葡萄球菌B型肠毒素突变体SEB(Y89A,C93S,Y94A)对小鼠的免疫保护作用

Protection Effect of Staphyloccocal Enterotoxin B Mutant SEB(Y89A,C93S,Y94A)on Mice

目的:评价葡萄球菌B型肠毒素(SEB)突变体SEB(Y89A,C93S,Y94A)作为超抗原疫苗候选分子对小鼠的免疫保护作用。方法:制备具有一定纯度和活性的突变体蛋白SEB(Y89A,C93S,Y94A)样品,灭活后免疫BALB/c小鼠,待小鼠抗体水平上升后,再以野生型SEB(wt-SEB)攻击用D-半乳糖胺致敏的BALB/c小鼠,评价该突变体蛋白的免疫保护作用。结果:突变体蛋白SEB(Y89A,C93S,Y94A)在重组大肠杆菌DH5α中得到表达,主要以包涵体形式存在,经变性、复性、SephacrylS200凝胶过滤,制备成较高纯度(95%)的、具有与wt-SEB相同抗原性的突变体蛋白样品,甲醛灭活后免疫BALB/c小鼠至4周,ELISA法测定小鼠抗体效价水平可达106;进而以wt-SEB攻毒,在达8倍LD50的攻击下,阴性对照小鼠在24h内全部死亡,而SEB(Y89A,C93S,Y94A)组与wt-SEB组小鼠至48h仍有存活。结论:突变体蛋白的保护效果与wt-SEB相类似,有望成为SEB减毒疫苗候选分子。

Objective： To evaluate the protection effect of staphylococcal enterotoxin B（SEB） mutant SEB（Y89A,C93S, Y94A） on mice. Methods： The mutant protein was expressed and purified. And this protein was inactived in 1% formaldehyde solution and immunized BALB/c mice. After the antibody titers of them raised, the protection of this mutant protein was evaluated in the D-galactosamine sensitized BALB/c mice model. Results： The mutant protein was over-expressed in recombinant E.coli DH5α. The product, which was existed in inclusion body, was renatured and purified in Sephacryl S200, and the higher purity（95%） protein sample with the same antigenicity as wild-type SEB（wt-SEB） was obtained. The antibody titre of the mouse immunized by toxoids raised to 106 by ELISA, the protection of SEB is similar to that of wt-SEB, even challenged with 8×LD50 Conclusion： The mutant protein SEB（Y89A,C93S,YggA） was a prospective candidate molecule of superantigen SEB vaccines.