INTERLEUKIN-1 RECEPTOR ANTAGONIST - CHARACTERIZATION OF ITS GENE-EXPRESSION IN RABBIT-TISSUES AND LARGE-SCALE EXPRESSION IN EUKARYOTIC CELLS USING A BACULOVIRUS EXPRESSION SYSTEM

The gene expression of rabbit interleukin-1 receptor antagonist (RbIL- 1ra) was examined in rabbit tissues. RNA was isolated from heart, lung , kidney, muscle, liver, spleen, brain, and peripheral blood monocytes (PBMs), and RbIL-1ra mRNA was identified as a single species by North ern analysis using a RbIL-1ra probe. RbIL-1ra was abundantly expressed in lung, brain, heart, and liver, expressed at low levels in spleen, and undetectable in kidney and unstimulated PBMs. Expression of large scale recombinant production of RbIL-1ra was achieved by subcloning th e cDNA into a baculovirus expression vector. Recombination of this vec tor was completed with the BacPAK6 baculovirus genome. The recombinant virus, containing the RbIL-1ra cDNA, was used to infect Spodoptera fr ugiperda (Sf21) insect cells in a spinner flask system and in monolaye rs in cell culture flasks. Recombinant rabbit IL-1ra (rRbIL-1ra) was s ecreted into the culture medium in this system at very high levels (35 mg/l). The protein was identified by reducing SDS-PAGE electrophoresi s, was variably glycosylated and had a molecular weight between 19-25 kDa. It was then purified by size exclusion HPLC on a Du Pont Gf-250 c olumn. The rRbIL-1ra was demonstrated to be functionally active by inh ibiting recombinant human IL-1 alpha in a mouse thymocyte proliferatio n assay. 20 ng/ml (6.7 U/ml) of rRbIL-1ra inhibited 95% of the activit y of 2 ng/ml IL-1 alpha.