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Expression, purification and identification of Mycobacterium tuberculosis RpfA
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- Author:
- No author available
- Journal Title:
- JOURNAL OF THE FOURTH MILITARY MEDICAL UNIVERSITY
- Issue:
- 18
- DOI:
- 10.3321/j.issn:1000-2790.2006.18.001
- Key Word:
- RpfA;表达;纯化;分枝杆菌,结核
Abstract： 目的:表达和纯化RpfA融合蛋白.方法:将含有RpfA基因片段的质粒用NdeI与BamHⅠ双酶切,然后将目的基因片段克隆入pcDNA3.1+载体构建重组载体pcDNA3.1+-RpfA,测序正确后再将目的基因片段亚克隆入pET19b原核表达载体并转化E.coli DE3,IPTG诱导表达融合蛋白,Western blot鉴定融合蛋白.在变性条件下Ni2+-NTA 亲和色谱柱纯化目的融合蛋白.结果:目的基因片段测序与Genbank报道一致(切去了5'端99 bp).SDS-PAGE显示,在Mr约为80 ku处有表达条带,Western blot鉴定为(His)6融合蛋白,并与小鼠抗Rv1884和抗Rv2389免疫血清有交叉反应.可溶性分析发现融合蛋白主要以包涵体形式存在.经Ni2+-NTA亲和色谱柱纯化得到了融合有6个组氨酸残基的RpfA融合蛋白.结论:成功构建了pET19b-RpfA表达载体,并在E.coli DE3中高效表达,亲和层析后获得了纯化目的蛋白.
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