莱茵衣藻IFT20的原核表达、纯化及多克隆抗体鉴定

Prokaryotic Expression, Purification, and Polyclonal Antibody Identification of Chlamydomonas reinhardtii IFT20

摘要:

莱茵衣藻鞭毛内运输（intraflagellar transport, IFT）蛋白IFT20是IFT蛋白复合物B（IFT-B）的组分之一。虽然IFT20参与调控细胞内大分子的胞内运输过程，但其作为一个鞭毛蛋白在鞭毛内的详细功能仍有待研究。为了开发高特异性的莱茵衣藻IFT20兔源多克隆抗体，作者首先在大肠杆菌中表达了N端6×His标签标记的IFT20融合蛋白（6×His::IFT20），并对其进行镍柱纯化，将纯化所得6×His::IFT20蛋白免疫新西兰大白兔。采集3次免疫后的抗血清，利用间接ELISA法测定其效价为1∶102 400。利用protein A纯化珠对所得抗血清进行了IgG亚型抗体富集，接着用大肠杆菌表达纯化所得N端MBP标签标记的IFT20（MBP::IFT20），并对IgG抗血清进行抗原抗体亲和纯化。经对CC-125藻种全细胞蛋白质提取物进行免疫印迹法鉴定，所得IFT20多克隆抗体特异性较高，适合用于后续莱茵衣藻IFT20鞭毛内运输功能的研究。

Abstract:

Intraflagellar transport protein 20 (IFT20) of Chlamydomonas reinhardtii is a component of the intraflagellar transport complex B(IFT-B). IFT20 is known to be involved in regulating intracellular transport processes of macromolecules, but its detailed function as a flagellar protein in flagella remains to be investigated. This study aimed to develop highly specific polyclonal antibodies against Chlamydomonas reinhardtii IFT20 from rabbit serum to lay the foundation for further investigation into its flagellar function. To achieve this goal, the N-terminal 6×His-tagged IFT20 fusion protein(6×His::IFT20) was first expressed in Escherichia coli, and 6×His::IFT20 was purified by Ni-column. The purified 6×His::IFT20 was then applied to immunize the New Zealand white rabbits. The antiserum was collected after three rounds of immunization, and the titer was determined to be 1∶102 400 by an indirect ELISA method. Subsequently, the obtained anti-serum was purified using protein A purification beads to enrich the IgG subtype antibodies. Next, N-terminal MBP-tagged IFT20(MBP::IFT20) expressed and purified from Escherichia coli was used for antigen-antibody affinity purification of IgG antiserum. The western blotting assay of whole cell protein extracts from CC-125 identified the high specificity of the obtained IFT20 polyclonal antibodies, making them suitable for future investigations on the flagellar function of IFT20 in Chlamydomonas reinhardtii.