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

Construction of expression plasmids: Yamaguchi-1 strain fragments were amplified using primer pairs, N1F (5′-TGG GAT CCA TGG CTG GAA ACG GAC CAC-3′) and N179R (5′-GAC TCG AGT TAG TTA TTG GAT CTA TTG TTG GAC-3′) for nt 1-537 encoding a.a. 1-179, and N180F (5′-TGG GAT CCA TTA ACA GTA ACA GTG GTG ATA T-3′) and N374R (5′-GAC TCG AGT TAG TTT AGT TCA TCA ATA ATT TCA-3′) for nt 538-1125 encoding a.a. 180-374. These forward and reverse primers contained BamHI and XhoI sites at the 5′-end, respectively. Fragments were purified using a MinElute PCR purification Kit (QIAGEN) and digested with restriction enzymes, BamHI and XhoI. Two fragments of the Yamaguchi-1 strain were electrophoresed on a 0.8% gel and extracted using a QIAEX II Gel Extraction Kit (QIAGEN). Fragments were then cloned into BamHI and XhoI sites of the expression plasmid pGEX-6P-1 vector (GE Healthcare, Piscataway, NJ, U.S.A.) using a DNA Ligation Kit Ver. 2.1 (TaKaRa). Plasmids were transformed into Escherichia (E.) coli strain DH5α (TOYOBO, Osaka, Japan).