Fusion Expression and Immunogenicity of EHEC EspA-Stx2A1 Protein: Implications for the Vaccine Development
Fusion Expression and Immunogenicity of EHEC EspA-Stx2A1 Protein: Implications for the Vaccine Development
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EHEC EspA-Stx2A1 蛋白的融合表达和免疫原性：对疫苗开发的影响
DOI：
10.1007/s12275-009-0116-8
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发表时间：
2009-08-01
影响因子：
3
通讯作者：
Mao, Xu-hu
中科院分区：
文献类型：
--
作者：
Cheng, Yan;Feng, Youjun;Mao, Xu-hu
滋贺毒素2（Stx 2）是肠出血性大肠杆菌（EHEC）的主要毒力因子，由整合在EHEC染色体上的λ溶原性噬菌体编码。Stx 2A 1是Stx 2毒素的所有亚单位，由于其潜在的开发成为候选疫苗而受到广泛关注。然而，由于缺乏合适的体外表达系统，其实质性进展受到阻碍。利用原核表达系统pET-28 a：：espA-Stx 2A 1/BL 21进行Stx 2A 1与大肠杆菌的融合表达。大肠杆菌在其N端分泌蛋白A（EspA）。在IPTG诱导下，成功获得了包涵体形式的EspA-Stx 2A 1融合蛋白，在25 ℃时表达量可达菌体总蛋白的40%左右，远高于37 ℃时的表达量。Western blot结果表明，复性后的融合蛋白与EspA和Stx 2A 1单克隆抗体均具有良好的免疫反应性。用EspA-Stx 2A 1融合蛋白免疫Balb/c小鼠，获得的抗血清在体内外均表现出较强的中和活性和保护能力。本研究为进一步研究肠出血性大肠杆菌O 157：147多价亚单位疫苗提供了一种新的、可行的体外大规模生产方法。
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. Stx2A1, All 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 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 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 O157:147 infections.