Serum investigation of antibodies against porcine circovirus 4 Rep and Cap protein in Jiangxi Province, China

Figures

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  Figure 1. Purification of PCV4 Rep and Cap protein. (A,B) His-tagged recombinant protein expression and purification. pET-32a-Rep (A) and pET-32a-Cap (B) recombinant plasmid individually transferred into BL21pLyss Escherichia coli were induced expression by IPTG at different times. Induced protein samples were subjected to SDS–PAGE and Coomassie brilliant blue staining. (C,D) Subsequently, expressed His-tagged Rep (C) and Cap (D) recombinant proteins were purified by Ni-NT assay, which were further analyzed by SDS–PAGE and Coomassie brilliant blue staining.

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  Figure 2. Immunogenicity examination of both Rep and Cap protein. (A,B) Four mice antibody titers against Rep (A) or Cap (B) after third round immunizations. The data were analyzed using Graphpad Prism software 5.0. OD450: optical density 450 nm. (C,D) Different antibodies against Rep protein (C) or Cap protein (D) through 1:500 to 1:8,000 were used to react with indicated proteins. The three lanes were as follows: 1, protein expression before induced; 2, protein expression after induced; 3, proteins after purification. (E) Both Rep and Cap antibody (1:1,000) were used to detect the endogenous corresponding protein derived from wild boars' tissues including liver, kidney, spleen, lymph node, and lung.

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  Figure 3. The cut-off value determination. (A) The evaluation of the cut-off OD450 value of the indirect Rep based ELISA. The black solid line presents the OD450 cut-off value (0.262). (B) The evaluation of the cut-off OD450 value of the indirect Cap based ELISA. The black solid line presents the OD450 cut-off value (0.327).

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  Figure 4. Sensitivity and specificity analyzing of the indirect ELISA. (A,B) Antibody against Rep protein (A) or against Cap protein (B) diluted from 1:50 to 1:6,400 was individually used to test the sensitivity of ELISA. (C,D) Eight indicated serum samples were used to analyze the specificity of Rep-based (C) and Cap-based (D) ELISA. The OD450 value higher than the value of black line was recorded as positive, if not, negative.