Purification Technology And Biological Activity Analysis Of Melittin-～(88)ArgIL-2 Chimeric Protein

Objective We aim to obtain partially soluble expression of the recombinant melittin-⁸⁸ArgIL-2 chimeric protein in an Escherichia coli(E.coli) system under certain conditions, establish purification technology of melittin-⁸⁸ArgIL-2 chimeric protein, obtain the melittin-⁸⁸ArgIL-2 with high purity and study the biological activities of it in vitro. Methods The E. coli DH5a containing the constructed recombinant pGEX-4T-2/ melittin-⁸⁸ArgIL-2 plasmid was induced to express pGEX-4T-2/ melittin-⁸⁸ArgIL-2 by IPTG. Then melittin-⁸⁸ArgIL-2 chimeric protein was purified by the combination of glutathione-containing affinity chromatography, thrombin cleavage and cation chromatography. The purified products were analyzed by SDS-PAGE. The bacteriostasis test of melittin-⁸⁸ArgIL-2 chimeric proteine was performed. The proliferation assay of peripheral blood monouclear cells(PBMC) and cytotoxic activity assay of NK cells were analyzed by using MTT method. The growth inhibiting assay of human ovarian cancer cells SKOV₃ was analyzed by using MTT method. The growth inhibiting assay of Hela cells was analyzed by using MTT method. Results Expression of melittin-⁸⁸ArgIL-2 was achieved by IPTG induction, partially soluble expression of it was achieved under certain conditions. By the combination of glutathione-containing affinity chromatography, thrombin cleavage and cation chromatography, purity of melittin-⁸⁸ArgIL-2 was 95% and concentration of it was 200mg/L. It can inhibit the generation of Staphylococcus aureus and E.coli. The melittin-⁸⁸ArgIL-2 chimeric protein can augment PBMC proliferation and cytotoxic activity of NK cells. It can inhibit the growth of human ovarian cancer cells SKOV₃ and Hela cells in vitro. Conclusin By this work, we established purification technology of melittin-⁸⁸ArgIL-2 chimeric protein. Recombinant melittin-⁸⁸ArgIL-2 chimeric protein with bioactivity was successfully prepared in small scale and purified. It possessed bacteriostatic action, enhancing immune function and anti-tumor activity in vitro. This work laid a foundation of study on the expression in eukaryocyte system, purification, biological activity in vivo, the procedure of pilot and large-scale preparations of the chimeric protein. The melittin-⁸⁸ArgIL-2 chimeric protein probably becomes an original immunomodulator and antitumor drug.