Over expression,purification and defection of antivirus activity and antiproliferative activity of a novel engineered mutant interferon alpha-m

This study was to express,purify,analyse anti virus activity and antiproliferative activity of a novel engineered mutant human interferon alpha m(IFNα m).Expression vector pBV220/IFNα m was obtained by subcloning IFNα m gene into vector pBV220 via polymerase chain reaction from PUC 18/IFNα m(IFNα m cDNA had been changed according to bacterial codon preference to direct toward over expression),and transformed in E.coli DH5α.Under thermal induction at 42℃ for 4 5 hours,cultures resulted in the production of IFNα m which was produced exclusively in the form of insoluble inclusion bodies.Inclusion bodies were solubilized under denaturing conditions and refolded with suitable methods.Following refolding,purification of IFNα m was carried out subsequently by ion exchange chromatography using CM Sepharose FF,DEAE Sepharose FF as well as by size exclusion chromatography using Sephacyral HR100.The antiviral(AV)activities of purified IFNα m were compared with those of IFNα m on human WISH cell line by a bioassay of a cytophathic effect reduction assay.The antiproliferative activities of purified IFNα m were compared with IFNα b by MTT test on Hela cell line.Dry weight of inclusion bodies accounted for 40% of total protein in genetically transformed Escherichia coli cells,as determined by SDS PAGE scanning.By purification,the purity was more than 95%,IFNα m near to homogeneity was produced.The established purification process is reproducible and leads to a total recovery of 34.4%.The specific antiviral activity of purified IFNα m is 1.26×10 7 IU/mg protein,which is comparable to that of its counterpart IFN α 1b.IFNα m possesses the antiviral activity same as IFNα b,and its antiproliferative activity is higher than that of IFNα b.