ABSTRACT
The aim of this study is to construct the expression vector of zot gene of Vibrio cholerae and to realize the expression of zot gene of Vibrio cholerae in E.coli and study the biological activity of its recombinant expression product.In order to express zot protein in E.coli,the full-length open reading frame of zot was amplified by PCR from the standard strains of Vibrio cholerae MO45 genome DNA.The PCR product was cloned into prokaryotic expression vector pET-32a(+) with restriction enaymes Bam H I and XhoI.The recombinant vector pET-32a(+)-zot was transformed into E.coli BL21 (DE3) and expressed by IPTG induction.The zot fusion protein was detected by SDS-PAGE and Western blotting and purified by Ni-NTA affinity chromatography.After expression and purification,the recombinant expression protein played as a target for human small intestinal epithelial cells.Restriction endonuclease digestion,PCR and DNA sequencing analysis showed that the zot gene of 1 200 bp was amplified from Vibrio cholerae DNA,and the recombinant plasmid pET-32a(+)-zot was constructed and detected its expression in prokaryotic cell successfully with SDS-PAGE and Western blot techniques.The zot recombinant protein was successfully expressed and purified.The purified zot recombinant protein can cause human intestinal epithelial cells apoptosis.