| Enterotoxigenic Escherichia coli (ETEC) is the cause of human and animal pathogens diarrhea. Heat-stable toxin gene (ST) eferred to the disease is a virulent unit. Vibrio cholera is a cholera epidemic caused the pathogen .It has caused several major worldwide pandemic,while the death rate is very high.Cholera toxin B subunit with avirulent can combine together with GM1. It contains most toxins antigen cluster and a strong antigen. In the immunization filed, Cholera toxin B subunit plays an important role.Cholera toxin B subunit gene and the tandem mutant heat-stable enterotoxin epitope gene were respectivly amplified by using PCR technique from PEOCI and PMC-ST2 plasmids, and they were fused with linker by using overlap PCR technique.These genes were subcloned into eukaryotic expression vectors, pET22-b and PITG-Trx ,and then transformed into E.coli BL21(DE3).Optimization of culture medium composition and culture conditions were studied by orthogonal test, and induction stage, induction temperature, IPTG concentration and expression time were also explored to optimize expression in engineering bacteria. When the engineering bacteria were cultured in optimized medium, under the optimized conditions, the maximum expression level of the objective protein was obtained at 320 mg/L in shaking flasks, accounting for about 40% of the total proteins. After being renatured, the fusion protein was purified by affinity chromatography column, and the purity exceeded 95%. Then antigenicity and immunogenicity of the fusion protein were estimated. Western blotting showed CTB-mST2 could react with anti-rabbit CTB antiboby.After immunizing mice with the fusion protein CTB-mST2 and dead-bacteria of O157:H7, high titer sera of anti-CTB and anti-ST were induced, and combined immunization could improve protective effect against O157:H7 in mice. This study was very useful to construct vaccine against ETEC and EHEC.
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