Pet32a vector

Utilizing resources from online database, the open reading frame (ORF) of cystatin-like gene (GenBank: CDJ92568.1) without signal peptide sequence was amplified by reverse transcription-polymerase chain reaction (RT-PCR) using designed specific primers (forward primer: 5′-TAG AAT TCG GTA TGG TCG GAG GAT TTA-3′ and reverse primer: 5′-TAC TCG AGG ACC TGC TCT CCT TCA GCG-3′), in which the EcoRI and XhoI restriction sites, respectively, were introduced and are shown underlined. Following ligation of the obtained RT-PCR product with the pMD19-T vector (Takara, Dalian, China) to form pMDcystatin, the cystatin fragment was cleaved from pMDcystatin with EcoRI and XhoI and subcloned into the corresponding sites of pET32a vector (Invitrogen, Carlsbad, CA, USA). The accuracy of the insertion in the resulting plasmid was confirmed by sequencing.

The SsAK-1 (GenBank accession number JXLN01011522.1) and SsAK-2 genes (GenBank accession number OP185092) were screened according to the scabies mite transcriptome and genome databases reported in the National Center for Biotechnology Information. Specific primers with restriction sites for amplification were designed using Primer Premier 5.0 (Table 1). SsAK-1 and SsAK-2 were amplified as follows: 94°C for 3 min, followed by 35 cycles of amplification at 94°C for 30 s, 55°C for 45 s, 72°C for 1 min, and 72°C for 10 min. The amplified fragments were individually linked with pET-32a(+) vector (Invitrogen) and transfected into Escherichia coli BL21 cells. Protein expression was examined with isopropyl-β-D-thiogalactopyranoside (IPTG) for 12 h at 22°C. The rSsAKs were purified by nickel chelate affinity chromatography columns and detected by 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE). The rSsAK concentrations were detected by bicinchoninic acid assay (Takara Bio) and their activity was analyzed by phosphorus determination.

Electrophoretic mobility shift assay was performed as described previously (Zheng et al., 2021 (link)). The full-length cDNA of PuCRZ1 was cloned into pET32a (+) vector (Invitrogen, CA, USA), and then transformed into the Escherichia coli strain BL21 (DE3) competent cell (TransGen Biotech, Beijing, China). An empty pET32a (+) vector was used as a negative control. Prokaryotic expression was performed at 16°C for 16 h with 0.5 mM isopropyl-β-d-thiogalactoside (IPTG), and then recombinant protein was purified using Ni-NTA column (TransGen Biotech, Beijing, China), and the SDS-PAGE result of purified PuCRZ1 protein in Supplementary Figure 5. Primers and probes are listed in Supplementary Table 2. Unlabeled probes were subjected to cold competition experiments. EMSA were performed using the Chemiluminescent EMSA kit (Beyotime, Shanghai, China) according to the manufacturer’s instructions. The binding reactions were performed using 1 μg of 6 × His-PuCRZ1 incubated with 7.5 nM probe in binding buffer for 30 min at room temperature. The 6 × His protein was used as a negative control. The reaction mixtures were separated in 6% native polyacrylamide gel electrophoresis (PAGE) gel. Biotin activity was detected according to the manufacturer’s instructions. The EMSAs were repeated three times, and representative results are shown.

Partial ORFs of Har-JNK, Har-FoxO, Har-CREB, Har-PRMT1, and Har-Actin were amplified and cloned into the pET32a vector (Invitrogen). The recombinant proteins were expressed in BL21 (DE3) induced by IPTG at 20 °C for 9 to 12 h. The cells were lysed by ultrasonication in binding buffer (20 mM Na₃PO₄, 500 mM NaCl, pH 7.8), followed by centrifugation. The fractions containing the recombinant proteins were applied to an NTA-Ni²⁺-agarose cartridge (Qiagen N.V.) and eluted using elution buffer with an imidazole gradient from 50 mM to 1000 mM. The purified proteins were quantified using the Bradford method (43 (link)) and then used to generate polyclonal antibodies in rabbits, as described previously (34 (link)).

Based on the sequence of the open reading frame (ORF) of the rhodanese-like gene retrieved from the online database (GenBank: CDJ82729.1), the following primers were designed to specifically amplify the gene using reverse transcription-polymerase chain reaction (RT-PCR): forward primer (5’-ACG GAT CCA TGA TGT GTC CAC CTC CA-3’) and reverse primer (5’-GCA AGC TTG GAG AAC TGT AAC TGC CT-3’). The underlined sequences indicate BamHI and HindIII restriction endonuclease sites. The RT-PCR products were ligated with pMD19-T vector (Takara, Dalian, China) to produce pMD-rhodanese. Fragments of rhodanese were then cleaved from the pMD-rhodanese plasmid using BamHI and HindIII before being subcloned into the relevant location of the pET32a vector (Invitrogen, Carlsbad, CA, USA). Sequencing analysis was used to confirm proper plasmid construction.

WT and each mutant mdkCSS cDNA were cloned using a pET32a vector (Invitrogen, USA) and expressed in E. coli cells. Five mL of transformant E. coli cells was prepared (see above). Transformant cells were harvested by centrifugation at 6,000 × g at 4 °C for 10 min, and suspended in 500 μL of 50 mM Tris–HCl (pH 8.0) prior to sonication. The supernatant of sonically disrupted cells was collected by centrifugation at 21,600 × g at 4 °C for 15 min and used as the crude enzyme fraction for the enzyme assay. For purification of recombinant enzymes, 5 mL of transformant cells from an overnight culture were incubated in 500 mL of LA at 37 °C for 4 h, followed by induction as described above. The supernatant obtained from the sonically disrupted cells was mixed with an equal volume of 10 mM imidazole, 1.0 M NaCl, and 50 mM Tris–HCl (pH 8.0), and applied to an Ni²⁺ chelating column (1 × 1.3 cm), which had been equilibrated with 5 mM imidazole, 0.5 M NaCl, and 50 mM Tris–HCl (pH 8.0). After washing with 30 mM imidazole, 0.5 M NaCl, and 50 mM Tris–HCl (pH 8.0), the column was eluted with 100 mM imidazole. The eluted fraction was desalted using VIVA SPIN20. SDS–PAGE, Coomassie Brilliant Blue staining, and western blotting were carried out with an anti-mdkCSS antibody (see above) or anti-His antibody.