Cloning, Expression And Analysis Of Biological Activity On The Fusion Protein Of Recombinant Mutant Human IL-2 And A Tumor-targeting Peptide

Objective:To obtain the fusion protein of recombinant mutant human IL-2 and a tumor-targeting peptide NGR and compare its biological activity with native human IL-2 in vitro.Methods: cDNA encoding rmhIL-2, including the mutant residues of Asn88Arg, Cys125Ala, in the clone vector pGEM-3zf(-) as a EcoR I–Pst I fragment, was cloned into the expression vector pET-22b(+)-NGR as a NdeI–BamHI fragment. Meanwhile, cDNA encoding NGR had been cloned into the expression vector pET-22b(+)-NGR as a BamHI-Pst I fragment. After identification by sequencing, the recombinant vector, named pET-22b(+)-rmhIL-2-NGR ,transformed into host E.coli BL21(DE3)for expression under induction of IPTG. The product of expression was disrupted, washed, refolded and purified by anion exchange chromatography. At last, we tested its immunological activity by Western blot and biological activity by CTLL-2 proliferative assay.Results: The recombinant fusion protein was shown to be insoluble and in an inclusion body form, which MW was about 17kDa as expected. After densitometry of gel identification, we concluded that after 4 hours induction, the targeted protein was highest expressed, which occupied more than 40% of the total bacterial protein. After identified its immunological activity by Western blot, the targeted protein showed immunogenicity with anti-IL-2 mAb and anti-NGR mAb.