Epidemiological Survey of Porcine Circovirus Types 2 and 3 in Liaoning Region of China and Preparation of Monoclonal Antibodies Against PCV3 Cap Protein

Figures

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  Figure 1. PCR amplification and restriction enzyme digestion analysis of the PCV3 Cap gene. (A) PCR amplification of the full-length PCV3 Cap gene from qPCR-positive clinical samples. M: DNA marker; lanes 1–3: PCV3-positive samples; lane 4: negative control. (B) Double restriction enzyme digestion of the recombinant plasmid pUCm-T-PCV3-Cap. M: DNA marker; lane 1: digested pUCm-T-PCV3-Cap plasmid. (C) Single restriction enzyme digestion of the recombinant plasmid pUCm-T-PCV3-Cap. M: DNA marker; lane 1: digested pUCm-T-PCV3-Cap plasmid.

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  Figure 2. Genetic Evolution Analysis of the PCV3 Cap Gene in Liaoning Province. (A) Nucleotide sequence identity analysis of the PCV3 Cap gene. Nucleotide sequence identities between six PCV3 sequences and representative reference strains from the United States, South Korea, and China are shown as percentage identity (%). (B) Amino acid sequence identity analysis of the PCV3 Cap protein. Amino acid sequence identities between the six sequences and reference strains are expressed as percentage identity (%). (C) Phylogenetic analysis based on the PCV3 Cap gene. Sequence alignment and phylogenetic tree construction were performed using the Neighbor-Joining method in MEGA software, with bootstrap values based on 1000 replicates. Filled circles (•) indicate PCV3 strains identified in this study; reference strains are labeled with their GenBank accession numbers. (D) Multiple sequence alignment and conservation analysis of the PCV3 Cap protein. Amino acid alignment was conducted using MegAlign software. Variable residues are highlighted, with mutated sites indicated in red and conserved residues shown in white. Conservation scores (bits) reflect amino acid conservation, and residue numbers are…

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  Figure 3. Construction and verification of the recombinant pET-32a-PCV3 Cap∆N plasmid. (A) PCR amplification of the truncated PCV3 Cap gene. M: DNA marker (8000 bp); lanes 1–3: positive samples. (B) Restriction enzyme digestion of the pET-32a vector for linearization. M: DNA marker; lanes 1–6: linearized pET-32a vector. (C) Double restriction enzyme digestion analysis of the recombinant pET-32a-PCV3 Cap∆N plasmid. M: DNA marker; lane 1: Recombinant plasmid pET-32a-PCV3 Cap∆N after double digestion; lane 2: recombinant pET-32a-PCV3 Cap∆N plasmid. Correct digestion patterns and sequencing analysis confirmed successful plasmid construction. The restriction enzymes used for vector linearization and double digestion are both BamHI and XhoI.

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  Figure 4. Expression, purification, and immunoblot analysis of the recombinant PCV3 Cap protein. (A) SDS–PAGE analysis of recombinant PCV3 Cap protein expressed in E. coli BL21 (DE3) at different induction temperatures. M: protein molecular weight marker (15–150 kDa); lane 1: supernatant induced at 16 °C; lane 2: pellet induced at 16 °C; lane 3: supernatant induced at 25 °C; lane 4: pellet induced at 25 °C; lane 5: supernatant induced at 37 °C; lane 6: pellet induced at 37 °C; lane 7: vector pET-32a Precipitate. (B) SDS–PAGE analysis of recombinant PCV3 Cap protein induced with different IPTG concentrations at the optimal induction temperature. M: protein molecular weight marker (15–150 kDa); lanes 1–7: induction with 0,0.2, 0.5, 0.8, 1.0, 1.2, and 1.5 mM IPTG, respectively. (C) SDS–PAGE analysis of purified recombinant PCV3 Cap protein. M: protein molecular weight marker (15–150 kDa); lane 1: flow-through fraction; lanes 2–3: wash fractions; lanes 4–7: elution fractions. (D) Western blot analysis of recombinant PCV3 Cap protein induced with different IPTG concentrations at the optimal temperature. M: protein molecular weight marker (15–150 kDa); lanes 1–7: induction with 0, 0.2, 0.5, 0.8,…

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  Figure 5. Optimized Conditions for Indirect ELISA. (A) Optimization of protein coating concentration versus serum dilution. (B) Optimization of antigen coating time. (C) Optimization of blocking time. (D) Optimization of positive serum reaction time. (E) Optimization of HRP-conjugated goat anti-mouse IgG dilution. (F) Optimization of HRP-conjugated goat anti-mouse IgG reaction time. (G) Optimization of TMB chromogen reaction time.

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  Figure 6. Preparation and Subtype Identification of Monoclonal Antibodies. (A) Immunization Schedule. Mice were immunized on days 0, 14, 28, and 35 according to the schedule. Blood was collected via tail vein on the day prior to each immunization, serum was isolated, and the titer of antibodies produced in vivo was determined. (B) ELISA results for antibody titers in mouse serum at 14 days post-immunization. (C) ELISA results for antibody titers in mouse serum at 28 days post-immunization. (D) ELISA results for antibody titers in mouse serum at 35 days post-immunization. S/N ratio = OD450 sample/OD450 negative sample. The dashed line indicates a ratio of 2.1. Results were considered positive when the ratio exceeded 2.1 (dashed line) and negative when below 2.1. (E) 3 days after cell fusion. (F) 14 days after cell fusion. (G) 7 days after subcloning. (H) Identification results for the 3E6 subclass and type of monoclonal antibody. OD450 values were measured using a microplate reader. Hybridoma cells were cultured in HAT medium for the first ten days. Expansion and subcloning were performed in HT medium, both at 37 °C in a cell culture incubator.

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  Figure 7. Specificity Characterization of Monoclonal Antibody 3E6. (A) Immunofluorescence assay. pcDNA3.1-PCV3 Cap plasmid was transfected into 293T cells, while the Mock group received the empty pcDNA3.1 vector. The primary antibody for both the 3E6 and Mock groups was monoclonal antibody 3E6; the primary antibody for the positive control group was positive serum diluted 1:200 from immunized mice. (B) Specific identification of monoclonal antibody 3E6 and PCV3 Cap protein. M: protein molecular weight marker; line 1: PCV3 Cap. (C) Specific identification of 3E6 and PCV2 Cap protein. M: protein molecular weight marker; line 1: PCV2 Cap. (D) Specific identification of 3E6 and PRRSV N protein. M: protein molecular weight marker; line 1: PRRSV N.