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.