Recombinant KRBV NS1 Protein Production

The NS1-encoding gene was amplified by high-fidelity RT-PCR from RNA extracted from KRBV prototype sample 1892 and cloned into a mammalian expression vector by infusion cloning (Clontech). The 3′ end of the gene was fused to the V5 epitope sequence followed by a polyhistidine sequence. Sequencing of the expression plasmid confirmed that the recombinant gene fragment was authentic and in frame for correct translation. COS-7L cells were transfected with Lipofectamine 3000 (Invitrogen) in accordance with the manufacturer’s protocol. After 3 days, the cells were harvested with a cell scraper into PBS with protease inhibitors. rNS1 was purified on a HisTrap column (GE), buffer exchanged into PBS, and concentrated on an Amicon Ultra centrifugal filter with a 10-kDa cutoff (Merck Millipore). The presence of rNS1 was confirmed by Western blotting of cell lysate, cell supernatant, and purified protein and IFA of transfected cells with an anti-V5 MAb (Invitrogen) as described in reference 14 (link). The identity of KRBV NS1 was confirmed by mass spectrometry in accordance with previously published protocols (53 (link)).