莱茵衣藻IFT25的原核表达、纯化及其多克隆抗体的制备

Prokaryotic Expression and Purification of Chlamydomonas reinhardtii IFT25 in Escherichia coli and Preparation of Polyclonal Antibody

摘要:

IFT25是维持纤毛运动和感知所必须元件纤毛内运输蛋白IFT复合物B中的重要组分之一，探索IFT25尤其在模式生物莱茵衣藻中的作用机制具有重要作用。作者采用经济和简单的方法，制备莱茵衣藻IFT25的兔源多克隆抗体。将ift25分别连接到pMAL-c2X-和pET-28a（+）-载体上，转入大肠杆菌BL21（DE3），用IPTG诱导大量表达，并经亲和纯化后蛋白质纯度超过90%。通过颈背部皮下多次免疫8周龄大的新西兰大白兔，制备多克隆抗体，取第五次免疫后血清用ELISA测定效价超过128 000。抗血清依次经过Protein A和硝酸纤维素膜纯化后，用Western blotting检测抗体特异性，结果表明制备的抗体可以特异性识别莱茵衣藻中的IFT25，为IFT25作用机制的阐明和纤毛相关疾病的诊断提供了重要理论和方法支持。

Abstract:

IFT25 is one of the important components of IFT-B complexes，which are necessary to maintain cilia movement and perception. To explore IFT25 mechanism especially in the model organism Chlamydomonas reinhardtii plays an important role. This experiment adopts economic and simple method to prepare the rabbit polyclonal antibody of the Chlamydomonas reinhardtii IFT25.The target gene ift25 was cloned into vectors of pMAL-c2X- and pET-28a（+）-，respectively. Then the plasmids were transformed into E. coli BL21（DE3） to induce highly expressedby IPTG. After purified by affinity adsorption purification，the purity of fusion protein is both higher than 90%.Through the back of the neck skin repeatedly immune eight weeks of the New Zealand white rabbit to prepare polyclonal antibody. After the fifth immune antiserum titer was determined by ELISA，which titer more than 12 800. Polyclonal antibodies were then successively prepared by Protein A affinity adsorption purification and nitrocellulose membrane purification. And the specificity of polyclonal antibodies was detected by Western blotting. Results showed that the polyclonal antibody prepared could specifically and precisely bind IFT25 in Chlamydomonas reinhardtii，which could provide important theoretical and methodological supports to clarify mechanisms of the action of IFT25 and diagnose diseases associated with cilia.