超抗原SED突变体的构建表达和鉴定

Construction and identification of expression of superantigen SED mutants

目的 在预测的SED免疫识别活性位点处引入定点突变 ,构建SED突变体。方法 分别设计侧翼引物和突变引物 ,用megaprimerPCR方法引入突变碱基 ,将PCR产物与pTrcHis B质粒连接 ,转化E .coliDH5α ,用IPTG进行诱导表达 ,金属螯合亲和层析法纯化 ,免疫印迹检测纯化的蛋白。结果 测序结果表明在相应的位置引入了正确突变 ,SDS PAGE和免疫印迹检测结果证实了目的蛋白的表达 ,将构建成功的突变体分别命名为SEDN2 3A、SEDN2 3A/H2 6R、SEDF45A、SEDL5 9A、SEDN61A、SEDI92A和SEDF2 0 3A。结论 成功构建了一系列SED突变体 。

Objective To construct staphylococcal enterotoxin D (SED) mutants expressed in Escherichia coli ( E. coli ). Methods The expected mutants were introduced into the SED DNA by megaprimer PCR method. The PCR products ligated to plasmid pTrcHis B were transformed into E. coli DH5α for IPTG induced expression. The target protein was purified by Ni NTA metal affinity chromatography and analyzed by SDS PAGE and immunoblotting. Results The sequencing results showed that mutant nucleic acids were successfully introduced at the expected sites of SED gene. SDS PAGE and immunoblotting confirmed that the proteins of SED mutants were obtained by Ni NTA metal affinity chromatography. The mutants were named as SEDN23A, SEDN23A/H26R, SEDF45A, SEDL59A, SEDN61A, SEDI92A, and SEDF203A, respectively. Conclusion Several SED mutants are successfully constructed, which lays a foundation for subsequent studies of immune recognition of SED.