幽门螺杆菌尿素酶B亚单位功能片段的纯化及活性研究

目的 建立一种有效的尿素酶功能片段的纯化方法.方法 应用已构建的尿素酶B功能片段表达载体,IPTG诱导表达,包涵体鉴定试验判断目的蛋白表达形式,AKTA100上分别采用亲和层析和离子层析等多级纯化方式优化纯化条件,HPLC检测纯化蛋白纯度,尿素酶活性阻断试验对纯化后功能片段进行活性鉴定.结果 IPTG诱导后目的蛋白高表达,包涵体鉴定试验证实目的蛋白以可溶形式存在宿主细胞中,优化后方案得到目的蛋白纯度＞90%且能被尿素酶B免疫的兔血清识别,纯化后蛋白免疫家兔后产生兔抗血清能够特异性中和Hp尿素酶活性.结论 成功建立了尿素酶功能片段的纯化方法.

Purification and functional analysis of Helicobacter pylori UreB protein fragment

Objective To establish an effective method for purification of Helicobacter pylori UreB fragment and conduct functional analysis of the purified protein.Methods The protein fragment expression was induced by IPTG and the expressed protein was purified through affinity chromatography and ion-exchange chromatography.The purity of the fragment was determined by high-performance liquid chromatography(HPLC),and the specific biological activity of the purified fragment was assayed by urease activity inhibition test.Results The protein fragment was highly expressed in E.coli with a purity over 91%.The protein fragment showed highly specific biological activity and the specific antibody induced by this fragment in rabbits could inhibit the activity of urease in a dose-dependent manner.Conclusion The UreB fragment with high purity and biological activity can be applied for further studies.