金黄色葡萄球菌新型肠毒素Ⅰ型蛋白的表达研究

Study on the protein expression of a new Staphylococcus aureus enterotoxin Ⅰ

为了研究金黄色葡萄球菌新型肠毒素Ⅰ型的检测方法,诱导融合表达并纯化金黄色葡萄球菌肠毒素Ⅰ型蛋白,试验根据Gen Bank中SEⅠ的序列设计一对特异性引物,采用PCR技术从金黄色葡萄球菌中扩增出新型肠毒素Ⅰ型基因,亚克隆入p EASY-E1载体中,构建重组质粒p EASY-E1-SEⅠ,以IPTG诱导表达,镍柱纯化表达蛋白,用His标签单克隆抗体进行免疫印迹分析验证融合蛋白的表达。结果表明:成功扩增出SEⅠ基因,片段大小为507 bp,重组质粒高效表达SEⅠ融合蛋白,经SDS-PAGE鉴定成功表达了分子质量约为26 ku的重组蛋白,Western-blot检测表明目的蛋白具有良好的免疫原性。说明表达系统p EASY-E1/BL21能高效表达金黄色葡萄球菌新型肠毒素Ⅰ蛋白。

To study the detection methods of a new Staphylococcus aureus enterotoxin I （SE I ）, and induce fusion expression and purify the protein of SE I , a pair of specific primers was designed according to SE I gene sequence in the GenBank. A new enterotoxin I gene was am- plified from Staphylococcus aureus using PCR technique, and then was subcloned into vector pEASY -E1 to construct a recombinant plasmid pEASY - El. IPTG was used to induce protein expression, and the expressed protein was purified by Nickel column, and the expression of fu- sion protein was identified by west - blotting analysis with anti - His - Tag monoclonal antibody. The results showed that the SE I gene was successfully amplified, and the size of gene fragment was 507 bp, and recombinant plasmid pEASY - E1 - SE I expressed SE I fusion protein efficiently. The recombinant protein with the molecular mass of about 26 ku was successfully expressed, which was identified by SDS - PAGE. The west - blotting detection showed that the target protein had good immunogenicity. The results indicate that the expression system pEASY - E1 / BL21 can express SE I protein efficiently.