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目的:亮氨酸拉链转录因子1(LZTFL1)是一种与纤毛信号转导相关的蛋白质,关于其如何在信号转导中发挥作用以及作用机制目前尚不清楚。莱茵衣藻(Chlamydomonas reinhardtii)是研究纤毛信号传导的模式生物,而目前对莱茵衣藻LZTFL1蛋白的研究甚少,尚未有相应的检测抗体,因此制备LZTFL1多克隆抗体用于后续实验研究。方法:采用RT-PCR技术从C.reinhardtii CC125中提取总RNA,扩增981bp的目的基因Lztfl1,插入到p ET-28a(+)原核表达载体,成功构建p ET-28a(+)-Lztfl1重组质粒,转入E.coli BL21(DE3)经IPTG诱导后成功表达6×His-LZTFL1融合蛋白,融合蛋白经亲和纯化后免疫新西兰大白兔制备多克隆抗体,最后采用间接ELISA法测得抗血清效价达到1∶512 000,经Western blot对C.reinhardtii CC125检测具有较高特异性。结果:首次实现了莱茵衣藻LZTFL1蛋白的原核表达,制备出一支兔抗莱茵衣藻LZTFL1蛋白的多克隆抗体,为后续研究LZTFL1蛋白在莱茵衣藻中的结构功能及在纤毛信号转导中的相互作用奠定了基础。
OBJECTIVE: Leucine zipper transcription factor 1 (LZTFL1) is a protein involved in ciliary signal transduction. It is unclear how it plays a role in signal transduction and its mechanism of action. Chlamydomonas reinhardtii is a model organism for studying ciliary signaling. At present, little research has been done on LZTFL1 protein of C. reinhardtii, and no corresponding detection antibody has been found yet. Therefore, a polyclonal antibody against LZTFL1 was prepared for further study. Methods: The total RNA was extracted from C. reinhardtii CC125 by RT-PCR and the 981 bp Lztfl1 gene was amplified and inserted into the prokaryotic expression vector p ET-28a (+). The recombinant plasmid p ET-28a (+) - Lztfl1 The recombinant plasmid was transformed into E.coli BL21 (DE3) and induced by IPTG. The recombinant protein was successfully expressed in 6 × His-LZTFL1 fusion protein. The purified fusion protein was used to immunize New Zealand white rabbits to prepare polyclonal antibody. Finally, The titer reached 1: 51200. Western blot analysis of C.reinhardtii CC125 showed high specificity. Results: The prokaryotic expression of LZTFL1 protein of C. reinhardtii was achieved for the first time, and a polyclonal antibody against LZTFL1 of C. reinhardtii was prepared. In order to study the structure and function of LZTFL1 in C. reinhardtii, The foundation of the interaction is laid.