ACTA Theriologica Sinica

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Generation and validation of a polyclonal antibody against recombinant Galectin-1 in sika deer (Cervus nippon)

WANG Zhen, WANG Datao, YU wei, LI Chunyi   

  1. (1 College of Life Science, Zhejiang Sci-Tech University, Hangzhou 310018, China)
    (2 Institute of Special Wild Economic Animal and Plant Science, the Chinese Academy of Agricultural Science, Changchun 130112, China)
    (3 Changchun Sci-Tech University, Changchun 130600, China)
  • Online:2019-03-30 Published:2019-03-26

梅花鹿重组Galectin-1的表达及多克隆抗体的制备与鉴定

汪珍 王大涛 于威 李春义
  

  1. (1 浙江理工大学生命科学学院 杭州, 310018)
    (2 中国农业科学院特产研究所 长春 130112)
    (3 长春科技学院 长春 130600)
  • 通讯作者: 于威 E-mail: yuwei@zstu.edu.cn; 李春义 E-mail: lichunyi1959@163.com
  • 基金资助:
    中国科学院A类战略性先导科技专项(XDA16010105)

Abstract: Galectin-1, a mammalian galactoside lectin is present in many cells and tissues. It is involved in many physiological processes, such as cell adhesion, proliferation, apoptosis, and inflammation. Galectin-1 is a member of the β-galactoside binding family, which are involved in the regulation of the immune system and development of tumors. Antler is a rare accessory organ that can periodically shed and regenerate from its pedicle in deer and has attracted attention as a new model for studying mammalian organ regeneration. Deer antlers offer a unique opportunity to explore how nature has devised a system of epimorphic regeneration in mammals. Antler regeneration is a stem cell-based process derived from antler stem cells that are located in the pedicle periosteum (PP). Elevated expression of galectin-1 in antler stem cells suggest that this molecule may play an important role in antler regeneration. To study the biological function of Galectin-1 in antler regeneration, it is necessary to generate a polyclonal antibody. In this study, the Galectin-1 gene of Sika deer (Cervus nippon) was inserted into the pET28a vector to obtain the recombinant plasmid, pET28a-Galectin-1. This recombinant plasmid was then transformed into Escherichia coli strain BL21(DE3) for induction and expression. The target protein was purified by using a Ni-NTA agarose affinity column. The purified protein was used to immunize rabbits to obtain the polyclonal antibody. Antibody titer was determined by enzyme-linked immunosorbent assay (ELISA) for Galectin-1. The specificity of antibody was confirmed by western blot analysis and the expression of Galectin-1 in the antler stem cells was detected by an immunofluorescence assay. The results showed that the titer of the antibody was 1:64000 with high specificity and that Galectin-1 was expressed in almost all of the antler PP cells. We have generated a specific polyclonal antibody of deer Galectin-1, which will serve as an important tool for the study of the regulation of Galectin-1 in antler regeneration.

Key words: Sika Deer, Antler, Galectin-1, Prokaryotic expression, Polyclonal antibody

摘要: 半乳糖凝集素-1(Galectin-1)是最先被报道的哺乳动物半乳糖凝集素,存在于多种组织和细胞内,参与细胞的粘附、增殖、凋亡和炎症反应等多种生理病理过程,并且与免疫系统的调节和肿瘤的发生发展密切相关。鹿茸是哺乳动物中罕见的能够周期性的脱落和再生的附属器官,作为研究哺乳动物器官再生的新模型受到关注。鹿茸再生是一个基于干细胞的过程,定位于角柄骨膜的干细胞是鹿茸再生的基础,Galectin-1在角柄骨膜细胞(pedicle periosteum cell, PPC)中高度表达,提示其在鹿茸再生中发挥着重要的作用。由于尚没有商用的鹿Galectin-1蛋白及其抗体,为进一步研究Galectin-1在鹿茸再生中的生物学功能,需要制备相应的蛋白和抗体,本实验将梅花鹿Galectin-1基因与pET28a连接,并将重组质粒pET28a-Galectin-1转入大肠杆菌(Escherichia coli)BL21(DE3)中诱导表达。用Ni-NTA Agarose亲和层析纯化融合蛋白,免疫兔子制备多克隆抗体。酶联免疫吸附(enzyme-linked immunosorbent assay, ELISA)法检测抗体效价、Western blot 检测抗体特异性、细胞免疫荧光检测Galectin-1在角柄骨膜细胞中的表达情况。结果表明,本实验成功诱导重组原核表达载体pET28a-Galectin-1在BL21(DE3)中表达,通过Ni纯化获得融合蛋白。ELISA结果显示,抗体效价达到1:64000,Western blot结果表明该抗体特异性良好,细胞免疫荧光显示Galectin-1在PPC全细胞中表达。本实验获得了纯化的鹿Galectin-1蛋白和特异性较好的多克隆抗体,为揭示Galectin-1在鹿茸再生调控中的作用提供了重要的实验材料。

关键词: 梅花鹿, 鹿茸, 半乳糖凝集素-1, 原核表达, 多克隆抗体