华东师范大学学报(自然科学版) ›› 2010, Vol. 2010 ›› Issue (4): 77-84.

• 生命科学 • 上一篇    下一篇

盘基网柄菌allC的克隆、表达和多克隆抗体的制备

陈能星, 魏晓静, 刘 伟, 侯连生   

  1. 华东师范大学 生命科学学院，上海 200062
  • 收稿日期:2009-03-01 修回日期:2009-07-01 出版日期:2010-07-25 发布日期:2010-07-25
  • 通讯作者: 侯连生

Clone, expression of allantoicase gene from Dictyostelium disc-oideum and preparation of polyclonal antibody against ALC

CHEN Neng-xing, WEI Xiao-jing, LIU Wei, HOU Lian-sheng   

  1. School of Life Science, East China Normal University, Shanghai 200062, China
  • Received:2009-03-01 Revised:2009-07-01 Online:2010-07-25 Published:2010-07-25
  • Contact: HOU Lian-sheng

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摘要： 运用RT-PCR技术从盘基网柄菌(Dictyostelium discoideum)总mRNA中克隆到了尿囊酸酶基因(allC)，该基因编码区开放读框长1 100 bp,编码的蛋白约42 kD.由于是在野生型和突变型细胞中差异表达的片段，表明该基因在盘基网柄菌多细胞发育中起到重要作用,因此将allC克隆入融合表达载体pET-32a(+) ,在大肠杆菌E.coliBL21(DE3) 中进行诱导表达带有6个组氨酸标签的尿囊酸酶(ALC)融合蛋白,经镍柱亲和层析，获得了电泳纯蛋白.用纯化的融合蛋白免疫新西兰大白兔，制备多克隆抗体.ELISA 测得制备的抗ALC蛋白的多克隆抗体的效价可达1∶64 000，Western Blotting检测证明该抗体有较强的针对ALC蛋白的专一性.这些数据表明重组质粒表达的ALC融合蛋白具有良好的抗原性,制备ALC的多克隆抗体有良好特异性和效价,能够满足针对ALC免疫印迹和细胞内定位检测等实验要求,为深入研究ALC蛋白在盘基网柄菌多细胞发育的功能作用提供了有力工具.

关键词: 盘基网柄菌, 尿囊酸酶, 原核表达, 蛋白纯化, 多克隆抗体, 盘基网柄菌, 尿囊酸酶, 原核表达, 蛋白纯化, 多克隆抗体

Abstract: The coding region of allCis obtained from total mRNA of Dictyostelium discoideumby RT-PCR. The results show that the length of allC is 1 100 bp with sequence analysis and coded protein is 42 kD.Becase it is a differentially expression fragments, indicating that the gene play an important role in multicellular development and allC was cloned into the fusion expression vector pET-32a(+) with 6 His tagged and expressed in E. coli BL21 (DE3) host cells. and then purified by Ni2+ affinity chromatography column and fusion protein purified was used to immune the New Zealand rabbits for preparing polyclonal antibody. The fusion protein was successfully expressed and polyclonal antibody was also successfully obtained. The potency of the antibody was as high as 1∶64 000. The specificity of antibody was proved by Western Blotting analysis of expression product of allC. These data suggest ALC fusion protein has the good antigenicity. The antibody with high titer and specificity was obtained in the satisfaction of Western blot and localization experiment request , which was the foundation to farther study the characters of the allantoicase protein and their function during Dictyostelium discoideumdevelopment.

Key words: allantoicase, prokaryotic expression, protein purification, polyclonal antibody, Dictyostelium discoideum, allantoicase, prokaryotic expression, protein purification, polyclonal antibody

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