【摘要】 目的 :建立经济有效的方法 ,从大肠杆菌表达、纯化重组幽门螺杆菌尿素酶A、B亚单位。方法 :含重组表达质粒pGEX- 2T ureA ,pGEX - 2T ureB大肠杆菌分别在不同浓度IPTG、不同温度条件下作诱导培养 ,以SDS -PAGE分析表达产物 ,采用谷胱甘肽 -Sepharose 4B亲和层析分离纯化表达产物 ,以Western -blotting和ELISA对纯化产物作抗原性分析鉴定。结果 :IPTG诱导浓度为 0 .1mmol L ;诱导温度为 2 8℃ ,含重组质粒大肠杆菌表达可溶性融合蛋白GST -ureA、GST -ureB各约占总表达产物的 78%和87%。经亲和层析 ,得纯度 90 %以上的GST -ureA约为 14mg L ,GST -ureB约为 18mg L ;融合蛋白呈ureA、ureB抗原性反应。结论 :建立了低温诱导及纯化高纯度ureA、ureB基因表达产物的方法 ,所得纯化产物具有ureA、ureB抗原活性 ,为进一步的应用研究打下基础。更多还原

【Abstract】 Objective:To estabilish an affective,economical and easy method for expression and purification Hp Urease A,B having antigenicity from E.coli .Methods:The E.coli transformed by plasmid pGST/ureA or pGST/ureB was induced with different concentration of IPTG and cultured in different low temperature.The GST-ureA,GST-ureB fusion protein expressed in E.coli JM 105 were purified by affinity chromatography of glutathione-Sepharose 4B.The expression level,extraction and purification result were estimated by SDS-PAGE.The antigenicity of GST-ureA,GST-ureB were detected by western-blotting and ELISA.Results:The soluble GST-ureA,GST-ureBin lytic supernatant were 78% and 87% in total expression fusion protein respectively,when the concentration of IPTG was 0.1mmol/L and induced temperature was 28℃.14 mg GST-ureA or 18 mg GST-ureB with purity over 90% was obtained from 1L culture by using affinity chromatography of glutathione-Sepharose 4B.The antigenicity of GST-ureA or GST-ureB was determined by Western-blotting and ELISA.Conclusion:The effective and easy method for low temperature expressing GST-ureA and GST-ureB was estabilished.It provids a foundation of further study and applying in diagnostic kit and vaccine.更多还原