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.