创伤弧菌铁调基因<i>fur</i>的原核表达及多克隆抗体制备

创伤弧菌铁调基因fur的原核表达及多克隆抗体制备

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450 bp，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33 kD。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。

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.