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幽门螺杆菌尿素酶B亚单位的克隆表达及纯化

DOI: 10.13560/j.cnki.biotech.bull.1985.2015.07.034, PP. 220-225

Keywords: 幽门螺杆菌,尿素酶B亚单位,UreB蛋白,蛋白纯化

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Abstract:

为获得高纯度具有生物活性的尿素酶B亚单位(UreB)蛋白,利用PCR方法扩增出ureB目的基因,将其插入到pET28a载体中,构建表达质粒pET28a-ureB。将鉴定正确的质粒转入大肠杆菌中培养,通过IPTG诱导表达获得UreB蛋白。采用QSepharoseHighPerformance阴离子交换层析纯化,G-25凝胶过滤层析脱盐,并通过SDS-PAGE和免疫双扩散法对UreB蛋白进行鉴定。结果表明,该蛋白相对分子质量约为64kD,与预期结果相符,脱盐后获得的UreB蛋白纯度为98.5%;免疫双扩散法证明该蛋白具有良好的生物活性和反应特异性。最终确定的纯化工艺,达到了一步纯化即得到高纯度、具有生物活性蛋白的目的,该纯化工艺简单、有效。

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