Journal of Microbiology

Research Support, Non-U.S. Gov't

Fusion Expression and Immunogenicity of EHEC EspA-Stx2A1 Protein: Implications for the Vaccine Development: Yan Cheng , Youjun Feng , Ping Luo , Jiang Gu , Shu Yu , Wei-jun Zhang , Yan-qing Liu , Qing-xu Wang , Quan-ming Zou , Xu-hu Mao; J. Microbiol. 2009;47(4):498-505. Published online September 9, 2009; DOI: https://doi.org/10.1007/s12275-009-0116-8

Abstract: 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. Stx2A1, A1 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-Stx2A1/BL21 to carry out the fusion expression of Stx2A1 which is linked to E. coli secreted protein A (EspA) at its N-terminus. Under the IPTG induction, EspA- Stx2A1 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 Stx2A1, respectively. Anti-sera from Balb/c mice immunized with the EspA-Stx2A1 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 Stx2A1 in large scale in vitro, which is implicated for the development of multivalent subunit vaccines candidate against EHEC O157:H7 infections.

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