2.2. Plasmid construction

TGEV and PDCoV N encoding genes were codon-optimized for high expression in mammalian cells and to avoid expression of PDCoV NS7 embedded in the PDCoV N gene (^{Fang et al., 2017}) (GenBank: {"type":"entrez-protein","attrs":{"text":"AAG30228.1","term_id":"11096193","term_text":"AAG30228.1"}}AAG30228.1, {"type":"entrez-protein","attrs":{"text":"AFD29191.1","term_id":"380005464","term_text":"AFD29191.1"}}AFD29191.1, respectively). PCR products of the corresponding N genes with indicated tags at the C-termini were ligated into pCAGGS vectors to give plasmids for expression of tagged CoV N (pCAGGS-TGEV N-FLAG and pCAGGS-PDCoV N-HA). pCAGGS-HA-PEDV N for N-terminally HA-tagged PEDV N expression was similarly constructed based on the same PEDV N DNA sequence in pCAGGS-PEDV N-Myc. pGEX-4T3-PEDV N was constructed by in-frame insertion of the PEDV N gene sequence, codon-optimized for bacterial expression based on the sequence from AVCT12 strain (accession number {"type":"entrez-nucleotide","attrs":{"text":"LC053455","term_id":"820947799","term_text":"LC053455"}}LC053455), into the pGEX-4T3 vector (Amersham) following the coding sequence of N-terminal GST. All plasmids were verified by DNA sequencing.