EspA-Stx2A1融合蛋白的表达、纯化及其免疫学活性

Expression, Purification and Immunological Activity of EspA-Stx2A1 Fusion Protein

目的在原核表达系统中表达肠出血性大肠杆菌O157∶H7(EHECO157∶H7)Ⅲ型分泌蛋白EspA与Stx2毒素A1亚单位(Stx2A1)的融合蛋白,并对表达蛋白进行纯化及免疫学活性检测。方法PCR扩增espA和stx2A1全长基因,利用重叠延伸PCR技术获得espA-stx2A1融合基因,T-A克隆后插入表达载体pET-28a(+),构建原核表达质粒pET-28a(+)-espA-stx2A1,转化大肠杆菌BL21(DE3),分别在37℃和25℃用IPTG诱导表达。以EspA单克隆抗体亲和层析柱纯化目的蛋白,免疫小鼠,检测其免疫原性及抗血清的反应原性,并以天然Stx2毒素攻击,观察保护效果。通过细胞毒试验检测免疫小鼠抗血清的体外中和作用。结果重叠延伸PCR方法扩增出1319bp的融合基因片段,重组表达质粒构建正确;目的蛋白在25℃时的表达量明显高于37℃,约占菌体总蛋白的40%。两种温度下,目的蛋白均主要以包涵体形式存在;纯化后蛋白纯度可达90%。Western blot结果证实,融合蛋白与EspA单克隆抗体和Stx2A1单克隆抗体均发生特异性反应。融合蛋白免疫小鼠制备的抗血清能分别与O157∶H7的EspA、Stx2A发生特异性免疫反应。融合蛋白免疫小鼠能够抵御致死剂量天然Stx2毒素的攻击,保护率达95%。免疫小鼠血清可以中和天然Stx2毒素对HeLa细胞的毒性作用。结论已成功表达了EspA-Stx2A1融合蛋白,纯化的蛋白显示出较好的免疫保护效果,为研制EHECO157∶H7基因工程多亚单位疫苗奠定了基础。

Objective To express the fusion protein of enterohemorrhagic Escherichia coli （EHEC） O157：H7 type Ⅲ secreted protein EspA and Stx2 toxin A1 subunit （Stx2A1） in prokaryotic expression system, purify the expressed product and determine its immunological activity. Methods Full-length espA and stx2A1 genes were amplified by PCR, based on which fusion gene espA- stx2A1 was obtained by overlapped PCR and, after T-A cloning, inserted into expression vector pET-28a（＋）. The constructed recom- binant plasmid pET-28a（＋ ）-espA-stx2A1 was transformed to E. coli BI21 （DE3） for expression at 37 and 25℃ under induction of IPTG respectively. The expressed protein was purified by affinity chromatography with McAb against EspA. BALB/c mice were im- munized with the purified protein and determined for titer and reactogenicity of antisera, then challenged with natural Stx2 toxin to observe the protective effect of fusion protein. The in vitro neutralizing effect of antisera of immunized mice was determined by toxicity test. Results The fusion gene fragment at a length of 1 319 bp was amplified by overlapped PCR, and recombinant expression vector pET-28a（＋）-espA-stx2A1 was constructed correctly. The expression level of target protein at 25℃ was significantly higher than that at 37℃. Both the expressed proteins at 25 and 37℃ existed mainly in forms of inclusion body, and that at 25℃ contained about 40% of total somatic protein. The fusion protein reached a purity of 90% after purification and reacted specifically with both McAbs against EspA and Stx2A1 as proved by Western blot. The antisera of mice immunized with the fusion protein showed specific immune reac- tions with both EspA and Stx2A of O157：H7. The protective rate of immunized mice against a lethal challenge with natural Stx2 toxin was 95%. The antisera of immunized mice neutralized the toxicity of natural Stx2 toxin to HeLa cells. Conclusion EspA-Stx2A1 fusion protein was successfully expressed and showed good immunological protective effect after purification, which laid a foundation of developing recombinant EHEC O157：H7 muhivalent subunit vaccine.