E coli transetta de3

The completed cDNA fragment of CgDM9CP-4 was amplified with the primers P7 and P8 (Supplementary Table 1) with NdeI and XholI (NEB) cleavage site sequences added to the 5’ end, respectively. The PCR fragment was digested by restriction enzymes NdeI and XholI, and ligated into the same restriction enzymes sites of expression vector pET-30a (Novagen). The recombinant plasmid (pET-30a-CgDM9CP-4) was transformed into E. coli Transetta (DE3) (TransGen). The positive transformants were incubated in LB medium containing 50 μg mL^-1 kanamycin at 37°C with shaking at 220 rpm for 4 h. When the culture mediums reached OD₆₀₀ of 0.5–0.7, IPTG was added to the LB medium at a final concentration of 1 mmol L^-1, and incubated at 16°C with shaking at 180 rpm for 20 h. The bacterial culture was sonicated and centrifuged to get the supernatant containing soluble target protein. The recombinant protein of CgDM9CP-4 (rCgDM9CP-4) was purified by Ni²⁺ chelating Sepharose column (Roche), and the purified protein was dialyzed out of imidazole against ddH₂O at 4°C for 24 h. The protein was resolved by 12% SDS-polyacrylamide gel electrophoresis (SDS-PAGE), and visualized with Coomassie Bright Blue R250. The concentration of purified soluble protein was quantified by BCA method. The obtained protein was stored at -80°C for subsequent experiment.

The cDNA sequence of CgGLS-1 containing glutaminase domain was cloned into pET-30a vector (Primers were shown in Table 1). Restriction enzymes BamH I and Hind III were used to construct recombinant plasmids. The recombinant plasmid was isolated by MiniBEST plasmid purification kit (Takara, Japan) and then transferred into E. coli Transetta (DE3) (Transgen, China). Isopropyl β-D-Thiogalactoside (IPTG) (1 mmol/L) was used to induce the expression of recombinant protein, and the recombinant protein CgGLS-1 (designated rCgGLS-1) was purified by a Ni²⁺chelating Separate column (Sangon Biotech, China). The purity of obtained rCgGLS-1 was evaluated by SDS–polyacrylamide gel electrophoresis. An enhanced BCA protein assay kit (Beyotime, China) was used to quantify the content of rCgGLS-1^{46 (link)}. The purified protein was stored at −80 °C before use.

The fragment encoding the SbIGF-1R fibronectin type III domains was amplified using gene-specific primers (Table 1) and was subsequently cloned into the pET-30 (a)+ vector (Novagen, Madison, WI, USA). The recombinant plasmid was introduced into E. coli Transetta (DE3) (TransGen). The recombinant fibronectin type III domains of SbIGF-1R protein (rSbIGF-1R-FNIII) were induced by 0.2 mM IPTG for 6 h at 37 °C and purified using Ni-Charged Resin (GenScript). A mixture containing 100 μg rSbIGF-1R-FNIII was used to immunize 4-week-old mice for the polyclonal antibody preparation, while the SbFn antiserum was prepared by immunizing a 6-month-old rabbit.

Primers (M2F, M2R) containing BamHI and SacI restriction enzyme sites were used to insert the ORF of AccMKK6 into the expression vector pET-30a(+) (Novagen, Darmstadt, Germany). The recombinant plasmid pET-30a(+)-AccMKK6 was transformed into E. coli Transetta (DE3) (TransGen Biotech, Beijing, China). The bacterial solution was cultured in Luria Bertani (LB) with 50 μg/ml kanamycin at 37°C for 1–2 h until the cell density reached 0.4–0.6 at 600 nm. The expression of the recombinant AccMKK6 protein was induced with 2 mM isopropyl-1-thio-β-d-galactopyranoside (IPTG) at 28°C for 6-8 h, and the protein was then separated by 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). To prepare an antibody against AccMKK6, the recombinant AccMKK6 protein band was excised from the gel, milled with 1 ml of normal saline and injected into white mice. The mice were injected once a week for a total of four injections. Blood was collected on the fourth day after the last injection and stored at 37°C for 1 h and then 4°C for 6 h. After centrifugation at 3000 g for 15 min, the supernatant antibodies were stored at −70°C.

The full length of CgLSD1 was obtained with the specific primers P1 and P2 (Table 1). Another pair of specific primers P3 and P4 (Table 1) with BamHI and XhoI cleavage sites was designed to amplify the cDNA of the SWIRM domain of CgLSD1 and cloned into pET-30a expression vector. E. coli Transetta (DE3) (TransGen Biotech, China) was used to express the recombinant protein of CgLSD1-SWIRM (designated as rCgLSD1-SWIRM). After isopropyl-β-D-thiogalactoside (IPTG) induction, the rCgLSD1-SWIRM protein was purified by Ni⁺ affinity chromatography (Sangon, China). The concentration of rCgLSD1-SWIRM protein was verified by the BCA method (Beyotime, China) and stored at −80°C for the preparation of antibody.

To determine the effect of NKLnc expression on host bacterial cells, the coding sequence of NKLnc was PCR amplified with primers NKLnc-F/R (Table S2), and the PCR product was inserted into pET-30a (+) vector (Novagen, Madison, WI, USA). The resulting recombinant plasmid was introduced into E. coli Transetta (DE3) (TransGen, Beijing, China). The transformant was cultured to OD600 0.6–0.8 in LB medium and the culture was then divided into groups. Isopropyl β-D-1-thiogalactopyranoside (IPTG) (0.3 mM) was added into one group to induce NKLnc expression. The two groups were cultured for various hours, and bacterial growth at different time points was recorded by measuring OD600. For plate counting, the above E. coli transformant was plated on LB agar plates supplemented with or without 0.3 mM IPTG. After incubation at 37 °C overnight, the colony numbers were calculated.

The pGEX-4T-1-NS3, pGEX-4T-1-mNS3, and pGEX-4T-1-OsDRB1a constructs, as well as empty pGEX-4T-1 vectors, were individually transformed into E. coli Transetta (DE3) (Transgene, Beijing, China) with protein expression induced by IPTG. The soluble GST fusion proteins were extracted and immobilized onto glutathione sepharose beads (Amersham). RNAs were labeled with ³²P–UTP using a T7 RNA Production system (Promega) and purified with a MEGAclear kit (Ambion, Waltham, MA, USA). Approximately 2,000 cpm of radioactive RNA was denatured and then annealed to form dsRNA. The dsRNA was incubated with individual GST fusion proteins in the presence of RNase inhibitor (1 U per group) for 30 min on ice. The mixtures were then run on an RNase-free native PAGE gel for 1 h at 4°C.