莱茵衣藻RSP10的多克隆抗体制备及应用

Preparation and Application of a Polyclonal Antibody to Chlamydomonas reinhardtii RSP10

摘要:

【目的】研究径向辐条蛋白RSP10如何完成纤毛组装，制备一种高特异性的RSP10多克隆抗体。【方法】首先构建原核表达载体pMal-C2X-rsp10，并在大肠杆菌BL21（DE3）中诱导表达MBP标签的RSP10，待融合蛋白纯化后切除MBP标签，免疫新西兰雄性大白兔获得抗血清，以间接ELISA法测定其效价。【结果】通过Protein A和抗原抗体纯化后，获得了特异性强、灵敏度高的兔抗莱茵衣藻RSP10抗体，通过免疫荧光检测其定位于细胞基体部位，进一步分离纤毛组分发现RSP10大量存在于纤毛轴丝中，少量存在于纤毛基质中。【结论】该试验为后续研究RSP复合物如何完成组装及其影响纤毛运动的深层分子机理提供了有力的数据支撑。

Abstract:

[Objective]This study aims to explore the cilium assembly of the radial spoke protein RSP10 and develop a highly specific polyclonal antibody to RSP10.[Method]Firstly, the prokaryotic expression vector pMal-C2X-rsp10 was constructed, and the expression of MBP-labeled RSP10 was induced in Escherichia coli BL21 (DE3). After the fusion protein was purified, and the MBP label was excised. The obtained protein was used to immunize New Zealand male white rabbits, and the antibody titer in the anti-serum was determined by indirect ELISA.[Result]The rabbit anti-Chlamydomonas reinhardtii RSP10 antibody with high specificity and high sensitivity was obtained after purification of Protein A and antigen antibody. The immunofluorence assay showed that RSP10 was mainly located in the kinetosome. The isolation of ciliary components showed that a large amount of RSP10 was detected in the axial filament and a small amount in the matrix of cilia.[Conclusion]This finding provides strong data support for the subsequent study of the assembly of RSP complex and the molecular mechanism underpinning its influence on cilium movement.