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李 静, 秦 勇, 顾 云, 刘 炎, 刘 梅. 2012: 多疣壁虎tubulin beta 3基因克隆和多克隆抗体制备. 动物学研究, 33(4): 395-401. DOI: 10.3724/SP.J.1141.2012.04395
引用本文: 李 静, 秦 勇, 顾 云, 刘 炎, 刘 梅. 2012: 多疣壁虎tubulin beta 3基因克隆和多克隆抗体制备. 动物学研究, 33(4): 395-401. DOI: 10.3724/SP.J.1141.2012.04395
LI Jing, QIN Yong, GU Yun, LIU Yan, LIU Mei. 2012: Molecular cloning of tubulin beta 3 (TUBB3) in Gekko japonicus and preparation of its polyclonal antibody. Zoological Research, 33(4): 395-401. DOI: 10.3724/SP.J.1141.2012.04395
Citation: LI Jing, QIN Yong, GU Yun, LIU Yan, LIU Mei. 2012: Molecular cloning of tubulin beta 3 (TUBB3) in Gekko japonicus and preparation of its polyclonal antibody. Zoological Research, 33(4): 395-401. DOI: 10.3724/SP.J.1141.2012.04395

多疣壁虎tubulin beta 3基因克隆和多克隆抗体制备

Molecular cloning of tubulin beta 3 (TUBB3) in Gekko japonicus and preparation of its polyclonal antibody

  • 摘要: Tubulin beta 3 (TUBB3) 是特异表达于神经元的微管蛋白tubulin beta家族成员, 被认为在维持轴突正常状态起着重要作用, 是神经元特异的标志蛋白。该研究旨在获得多疣壁虎TUBB3全长cDNA序列并制备其多克隆抗体, 为进一步研究多疣壁虎断尾再生提供基因和抗体工具。根据多疣壁虎中枢神经组织cDNA文库中TUBB3的EST片段序列, 采用RACE-PCR方法, 获得了全长cDNA, 序列全长1 790 bp, 编码450个氨基酸, 与其他物种TUBB3蛋白高度同源; RT-PCR方法 和Northern blotting检测了TUBB3组织表达谱及其转录本的大小; 在GenBank登录号为JQ080315; NJ法构建的系统进化树分析表明,多疣壁虎的TUBB3蛋白与鸡的TUBB3蛋白在进化距离上较近; 构建pET-32a-TUBB3原核表达载体, 诱导表达并纯化TUBB3蛋白, 获得兔抗壁虎TUBB3多克隆抗体, ELISA检测其效价大于1:65 536, Western blotting方法证实所制备的多克隆抗体可特异识别多疣壁虎神经组织的的TUBB3蛋白。

     

    Abstract: The tubulin beta III (TUBB3) gene encodes a class III member of the beta tubulin protein family that is primarily expressed in neurons and is considered to play a critical role in proper axon guidance and maintenance. This protein is generally used as a specific marker of neurons in the central nervous system. We obtained the full length cDNA sequence of TUBB3 by using the RACE method based on the EST fragment from the brain and spinal cord cDNA library of Gekko japonicus. We further investigated the multi-tissue expression pattern by RT-PCR and identified one transcript of TUBB3 about 1.8 kb in the central nervous system of Gekko japonicus by Northern blotting. The completed cDNA of gecko TUBB3 is 1 790 bp with an open reading frame of 1 350 bp, encoding a 450 amino-acid protein. The recombinant plasmid of pET-32a-TUBB3 was constructed and induced to express His-tagged TUBB3 protein in prokaryotic BL21 cells. The purified TUBB3 protein was then used to immunize rabbits to generate polyclonal antisera. The titer of the antiserum was more than 1:65 536 determined by ELISA. The result of western blotting showed that the TUBB3 antibody could specifically recognize the recombinant TUBB3 protein and endogenous TUBB3 protein. Our findings provide the tools to further understand the TUBB3 gene and investigate the regeneration of the central nervous system in Gekko japonicas.

     

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