碳青霉烯酶OXA-72在毕赤酵母中的表达和纯化

Expression and purification of carbapenemase OXA-72 in pichia pastoris

目的对我国临床分离的第一株鲍曼不动杆菌产生的碳青霉烯酶OXA-72在毕赤酵母表达系统中进行重组表达及分离纯化。方法提取临床分离的鲍曼不动杆菌菌株40的基因组DNA,PCR扩增oxa-72全基因;将oxa-72基因双酶切回收后与毕赤酵母表达载体连接,重组质粒分别以PCR、酶切及测序鉴定;电转化法将线性化的重组表达DNA转导入GS115感受态细胞,在Zeocin平板上筛选阳性转化子,并通过PCR和Western进行验证;对阳性克隆进行头孢硝噻吩实验,检测表达OXA-72的活性。用镍柱分离纯化OXA-72。结果以该株鲍曼不动杆菌的基因组DNA为模板,用目的引物进行PCR可获得843 bp的特异扩增条带;DNA测序证明核苷酸序列与Genbank公布的oxa-72基因序列100%一致;电转化后经Zeocin平板筛选得到一株阳性克隆,SDS-PAGE表明重组蛋白的相对分子量在34~43 kDa之间,Westem blot分析显示,重组蛋白能特异地与抗His-Tag抗体结合。头孢硝噻吩试验为阳性。纯化得到纯度为95%的重组蛋白,纯化回收率达40%。结论成功构建了OXA-72的表达载体,在毕赤酵母中成功地表达并分离纯化了具有...

Objective To amplify and identify the chromosomal DNA of Acinetobacter Baumanii which is the first isolate producing carbapenemase OXA-72 in our country and to express and purify OXA-72 in Pichia pastoris.Methods This study amplified the oxa-72 gene by PCR using the extracted genomic DNA of the isolate 40 which was obtained from the clinic as a template.After the amplified gene was cloned into vector pGAPZalphaA,linearized recombinant plasmid oxa72-pGAPZalphaA was transformed to Pichia pastoris GS115 by ele...