MOLECULAR-CLONING AND EXPRESSION OF DNA ENCODING OVINE INTERLEUKIN-2

We have generated DNA encoding the mature form of ovine interleukin 2 (IL-2) by polymerase chain reaction (PCR) using primers complementary to sequences at the 5' and 3' ends of human, murine and bovine IL-2 cDNA, The predicted PCR product of 400 bp was ligated into the yeast Ty-P1 galactose-inducible expression vector pOGS40 which was used to transform yeast spheroplasts. The fusion protein, with a Factor Xa proteolytic cleavage site between ovine IL-2 and the F1 fusion partner, was expressed from galactose-induced transformed yeast. P1:IL-2 fusion protein, which self-assembles into virus-like particles (VLPs) due to the interaction of the F1 protein, was purified from lysates of mechanically disrupted yeast by centrifugation on a discontinuous sucrose gradient, Fusion protein was detected in Western blot analysis with polyclonal antisera raised to recombinant bovine IL-2, Soluble recombinant ovine IL-2 was released from the F1 fusion protein by cleavage with Factor Xa enzyme, After purification recombinant ovine IL-2 was functionally active as shown by its ability to support the proliferation of Con A-activated T cells and was capable of generating maedi visna virus-specific cytotoxic T cells from primed precursor cells, The availability of recombinant ovine IL-2 will greatly help the analysis of the specificity of pathogen-specific cells in the sheep.