关键词: 蓝氏贾第鞭毛虫, 沉默信息调节因子2, 克隆, 表达, 纯化

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 M_r 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