Establishment of serological test to detect antibody against ferret coronavirus

MINAMI, Shohei; TERADA, Yutaka; SHIMODA, Hiroshi; TAKIZAWA, Masaki; ONUMA, Mamoru; OTA, Akihiko; OTA, Yuichi; AKABANE, Yoshihito; TAMUKAI, Kenichi; WATANABE, Keiichiro; NAGANUMA, Yumiko; KANAGAWA, Eiichi; NAKAMURA, Kaneichi; OHASHI, Masanari; TAKAMI, Yoshinori; MIWA, Yasutsugu; TANOUE, Tomoaki; OHWAKI, Masao; OHTA, Jouji; UNE, Yumi; MAEDA, Ken

Expression and purification of glutathione-S transferase (GST)-fusion proteins: Two N protein fragments, N1-179 and N180-374, were expressed as fusion proteins with GST, GST-N (1-179) and GST-N (180-374), respectively. E. coli containing recombinant or control plasmid was cultured in 2 × yeast extract and tryptone (YT) medium (1.6% tryptone, 1% yeast extract and 0.5% Nacl, pH 7.0) containing 50 µg ampicillin ml −1 . Expression of recombinant proteins was induced by the addition of 1 mM isopropyl β-d-1thiogalactopyranoside (Wako, Osaka, Japan) for 4 hr. The bacterial cells were suspended in sonication buffer (50 mM Tris-Hcl, pH 8.0, 50 mM Nacl, 1 mM EDTA and 1 mM dithiothreitol) and lysed using a Multi-beads shocker (YASUI KIKAI, Osaka, Japan). After centrifugation, supernatants were mixed with Triton X-100 at a final concentration of 1% for 30 min and then centrifuged at 20,630 × g at 4°c for 30 min. The supernatants were collected, mixed with glutathione sepharose 4B beads (GE Healthcare) and incubated at 4°c for 30 min. After centrifugation, beads were washed four times with phosphate-buffered saline (PBS) containing 0.5% Triton X-100 and once with sonication buffer. The beads were mixed with 300 µl of 10 mM glutathione and incubated at 4°c for 1 hr. After incubation, supernatants were harvested as purified recombinant proteins and used for ELISA and immunoblot analysis. The purified proteins were confirmed to be single bands by coomassie-brilliant blue (cBB) staining after sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis.