Pet32a expression vector

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′-CGGGATCCCCAATGCCTTCAGGTG-3′ (forward; BamHI restriction site underlined) and 5′-CCCTCGAGTCACATTGTTTTTTCCATT-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.

The Sol g 4.1 gene was subcloned from the pGEM-T easy vector into the pET-32a expression vector (Invitrogen, UK). Briefly, the vectors were double digested with NcoI and XhoI restriction enzymes, and the Sol g 4.1 gene was ligated into the same restriction sites of the pET-32a expression vector. Recombinant plasmids were transformed into E. coli BL21 (DE3) pLysS competent cells (Promega, Malaysia). A single colony from a freshly streaked plate was picked, inoculated in LB (Sigma-Aldrich, USA) starter media containing 50 μg/mL ampicillin and incubated at 37 °C overnight with shaking until the culture was turbid but not saturated.
The 5-mL starter cultures were transferred into 500 mL of LB expression medium containing 100 μg/mL ampicillin and incubated at 37 °C until the cell density reached to OD₆₀₀ of ~ 0.5. Afterwards, the temperature was reduced to 30 °C and the culture was induced with 0.4 mM IPTG. The induced cultures were grown for 8 h. Cell pellets were collected and washed with 10 mL of lysis buffer (20 mM sodium phosphate, pH 7.4, 100 mM NaCl, 1 mM DTT, and 0.1 mM PMSF) and disrupted by sonication on ice. After centrifugation at 15,000×g for 20 min at 4 °C, the supernatants were separated on 13% SDS-PAGE gels.

Based on genomic data (GenBank: JXLN01012673.1) and proteomics data (GenBank: KPM08736.1), we identified the S. scabiei chitinase-like protein 5 (SsCLP5) and amplified regions showing high sequence homology with other species according to the results of epitope analysis. Primers for amplification were designed using Primer 5.0 software and were as follows: forward (5'-CGG GAT CCA TGC AAG AGC TTC GTA A-3'), with a BamHI restriction site (underlined), and reverse (5'-CCC TCG AGA TCA TAG AAG ATC ATA GAA AT-3'), with an XhoI restriction site (underlined; Invitrogen, Beijing, China). SsCLP5 was amplified using the following PCR cycling conditions: 94 °C for 5 min; 30 cycles of amplification at 94 °C for 45 s, 55 °C for 45 s, and 72 °C for 45 s; followed by a final extension at 72 °C for 10 min. The fragment was cloned into the pET-32a (+) expression vector (Invitrogen), which was transformed into Escherichia coli BL21 (DE3) cells. Protein expression was induced with 1 mM isopropyl-β-D-thiogalactoside (IPTG) at 37 °C for 6 h. Recombinant S. scabiei chitinase-like protein 5 (rSsCLP5) was purified using a Ni-NTA His-tag affinity chromatography kit (Bio-Rad Laboratories, California, USA), according to the manufacturer’s instructions.