101. Preparation of recombinant rat interleukin-5 by baculovirus expression system and analysis of its biological activities
- Author
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K, Ishihara, I, Satoh, T, Nittoh, T, Kanaya, H, Okazaki, T, Suzuki, T, Koyama, T, Sakamoto, T, Ide, and K, Ohuchi
- Subjects
B-Lymphocytes ,Cell Survival ,Blotting, Western ,Cell Differentiation ,Bombyx ,Recombinant Proteins ,Cell Line ,Rats ,Colony-Forming Units Assay ,Eosinophils ,Hemolymph ,Animals ,Electrophoresis, Polyacrylamide Gel ,Cloning, Molecular ,Interleukin-5 ,Baculoviridae ,Cell Division - Abstract
Rat interleukin-5 (IL-5) cDNA was subcloned from peritoneal cells collected 4 h after intraperitoneal injection of Ascaris suum antigen solution into the immunized rats. Cysteine proteinase-deleted (CPd) rat IL-5 recombinant virus was constructed by inserting rat IL-5 cDNA into CPd virus having a deletion in the cysteine proteinase gene of the silkworm Bombyx mori nuclear polyhedrosis virus. On infection with the CPd rat IL-5 recombinant virus, the silkworm B. mori larvae produced rat IL-5 as a dimeric form in hemolymph. Recombinant rat IL-5 was purified more than 95.5% by anion-exchange chromatography and hydrophobic chromatography. The purified recombinant rat IL-5 promoted the proliferation of T88-M cells in a concentration-dependent manner, and its effect was inhibited by an anti-murine IL-5 neutralizing polyclonal antibody. When bone marrow cells from normal rats were incubated with recombinant rat IL-5 in medium containing methylcellulose, the colony formation by eosinophilic cells was induced. Furthermore, when rat peritoneal eosinophils were incubated with recombinant rat IL-5, the spontaneous decrease in the eosinophil viability was inhibited in time- and concentration-dependent manners. In addition, the recombinant rat IL-5-induced eosinophil survival was inhibited by an anti-murine IL-5 neutralizing polyclonal antibody. These findings suggest that rat IL-5 acts as B-cell growth factor II (BCGF-II), eosinophil differentiation factor (EDF), and eosinophil survival-enhancing factor.
- Published
- 1999