Purification of Naturally Produced Avian Cytokines

Recombinant cytokines could be used to augment immune responses during periods of bird production such as the embryonic/early posthatch stage when birds are most susceptible to disease. However, only seven avian cytokines have been cloned and no recombinant avian cytokines are presently available in sufficient quantities for testing in the prevention and therapy of poultry diseases. Cytokines from other species have limited or no activity in avians. Attempts to clone the avian cytokine cDNA's for Interleukin-2 and interferon-y based on the known sequences of mammalian cytokines have been remarkably unsuccessful suggesting that the avian homologs have little sequence homology to their mammalian counterparts. Therefore, a cloning strategy employing primers/probes based on the amino acid sequences of purified natural cytokines appears to be necessary. The purification of naturally produced avian cytokines from cell conditioned media sources for the purpose of generating amino acid sequence data and/or antibodies is described in this Phase I proposal. EMBREX is unique in having available in the form of its immunomodulator product ample quantities of cytokine containing cell conditioned media derived from Con A stimulated chicken spleocytes as source material for further cytokine purification. Reverse phase high performance liquid chromatography (RP-HPLC) will be used as the primary step to fractionate cytokine activities. Cytokines will be purified from RP-HPLC fractions by additional chromotography steps monitored by a combination of protein determinations, bioassays and SDS-PAGE.