结核分枝杆菌glcB基因原核表达质粒的构建、表达和纯化

中国防痨杂志 ›› 2009, Vol. 31 ›› Issue (9): 526-529.

结核分枝杆菌glcB基因原核表达质粒的构建、表达和纯化

陈曦；贾红彦；古淑香；李自慧；刘忠泉；郑晓静；邢爱英；杜博平；张继增；张宗德

北京结核病胸部肿瘤研究所北京 101149

出版日期:2009-09-10 发布日期:2011-11-03

Construction and expression of the prokyaryotic expression plasmid of M.tb glcB gene and protein purification

Chen Xi, Jia Hongyan, Gu Shuxiang，Li Zihui,Liu Zhongquan,Zheng Xiaojing,Xing Aiying,Du Boping,Zhang Jizeng,Zhang Zongde

Beijing Tuberculosis and Thoracic Tumor Research Institute, Beijing 101149,China

Online:2009-09-10 Published:2011-11-03

摘要/Abstract

摘要： 目的构建结核分枝杆菌glcB基因原核表达工程株，并进行表达、纯化。方法用PCR 扩增结核分枝杆菌glcB基因, 并克隆入pTA2质粒。测序正确后, 再亚克隆入pET30a(+)质粒, 构建pET30a(+):glcB重组体，转化宿主菌大肠埃希菌BL21 (DE3)。结果结核分枝杆菌glcB基因工程株经0.4mM异丙基硫代-β-D-半乳糖苷(IPTG)诱导后,表达出相对分子质量为92kD重组蛋白。SDS-PAGE 分析显示,IPTG诱导4h重组蛋白的表达量最高。表达蛋白以包涵体形式存在于胞质中, 表达量占全菌蛋白质的50％。经Ni-NTA 柱纯化,获得纯度为90%的重组蛋白。结论成功地构建了结核分枝杆菌glcB基因工程表达株，并获得GlcB重组蛋白, 为研究新型血清学诊断活动性结核病奠定了基础。

关键词: 分枝杆菌, 结核, 重组蛋白质类

Abstract: ObjectiveTo obtain recombinant GlcB protein efficiently expressed in E. coli. MethodsThe M. tuberculosis (M.tb) glcB gene was amplified by PCR and then cloned into plasmid pTA2. After sequencing, the glcB gene was cloned into plasmid pET30a(+), and expressed in E. coli BL21 (DE3). ResultsWhen E. coli BL21 (DE3) containing recombinant plasmid pET30a(+):glcB was induced by 0.4mmol/L of IPTG for 4h, the quantity of recombinant protein expressed in E. coli was maximum. The recombinant protein GlcB with molecular weight 92KD was expressed in incusion bodies of E. coli, and amounted to 50% of total bacterial protein. The purity of the protein purified through the Ni-NTA resin reached 90%. ConclusionsThe prokaryotic expression plasmid pET30a(+):glcB was constructed successfully, and the recombinant protein GlcB was obtained.

Key words: Mycobacterium tuberculosis, Recombinant proteins

陈曦,贾红彦,古淑香,李自慧,刘忠泉,郑晓静,邢爱英,杜博平,张继增,张宗德. 结核分枝杆菌glcB基因原核表达质粒的构建、表达和纯化[J]. 中国防痨杂志, 2009, 31(9): 526-529.

Chen Xi, Jia Hongyan, Gu Shuxiang，Li Zihui,Liu Zhongquan,Zheng Xiaojing,Xing Aiying,Du Boping,Zhang Jizeng,Zhang Zongde. Construction and expression of the prokyaryotic expression plasmid of M.tb glcB gene and protein purification[J]. Chinese Journal of Antituberculosis, 2009, 31(9): 526-529.

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