黄鳝血清转铁蛋白多克隆抗体的制备及检测

黄鳝血清转铁蛋白多克隆抗体的制备及检测
雷华明;李伟
【期刊名称】《水产科学》
【年(卷),期】2017(036)002
【摘要】This study was performed to express swamp eel transferrin C-lobe in Escherichia coli in swamp eel Monopterus albus and to generate rabbit polyclonal antibody of the protein.A pair of primers was used to amplify the C-lobe sequence of swamp eel tranferrin gene from the liver cDNAs.Subsequently, the purified fragments were subcloned into pET
28a(+), and then, the constructed plamid was introduced into
BL21(DE3).After it was induced and expressed, recombinant protein was purified by one step affinity chromatography with a Ni-NTA agarose column.New Zealand white rabbits were immunized with the purified protein to generate polyclonal antibodies.Indirect enzyme-linked immunosorbent assay (ELISA) was applied to examine the titers of the polyclonal antibody, and Western-blotting was used to detect the specificity of the antibody.Results showed that the titer of the polyclonal antibody was more than 1∶25 600 with indirect ELISA and combined specifically with the overexpressed protein in Escherichia coli and crude proteins from different tissues of swamp eel.The polyclonal antibody with high affinity and specificity was generated successfully and used for further research on biological function of transferrin in fish.%为实现黄鳝血清转铁蛋
白基因的原核表达并制备其多克隆抗体,利用基因特异性引物从黄鳝肝脏cDNA中扩增黄鳝转铁蛋白的C端序列,亚克隆至原核表达载体pET-28a(+)中,构建
pET/Tf-C重组表达载体;转化大肠杆菌BL21(DE3)后进行IPTG诱导.利用Ni离子亲和层析技术纯化Tf-C蛋白,并免疫新西兰兔制备多克隆抗体;通过间接ELISA技术和组织蛋白印迹对制备的多克隆抗体进行检测.试验结果表明,成功构建pET/Tf-C 原核表达载体,并实现了蛋白的表达和纯化;制备的多克隆抗体效价大于1∶25 600,并能特异性地识别来源于黄鳝不同组织的血清转铁蛋白.研究结果对黄鳝血清转铁蛋白功能的研究奠定了基础.
【总页数】4页(P220-223)
【作者】雷华明;李伟
【作者单位】长江大学湿地生态与农业利用教育部工程研究中心,湖北荆州434025;长江大学湿地生态与农业利用教育部工程研究中心,湖北荆州 434025【正文语种】中文
【中图分类】S917.4
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