| 创伤弧菌铁调基因fur的原核表达及多克隆抗体制备 |
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| 引用本文： | 李素一,张丽娟,陈华,柯翎,陈叙,林晨韬.创伤弧菌铁调基因fur的原核表达及多克隆抗体制备[J].福建农业学报,2016(9):912-916. |
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| 作者姓名： | 李素一 张丽娟 陈华 柯翎 陈叙 林晨韬 |
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| 作者单位： | 福建省农业科学院生物技术研究所,福建 福州,350003 |
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| 基金项目： | 福建省科技计划项目---省属公益类科研院所基本科研专项(2014R1019-9)，福建省农业科学院引进海外人才科研启动基金项目(HWRC2011-03、HWRC2011-02)，国家自然科学基金项目(31100658)，福建省农业科学院青年英才计划项目(YC2015-20)，福建省农业科学院杰出青年人才基金项目(2014JQ-4) |
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| 摘 要： | 应用PCR方法克隆了创伤弧菌Vibrio vulnificus FJ03-X2株的铁调基因fur(Ferric uptake regulator),该基因片段大小为450bp,编码149个氨基酸;以pET32a为表达载体,构建了原核表达质粒pET32a-FUR,表达质粒测序结果表明目的基因与GenBank中报道的创伤弧菌fur基因的同源性达98%以上;诱导表达获得可溶性的重组表达蛋白rFUR。镍离子金属螯合亲和层析介质(Ni-NTA)纯化rFUR,SDS-PAGE电泳分析其分子量约33kD。以纯化后的融合蛋白rFUR为抗原,4次免疫SD大鼠,制备抗rFUR蛋白大鼠多克隆抗体。用ELISA方法检测鼠多克隆抗体的效价达到1∶256 000,表明融合蛋白rFUR具有良好的免疫原性。
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| 关 键 词： | 创伤弧菌 铁调基因fur 克隆 原核表达 多克隆抗体 |
Prokaryotic Expression and Polyclonal Antibody for Ferric Uptake Regulator Prepared from Vibrio vulnificus FJ0 3-X2 |
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| Abstract: | Ferric uptake regulator (fur)gene from Vibrio vulnificus FJ03-X2 was cloned,and inserted into the prokaryotic expression vector,pET32a. Coding sequence of the gene contained 450 bps,encoding 149 amino acids. Its sequencing showed a greater than 98% homology with that of the fur gene from GenBank. Subsequently,the plasmid,pET32a-FUR,was transformed into E.coli BL21 to express the recombinant protein,rFUR. The soluble rFUR was then subject to the Ni-NTA His Binding affinity purification. The purified rFUR was injected into SD rats to produce polyclonal antibody. The obtained highly specific antibodies of FUR was confirmed by ELISA assay. |
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| Keywords: | Vibrio vulnificus ferric uptake regulator cloning prokaryotic expression polyclonal antibody |
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