Abstract

The ability to detect inflammatory disease at an early stage in marine mammal species would have a useful application in captive, rehabilitation and field situations. To date there are almost no marine mammal specific reagents readily available to detect the presence of inflammatory mediators (cytokines, C-RP, etc.) in peripheral blood. Additionally, cytokine-specific antibody reagents established for other species rarely cross-react with marine mammals. This research report describes the first expression of recombinant killer whale (Orcinus orca), harbor seal (Phoca vitulina) and sea otter (Enhydra lutris) interleukin 6 (rIL-6). These proteins are being used to produce IL-6 specific antibodies for development of rapid antigen capture ELISA's (enzyme linked immunosorbent assay).

IL-6, a well studied cytokine, exhibits a number of effects in mammals, which together contribute to the early acute phase response. The acute phase response can be defined as the reaction of animals to disturbances of their homeostasis (internal environment) due to infection, tissue injury or immunologic disorder. Typical systemic effects of IL-6 are synthesis of acute phase proteins, platelet production, T- and B-lymphocyte activation and the increase in immunoglobulin synthesis.

Based upon our previous studies, the nucleotides coding for killer whale, harbor seal and sea otter IL-6 were cloned into pET vectors. A well established prokaryotic expression system (BL21[DE3]LysS) was transfected with the vector containing the fragment encoding IL-6. Synthesis of killer whale (rKWIL-6), harbor seal (rHSIL-6) and sea otter (rSOIL-6) recombinant IL-6 was induced by IPTG, a commonly used reagent that controls the expression of the target protein. SDS-polyacrylamide gel electrophoresis demonstrated that recombinant IL-6 of appropriate molecular weight represented the majority of protein present in isolated bacterial inclusion bodies. After purification, biological activity of the recombinant proteins was demonstrated by their ability to sustain the growth of an IL-6 dependent mouse cell line.

In order to develop a fast, sensitive and easy to perform assay to detect IL-6 levels in body fluids, the production of antibodies to rIL-6 is underway. These IL-6 specific monoclonal antibodies are necessary to develop species specific antigen-capture ELISA. These ELISA's will be used as diagnostic tests that can be easily applied in captive, rehabilitation and field situations.