Expression,purification and biofunctional analysis of mouse soluble IL-15 receptor α-chain

Objective:To express and purify soluble fragment(extracellular domain) of mouse IL-15 receptor α-chain(sIL-15Rα),and to study its ligand binding property and biological function.Methods:The sIL-15Rα gene encoding the extracellular domain of mouse IL-15Rα was obtained by RT-PCR,using total RNA extracted from mouse spleen as the template,and cloned into pQE-30 expressing vector to form pQE30/sIL-15Rα.The plasmid was transfected into Escherichia coli M15 strain.To simplify the following purification procedure,the recombinant strain was induced to express recombinant protein in soluble form under modified conditions.After extracted from induced bacteria,the recombinant 6×His-tagged protein was purified using Ni-NTA affinity chromatography,and further identified and determined by SDS-PAGE and Wes-tern blot.Solid-phase receptor binding assay was used to determine the binding affinity of the recombinant protein to IL-15.CTLL-2 cells were selected as target to perform biological function analysis.Results:The recombinant vector pQE30/sIL-15Rα could release a single DNA fragment of 500 bp after double-digested by restricted endoenzymes.Western blot analysis showed specific band,with using either anti-His monoclonal antibody or anti-IL-15Rα antibody as the first antibody, respectively.Under the conditions of 25℃ and 0.5 mmol/L IPTG,the recombinant protein was expressed in soluble form.After one-step Ni-NTA affinity purification,the recombinant protein with a molecular weight about 29 kD with purity more than 95% was obtained.The recombinant protein,with an affinity about 4.9×10-11 mol/L to IL-15,could be synergetic with IL-15 to stimulate the proliferation of CTLL-2 at the concentration as low as 1 ng/ml.Conclusion:Biofunctional recombinant protein sIL-15Rα with biofunctions is successfully obtained.This provides a useful platform for further research on sIL-15Rα and study targeted to IL-15.