Nanoparticle orientationally displayed antigen epitopes improve neutralizing antibody level in a model of porcine circovirus type 2

Ding, Peiyang; Zhang, Teng; Li, Yafei; Teng, Man; Sun, Yaning; Liu, Xiao; Chai, Shujun; Zhou, Enmin; Jin, Qianyue; Zhang, Gaiping

The genomic DNA of PCV2 Cap protein without nuclear localization signal was codon optimized for Escherichia coli (E. coli) and cloned in pUC57 vector, which was synthesized by Sangon Company (Shanghai, People's Republic of China) and stored in our lab. The coding sequence of Cap protein was PCR amplified. The PCR product and PET28a vector were doubly digested with BamHI and HindIII (NEB, Ipswich, MA, USA), treated with T4 DNA ligase (NEB), and then transformed into E. coli BL21 (DE3). For protein expression, bacteria were cultured in Luria-Bertani liquid medium (containing 0.1 mg/mL kanamycin sulfate) at 37°C with shaking until the optical density (OD) of the culture at 600 nm reached 0.6. Then, isopropyl-β-d-thiogalactoside (IPTG) was added to a final concentration of 0.1 mM to induce expression at 30°C for 10 h. The purification of Cap was performed by using a Ni-NTA His⋅Bind Resin (Novagen, Madison, WI, USA). Purity and bioactivity of Cap protein were determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting, respectively. The concentration was determined with a BCA protein assay kit (Thermo Fisher Scientific). The endotoxin content of Cap proteins was detected by ToxinSensor™ Chromogenic LAL Endotoxin Assay Kit (GenScript, Piscataway, NJ, USA).