Construction, Expression And Purification Of Human Interleukin-24

Hman interleukin-24 (hIL-24), also known as melanoma differentiation antigen 7 (mda-7), is a member of the IL-10 family of cytokines. is stably expressedin human tissues associated with the immune system such as the spleen,thymus,peripheral blood leukocytes and normal melanocytes.Subtraction hybridization applied to human melanoma cells induced to terminally differentiate by treatment with fibroblast interferon (IFN-β) plus mezerein (MEZ) permitted cloning of melanoma differentiation associated (mda) genes. Founded on its novel properties, one particular mda gene, mda-7, now classified as a member of the interleukin (IL)-10 gene family (IL-24) because of conserved structure, chromosomal location, and cytokine-like properties has become the focus of attention of multiple laboratories. When administered by transfection or adenovirus-transduction into a spectrum of tumor cell types, melanoma differentiation associated gene-7/interleukin-24 (mda-7/IL-24) inhibiting cell growth, inducing apoptosis, inducing PMBC to secrete IL-6,TNF-α,IFN-r,IL-12 and inhibiting blood vessel formation whereas no toxicity is apparent in normal cells. Based on these remarkable attributes and effective antitumor therapy in animal models, this cytokine has taken the important step of entering the Phase I clinical trial and he Phaseâ…¡clinical trial. Our trial has been done for lots of the protein of IL-24.Objective To construct a recombinant expression vector of (IL-24) gene and express it in E. coli M15, and to evaluate the bioactivity of IL-24 fusion protein.Methods: The encoding sequence for mature peptide of human h-IL-24 was Amplified from plasmid by polymerase chain reaction (PCR) and inserted into pQE30 vector to establish the prokaryotic expressing system coupled with 6His. The competent cells of host strain of M15 were transformed by the recombinant plasmid (pQE-H-IL-24). Expression of the target protein was induced with IPTG and assayed by SDS-PAGE and immunoblot after sequencing. The recombin-ant products were purified by Ni²⁺-NTA agarose column.Results: The cloned fragment of human interleukin-24 was 100% consistent with that in genebank (NM006850), which was deduced to express 174 animo acids mature peptide of human interleukin-24 correctly. The expressed fusion-protein was 18 400kD in SDS-PAGE as expected.Conclusion: The recombinant expression vector PQE-IL-24 has been constructed successfully and has efficency expression in E coli.M15. The experiment is helpful for the further study of the biological function of IL-24.