Scientia Agricultura Sinica ›› 2012, Vol. 45 ›› Issue (1): 178-183.doi: 10.3864/j.issn.0578-1752.2012.01.021

• VETERINARY SCIENCE • Previous Articles     Next Articles

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

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

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