Ni2 affinity chromatography

Total RNA was extracted using an RNA prep Pure Tissue Kit (Nanjing Vazyme Biotech, Nanjing, China). First-strand cDNA was synthesized using a reverse transcription system kit (Nanjing Vazyme Biotech). The full coding sequence of the non-membrane region of EgHCDH was amplified using the primers 5′- CGG GAT CCA TGT CAG CCG GTG CTG G-3′ (BamHI) and 5′-GAC GTC GAC TCA CTG TTT TTC CTT GAC AAT GCG C-3′ (SalI). Amplification reactions were performed using the following cycling conditions: pre-denaturation at 95 °C, 5 min; then denaturation at 95 °C/30 s, 62 °C/30 s, 72 °C/1 min; with a final extension at 72 °C, 5 min. Through sequencing, digestion and ligation, EgHCDH was ligated into the pET32a (+) plasmid (Novagen, Darmstadt, Germany) and transformed into Escherichia coli BL21 (DE3) cells (Tiangen, Beijing, China). Protein expression was induced with 1 mM isopropyl-1-thio-β-d-galactopyranoside at 37 °C for 6 h. The rEgHCDH protein was purified using Ni²⁺ affinity chromatography (Bio-Rad, Hercules, CA, USA), with the the purity of the final product determined by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE). The concentrations of the purified protein were determined using a NanoDrop 2000c spectrophotometer (Bio-Rad).

The region encoding mature Eg-DHFR was amplified from E. granulosus cDNA using the primers 5′-CGCGGATCCATGGGGCTGAAGCGTCT-3′ and 5′-CCGCTCGAGATGATCATTAAGGGGATGCG-3′, and then ligated into the BamHI/XhoI restriction sites of vector pET-28a(+) (Novagen, Madison, WI, USA). Recombinant protein was expressed and purified as previously described^{33 (link)}. Briefly, the recombinant plasmid was transformed into E. coli BL21 (DE3) cells (Invitrogen, Carlsbad, CA, USA) and expression was induced with 1 mM isopropyl- thio-β-D-1-thiogalactopyranoside at 37 °C for 5 h. The bacterial cells were harvested and suspended in lysis buffer, followed by ultrasonic lysis. The recombinant protein was purified by Ni²⁺ affinity chromatography (Bio-Rad, Hercules, CA, USA) following the manufacturer’s instructions. Recombinant protein was detected by 12% SDS-PAGE and the concentration of protein was estimated using a bicinchoninic acid protein assay kit (Pierce, Rockford, IL, USA).

TRIzol reagent (Tiangen, Beijing, China) was used to extract total RNA from adult worms, protoscolices and oncospheres. cDNA was reverse-transcribed using a RevertAid First Strand cDNA Synthesis Kit following the manufacturer’s instructions (Thermo Fisher Scientific, Vilnius, Lithuania). Target fragments were amplified, ligated into the pET32a(+) vector (TaKaRa, Dalian, China) and transformed into BL21 (DE3) competent cells. Expression of the recombinant proteins was induced by 1 mM isopropyl β-d-1-thiogalactopyranoside (IPTG) and purification was achieved using Ni²⁺ affinity chromatography (Bio-Rad, Hercules, CA, USA).