肠出血性大肠埃希菌O157:H7 espA基因的克隆与表达

目的 通过DNA重组技术表达肠出血性大肠杆菌（EHEC）O157：H7的EspA蛋白，并分析其免疫学性质。方法 采用PCR技术从EHEC O157：H7基因组中扩增espA基因，T／A法克隆，连接至pET-28a（＋）载体上，转化宿主细胞E．coli BL21（DE3），IPTG诱导表达，亲和层析法纯化目的蛋白，SDS-PAGE测定其相对分子质量，同时分析其N端氨基酸序列，免疫家兔鉴定其抗原性。结果 重组espA基因片段的测序结果与GenBank上基因序列只有1个碱基不同，一致性为99．83％，所编码的氨基酸序列没有改变。重组EspA蛋白在工程菌中以包涵体的形式表达，表达量约30％。免疫家兔所得抗体滴度为1：32。结论构建高效表达EspA蛋白的重组载体pET-28a（＋）-espA，表达的蛋白具有良好的免疫原性，为进一步制备亚单位疫苗奠定基础。

Cloning and expressing espA gene from enterohemorrhagic E. coli O157: H7 and study on antigenicity of the recombinant EspA protein

Objective To clone and express the espA gene from EHEC 0157: H7 by DNA recombinant technology, and analyze the immunologic properties of the EspA protein. Methods The espA gene was amplified by PCR with the genomic DNA from enterohemorrhagic E. coli O157: H7 strain as template. The amplified product was cloned into pET-28a( + ) vector, then transferred into the host cells E. coli BL21(DE3) strain. The EspA protein was expressed induced by IPTG, and purified by affinity chromatography. The molecular weight of purified protein was determined by SDS-PAGE. The sequence of amino acids on the N-terminal of purified protein was analyzed . Its antigenicity was analyzed by the immunization of rabbits. Results The nucleonde sequence of the espA gene is consistence with the sequence from GenBank by 99.83% (only one base different) , but there is no change on sequence of the amino acid. The recombinant EspA protein was expressed by approximately 40% with inclusion body in E. coli. The highest antibody titer of immunized rabbits sera is 1:32. Conclusion A recombinant expressed EspA high performantly was successfully constructed, and the recombinant protein has good antigenicity. These results may provide the foundation for the further development on EHEC O157: H7 subunit vaccine.