Expression and bioactivity of staphylococcal enterotoxin O / 中华微生物学和免疫学杂志

ABSTRACT

Objective To clone the mature peptide gene of seo, construct two kinds of prokaryotic fusion expression vector, obtain recombinant staphylococcal enterotoxin O(SEO) and evaluate its biological activity. Methods The seo gene was amplified from ZNZ2-3 strain of Staphylococcus xylosus by designed primers and it was cloned into fusion vector pGEX-6P-1 and pET28a. The soluble recombinant protein(GST-SEO) was expressed in E. coli BL21 host cells, the purified GST-SEO was obtained by a single step of affini-ty chromatography using Glutathinione Sepharose 4B colume. Another fusion protein(P28-SEO) with his-tag was expressed in E. coli BL21 ( DE3), the purified P28-SEO protein was harvested by nickel chelate affinity chromatography method. The antigenicity of recombinant SEO was tested by Western blot. The stimulating effects of recombinant SEO on mouse lymphocytes was tested by MTT. The activity of Caspase 3 and the ap-optosis of DNA ladder were tested. Results The nucleotide sequence of the cloned seo gene was the same as that of reported in GenBank. The soluble recombinant protein GST-SEO was expressed at 25% expression level, and P28-SEO was 22%. The Western blot by GST-SEO-positive sera demonstrated that the recombi-nant SEO had good immunogenicity. The MTY assay of the enterotoxin activity showed that low dose of re-combinant SEO stimulated the proliferation of mice spleen lymphocytes, higher doses will lead to apoptosis. Conclusion Two of the recombinant SEO still have superantigen activity, and can lead to apoptosis of mice splenocytes.