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中国生物工程杂志

CHINA BIOTECHNOLOGY
中国生物工程杂志
中国生物工程杂志  2017, Vol. 37 Issue (11): 109-115    DOI: 10.13523/j.cb.20171115
技术与方法     
莱茵衣藻LZTFL1蛋白的多克隆抗体制备及应用
刘雁霞, 樊振川
天津科技大学大健康生物技术研究所 天津市大健康生物技术国际联合研究中心 天津科技大学食品工程与生物技术学院 天津 300457
Polyclonal Antibody Preparation and Application of C.reinhardtii LZTFL1 Protein
LIU Yan-Xia, FAN Zhen-Chuan
Institute of Health Biotechnology, International Collaborative Research Center for Health Biotechnology, College of Food Science and Biotechnology, Tianjin University of Science and Technology, Tianjin 300457, China
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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pET-28a（+）原核表达载体，成功构建pET-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。
关键词： 蛋白质纯化;  免疫荧光;  莱茵衣藻;  LZTFL1;  多克隆抗体    
Abstract: 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 Lztfl1 gene in Escherichia coli (E. coli). The Lztfl1 gene of C.reinhardtii CC125 was amplified by RT-PCR, then was sub-cloned and heterologous expressed in E.coli BL21(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 Lztfl1 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.
Key words: Polyclonal antibody    Immunofluorescence    Chlamydomonas reinhardtii    LZTFL1    Protein affinity purification
收稿日期: 2017-05-08 出版日期: 2017-11-15
ZTFLH:  Q819  
基金资助: 国际遗传工程与生物技术中心联合研究资助项目（CRP/CHN15-01）
通讯作者: 樊振川     E-mail: fanzhen@tust.edu.cn
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引用本文:

刘雁霞, 樊振川. 莱茵衣藻LZTFL1蛋白的多克隆抗体制备及应用[J]. 中国生物工程杂志, 2017, 37(11): 109-115.

LIU Yan-Xia, FAN Zhen-Chuan. Polyclonal Antibody Preparation and Application of C.reinhardtii LZTFL1 Protein. China Biotechnology, 2017, 37(11): 109-115.

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https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20171115    或    https://manu60.magtech.com.cn/biotech/CN/Y2017/V37/I11/109

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