Molecular cloning and expression of rat IL-10cDNA

Objective:To clone the full length of rat interleukin-10 cDNA and make it express in E.coli. Methods: The rat spleen was removed and spleenocytes were harvested under sterilizing condition. Cultured under the stimulation of LPS for 4 hours, the spleenocytes were collected and were used for extraction of total UNA.The cDNA of rat IL-10 was cloned from RNA by RT-PCR and subcloned into pJW2 vectors. By transferring E.coli DH5a, recombinant pJW2-IL,(encoding IL-10)vectors were prepared for identification and sequensing.and its expression was carried out under heat induction.Results:The transcription of IL-10 in spleenocytes stimulated by LPS was increased and the expected band was readily produced by RT-PCR. It was confirmed that the sequence of rat IL-10 cDNA cloned was identical to that reported in Genbank by gene sequensing. SDS-PAGE analysis showed that the recombinant pJW2 IL-10 vectors could express a 18.6 kD protein which accounted for about 20% of the total cellular protein. Western blot analysis showed that anti-IL-10 specifically bound to the 18.6 kD band of expression product. Conclusion: The full-length sequence of rat IL-10 cDNA is successfully cloned and it can express in E.coli.