Ni2 affinity column

The full coding sequence of Eg-Grx1 was amplified from E. granulosus cDNA using primers 5ʹ-CCG GAA TTC ATG TGG CGC TTT TTA TC-3ʹ and 5ʹ-CCG CTC GAG CTC TAA AAG TTC AGC AAG TG-3ʹ, and then integrated into the EcoRI/XhoI restriction sites of vector pET28a (Novagen, Heidelberg, Germany). Recombinant protein was expressed and purified as previously described [30 (link)]. Briefly, the plasmid was transformed into Escherichia coli BL21 (DE3) cells (Cowin Biotech, Beijing, China) and induced with 0.8 mM isopropyl-1-thio-β-D-galactopyranoside at 37 °C for 6 h. Proteins were then purified using a Ni²⁺ affinity column (Bio-Rad, Hercules, CA) following the manufacturer’s instructions. The purified rEg-Grx1 protein was monitored by 15 % SDS-PAGE, and the protein concentration was estimated with a bicinchoninic acid protein assay kit (Pierce, Rockford, IL).

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.

The total RNA of PSCs was isolated using Trizol reagent (Invitrogen, Carlsbad, CA, USA) and then reverse transcribed into cDNA using the ThermoScript™ RT-PCR System for First-strand cDNA Synthesis Kit (Invitrogen). The nucleotide sequence encoding the LEL domain of Eg-TSP1 was amplified from cDNA using a sense primer (5′-CGCGGATCCCCTGATAACCTAAACAAAGC-3′) containing a BamHI site (underlined) and an antisense primer (5′-CCCAAGCTTGAGGGTTTTGTTCTCTGCCAA-3′) containing a HindIII site (underlined). The PCR products were ligated into the pET32a(+) plasmid (Novagen, Madison, WI, USA). The recombinant plasmid with correct sequence was transformed into Escherichia coli BL21 (DE3) cells (Invitrogen) and subsequently grown at 37 °C in Luria-Bertani broth containing 50 μg.mL⁻¹ ampicillin. When the OD_600nm value of bacteria reached 0.6, 1 mM isopropyl-β-D-thiogalactopyranoside (IPTG) was added for 5 h induction at 37 °C. The recombinant protein was harvested from E. coli lysates, followed by purification with a Ni²⁺ affinity column (Bio-Rad, USA) under denaturing conditions in 8 M urea. The purified protein was refolded by dialysis against PBS and concentrated using an Amicon Ultra-15 10,000 MWCO centrifugal filters (Millipore, USA), and then analyzed by SDS-PAGE.