Recombinant Protein Expression and Purification

Total RNA from PSCs was extracted using the RNA-prep Tissue Kit (Cowin Biotech, Beijing, China) and transcribed into cDNA by the Reverse Transcription System Kit (Thermo Fisher, USA) according to the manufacturers' instructions. Primers for PCR were designed based on transcriptome data (Table 1). The amplified PCR products were ligated into expression vector pET32a(+) (Novagen, Madison, WI) and transformed into Escherichia coli BL21 (DE3) (Cowin Biotech). Protein expression was induced with 0.1 mM isopropyl-β-D-thiogalactopyranoside (IPTG; Applied Biosystems, United Kingdom) at 37°C for 6 h with gentle shaking. Recombinant protein was purified from bacterial lysate using a Ni²⁺ affinity column (Bio-Rad, Hercules, CA) following the manufacturer's instructions. Crude HF antigen was extracted from hydatid cysts as previously described (Song et al., 2016 (link)). rEg-DHFR and rEg-P29 were provided by the Department of Parasitology of Sichuan Agricultural University.