Molecular Cloning of Harbor Seal Interleukin-1 Beta (IL-1β) From a LPS-Stimulated Mononuclear Cell CDNA Library
IAAAM Archive
Mary Bozza1; Shannon Atkinson2
1Alaska SeaLife Center, Seward, AK, USA; 2Alaska SeaLife Center and Institute of Marine Science, University of Alaska Fairbanks, Seward, AK, USA


The harbor seal (Phoca vitulina richardsi) population in the Gulf of Alaska has decreased dramatically in the past 3 decades. Since the mid-1970s, a nearly 90% decline has been documented at Tugidak Island.1 The causes for their continued decline remain unknown, although environmental stress, including exposure to contaminants, is a potential contributing factor. Environmental contaminants, including organochlorines, are known to induce immune suppression in mammals.2 Correlation of body burdens of contaminants with possible toxic effects, including immune function disruption, will elucidate the potential role of contaminants in the harbor seal population decline. To further characterize the harbor seal immune system, we are developing species-specific immunoassays to measure concentrations of cytokines including interleukin-1 beta (IL-1β) in harbor seal serum and in vitro mononuclear cell cultures. Cytokines are immune modulators produced in response to infection or inflammation and are important indicators of immune function in humans, experimental and domestic animals, and increasingly in wildlife species.

Mononuclear cells from an adult female harbor seal at the Alaska SeaLife Center (Seward, AK) were stimulated in vitro with lipopolysaccharide (LPS). Total RNA was isolated, and a cDNA library was constructed in the plasmid pDNR-LIB and transformed into E. coli DH5α. Plasmid from the cDNA library was analyzed by PCR (polymerase chain reaction) using oligonucleotide primers specific for Steller sea lion (Eumetopias jubatus) IL-1β (Bozza, unpublished). These primers amplified several over-lapping PCR products spanning the protein coding region of the cDNA. The PCR products were sequenced and a 813 base pair, full-length cDNA was identified as harbor seal IL-1β based on sequence homology to Steller sea lion (97%), dolphin (Tursiops truncatus) (78%), beluga (Delphinapterus leucas) (78%), and human (Homo sapiens) (77%) IL-1β at the nucleotide level. The predicted amino acid sequence for the 270 amino acid harbor seal IL-1β pro-protein was 94% (Steller sea lion), 63% (dolphin), 63% (beluga), and 63% (human) homologous (dolphin and beluga are 96% homologous). Subsequent expression and purification of IL-1β protein will allow validation of commercially available, cross-reactive antibodies for immunoassay development. Assays will be valuable worldwide in assessing the potential effects of environmental contaminants on immune function in harbor seal populations and as a diagnostic tool for veterinarians managing captive or stranded harbor seals.


The authors thank the Alaska SeaLife Center Veterinary Services and Mammal Departments, Anne Hoover-Miller and Carol Stephens.


1.  Pitcher KW. 1990. Major decline in numbers of harbor seals (Phoca vitulina richardsi) in the Gulf of Alaska. Marine Mammal Science 6; 121-134.

2.  Safe S. 1994. Polychlorinated biphenyls (PCBs): Environmental impact, biochemical and toxic responses, and implications for risk assessment. Critical Reviews in Toxicology; 24(2):87-149.

Speaker Information
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Shannon Atkinson

Mary Bozza