| 霍乱弧菌中弧菌素合成酶VibF的A结构域基因的克隆、表达与纯化研究 |
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| 引用本文： | 刘秀华,王执,徐素娟.霍乱弧菌中弧菌素合成酶VibF的A结构域基因的克隆、表达与纯化研究[J].河南农业科学,2012,41(3):149-153. |
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| 作者姓名： | 刘秀华 王执 徐素娟 |
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| 作者单位： | 1. 河北大学生命科学学院,河北保定071002;山东大学微生物技术国家重点实验室,山东济南250100
2. 山东大学微生物技术国家重点实验室,山东济南,250100 |
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| 基金项目： | 保定市科学技术研究与发展指导计划项目(11ZF112) |
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| 摘 要： | 弧菌素合成酶的A结构域在弧菌素的合成过程中发挥着重要作用。使用DNAStar软件对A结构域的蛋白质二级结构进行预测,截取了多组不同氨基酸长度的A结构域片段。使用PCR方法从霍乱弧菌N16961的基因组中克隆了A结构域基因,通过酶切、连接等方法将其构建到表达载体pET-21b(+)上,并在大肠杆菌BL21(DE3)中得到高效表达。在30个不同氨基酸长度的A结构域蛋白片段中获得5个可溶性表达的片段;并且通过三步分离纯化的方法获得了一性状良好、均一和高纯度的A结构域蛋白(N861-1410),从而为A结构域蛋白质晶体生长提供了基础。
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| 关 键 词： | 霍乱弧菌 A结构域 克隆 表达 纯化 |
Cloning, Expression and Purification of the A Domain of Vibriobactin Synthetas VibF from Vibrio cholerae |
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| LIU Xiu-hua
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WANG Zhi
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XU Su-juan.Cloning, Expression and Purification of the A Domain of Vibriobactin Synthetas VibF from Vibrio cholerae[J].Journal of Henan Agricultural Sciences,2012,41(3):149-153. |
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| Authors: | LIU Xiu-hua WANG Zhi XU Su-juan |
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| Institution: | 1.College of Life Sciences,Hebei University,Baoding 071002,China; 2.State Key Laboratory of Microbial Technology,Shandong University,Jinan 250100,China) |
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| Abstract: | The A domain of vibriobactin synthetas VibF plays a key role in the biosynthesis of vibriobactin in the pathogenic bacteria Vibrio cholerae.DNAStar software was used to predict thesecondary structure of the A domain for chosing several fragments with various amino acid lengths.The A domain gene was cloned from the genome of Vibrio cholerae strain N16961 and constructed into the expression vector pET-21b(+),and then was overexpressed in E.coli BL21(DE3).Five soluble A domain fragments from 30 different fragments with various molecular weight were obtained.One A domain protein,N861-1410,with homogeneous state and high purity,was purified via three steps of chromatography,providing fundamental step for its crystallization. |
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| Keywords: | Vibrio cholerae A domain cloning expression purification |
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