关键词: 蓝氏贾第鞭毛虫, 核苷二磷酸激酶, 克隆, 表达, 纯化

Abstract:

The nucleoside diphosphate kinase of Giardia lamblia(GlNDPK) (Chinese strain C2) coding sequence was amplified by PCR using the genomic DNA as the template. The PCR product was inserted into the pET28a plasmid and transformed into E. coli JM109. The positive clones were picked up for identification and sequencing. The pET28a-GlNDPK recombinant plasmid was transformed into E. coli BL21 (DE3), and expressed under IPTG induction. The proteins expressed were analyzed by SDS-PAGE. Inclusion bodies were dissolved with 8 mol/L urea and the supernatant was collected and purified by Ni2+ affinity chromatography. The recombinant protein was verified by Western blotting using anti-His6 tag antibody. The results showed amplification of the GlNDPK coding sequence of Giardia lamblia (Chinese strain C2)  by PCR, resulting in a product of 1 200 bp. The recombinant plasmid pET28a-GlNDPK was verified by PCR, single enzyme digestion, and sequencing. As expected, SDS-PAGE showed that the recombinant GlNDPK protein was expressed as an inclusion body with a Mr of 40 000. Western blotting showed that the recombinant GlNDPK could be recognized by anti-His6 tag antibody. Therefore, the coding sequence of GlNDPK gene and its recombinant protein have been obtained.
  

Key words:  Giardia lamblia, Nucleoside diphosphate kinase, Clone, Expression, Purification