Streptrap beads

Following optimization of the culture conditions, the E. coli cells were centrifuged (5000 rpm, 30 min, 4 °C), pellets obtained, and then re-suspended in pre-cold PBS on ice for ultrasonication. The lysate was centrifuged at 6682 rpm and 4 °C for 20 min and the recombinant k3 was purified using StrepTrap beads according to the manufacturer’s instructions (General Electric Company, Boston, MA, USA). The collected samples were identified by SDS–PAGE and transferred to polyvinylidene fluoride membrane (Millipore, Darmstadt, Germany). After a 2 h blocking step with 5% BSA, the target protein underwent probing with an anti-His tag polyclonal antibody for an additional 2 h. Protein bands were visualized using Clarity Western ECL substrate (Bio-Rad, Hercules, CA, USA) in conjunction with NcmECL Ultra (NCM Biotech Co., Ltd., Suzhou, China).

Based on the K205R gene sequence of the ASFV SY18 strain, specific primers were designed using Oligo7 software, and pcDNA3.4-K205R-strep plasmid was constructed using the pcDNA3.4 TOPO™ TA Kit (Thermo Fisher Scientific). The recombinant eukaryotic expression vector pcDNA3.4-K205R-strep was transiently transfected into 293i cells and maintained in a shaking incubator at 37°C and 8% CO₂ at 125r/min. The cells were supplemented with enhancers and auxiliaries after 18-22 hours, and incubated in a shaking incubator for 5 days. Cells were identified 1, 3, and 5 days post-transfection (dpt). Cells were collected at 5 dpt, and the recombinant pK205R was purified using StrepTrap beads according to the instructions provided by the manufacturer (General Electric Company, Boston, MA, USA). The collected samples were identified by SDS-PAGE and Western blotting (WB) using an anti-Strep tag antibody (1:4000, ab180957, Abcam, Cambridge, MA, USA).

Based on D117L sequence of the ASFV SY18 strain, pcDNA3.1-D117L-strep plasmid was constructed using the pcDNA™3.1 (+) vector (Thermo Fisher Scientific, Waltham, MA, USA). The recombinant eukaryotic expression vector pcDNA3.1-D117L-strep was transiently transfected into CHO cells and maintained in a shaking incubator at 37 °C and 8% CO₂ at 125 r/min. The cells were supplemented with enhancers and auxiliaries after 18–22 h and incubated in a shaking incubator for 5 days. Cells were identified 1, 3, and 5 days post-transfection (dpt). Cells were collected at 5 dpt, and the recombinant p17 was purified using StrepTrap beads according to the instructions provided by the manufacturer (General Electric Company, Boston, MA, USA). The collected samples were identified by SDS-PAGE and Western blotting (WB), using an anti-strep tag antibody (1:4000, ab180957, Abcam, Cambridge, MA, USA).