Cloning, Expression, Purification And Identification Of IFN-α2a-α-MSH Fusion Protein And Its Biological Function Research

Objective: Interferons-alpha2a (IFN-α2a), a multifunctional regulatory cytokines, has many biological functions, such as antiviral, anti tumor and immunoregulatory effects. It has been approved for the treatment of variety of malignancies and viral diseases. Unfortunately, significant toxicities of IFN-α2a impact on its spreading of clinical application.α-MSH(α-melanocyte-stimulating hormone ) peptide (SYSMEHFRWGKPV)is a kind of peptide hormone, which is secreted by many kinds of cells naturally. It can specific bind to MC-1R (melanocortin receptor 1 ) of melanocyte and melanoma cells,and promote malignant melanoma cell apoptosis. Andα-MSH can inhibit the transfer of skin melanoma through rebuild skin basal membrane. Present studies have shown that all melan- and unmelan-melanoma cells are express MC-1R, and the MC-1R is up-regulated in melanoma cells. There are about 900-5700 binding sites of MC-1R on every human melanoma cell. The express of MC-1R on every normal cell is lower than 100 binding sites .We prepared to use the technique of gene recombination to construct targeting fusion gene expression vector IFN-α2a-α-MSH, and induce it in E.coli to establish a high performance prokaryotic expression system. Methods: Plasmid pET-22b(+)IFN-α2a-NGR was digested with BamH I and Sal I restriction enzymes,and then insertedα-MSH peptide sequences to construct prokaryotic expression vector pET-22b(+)IFN-α2a-α-MSH. The plasmid was transfected into Rosetta-gamiTM2(DE3) cell. IPTG was used to induce fusion protein IFN-α2a-α-MSH expression. The expression of IFN-α2a-α-MSH was detected by SDS-PAGE and Western blot. Then IFN-α2a-α-MSH protein was purified by hydrophobic interaction chromatography (HIC). Used purified IFN-α2a-α-MSH fusion protein to treat malignant melanoma mice model. Then observed and measured the changes of tumor volume, calculated tumor inhibition rates, and statistics analysis. Results: The IFN-α2a-α-MSH fusion protein in form of inclusion body, was successfully and stably expressed in E.coli. After the E.coli being lysed by ultrasonic wave, Phenyl Sepharose 6 Fast Flow (high sub) were used to get the purified recombination protein. Tumor inhibition experiment showed that the inhibition rate of IFN-α2a-α-MSH is obviously higher than IFN-α2a and control group. Conclusions: Succeed acquired recombinant pET-22b(+)IFN-α2a-α-MSH vector, and the stable prokaryotic expression strain, purified IFN-α2a-α-MSH fusion protein, and used the purified protein to treat tumor bearing mice model. In the treatment, we got a high tumor inhibition rates. All the results indicated that it may be a new idea to use recombinant pET-22b(+)IFN-α2a-α-MSH protein to target treat malignant melanoma diseases.