莱茵衣藻IFT139蛋白抗原的原核表达、纯化及多克隆抗体的制备

Prokaryotic Expression,Purification and Polyclonal Antibody Preparation of Chlamydomonas reinhardtii IFT139 Protein Antigen

IFT139是莱茵衣藻(Chlamydomonas reinhardtii)纤毛内运输蛋白IFT复合物A中的一个重要组分.其编码基因的突变、缺失或沉默将会影响纤毛正常的组装和解聚,进而导致纤毛病的产生.为深入研究IFT139的作用机制,本实验首先PCR获得ift139 5′端458 bp的EST片断,成功构建了p MAL-c2-ift139与p ET-28a(+)-ift139重组质粒.然后将重组质粒转入大肠杆菌(E.coli)BL21(DE3),通过IPTG诱导表达,分别得到相对分子质量为6.0×104和2.5×104的重组MBP/HIS-IFT139融合蛋白.进一步将亲和纯化的MBP-IFT139融合蛋白(纯度95%,以上),免疫新西兰大白兔获得抗血清,间接ELISA法测得血清效价为1﹕256,000.最后将抗血清依次经Protein A和硝酸纤维素膜纯化后,用Western blot检测抗体特异性,结果显示所制备抗体能够正确特异性识别莱茵衣藻中的IFT139,从而为IFT139作用机制的阐明和纤毛相关疾病的研究奠定了重要基础.

IFT139 is a key component of the intraflagellar transport （IFT） complex A in Chlamydomonas reinhardtii. Mutations, deletions or silence of ift139 gene can affect cilia assembly and depolymerization, and thus lead to ciliopathy. To further investigate the role of IFT139 in ciliogenesis, ift139 5＇-458 bp EST eDNA fragment was firstly amplified and the recombined plasmids pMAL-c2-ift139 and pET-28a （ ＋ ） -ift139 were constructed and transferred into Escherichia coli BL21 （DE3）. Under IPTG induction, fusion proteins MBP-IFT139 and HIS-IFT139 with a molecular weight of 6.0 × 104 and 2.5 ×104, respectively, were obtained. The fusion protein MBP-IFT139 was purified by affinity adsorption purification （more than 95% purity） and then used for immunizing New Zealand white rabbits to produce antiserum. When the titer of antiserum reached 256 000, it was purified by Protein A, and then affinity adsorption purification was followed by nitrocellulose membrane purification procedure. The specificity of polyclonal antibodies was evaluated with Western blot. The result showed that the polyclona/antibody can specifically recognize IFT139. The research has provided an important tool for further clarifying the role of IFT 139 in ciliogenesis and cilipathy,