超抗原SED在大肠杆菌中的表达

Expression of superantigen SED in Escherichia coli

目的构建稳定的SED原核表达系统。方法采用PC R技术扩增葡萄球 菌D型肠毒素（SED）超抗原的成熟蛋白编码区DNA序列，构建SED DNA与6个组氨酸基因融合的表达载体pTrcHis-SED，转入E.coli DH5α，IPTG诱导后，用SDS-PAGE和免疫印迹 检测融合蛋白的表达情况。目的蛋白用Ni-NTA金属亲和层析法进行纯化，SDS-PAGE和毛细管电泳检测蛋白纯度。结果成功构建了原核表达载体pTrcHis-SED。分子量 约 为28 000 u的6His-SED融合蛋白可在E.coli DH5α中稳定表达。Ni-NTA金属亲和层析 后得到 纯度较高（>95%）的SED融合蛋白，且具有免疫学活性。结论本研究为SE D免疫识别研究奠定了实验基础。

Objective This study was designed to construct prokaryotic expression system of superantigen staphylococcal enterotoxin D( SED ). MethodsDNA encoding mature protein of SED was cloned into vector pTrcHis-B to fuse with 6His-tag gene and transformed into E.coli DH5α.Following IPTG induction, the expression of 6His-SED fusion protein was analyzed by SDS-PAGE and immunoblot. The purity of target protein purified by Ni-NTA metal-affinity chromatography was determined by SDS-PAGE and capillary electrophoresis. Results We constructed successfully the express vector pTrcHis-SED. 6His-SED fusion protein, having a molecular weig ht about 28 000 u, was expressed stably in E.coli DH5α.The high purity(>95%)of protein was obtained by Ni-NTA metal-affinity chromatography. Conclusions The prokaryotic expression of superantigen SED might provide experimental materials for further study on immune recognition of SED.