ABSTRACT
<p><b>BACKGROUND</b>Based on the earlier mapping of the epitope recognized by neutralizing antibody, the authors directly replaced binding domain of IFN in AB-loop for enhancement of biological activity.</p><p><b>METHODS</b>Two unique restriction sites (EcoR? and BsE?) were created into region flanking LoopAB. Casette mutagesis, restriction enzyme digestion, DNA sequencing, antiviral activity assay and antiproliferative activity assay have been used in the project.</p><p><b>RESULTS</b>The mutated residues M31?D,D32?P of LoopAB in parent IFN were produced. The recombinant phagemid pCANTAB5E/3132IFN 1c/86D and expression plasmid PBV322-132IFN 1c/86D were constructed respectively by replacing the corresponding LoopAB with DNA fragment mutated in the residues M31?D,D32?P, which have been confirmed. The recombinant protein has been expressed in E.coli JM103. The crude 3132IFN 1c/86D has been assayed on human WISH cells challenged with VSV and on HeLa cells by colorimetric MTT. 3132IFN 1c/86D showed 8-old antiviral activity compared to that of parent IFN 1c/86D, while IFN?induced growth inhibition of both types had no difference.</p><p><b>CONCLUSIONS</b>The authors concluded that a mutant IFN with enhanced antiviral activity can be obtained via a targeted replacement of receptor binding domain in AB-loop.</p>
Subject(s)
Humans , Amino Acid Substitution , Antibodies, Viral , Antiviral Agents , Pharmacology , Cells, Cultured , DNA Mutational Analysis , Interferon Type I , Genetics , Pharmacology , Interferon-alpha , Mutagenesis, Site-Directed , Mutation , Peptide Fragments , Genetics , Pharmacology , Plasmids , Genetics , Receptors, Interferon , Metabolism , Recombinant ProteinsABSTRACT
Aim To express and purify a new sub-type of interferon α , α 1c in E.coli. Methods To establish a brief protocol for expression and purification of IFN-α 1c. In vitro assay for the IFN activity is required for the measurement of antiviral, antiproliferative and immunoregulatory properties. Results Expression of IFN-α 1c was verified by its reactivity to IFN-α 1-specific neutralizing antibodies by ELISA. Western blot also detected a band with relative molecular mass(Mr) of 19 000. IFN-α 1c possessed antiviral biological activity(3.2× 107) higher than IFN-α 1b(1.0× 107～ 1.8× 107) did on VSV challenged WISH cells. IFN-α 1c also inhibited growth rate of A549 comparable to IFN-α 1b. IFN-α 1c as an immunologic effector also augmented the cytotoxic activity of NK cells. Conclusion These results demonstrate that IFN-α 1c can be successfully expressed in E.coli. This interferon with high bioactivity may be a good candidate for clinical use.