Construction,expression,and immunogenicity identification of STX2A1 subunit protein of EHEC O157∶H7

Objective To express GST-STX2A1 fusion protein and identify the immunogenicity of the protein.Methods The STX2A1 gene was amplified from the EHEC O157∶H7 EDL933 type strain with specific primers.The PCR product was inserted into the prokaryotic expression plasmid PGEX-6p-1 and confirmed by DNA sequencing.The constructed prokaryotic expression vector PGEX-6p-1/STX2A1was transformed into the competent E.coli BL21.The supernatant of the lysates was collected after the resuspension and sonication of the bacteria which were collected after IPTG induction.Then the recombinant GST-STX2A1 fusion protein was purified by GSTrap FF affinity column,desalted by HiTrap desalting column,and analyzed by SDS-PAGE.The antiserum against GST-STX2A1 fusion proteins was prepared by immunizing Balb/c mouse with purified GST-STX2A1 fusion protein.The specificity of the antiserum against GST-STX2A1 fusion proteins was identified by Western blotting.Results The recombinant plasmid PGEX-6p-1/STX2A1 was constructed successfully.The soluble fusion protein GST-STX2A1,whose Mr was about 5.3 ×104,was expressed in E.coli.After purification by affinity column,the purity of the recombinant protein was about 90%.The antiserum against GST-STX2A1 fusion proteins could specifically react with the A subunit of native shiga toxion Ⅱ.Conclusion The high purity fusion protein GST-STX2A1 has been produced successfully,which provides a necessary foundation for preparing O157∶H7 subunit vaccine and monoclonal antibodies against STX2A1 subunit of O157∶H7.