cfIL-22 Cloning and Construct Design
Catfish IL-22 cDNA sequence was obtained from Dr. Sylvie Quiniou (USDA-ARS-Catfish Genetics Research Unit, Stoneville MS; Accession #MK956102). Two gene constructs, one codon-optimized for expression inN. benthamiana (cfIL-22 opt) and a second encoding native catfish IL-22 sequence (cfIL-22), were synthesized (GeneArt®, Thermo Fisher Scientific, Waltham, MA) with the native catfish signal peptide, a 3’ 6X-histidine tag and cloned into an ampicillin resistant pMA vector. Gene cassettes were cloned into a pBIB-Kan plant expression vector (Becker, 1990) downstream of the constitutive dual-enhanced 35S Cauliflower Mosaic Virus promoter (35S; Lam, Benfey, & Gilmartin, 1989), and a translational enhancer from the tobacco etch virus (TEV; Carrington and Freed, 1990) and upstream of the Tnos terminator. Gene constructs were confirmed by Sanger sequencing (CRC DNA Sequencing Facility, University of Chicago). Coding sequence for native cfIL-22 is provided in Supporting material Figure S1.