Prokaryotic Expression and Polyclonal Antibody for Ferric Uptake Regulator Prepared from Vibrio vulnificus FJ03-X2
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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.
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Key words:
- Vibrio vulnificus /
- ferric uptake regulator /
- cloning /
- prokaryotic expression /
- polyclonal antibody
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图 1 基因的PCR扩增电泳结果
1~3 为fur基因；4为阴性对照；5为DL2000分子量标准。
Figure 1. PCR amplification of fur gene
图 2 质粒的双酶切鉴定
1为 λ-EcoT14 分子量标准； 2~6为重组子酶切结果；7为 DL2000分子量标准。
Figure 2. Results of dual-enzyme digestion
图 4 Ni-NTA纯化的rFUR蛋白的SDS-PAGE结果
注：1为 BL21/pET32a空载诱导全菌蛋白；2为BL21/pET32a-FUR未诱导的全菌蛋白；3为BL21/pET32a-FUR经诱导后超声的菌体破碎物上清蛋白；4为纯化后的rFUR融合蛋白；5为蛋白质低分子量标准。
Figure 4. SDS-PAGE analysis of purified rFUR protein by Ni-NTA
表 1 ELISA 测定抗体效价(λ=490nm)
Table 1. Determination of antibody titer by ELISA
项目 1∶4000 1∶8000 1∶16000 1∶32000 1∶64000 1∶128000 1∶256000 抗血清 2.5755 2.3855 2.2835 2.034 1.722 1.176 0.783 阴性血清 0.1335 0.116 0.1135 0.1295 0.1095 0.116 0.1155 
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