Figure 1. Phylogenetic tree of many sequenced IL-22 genes.
Tree made with Geneious Tree Builder. Cost matrix: Blosum62, Alignment type: Global alignment with free end gaps, Genetic Distance model: Jukes-Cantor, Tree Build Method: Neighbor-joining, Gap open penalty: 12, Gap extension penalty: 3
Figure 2. Cloning strategy for cfIL-22 expression constructs.Coding region of cfIL-22 was synthesized using both the native andN. benthamiana codon optimized sequence with the native signal peptide (SP) and a 3’ histidine tag. The gene was cloned into the pBIBKan plant expression vector containing the 35S duel enhanced CaMV promoter and the tobacco etch virus translational enhancer (TEV).
Figure 3. Three-step purification process of plant-expressed cfIL-22. Western blot detected using an anti-His antibody (A) and Coomassie blue total protein stain (B) were used in monitoring purification of cfIL-22. Crude leaf extracts (1) from transient tobacco plants expressing cfIL-22 were subjected to a two-step ammonium sulfate (AS) precipitation. IL-22 recovered in the 35% saturated supernatant (2) was precipitated with 63% AS saturation (3). IL-22 containing fraction was subjected to nickel affinity chromatography; Flow through (4), wash (5), and elution (6-9) fractions. The 300mM imidazole elution fraction (8) enriched with IL-22 was concentrated (10) and further purified by size exclusion chromatography (11). The purified cfIL-22 protein (C) was concentrated, sterilized and analyzed by coomassie (a), silverstain (b), and western (c) for downstream bioassays. All samples were resolved on a 12% SDS-PAGE.
Figure 4. Cell proliferation as a measure of cfIL-22 bioactivity. Catfish fibroblast cells ( CCO) seeded on a 96-well plate at 8x104 cells/well were incubated with varying amounts of cfIL-22 and equivalent volumes of plant control protein, pBK, (as a negative control) for 24 hours. Resazurin assay and monitoring fluorescence intensity is used as a measure of cell growth and metabolism. Data represent eight biological replicates, error bars represent standard error mean. Difference between pBK and cfIL-22 significant from 100-1500 ng treatments P< 0.05).
Figure 5. IL-22 induced gene expression in catfish cell line.Recombinant cfIL-22 induced the expression of three key response genes-fibronectin, interferon, and NK Lysin-1 in CCO fibroblast cells. IL-22 induction of these genes was dose-dependent (100ng vs 500ng) and compared relative to media only treated cells (NT) and cells treated with a plant protein negative control (pBK). *=p<0.05, **=p<0.01, ***=p<0.001