Development and evaluation of a competitive enzyme-linked immunosorbent assay using a monoclonal antibody for diagnosis of severe fever with thrombocytopenia syndrome virus in bovine sera

Lee, Hyojin; Kim, Eun-Ju; Song, Jae-Young; Choi, Jeong Soo; Lee, Ji Youn; Cho, In-Soo; Shin, Yeun-Kyung

For antigen production, the gene encoding the full-length NP protein of SFTSV strain LN3 (GenBank accession No. HQ141612) was expressed in a bacterial overexpression system. The S fragment of SFTSV was chemically synthesized using the Bioneer gene synthesis service (Bioneer, Korea). The 735 bp gene encoding N-protein was amplified using the following primers: SFTS-NP-1 CTCGGAATTCACATGTCA GAGTGGTCC and SFTS-NP-735 CTTCAAGCTTCAGGTT CCTGTAAGCAG. The PCR amplification was performed using a T3000 thermocycler (Biometra, Germany). The NP gene was cloned into pET-30a(+) (Invitrogen, USA) using the BamHI and XhoI restriction enzymes (New England Biolabs, UK), and subsequently transformed into Escherichia coli BL21 (DE3) (Yeastern Biotech, Taiwan) to express 6xHis-tagged fusion proteins. Following induction with 0.2 mM isopropyl -D-1thiogalactopyranoside (AMRESCO, USA) for 20 h at 25 o C, the bacterial cell pellet was sonicated in chromatography buffer (20 mM sodium phosphate, 500 mM NaCl, 8 M Urea, and 20 mM imidazole, pH 7.4) and purified using Ni-NTA agarose (Qiagen, Germany) [22] . The affinity-purified protein was urea gradient dialyzed prior to use. The recombinant NP protein was solubilized in 8 M urea buffer, which was then exchanged for 150 mM Tris-HCl (iNtRon Biotechnology, Korea) for further use in subsequent experiments.