Objective: To express and identify enterohemorrhagic Escherichia coli (EHEC) O157:H7 Shiga toxin 1 A subunit (Stx1A).
Methods: Stx1A encoded gene was amplified from EHEC O157:H7 genome by PCR, confirmed by sequencing and cloned into vector pET-22b(+). The recombinant plasmid pET-22b(+)-Stx1A was transformed into E.coli BL21(DE3) which was induced by IPTG to express the target protein. After purified by AKTA(TM);-His affinity chromatography, the recombinant protein was identified by mass spectrometry. With the recombinant protein, BALB/c mice were immunized to develop the anti-sera and evaluate its specific reaction with the natural Stx1A by Western blotting.
Results: The Stx1A gene with a size of 945 bp was amplified and cloned into prokaryotic expression vector pET22b(+) to form pET-22b(+)-Stx1A. The recombinant protein was effectively expressed in E.coli BL21(DE3) and purified by 6×His-based affinity chromatography. The mass spectrometry analysis showed that the target protein was Stx1A. Western blotting demonstrated that its immunized sera could react specifically with the natural Stx1A.
Conclusion: The EHEC O157:H7 Stx1A gene was successfully cloned and expressed, which laid a solid foundation for the following researches.