Sequence and characterization of phocine interleukin 2.
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To improve assessment of cellular immune responses in seals, northern elephant seal (Mirounga angustirostris) interleukin 2 (IL-2) has been characterized. The gene was cloned and sequenced from a 658 base pair (bp) cDNA generated from total RNA by reverse transcription-polymerase chain reaction (RT-PCR). The sequence encoded a 154 amino acid (aa) polypeptide that included a 20 aa putative signal peptide. Seal IL-2 was found to share considerable identity with published sequences. Nucleotide sequence analysis of phocine (seal) IL-2 with canine, feline, human, trichechine (manatee), bovine and murine sequences demonstrated 93, 92, 86, 82, 78 and 71% identity, respectively. Analysis of the derived amino acid sequences demonstrated 88, 89, 78, 71, 66 and 60% identity, respectively. Interleukin-2 sequence identities appear to reflect evolutionary proximity among the analyzed species, and importantly, those residues identified as critical to IL-2 biological activity and receptor binding are largely conserved. To examine the kinetics of IL-2 mRNA expression, northern elephant seal lymphocytes were stimulated with the mitogen concanavalin A (Con A), and RNA was collected at several time points thereafter. The RT-PCR demonstrated that seal IL-2 mRNA expression peaks in the first 8 hr following Con A stimulation. Lastly, genomic DNA from northern elephant seal, harbour seal (Phoca vitulina) and California sea lion (Zalophus californianus) was used as template to identify and clone genomic IL-2. Partial sequence of the genomic clones demonstrated nearly complete identity among the three species. Sequence identity indicates that probes constructed from the northern elephant seal IL-2 gene will be effective in assessing IL-2 in other pinniped species.
