Molecular cloning, expression, and purification of pig interleukin-5

Interleukin-5 (IL-5) is thought to be a key cytokine in allergic inflammati on. Pig IL-5 was cloned, sequenced, and expressed to enable us to study of the biological role of IL-5 in pigs used in a model for allergen-induced la te-phase reactions. These pigs were sensitized to proteins extracted from A scaris suum, resulting in hypersensitivity to this antigen in both the skin and airways, and a slight blood eosinophilia. Peripheral blood mononuclear cells from antigen-sensitized pigs were isolated and polyclonally stimulat ed. Total RNA was extracted and reverse transcribed into cDNA. IL-5 primers based on the cow IL-5 cDNA sequence were used to obtain an initial polymer ase chain reaction product. 3' rapid amplification of cDNA ends (3'RACE) an d 5'RACE procedures were applied to identify the 3' and 5' ends, respective ly. The full-length pig IL-5 cDNA is 405 base pairs long. Mature pig IL-5 w as expressed in Escherichia coli with a His-tag for purification, The IL-5 protein is 115 amino acids long, has an estimated molecular weight of 14000 M-r and forms a biologically active homodimer of 28000 M-r. Pig IL-5 shows 65% amino acid identity to the human IL-5 sequence and 90, 88, 83, 62, and 61% identity to the cow, sheep, horse, mouse, and rat counterparts.