莱茵衣藻纤毛内运输蛋白IFT46的原核表达纯化及其多克隆抗体的制备

Prokaryotic expression and purification of Chlamydomonas reinhardtii intraflagellar transport protein 46(IFT46) and preparation of polyclonal antibody

摘要:

IFT46是纤毛内运输蛋白IFT复合物B (IFT-B) 的一个重要组分，对于纤毛的组装、运动和感知发挥着重要作用。为深入研究IFT46的作用机制，利用ift46基因全序列分别构建了带有GST和MBP标签的原核表达载体pGEX-2T-ift46和pMAL-C2X-ift46，并转入大肠杆菌BL21 (DE3) 诱导表达，以15% SDS-PAGE鉴定，分别获得了分子量为70、86 kDa的重组GST/MBP-IFT46融合蛋白。将亲和纯化的GST-IFT46融合蛋白 (纯度95%以上) 免疫新西兰大白兔，采集第5次免疫后血清用ELISA测定效价为1∶256 000。抗血清依次经Protein A和固定在MBP-IFT46纯化后，用Western blotting 和免疫荧光检测抗体特异性，结果表明制备的多克隆抗体能很好地识别莱茵衣藻中的IFT46，而且发现IFT46绝大部分定位在纤毛基体，极少部分沿纤毛呈点状分布，为继续开展 IFT46在肥胖症、糖尿病、多囊肾病等纤毛相关疾病中作用机制的研究奠定了重要基础。

Abstract:

IFT46 is one of the important components of intraflagellar transport complex B in Chlamydomonas reinhardtii, and plays important roles in the assembly, movement and perception of ciliary. To study its functional mechanism, a GST-tagged and an MBP-tagged prokaryotic expression plasmid, pGEX-2T-ift46 and pMAL-C2X-ift46 were constructed, respectively, by inserting ift46 into the pGEX-2T and pMAL-C2X vector, and then transformed into Escherichia coli BL21 (DE3) for protein expression. SDS-PAGE (15%) analysis results showed that the molecular weights of the fusion protein GST-IFT46 and MBP-IFT46 were 70 kDa and 86 kDa, respectively. We used the fusion protein GST-IFT46 purified by affinity adsorption purification (more than 95% purity) for immunity to New Zealand white rabbits. The 5th immune serum was collected and the antibody titer was determined to be 256 000 by ELISA. The antiserum was purified by Protein A affinity adsorption purification and immobilized MBP-IFT46 purification, and the specificity of polyclonal antibodies was evaluated by Western blotting and immunofluorescence. Results showed that the polyclonal antibody prepared could specifically and precisely bind IFT46 in C. reinhardtii, and IFT46 was mainly concentrated at basal body regions and few localized along the entire length of the flagellum as punctuated dots, which will make a foundation to further study the mechanism of IFT46 in cilia related diseases such as obesity, diabetes and polycystic kidney disease.