葡萄球菌肠毒素B突变体的构建及其抗肿瘤活性分析

Construction and Anti-Tumor Activity Analysis of Staphylococcus Enterotoxin B Mutant

目的:通过对葡萄球菌肠毒素B(SEB)32位His进行定点突变,获得抑瘤效果显著增强的SEB突变体。方法:利用基因定点突变的方法,将SEB的32位His替换为Asn,将重组质粒转入大肠杆菌中诱导表达,用CM弱阳离子层析柱纯化获得重组蛋白,用SDS-PAGE和Western印迹对其进行鉴定,并用增殖实验检测其丝裂原活性,通过MTS法检测其体外抗肿瘤活性。结果:构建并高效表达了突变体蛋白SEB(H32N),纯化获得了足够纯度的突变体蛋白;体外实验表明,在相同浓度下,SEB(H32N)的丝裂原活性及体外抗肿瘤活性明显优于野生型SEB。结论:同野生型SEB相比,突变体蛋白SEB(H32N)对肿瘤生长的抑制作用得到了提高。

Objective： The anti-tumor activity of staphylococcal enterotoxin B（SEB） was enhanced, by substitut- ing His32 of SEB for Ash through site-directed mutagenesis. Methods： A mutant superantigen SEB（H32N） was constructed by site-directed mutagenesis, the protein was expressed in E.coli BL21 after induced, purified by CM ion-exchange chromatography and identified by SDS-PAGE and Western blot. Mitogen biological activity was ana- lyzed by cell proliferation assay in vitro, and the anti-tumor activity was analyzed using MTS method in vitro. Re- sults： The mutated protein SEB（H32N） was expressed in E.coli efficiently and purified. The results showed that at the same concentration, the mitogen biological activity and anti-tumor effect of SEB（H32N） were better than SEB on a variety of tumor cells in vitro. Conclusion： Compared with the wild type SEB, SEB（H32N） mutant ex- hibited an enhanced anti-tumor effect.