Expression, purification and effect of different immunization approaches of Tir from EHEC O157:H7 / 中华微生物学和免疫学杂志

ABSTRACT

Objective To clone and express translocation intimin receptor(Tir)of enterohemorrhagic Escherichia coli(EHEC)O157H7,and to analyze the effect of different routes on the induction of immunity to the recombinant protein.Methods The tir eucoding genes were amplified from EHEC O157H7 strain guangzhou 246 genome,and genes were cloned into the vector pET-30a(+).The pET-30a(+)tir recombinant was transformed into E.coli BL21.and expression was induced bv IPTG.The expressed product was analyzed by SDS-PAGE and purified by Ni-IDA affinity chromatography.The immunized mice sera and fecal against the recombinant protein was detected.Resuits The length of the tir is 1677 bp,with the initiation codon ATG and the termination codon TAA.Double enzyme digestion and DNA sequencing confirmed that the recombinant expression plasmid pET-30a(+)-tir was constructed.The recombinant protein was expressed in Escherichia coli expression system,and was purified by Ni-IDA affinity chromatography.The mice were able to produce a high serum IgG antibody titer after both subeutaneous and intranasal immunizations.Meanwhile,the intranasal immunization induced serum and fecal IgA antibody titer was significantly higher than that of the subcutaneous immunization group.Conclusion Tir molecule is potential vaccine candidate for preventing EHEC disease.