| 研究报告 |
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| Construction，expression and purification of Recombinant Consensus Interferon Mutant Ⅱ |
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Abstract Interferon alpha are used clinically to treat a variety of viral diseases and cancers. They have a short circulating half-life, which necessitates frequent administration to patients. Previous studies showed that it is possible to extend the circulating half-life of interferon alpha by modifying cysteine residues of the protein with poly(ethylene glycol)(PEG) reagents. But protein seldom have unpaird -SH, so we introduce a free cysteine residue into the protein by recombinant DNA technique. Recombinant consensus interferon mutant Ⅰ（IFN-Con-m1）belongs to the family of interferon alpha. It has a wide variety of biological effects that include antiviral, antiproliferative, and immunomodulatory activities. Recombinant consensus interferon mutant Ⅱ (IFN-Con-m2 ) was a cysteine mutant of IFN-Con-m1. The protein has higher specific activity and may be Site-directed modified by PEG. It was constructed by substitution of Tyr at position 86 with Cys using site-directed mutagenesis. The DNA was constructed in pET-23b expression vector, and transformed into E.coli BL21（DE3）. IFN-Con-m2 was expressed as inclusion bodies with the yield of more than 30% of total bacterial protein. The recombinant protein was expressed after IPTG induction and purified by Phenyl Sepharose, DEAE Sepharose FF and Sephacryl S-100. After purification, the purity of IFN-Con-m2 was higher than 95%, and the biological activity was more than 5.0×108IU/mg. Western blot displayed the recombinant product had strong immunological activity with mouse anti-human IFN-Con-m2 monoclonal antibody.
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Received: 11 August 2006
Published: 25 December 2006
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