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∶256000,表明融合蛋白 rFUR具有良好的免疫原性。%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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