Gene Cloning, Fusion Expression and Purification of Human Interleukin-24

Objective To clone and express human interleukin-24 gene and purify the expressed product.Methods Isolate human peripheral blood mononuclear cells(PBMCs) and culture under stimulation of Con A.Extract the total RNA of the cultured PBMCs for amplification of the gene encoding the mature peptide of hIL-24 by RT-PCR.Directly clone the amplified gene into fusion expression vector pGEX-4T-1.Identify the recombinant plasmid pGEX-4T-1/hIL-24 by DNA sequencing,then transform to E.coli BL21(DE3) for expression under induction of IPTG.Purify the expressed product by anion exchange chromatography and identify by SDS-PAGE and Western blot.Results The sequence of the cloned hIL-24 gene was consistent with that reported in GenBank.The identification by sequencing showed that fusion expression vector pGEX-4T-1/hIL-24 was correctly constructed.The fusion protein,with a relative molecular weight of 44 000,was expressed in form of inclusion body and contained 30.63% of total somatic protein.The purity of the expressed protein after purification reached more than 85%.Western blot showed specific reaction of the expressed fusion protein with rabbit anti-human IL-24 polyclonal antiserum.Conclusion The fusion expression vector pGEX-4T-1/hIL-24 was successfully constructed,and hIL-24 was highly expressed in E.coli.The fusion protein with high purity was obtained.It laid a foundation of study on function and activity of hIL-24.