Construction and identification of expression of superantigen SED mutants / 第三军医大学学报

ABSTRACT

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 DH5? 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.