Expression and bioactivity analysis of an soluble extracellular fragment of human AITRL

Objective:To obtain recombinant protein of an soluble extracellular fragment of activation-inducible tumor necrosis factor ligand ( sAITRL), and to analyze its bioactivity.Methods:sAITRL gene was amplified by PCR from the full-length plasmid AITRL-pMD18-T and subcloned into the pQE30 vector. The recombinant plasmid was transformed into E.coli M15, and the expression of recombinant protein was induced by IPTG. After purified and refolded with Ni2+-IMAC column, identification of the recombinant protein by Western blot was performed. The binding ability of sAITRL protein to natural AITR was evaluated by FCM, and the bioactivity was estimated by proliferation assay in vitro.Results:The prokaryotic expression vector for sAITRL-pQE30 was constructed successfully. sAITRL protein with molecular mass about 15 kD was expressed in E.coli M15 after IPTG induction. This recombinant sAITRL,which was able to bind to natural AITR on surface of activated lymphocytes,significantly stimulated proliferation of lymphocytes at low doses, but had an opposite effect at higher concentrations.Conclusion:Recombinant sAITRL with bioactivity is expressed successfully. AITR-AITRL interaction appears to be crucial in regulation the ongoing immune responses.