A sensitive and specific antigen detection assay for Middle East respiratory syndrome coronavirus

Chen, Yixin; Chan, Kwok-Hung; Kang, Yahong; Chen, Honglin; Luk, Hayes KH; Poon, Rosana WS; Chan, Jasper FW; Yuen, Kwok-Yung; Xia, Ningshao; Lau, Susanna KP; Woo, Patrick CY

Cloning and purification of (His) 6 -tagged recombinant NP (rNP) of MERS-CoV A clinical isolate of MERS-CoV was kindly provided by RA Fouchier and colleagues. 2 All work with infectious MERS-CoV was performed inside a biosafety level-2 cabinet with standard operating procedures in approved biosafety level-3 facilities. [16] [17] [18] Viral culture and RNA extraction were performed as we described previously. 9, 16 To produce a plasmid for protein purification, primers (59-GAA TTC ATG GCA TCC CCT GCT GCA CCT-39 and 59-GTC GAC ATC AGT GTT AAC ATC AAT CAT-39) were used to amplify the gene encoding the NP of MERS-CoV by RT-PCR. The sequence coding for amino acid residues 1-413 of the NP was amplified and cloned into the EcoRI and SalI sites of expression vector pET-28a(1) (Novagen, Merck Millipore, Billerica, MA, USA) in frame and upstream of the series of six histidine residues. The rNP protein was expressed in Escherichia coli ER2566 cells (Invitrogen, Life Technologies, Grand Island, NY, USA) and purified using Ni-NTA affinity chromatograph column (Novagen, Merck Millipore, Billerica, MA, USA) according to the manufacturer's instructions.