CTB-mST2融合蛋白在大肠杆菌表达及其生物活性鉴定  

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

作　　者：赵聪[1] 张艳红[1] 张部昌[2] 马清钧[1] 

机构地区：[1]军事医学科学院生物工程研究所,北京100850 [2]安徽大学生命科学学院,合肥230039

出　　处：《军事医学科学院院刊》2007年第3期208-212,共5页Bulletin of the Academy of Military Medical Sciences

摘　　要：目的:表达霍乱毒素B亚单位(CTB)和大肠杆菌突变的热稳定肠毒素(mST)的融合蛋白,并进行抗原性和免疫原性的分析。方法:通过连接肽(linker)将CTB基因和串连的mST连接,然后插入表达载体pET-22b(+),获得克隆,经IPTG诱导获得CTB-mST2融合蛋白。采用多因素正交优选法优化了培养基及培养条件,对表达产物进行包涵体复性后,经过亲合层析纯化得到融合蛋白,并对该融合蛋白抗原性和免疫原性进行了分析。结果与结论:构建了高效表达工程菌,经摇瓶培养,在优化培养基及培养条件下融合蛋白表达量可达320 mg/L,占总蛋白40%。融合蛋白免疫小鼠,可获得高滴度抗CTB和ST血清;将融合蛋白与灭活的O157∶H7菌体组合免疫小鼠,可产生对O157∶H7毒株攻击的保护力。本研究可为构建抗ETEC和EHEC复合疫苗提供依据。Objectives： To express the fusion protein of cholera toxin B subunit and mutant heat-stable euterotoxin, 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 anff-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 con- struction of vaccine against ETEC and EHEC.

关 键 词：霍乱毒素B亚单位 大肠杆菌热稳定肠毒素 大肠杆菌O157:H7 疫苗 

分 类 号：R392[医药卫生—免疫学]