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Cloning of Mycobacteria tuberculosis furA and expression of its coding protein fused with (His)6 tag
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- Author:
- No author available
- Journal Title:
- JOURNAL OF THE FOURTH MILITARY MEDICAL UNIVERSITY
- Issue:
- 18
- DOI:
- 10.3321/j.issn:1000-2790.2004.18.002
- Key Word:
- 分枝杆菌,结核;FurA;克隆;表达;纯化
Abstract： 目的: 获得结核分枝杆菌furA基因片段并高效表达、纯化. 方法: 用PCR技术从MTB毒株H37Rv-DNA中扩增出相应大小的DNA片段,将片段与pGEM-T-easy 载体连接后测序. 将测序正确的furA按照BamHⅠ和HindⅢ酶切位点克隆入原核表达载体pRSET-A,将连接产物转化入E.coli BL21,挑出阳性克隆,IPTG诱导表达重组的(His)6融合蛋白. 对重组融合蛋白通过镍柱进行纯化. 结果: PCR获得的furA序列与Genbank报道的一致(为453 bp). 重组载体在E.coli BL21中以可溶形式高效表达,融合蛋白经SDS-PAGE和Western blot分析,在Mr为23.0×103处有特异的蛋白条带,目的蛋白表达量占菌体总蛋白量的10%,重组蛋白经镍柱进行纯化后,得到了高纯度的FurA融合蛋白. 结论: 成功克隆结核分枝杆菌furA基因片段,并在E.coli BL21中高效表达,亲和层析纯化后获得高纯度FurA融合蛋白.
- [1]师长宏,范雄林,徐志凯,等.融合表达Ag85B-ESAT6的结核分枝杆菌真核表达载体的构建及其免疫原性[J].2004,(2).
- [2]柏银兰,李元,李别虎,等.结核分枝杆菌分泌蛋白MPT64的克隆、表达与鉴定[J].2003,(1).
- [3]Zahrt TC,Song J,Deretic V,etc.Mycobacterial FurA is a negative regulator of catalase-peroxidase gene katG.[J].2001,39(5).
- [4]Garman E,Mijovilovich A,Vasil ML,etc.Architecture of a protein central to iron homeostasis: crystal structure and spectroscopic analysis of the ferric uptake regulator.[J].2003,47(4).
- [5]Ratledge C.Iron, mycobacteria and tuberculosis.[J].2004,84(1).
- [6]Escolar, Lucia,Perez-Martin, Jose.Opening the Iron Box: Transcriptional Metalloregulation by the Fur Protein.[J].1999,181(20).