2.3. Expression of PCV3 Cap Protein

The complete coding sequence of the PCV3 Cap protein (GenBank accession No. {"type":"entrez-nucleotide","attrs":{"text":"MK580468","term_id":"1733431165","term_text":"MK580468"}}MK580468) was synthesized and optimized (Sangon Biotech, Shanghai, China) into pGEX-4T-1 and pET-32a vectors, and the recombinant plasmids pGEX-4T-1-Cap and pET-32a-Cap were thus obtained, respectively. The expression of GST-fused Cap and 6×His-tagged Cap proteins (namely GST-Cap and His-Cap, respectively) was achieved by transforming into E. coli BL21. Protein expression was induced with 1 mM isopropyl-β-galactopyranoside for 6 h at 37 °C. The bacterial precipitate was collected by centrifugal force at 5000× g and diluted in PBS. The target proteins were lysed via ultrasonication and collected via centrifugation. The recombinant proteins in the supernatant after sonication were purified using the GST-tag and His-tag Protein Purification Kits (CoWin, Taizhou, China) following the steps outlined in the instructions. The proteins were characterized using SDS-PAGE in 12.5% polyacrylamide gels. The determination of protein concentration was conducted via a BCA protein detection kit (Solarbio, Beijing, China).

In addition, to characterize the ability of mAbs to bind to the PCV3 Cap protein in multiple ways, the PCV3 Cap protein gene was synthesized and subcloned into the pCAGGS vector (Sangon Biotech, Shanghai, China), and the resultant protein was named pCAGGS-Cap.