Expression of fusion protein CTB-mST2 in E. coli and its bioactivity assay

Objectives: To express the fusion protein of cholera toxin B subunit and mutant heat-stable enterotoxin,and to estimate its antigenicity and immunogenicity.Methods: Fused to 3-terminus of CTB gene by linker,the tandem mutant heat-stable enterotoxin epitope gene(mST) was efficiently expressed in BL21(DE3) after induced by IPTG.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.After being renatured,the fusion protein was purified by affinity chromatography column,and its antigenicity and immunogenicity were estimated.Results and Conclusion: When the engineering bacteria were cultured in optimized medium,under the optimized conditions,the maximum expression level of the trarget protein was obtained at 320 mg/L in shaking flasks,amounted to about 40% of the total proteins.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 obtained,and combined immunization with the fused protein and inactivated O157∶H7 bacteria could improve protective effect against O157∶H7 in mice.This study is very useful to construction of vaccine against ETEC and EHEC.