Fusion expression and immunogenicity of EHEC EspA-Stx2Al protein: Implications for the vaccine development

Shiga toxin 2 (Stx2) is a major virulence factor for enterohemorrhagic Escherichia coli (EHEC), which is encoded by λ lysogenic phage integrated into EHEC chromosome. Stx2Al, Al subunit of Stx2 toxin has gathered extensive concerns due to its potential of being developed into a vaccine candidate. However, the substantial progress is hampered in part for the lack of a suitable in vitro expression system. Here we report use of the prokaryotic system pET-28a::espA-Stx2Al/BL21 to carry out the fusion expression of Stx2Al which is linked to E. coli secreted protein A (EspA) at its N-terminus. Under the IPTG induction, EspA-Stx2Al fusion protein in the form of inclusion body was obtained successfully, whose expression level can reach about 40% of total bacterial protein at 25°C, much higher than that at 37°C. Western blot test suggested the refolded fusion protein is of excellent immuno-reactivity with both monoclonal antibodies, which are specific to EspA and Stx2Al, respectively. Anti-sera from Balb/c mice immunized with the EspA-Stx2Al fusion protein were found to exhibit strong neutralization activity and protection capability in vitro and in vivo. These data have provided a novel feasible method to produce Stx2Al in large scale in vitro, which is implicated for the development of multivalent subunit vaccines candidate against EHEC 0157:H7 infections.