Cloning, expression and purification of recombinant cotton rat interleukin-5

The coding sequence of the hispid cotton rat (Sigmodon hispidus) interleuki n-5 (IL-5) was isolated by a combination of reverse transcription (RT)-PCR and RACE protocols from concanavalin A stimulated spleen cells. The open re ading frame of 399 bp encodes a polypeptide of 132 amino acids. Comparison with the rat, mouse, gerbil and human counterparts revealed 88, 88, 87 and 75% identity at the nucleotide level and 88, 90, 89 and 70% at the amino ac id level, respectively. The entire coding sequence, minus the putative sign al peptide sequence, was inserted into an inducible Escherichia coli expres sion vector. The recombinant protein possessed an expected molecular mass o f 14 kDa and was located in bacterial inclusion bodies. A purification sche me under reducing and denaturing conditions followed by subsequent successi ve dialysis steps led to the recovery of a recombinant dimeric cotton rat I L-5. The biological activity of the recombinant protein was demonstrated in a murine cell line proliferation assay. This activity was specifically inh ibited by rat monoclonal antibodies directed against mouse IL-5. Together with specific antibodies that can be generated easily, cotton rat IL-5 constitutes a useful tool for extending the use of the cotton rat anim al model in the study of various human pathogens. (C) 2000 Elsevier Science B.V. All rights reserved.