创伤弧菌铁调基因fur的原核表达及多克隆抗体制备  

Prokaryotic Expression and Polyclonal Antibody for Ferric Uptake Regulator Prepared from Vibrio vulnificus FJ03-X2

作　　者：李素一[1] 张丽娟[1] 陈华[1] 柯翎[1] 陈叙[1] 林晨韬[1] 

机构地区：[1]福建省农业科学院生物技术研究所,福建福州350003

出　　处：《福建农业学报》2016年第9期912-916,共5页Fujian Journal of Agricultural Sciences

基　　金：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);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);国家自然科学基金项目(31100658)

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。Ferric uptake regulator （fur） gene from Vibrio vulni3Cicus 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.

关 键 词：创伤弧菌 铁调基因fur 克隆 原核表达 多克隆抗体 

分 类 号：S941[农业科学—水产养殖]