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目的:通过DNA重组技术表达肠出血性大肠杆菌(EHEC)O157∶H7的EspA和EspB蛋白,并分析它们的免疫保护性。方法:采用PCR技术从EHEC O157∶H7基因组中扩增espA和espB基因,连接至pET-22b(+)载体上,转化至宿主细胞大肠杆菌BL21(DE3),经IPTG诱导表达,用亲和层析纯化目的蛋白,SDS-PAGE测定其相对分子质量,免疫小鼠分析其免疫保护性。结果:重组espA和espB基因片段的测序结果与GenBank中的相应基因序列完全一致,一致性均为100%;得到了纯度为95%以上的重组EspA和EspB蛋白,免疫小鼠所得到的抗体效价均为106。结论:重组EspA和EspB蛋白获得了可溶性表达,表达的蛋白具有良好的免疫保护性,为进一步制备疫苗奠定了基础。
OBJECTIVE: To express EspA and EspB proteins of enterohemorrhagic Escherichia coli (EHEC) O157: H7 by DNA recombination technology and analyze their immunoprotective properties. Methods: The espA and espB genes were amplified from EHEC O157:H7 genome by PCR and ligated into pET-22b (+) vector. The recombinant plasmid was transformed into E. coli BL21 (DE3) and induced by IPTG. Purification of the target protein, SDS-PAGE determination of its relative molecular mass, immune mice were analyzed for their protective immunity. Results: The sequencing results of recombinant espA and espB gene fragments were identical with the corresponding gene sequences in GenBank, the concordances were 100%, and the recombinant EspA and EspB proteins with the purity of more than 95% were obtained. The antibody titer obtained from the immunized mice The price is 106. Conclusion: The recombinant EspA and EspB proteins obtained soluble expression, the expressed protein has good immunoprotection, which lays the foundation for further vaccine preparation.