Total RNA was extracted from P. ovis var. cuniculi mites using a MiniBest universal RNA extraction kit (TaKaRa, Dalian, China) and reverse-transcribed into cDNA using a PrimeScript RT reagent Kit with gDNA Eraser (TaKaRa). According to the annotated P. ovis var. cuniculi transcriptome sequence (GenBank No. PRJNA317241 ) (16 (link)), the full-length sequence encoding Poc-AK was amplified from cDNA using primers 5′-CGGGATCC CCAATGCCTTCAGGTG-3′ (forward; BamHI restriction site underlined) and 5′-CCCTCGAG TCACATTGTTTTTTCCATT-3′ (reverse; XhoI restriction site underlined). The PCR product was ligated into the pET32a (+) expression vector (Invitrogen, Beijing, China), and the resulting construct was transformed into Escherichia coli BL21 (DE3). The recombinant protein expression was induced by 0.5 mM isopropyl-β-D-thiogalactoside (IPTG) at 37°C for 12 h. The recombinant Poc-AK (rPoc-AK) was harvested in form of the inclusion body, solubilized in 8 M urea, and purified by a Ni-NTA His-tag affinity kit (Bio-Rad, California, USA) using a step-wise elution with 20, 50, and 100 mM imidazole. The purified protein was further dialyzed against phosphate buffered saline (PBS) and concentrated using Amicon Ultra Centrifugal Filter devices (Millipore, Billerica, MA, USA) according to the manufacturer's protocol.
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