肠出血性大肠杆菌O157∶H7 EspA和EspB蛋白的重组表达及其免疫效果  

Recombinant Expressing and Protective Immunity of EspA and Es pB from Enterohemorrhagic E.coli O157:H7

作　　者：韩燃[1,2] 李涛[2] 王琴[2] 房华丽[2] 罗森[2] 郭峰[2] 李崭[2] 王德慧[2] 李文平[1] 王慧[2] 

机构地区：[1]湖南农业大学动物医学院,湖南长沙410128 [2]军事医学科学院微生物流行病研究所,病原微生物生物安全国家重点实验室,北京100071

出　　处：《生物技术通讯》2013年第3期351-354,共4页Letters in Biotechnology

基　　金：国家自然科学基金(81072677);北京自然科学基金(7122134)

摘　　要：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 recombinant EspA and EspB from enterohemorrhagic Escherichia coli（EHEC） O157：H7, and analyze their protective immunity. Methods： The espA and espB gene were amplified by PCR with the genomie DNA from EHEC O157：H7 strain, the amplified products were cloned into pET-22b（＋） vector, then transferred into the host cells E.coli BL21（DE3） strain. The EspA and EspB proteins were expressed induced by IPTG, and purified by affinity chromatography. The relative molecular mass of purified proteins were determined by SDS-PAGE. The immune protection of the two proteins was analyzed by the immunization of mice. Results： The nucleotide sequences of the espA and espB were consistence with the sequence from GenBank by 100%. The purity of recombinant EspA and EspB were above 95%. The highest antibody titer of immunized mice sera was 10^6. Conclusion： The recombinant EspA and EspB protein of the EHEC O157：H7 were successfully expressed in soluble form and the recombinant proteins have good protective immunity. These results may provide the foundation for the further development on EHEC O157：H7 vaccine.

关 键 词：肠出血性大肠杆菌O157∶H7 EspA EspB 重组蛋白 免疫效果 

分 类 号：R392.1[医药卫生—免疫学] Q78[医药卫生—基础医学]