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 P1 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 P1 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 P1 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.
Bujdoso
R
R
Department of Veterinary Pathology, Summerhall, University of Edinburgh.
Williamson
M
M
Roy
D
D
Hunt
P
P
Blacklaws
B
B
Sargan
D
D
McConnell
I
I
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D003602:Cytotoxicity, Immunologic; D004247:DNA / Q000096:biosynthesis*; D000939:Epitopes; D005815:Genetic Code; D007376:Interleukin-2 / Q000096:biosynthesis / Q000235:genetics*; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000096:biosynthesis; D012441:Saccharomyces cerevisiae; D017386:Sequence Homology, Amino Acid; D012756:Sheep; D013601:T-Lymphocytes / Q000276:immunology*
D000939:Epitopes; D007376:Interleukin-2; D011994:Recombinant Proteins; D004247:DNA