You Position: Home > Paper
Cloning, expression and purification of Mycobacterium tuberculosis furB
( views:184, downloads:0 )
- Author:
- No author available
- Journal Title:
- JOURNAL OF THE FOURTH MILITARY MEDICAL UNIVERSITY
- Issue:
- 18
- DOI:
- 10.3321/j.issn:1000-2790.2004.18.003
- Key Word:
- furB;表达;纯化;结核分枝杆菌
Abstract： 目的: 从H37Rv基因组中扩增furB并高效表达和纯化. 方法: 用PCR技术从结核分枝杆菌H37Rv基因组中扩增furB序列,将其与pGEM-T-Easy载体连接转化E.coli DH5α,构建重组克隆载体pGEM-T-Easy/furB,测序正确后将目的基因片断克隆入pQE-80L原核表达载体并转化E.coli DH5α,IPTG诱导目的蛋白表达. 经Western blot鉴定目的基因与(His)6融合表达,将已表达的蛋白质通过Ni2+-NTA亲和色谱柱进行纯化. 结果: PCR得到结核分枝杆菌furB,测序结果与Genbank中报道的完全一致. SDS-PAGE显示,在Mr为15.0×103处有相应的蛋白质表达条带,Western blot鉴定为(His)6融合表达蛋白. 经Ni2+-NTA亲和色谱柱进行纯化后可得到纯化的蛋白. 结论: 成功克隆了铁调节蛋白基因furB序列,并在E.coli DH5α高效表达,亲和层析后获得了纯化目的蛋白.
- [1]范雄林,徐志凯,李元,等.结核分枝杆菌Ag85B成熟蛋白基因免疫[J].2001,(14).
- [2]江山,朱道银,蒋英,等.结核分枝杆菌Ag85B-Ag85A双抗原融合真核表达质粒的构建及表达[J].2003,(21).
- [3]S T, Cole,R, Brosch,J, Parkhill,etc.Deciphering the biology of Mycobacterium tuberculosis from the complete genome sequence.[J].1998,393.
- [4]Timo, Ulrichs,Stefan H E, Kaufmann.Mycobacterial persistence and immunity.[J].2002,7.
- [5]Touati D..Iron and oxidative stress in bacteria[J].2000,373(1).
- [6]C M, Litwin,S B, Calderwood.Role of iron in regulation of virulence genes.[J].1993,6(2).