Cloning And Expression Of Zot Gene Of Vibrio Cholerae And Its Effect On Human Small Intestinal Epithelial Cells Apoptosis

Objective:To further explore research symbiotic relationship between the two virulence genes of ctxAB and zot. To clone the full-length gene of zot from the standard strains of Vibrio cholera M045 genome DNA and construct the prokaryotic expression vector and transform it into E.coli BL21(DE3) to express protein. After expression and purification, the zot-protein provide active protein as a target for human small intestinal epithelial cells. Methods:1.PCR techniques were applied to detect ctxAB and zot of the 239 strains of Vibrio cholerae isolated from 2000 to 2012 in Jiangxi Province;2. zot gene was amplified by PCR from genomic DNA of Vibrio cholerae M045;3.The zot gene directionally inserted into vector pET-32a(+). Then the recombinant plasmid pET-32a(+)-zot was identified by restriction endonucleases digestion as well as sequencing;4.The recombinant plasmid pET-32a(+)- zot transformed into E. coli BL21(DE3) filter expression strain E.coli BL21(DE3) / pET-32a(+)- zot, the E.coli BL21(DE3) / pET-32a(+)- zot was inducted by IPTG and analysised by SDS-PAGE;5.The recombinant protein was purified by affinity chromatography(Ni-IDA Resin) and identified by Western blot;6.Already adherent growth of human intestinal epithelial cells were stimulated by purified zot protein and detected by flow cytometry apoptosis. Result:1.ctxAB virulence genes were detected positive and zot gene was detected negative of the 41 strains of Vibrio cholerae,indicating ctxAB and zot virulence genes are not strictly co-existence.2.zot gene was successfully amplified 1 200 bp products by PCR detection technology.3.zot gene was successfully inserted into the vector pET-32a(+) confirmed by double enzyme-digestion and sequencing.4.pET-32a(+)- zot was transformated into E.coli BL21(DE3),as well as to be inducted by IPTG.Recombinated bacterium was analysised by SDS-PAGE. It was showed that a a bright band at the location with the size of the target protein.5.Purified zot ecombinated protein was acquired after Ni-IDA Resin chromatography.The molecul are weight of zot recombinated protein 47 kD,which eoncentration is 0.324mg/ml analysised from SDS-PAGE.Recombinant protein is the target protein identified by Western blot.6.zot recombinant protein can cause human intestinal epithelial cells apoptosis was determined by flow cytometry. Conclusions:The whole gene sequence of zot from M045 was cloned succesfull.Which was used to constructured the recombinated bacterium E.coli BL21(DE3) / pET-32a(+) – zot.The zot recombinant protein was successfully expressed and purified.The purified zot recombinant protein can cause human intestinal epithelial cells apoptosis.