*2.9. Recombinant Protein Expression and Purification*

The engineered *dcapsid* (capsid with N-terminal nuclear localization sequence deleted) gene of PCV2 strain HZ0201 was cloned and inserted into pET28 vector encoded with a hexahistidine tag at the N-terminus. The dcapsid mutants with alanine substituted for specific amino acids were constructed using site-specific mutagenesis PCR technology. The vectors were transformed into *Escherichia coli* BL21 (DE3) and the protein expression was induced using 1 mM Isopropyl β-d-1-Thiogalactopyranoside at 16 ◦C overnight. The recombinant proteins were purified using Ni-NTA super-flow matrix (30410, Qiagen, Germantown, MD, USA), followed by PD10 column (17-0851-01, GE healthcare, Los Angeles, CA, USA) to remove the excess imidazole. The concentration of recombinant protein was measured using the BCA method and the protein samples were stored at −80 ◦C.
