To prepare the antigen used for camel immunization, a pET-30 prokaryotic expression system was used to express recombinant p54 protein of the ASFV China/2018/AnhuiXCGQ strain (GenBank accession no. AYW34096.1). Following the construction of the recombinant plasmid, pET-30-p54-His, the plasmids were sequenced, and the correct plasmids were transferred into Transetta (DE3) expression competent cells (TransGen Biotech, Beijing, China). A single clone was selected and treated with 1.0 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) at 37 °C for 12 h. Following ultrasonication, the lysates were centrifuged at 12,000 × g for 30 min at 4 °C, and precipitates and supernatants were subjected to SDS-PAGE and western blot analysis, respectively.
The ASFV p54 protein was purified using cOmplete His-Tag Purification Resin (Roche, Basel, Switzerland). Before protein purification, the resin was equilibrated with 10 times column volumes of buffer A (50 mM NaH2PO4 pH = 8, 300 mM NaCl). Then the contaminated proteins were eluted with buffer A containing 20 mM imidazole, and the p54 protein was eluted with buffer A containing 250 mM imidazole. The eluted product was collected and analyzed by SDS-PAGE.
The ASFV p54 protein was purified using cOmplete His-Tag Purification Resin (Roche, Basel, Switzerland). Before protein purification, the resin was equilibrated with 10 times column volumes of buffer A (50 mM NaH2PO4 pH = 8, 300 mM NaCl). Then the contaminated proteins were eluted with buffer A containing 20 mM imidazole, and the p54 protein was eluted with buffer A containing 250 mM imidazole. The eluted product was collected and analyzed by SDS-PAGE.