中国农业科学 ›› 2012, Vol. 45 ›› Issue (1): 178-183.doi: 10.3864/j.issn.0578-1752.2012.01.021

• 兽医 • 上一篇    下一篇

山羊SOX2多克隆抗体制备

 刘平, 张昀, 郑喜邦, 李恭贺, 岑小妹, 岳磊磊, 宗自杰, 卢晟盛, 卢克焕, 张明   

  1. 1.广西大学动物科学与技术学院,南宁 530005
  • 收稿日期:2010-09-15 出版日期:2012-01-01 发布日期:2011-11-10
  • 通讯作者: 通信作者郑喜邦,Tel:0771-3235690;E-mail:zhxibang2005@126.com
  • 作者简介:刘 平,Tel:0771-6189820;E-mail:lpzl82@163.com
  • 基金资助:

    广西自然科学基金项目(桂科自0728019,桂科自0991043)、广西亚热带生物资源保护与利用重点实验室开放课题(SB0907)

Preparation of Polyclonal Anti-Sox2 Antibody in Capra hircus

 LIU  Ping, ZHANG  Yun, ZHENG  Xi-Bang, LI  Gong-He, CEN  Xiao-Mei, YUE  Lei-Lei, ZONG  Zi-Jie, LU  Sheng-Sheng, LU  Ke-Huan, ZHANG  Ming   

  1. 1.广西大学动物科学与技术学院,南宁 530005
  • Received:2010-09-15 Online:2012-01-01 Published:2011-11-10

摘要: 【目的】构建山羊 Sox2 原核表达载体—pRSET-Sox2,并将诱导表达、纯化的 His-Sox2 融合蛋白免疫新西兰大白兔,制备 Sox2 多克隆抗体。【方法】从 pMD18T-Sox2 载体上以 Bam H I和 Xho I 双酶切截取 Sox2 片段,然后将其亚克隆到 pRSET-A 表达载体上,获得 pRSET-Sox2 重组质粒。转化了 pRSET-Sox2 的大肠杆菌 BL21(DE3),1 mmol•L-1 IPTG  37℃ 诱导 4 h,SDS-PAGE电泳及 Western blotting 检测融合蛋白表达。相同条件下大量增菌诱导,用Ni- NTA argrose 介质分离纯化 His-Sox2 重组蛋白。将体外复性的融合蛋白皮下注射新西兰大白兔,间隔 2—3 周注射一次,共 4 次。最后一次注射后 7 d,采血分离血清,用 Western blotting 检测抗体特异性。【结果】(1)原核表达载体 pRSET-Sox2 在大肠杆菌 BL21(DE3) 得到了高效表达;(2)纯化的 His-Sox2 融合蛋白能够满足多克隆抗体制备的要求;(3)经 Western blotting 检测,Sox2 多克隆抗体能与 His-Sox2 融合蛋白特异性结合。【结论】制备了高特异性山羊 Sox2 多克隆抗体,为深入探讨山羊 Sox2 基因的生物学功能奠定了基础,也为山羊(iPS)细胞检测创造了良好条件。

关键词: Sox2, 原核表达, 蛋白纯化, 多克隆抗体, 山羊

Abstract: 【Objective】 The present study was to construct a prokaryotic expression vector of Capra hircus Sox2 gene, pRSET-Sox2, to induce expression and purification of His-Sox2 fusion protein, which was used to immunize New Zeland white rabbits to prepare polyclonal anti-Sox2 antibody. 【Method】 Removed from plasmid pMD18-Sox2 by double digestion of BamH I and Xho I, Sox2 fragment was subcloned to pRSET-A vector to construct recombinant plasmid pRSET-Sox2. The plasmid was transformed into E. coli BL 21 (DE3), and His-Sox2 fusion protein was induced to expess with 1 mmol•L-1 IPTG at 37℃ for 4 h, which was identified with SDS-PAGE analysis and Western blotting. In the same way, large volume of expressing culture was prepared to purify His-Sox2 fusion protein with NI-NTA argrose under denaturing condition. The refolded fusion protein in vitro was injected subcutaneously into New Zeland white rabbits for four times at intervals of 2-3 weeks. Seven days after the last injection, blood samples were collected, serum was isolated, and specificity of polyclonal anti-Sox2 antibody was determined by Western blotting assay.【Result】 The prokaryotic expression vector pRSET-Sox2 was expressed efficiently in E. coli. BL21. The purified His-Sox2 was qualified for preparation of polyclonal antibody. The polyclonal anti-Sox2 antibody was prepared, and it could bind His-Sox2 fusion protein specifically, which was illustrated by Western blotting assay. 【Conclusion】 The polyclonal anti-Sox2 antibody with strong specificity was prepared, which will lay a solid biological foundation for study of Sox2, and for its application in detection of Capra hircus iPS cells (induced pluripotent stem cells).

Key words: Sox2 gene, prokaryotic expression, protein purification, polyclonal anti body, Capra Hircus