Epidemiological investigation of goose circovirus based on a newly developed indirect ELISA method

Figures

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  Figure 1. Identification of the recombinant plasmid pET-30a-△Cap. Lane M DL2000 Puls DNA marker; Lane 1 pET-30a-△Cap enzyme double digestion, Lane 2 pET-30a-(+) (NdeI + HindIII).

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  Figure 2. SDS-PAGE analysis of solubility and the optimization of expression conditions of the recombinant protein Cap. (A) SDS-PAGE analysis of △Cap protein solubility. Lane M, protein molecular quality standard. Lanes 1–2, the supernatant and precipitate after crushing and centrifugation of pET30a-△Cap-BL21 (DE3), respectively. Lanes 3–4, the supernatant and precipitate after crushing and centrifugation of pET30a--BL21 (DE3), respectively; Lanes 5–6: the supernatant and precipitate after crushing and centrifugation of BL21 (DE3), respectively. (B) SDS-PAGE analysis of the △Cap protein at different IPTG-induced concentrations. Lane M, protein molecular quality standard. Lanes 1–4, the samples with IPTG concentrations of 0.25, 0.5, 0.75, and 1 mM. (C) SDS-PAGE analysis of the △Cap protein at different induction times. Lane M, protein molecular quality standard. Lanes 1–8: Samples induced for IPTG for 1, 2, 3, 4, 5, 6, 7, and 8 h. The arrows pointed band are the expected protein.

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  Figure 3. SDS-PAGE analysis of recombinant proteins under different purification conditions. (A) SDS-PAGE analysis of △Cap protein purified by nickel column affinity chromatography. Lane M, protein molecular quality standard. Lanes 1–9, the collected △Cap proteins at different elution concentrations. (B) SDS-PAGE analysis of the △Cap protein purified by gel purification protein. Lane M, protein molecular quality standard. Lanes 1–3, △Cap protein samples of different concentrations were purified by cutting rubber. The arrows pointed band are the expected protein.

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  Figure 4. Western blot analysis of the △Cap fusion protein. M, protein molecular weight marker. (A) Lane 1, purified fusion protein blot incubated with mouse anti-His tag monoclonal antibody. (B) Lane 1, purified fusion protein blot incubated with prepared rabbit polyclonal antibody. The arrows pointed band are the expected protein.

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  Figure 5. The specificity analysis of △Cap-iELISA against the common pathgens in geese. The reference anti-serum samples included GoCV positive anti-serum (GoCV-positive), GoCV negative anti-serum (GoCV-negative), Newcastle disease virus positive anti-serum (NDV), Goose parvovirus positive anti-serum (GPV), Goose paramyxovirus positive anti-serum (GPMV), Tembusu virus positive anti-serum (TMUV), Avian reovirus positive anti-serum (ARV) and H5 and H7 subtypes of Avian influenza virus positive anti-serum (AIH5, AIH7). **** for p < 0.0001.