摘要
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。
Leucine zipper transcription factor 1 （LZTFL1） is a protein associated with cilia signaling, and it is unclear about its function and mechanism in signal transduction. While Chlamydomonas reinhardtii as a model organism that studies the signal transmission of cilia, there is little research on the LZTFL1 protein, and no corresponding antibody has been detected. Based on this phenomenon, polyclonal antibody of LZTFL1 was prepared for subsequent study. The aim was to clone and express C. reinhardtii Lztfll gene in Escherichia coli （E. coli）. The Lztfll gene of C. reinhardtii CC125 was amplified by RT-PCR, then was sub-cloned and heterologous expressed in E. coli BI21 （ DE3 ） using pET-28a （ ＋ ） expression vector. Fusion protein 6 × His- LZTFL1 was purified and immunized into New Zealand white rabbit so as to prepare polyclonal antibody. Finally, the sensitivity and specificity of the polyclonal antibody were detected through indirect ELISA and Western blot, respectively. The anti-LZTFL1 showed high sensitivity （1：512 000） and specificity. The above results indicate that Lztfll gene has been successfully cloned and expressed, and the rabbit anti-C, reinhardtii LZTFL1 polyclonal antibody has been prepared, which laid the foundation for the study of the structure and function of LZTFL1 protein in C. reinhardtii and its interaction in cilia signal transduction.
出处
《中国生物工程杂志》
CAS
CSCD
北大核心
2017年第11期109-115,共7页
China Biotechnology
基金
国际遗传工程与生物技术中心联合研究资助项目(CRP/CHN15-01)
关键词
莱茵衣藻
LZTFL1
蛋白质纯化
多克隆抗体
免疫荧光
Chlamydomonas reinhardtii LZTFL1 Protein affinity purification Polyclonal antibody Immunofluorescence
