莱茵衣藻纤毛内运送蛋白IFT57抗原的原核表达、纯化及其多克隆抗体的制备

Prokaryotic Expression and Purification ofChlamydomonas reinhardtiiIntraflagellar Transport Protein 57（IFT57） Antigen and Preparation of Polyclonal Antibody

DOI：10.3969/j.issn.1673-1689.2019.11.015

中文关键词: 莱茵衣藻 纤毛 IFT57 原核表达 多克隆抗体

英文关键词: Chlamydomonas reinhardtii,cilia,IFT57,prokaryotic expression,polyclonal antibody

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中文摘要:

IFT57是纤毛内运送蛋白IFT B复合物的一个重要组分，在鞭毛组装中发挥着重要作用。利用莱茵衣藻ift57基因中一段编码亲水性氨基酸序列的片段构建了带有6×His标签的原核表达载体pET-28a（+）-ift57，并转入大肠杆菌BL21（DE3），通过IPTG诱导表达蛋白质，以12 g/dL SDS-PAGE鉴定，获得相对分子质量大小为17 200的ITF57重组蛋白质片段。对6×His-IFT57融合蛋白质进行纯化，并用其免疫新西兰大白兔，采集第5次免疫后血液并分离血清。利用Western blotting对所获抗体进行特异性鉴定。ELISA测定结果表明，抗血清效价为512 000。在抗血清经Protein A纯化后，Western blotting检测显示所制备的IFT57多克隆抗体能够特异性识别莱茵衣藻中的IFT57蛋白质。这一结果表明，为表达纯化溶解性较差的蛋白质抗体制备提供了借鉴意义，所获抗体为深入研究IFT57在鞭毛组装中的作用机理奠定了基础。

英文摘要:

IFT57 is a subunit of the intraflagellar transport protein B complex and plays an important role in cilium assembly. To expressift57gene，DNA fragments were amplified by PCR and cloned into an in-house modified version of the pET28a vector. The resulting proteins contained a 6×His tag at their N terminus. BL21（DE3） Escherichia coli cells harboring the expression plasmid were grown in lysogeny broth（LB） medium at 37 ℃ and then induced with isopropyl-b-D-thiogalactoside，SDS-PAGE（12%） results showed that the molecular weights of the fusion protein 6×His-IFT57 was 17.2 KDa. After purified by affinity chromategraphy，the fusion protein 6×His-IFT57 was used as antigen to immune rabbits to prepare polyclonal antibody. After 5 hours we collected the immune blood，then separated the serum and identified the antibody activity. The ELISA result showed that the antibody titer of serum was determined to be about 512 000. After the affinity purification of Protein A，we got a high specificity and sensitivity polyclonal antibody. The results of Western blotting showed that the polyclonal antibody was specificity recognition to the IFT57 protein in C. reinhardtii. It provided the certain reference value for expression and purification of difficult soluble protein，and antibody preparation of polyclonal antibody. It laid the foundation to continue to study the functional mechanism of IFT57 in cilium assembly.

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