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Journal of Animal and Veterinary Advances

Year: 2013  |  Volume: 12  |  Issue: 2  |  Page No.: 201 - 207

Cloning, Expression and Purification of Nanog Protein from Capra hircus in Escherichia coli

X.B. Zheng, X.M. Cen, L.X. Wang, G.Y. Xin, X.H. Fu, P. Liu, G.H. Li, M. Zhang, S.S. Lu and K.H. Lu

Abstract

Nanog is one of important transcription factors to maintain characteristics of pluripotent stem cells. The present study was to clone Nanog of Capra hircus to express His-Nanog protein in E. coli BL 21 cells and further to purify it. The total RNA was extracted from primordial genital ridge tissues of a fetal lam and by means of RT-PCR, Nanog gene was amplified which was subcloned to pET32a to construct its prokaryotic expression vector. Confirmed by restrictive endonuclease digestion and DNA sequencing, the recombinant plasmid was transformed into E. coli BL21(DE3) and His-Nanog fusion protein was expressed by the induction of IPTG and identified with SDS-PAGE analysis. Under denaturing condition, the His-Nanog protein was purified by using Ni-NTA resin and verified by Western blotting assay. The results showed that The Open Reading Frame (ORF) of Nanog gene in Capra hircus is composed of 903 nucleutide acids, coding 320 amino acids; SDS-PAGE assay showed that His-Nanog fusion protein was efficiently expressed in form of inclusion bodies in E. coli BL21 (DE3) inclusion bodies were solubilized in 6 mol L-1 GuHCl, His-Nanog fusion protein with higher purity was purified by using Ni-NTA resin; Western blotting assay showed that the purified His-Nanog could bind to anti-His tag antibody specifically indicating the expected immunogenicity. This recombinant protein could be used directly to prepare polyclonal or monoclonal anti-Nanog antibody which will lead to study Nanog’s function or characteristics of pluripoten stem cells (such as iPS cells) in Capra hircus.

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