Title Fusion Expression and Immunogenicity of EHEC EspA-Stx2A1 Protein: Implications for the Vaccine Development
Author Yan Cheng, Youjun Feng, Ping Luo, Jiang Gu, Shu Yu, Wei-jun Zhang, Yan-qing Liu, Qing-xu Wang, Quan-ming Zou, and Xu-hu Mao*
Address Department of Clinical Microbiology and Immunology, College of Medical Laboratory, The Third Military Medical University, Chongqing 400038, P. R. China
Bibliography Journal of Microbiology, 47(4),498-505, 2009,
Key Words EHEC O157:H7, EspA, Stx2A1, fusion protein, vaccine
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.