FIG. 7.

Construction of the truncated NS1 in expression vector pSecTag. (A) Full length of the amino acid sequence for JEV NS1 protein. The boxed amino acids are the sequence recognized by monoclonal antibody D2/39.1, as described in Materials and Methods. Amino acids underlined and marked with an asterisk are the predicted sequences for glycosylation. The truncated NS1, marked with an arrow at the start position 121, was created by artificial deletion of the first 120 amino acids of NS1 and PCR cloned into expression vector pSecTag (Invitrogen). (B) Expression of the truncated NS1 protein from plasmid t-NS1, indicated by an arrow, was confirmed by an in vitro coupled transcription-translation assay (TNT; Promega). (C) Expression of truncated NS1 proteins in a stable cell line, T1. Plasmid t-NS1 was used to transfect BHK-21 cells to establish a permanent cell line (T1) stably expressing truncated NS1. Cell lysates from B2-5/RP9 cells (lanes 1 and 4), primarily infected BHK-21 cells (lane 2), and T1 cells (lanes 3 and 5) treated with (lanes 4 and 5) or without (lanes 1 to 3) heat denaturation were immunoblotted with anti-NS1 JE7/45-2.