Site-directed mutagenesis of staphylococcal enterotoxin C2 His118

Objective To obtain staphylococcal enterotox in(SE)without any side-efffects by selecting His118 of SEc2 as substitutional site through site-directed mutagenesis so as to preserve superantigenic activity.Methods His118 was substituted through PCR.The resultant plasmid harboring the mutated SEc2 was expressed in E.coli under the existance of IPTG.The proteins purified through the Ni-NTA affinity chromatography and Gel filtration chromatography were used to analyze superantigenic activity and anti-tumor effect.Results Site-directed mutagenesis was perfo rmed using MutanBEST kit through PCR.The resultant plasmid pET-28a-SEC2(H118Y)was efficiently expressed in Escherichia coli with 1 mMIPTG.The purified SEC(H118Y)exhibited the same superantigenic activity as SEC2 according to the results of PBMC proliferation activity when the dose was between 2 ng and 200 ng.SEC(H118Y)also exhibited the same anti-tumor effect as SEC2 when the dose was between 2 ng and 200 ng.The anti-tumor effect of SEC(H118Y)was better than SEC2 when the dose was 500 ng.Conclusion A lthough His118 of SEC2 was mutated,superantigenic activity and anti-tumor effect of SEC(H118Y)was not changed.