›› 2011, Vol. 29 ›› Issue (3): 9-204-207.

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Cloning, Expression and Purification of Silent Information  Regulator 2 from Giardia lamblia

 ZHANG  Xia, JU  Hong-Mei, WANG  Yun-Hua, LI  Ya-Jie   

  1. Medical School,Dalian Uninversity,Dalian 116622,China
  • Online:2011-06-30 Published:2012-09-27

Abstract: Objective  To clone and express silent information regulator 2 (Sir2) gene from Giardia lamblia.  Methods   The GlSir2 gene was amplified by PCR from genomic DNA of Giardia lamblia(Chinese strain C2 clone). PCR product was cloned into pMD-19T vector and transformed into E. coli JM109. The recombinant plasmid was sequenced and then cloned into the pET28b vector. The pET28b-GllSir2 recombinant plasmid was transformed into E. coli BL21(DE3), followed by expression of the protein induced by IPTG. The recombinant protein was analyzed by SDS-PAGE. Inclusion bodies were dissolved with 8 mol/L urea, and the supernatant was collected and applied to Ni2+ affinity chromatography. The purified recombinant protein was renatured by dialysis and verified by Western blotting using anti-His tag antibody.  Results  GlSir2 gene sequence was cloned. The GlSir2 open reading frame (1 680 bp) encoded a 559-amino acid protein with Mr 62 800. The recombinant plasmid pET28b-GlSir2 expressed an inclusion body protein of GlSir2 after being induced with IPTG. The protein purity reached above 80% after purification. The purified protein was renatured by dialysis. The recombinant GlSir2 was recognized by anti-His tag antibody.  Conclusion  The coding sequence of GlSir2 gene was cloned and expressed in vitro. The recombinant protein was identified by anti-His tag antibody.

Key words: Giardia lamblia, Silent information regulator 2, Clone, Expression, Purification