Genetic and antigenic characterization of recombinant nucleocapsid proteins derived from canine coronavirus and canine respiratory coronavirus in China

Lu, Shuai; Chen, Yingzhu; Qin, Kun; Zhou, Jianfang; Lou, Yongliang; Tan, Wenjie

Plasmids containing the N gene of CCoV and CRCoV were digested by the corresponding restriction enzymes and inserted into the bacterial expression vector pET28a (Novagen, Germany). All constructs were confirmed by enzyme digestion and DNA sequencing. The recombinant plasmids were transformed into expression E. coli competent cells BL21 (DE3). The E. coli culture was induced by adding isopropyl -D-1-thiogalactopyranoside (IPTG) at a final concentration ~0.5 mmol L 1 and incubating at three different conditions: 37°C for 4 h; 25°C for 10 h and 18°C for 16 h. After induction, the bacterial cells were harvested and suspended in binding buffer (Tris, 50 mmol L 1 ; NaCl, 500 mmol L 1 ; pH 8.0), and lysed by sonication for 20 min in an ice water bath, followed by centrifugation at 34,900×g for 30 min at 4°C.The supernatants and pellets were collected separately, and analyzed by 12% SDS-PAGE. The recombinant NPs in the supernatant were purified using a nickel-affinity column using the AKTA system, and further processed by size exclusion chromatography (Superdex 75 column, GE, USA). Briefly, the protein was eluted using different gradients of elution buffer (Tris, 50 mmol L 1 ; NaCl, 500 mmol L 1 ; imidazole, 500 mmol L 1 ; pH 8.0). The concentrated peak eluates were then subjected to an equilibrated S75 column and the purified soluble recombinant NPs were analyzed by SDS-PAGE.