Expression in E. coli and purification of the sea bass (Dicentrarchus labrax) interleukin-1β, a possible immuno-adjuvant in aquaculture

摘要

Interleuki-1β (IL-1β) is a pleiotropic cytokine that plays a pivotal role in regulating immune responses and shows a wide range of biological activities. Consistent with a central role in host defence, IL-1β has been postulated as an immuno-adjuvant. This molecule was extensively studied in mammals and in recent years many studies have confirmed the presence of functional IL-1β homologues in fish. Our group has cloned IL-1β from sea bass (Dicentrarchus labrax), the main Mediterranean aquacultured seawater fish species. The cDNA is 1292 bp and codes for a deduced peptide of 29.4 kDa with a pI of 5.1. As for trout and carp IL-1β precursor sequence, no candidate cut site for ICE enzyme was apparent in the alignments of sea bass IL-1β with other mammalian IL-1βs. Nevertheless, a possible mature peptide could start at Ala~(86), giving a protein of 176 aa (with a MW of 19750.17), larger than that seen in mammals. By RT-PCR we have added to the nucleotide sequence coding for this polypeptide Sal I and Sph I restriction sites and the construct was cloned into a Sal I/Sph I cut pQE-30 expression vector. The plasmid was transformed in E. coli and the recombinant protein was partially purified with metal-affinity chromatography in non-denaturing conditions. The rIL-1β has been tested for the induction of phagocytosis to study its biological activity.