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 lambda 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 degrees C, much higher than that at 37 degrees 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.