated in the dark for 5?min at room heat. by Baculovirus Expression Vector System (BEVS) as a high-quality covering antigen for detection of PCV3-associated PF-4618433 antibodies in serum samples; and 2) use this ELISA to conduct a serological survey for PCV3 in Alas2 various regions of Hunan province, China. Results The PCV3 positive rate to the ELISA assay (total of 190 serum samples) was higher in sows with reproductive failure compared to healthy sows (34/85, 40.0% versus 30/105, 28.6%), with similar results using qPCR assays. Further, in an additional 1038 serum samples collected from January 2016 to May 2018 in various regions of Hunan province and tested with this established ELISA, 20 to 84% were positive for PCV3 (according to region of sera collection), with high PCV3 seroprevalence ( ?50%) in herds in Changde, Hengyang and Yueyang. Moreover, among serum samples from herds in Shaoyang and Changde, PCV3 seroprevalence was higher in sows than in other classes of pigs (i.e., suckling piglets, nursery pigs, gilts, growing-finishing pigs and boars). Conclusions We developed a full-length PCV3 Cap-based ELISA using a eukaryotic expression system with excellent potential to elucidate PCV3 epidemiology. Based on this assay, PCV3 has been circulating in Hunan province. PCV3 prevalence was lower in healthy sows than in those with reproductive failure. Further studies are warranted to identify the PCV3 responsible for high seroprevalence in sows and determine pathogenesis of PCV3 in sows with reproductive failure. Electronic supplementary material The online version of this article (10.1186/s12917-019-1810-3) contains supplementary material, which is available to authorized users. gene was successfully constructed and amplified in a Bac-to-Bac baculovirus expression system, expression of PCV3 Cap in Sf9 cells was successfully recognized by SDS-PAGE and western blot using swine PCV3 positive serum and anti-His tag antibody, respectively. A clear band revealed that PCV3 Cap with an apparent molecular excess weight of ~?26 KDa was present in cell lysates from Sf9 cells infected by a recombinant baculovirus (Lane 2 in Fig. ?Fig.1B1B and C), but not in the Control (Lane 1 in Fig. ?Fig.1B1B and C). Subsequently, PCV3 Cap (40?g/mL) was successfully purified from your cell lysates via Ni-NTA affinity chromatography (Lane 3 in Fig. ?Fig.11A). Open in a separate window Fig. 1 Expression and purification of PCV3 Cap with a Baculovirus expression system. PCV3 Cap was expressed in Sf9 cells and purified with a Ni-NTA resin, separated on a 12% SDS-PAGE gel and stained PF-4618433 with Coomassie blue (a), and recognized using western blot with swine PCV3-specifc positive serum (b) and anti-His tag antibody (c). M, protein marker in KDa. Lane 1: Total extract in non-infected SF9 cells. Lane 2: Total extract in Sf9 cells expressed PCV3 Cap; Lane 3 Ni-NTA affinity-purified Cap protein Establishment of ELISA using purified PCV3 cap as antigen A checkerboard titration was performed to determine the optimal Cap concentration for plate covering and serum dilution in the ELISA. Based on our results (Table ?(Table1),1), 0.5?g/mL of Cap for plate covering, along with PF-4618433 a serum dilution of 1 1:100 was an optimal combination and chosen for subsequent ELISA assays. Furthermore, the optimal dilution of the secondary antibody was 1:7000 (Additional file 1: Table S3). The OD450 nm values decreased with successive dilutions of six positive serum samples, indicating that our PCV3 Cap ELISA experienced high sensitivity (Fig. ?(Fig.2).2). All five non-PCV3 serum samples used to determine specificity were unfavorable for PCV3 Cap ELISA (Additional file 1: Table S4). Table 1 Optimal dilutions of covering antigen and serum sample antibodies for ELISA gene of PCV3 in our study, PF-4618433 the PCV3 Cap was well expressed in BEVS. Further, this Cap was rapidly purified via one-step NTA-Ni affinity chromatography (Fig. ?(Fig.1).1). Importantly, Cap was exploited as a covering antigen responsible for detecting PCV3-specific antibodies present in serum samples (Figs. ?(Figs.2,2, PF-4618433 ?,4,4, ?,6).6). Previous studies also used a truncated PCV3 Cap harvested from bacteria expression system as a covering antigen to detect PCV3 antibodies in sera [26]. Of notice, the NLS, an arginine-rich domain name at the NH2-terminal end of the Cap, was deleted in the truncated Cap. The difference of antigenicity between full-length and NLS-truncated PCV3 Caps remains to be decided, as epitope mapping of PCV3 Cap has yet to be reported. Epitope prediction strongly indicated presence of a potential linear epitope in the NLS of PCV3 Cap (Additional file 1: Table S1 and Physique S1). In contrast,.
