Mechanism Of Cortactin-mediated Helicobacter Pylori VacA Protein Induced Apoptosis Of Gastric Epithelial Cells

Objiective：To study the effect and mechanism of cortactin on Helicobacter pylori VacAprotein-induced apoptosis of gastric epithelial cells.Methods:1.Purification of Helicobacter pylori VacA protein. The frozen H.pylori ATCC26695strains was seeded on skirrow’s medium plates and cultured in micro-aerobic environmentfor48h. Fresh cultured medium H.pylori colony were scraped, suspended in skirrow’sliquid medium, and cultured in microaerophilic environment for24h. Approximately2LH.pylori bacterium solution was collected,50%saturation of ammonium sulfate was addedinto the supernatant collected from bacterium solution at4℃to precipitate protein, theprotein precipitate was collected by centrifugation and dissolved in5ml PBS buffer.Protein samples was purified using HiTrap SP HP column, HiTrap Phenyl HP column,HiTrap Q HP column in sequence. The purified VacA protein was identified byimmunoblotting.2. Expression and purification of the recombinant VacA protein. Using VacA gene ofH.pylori60190as a template, VacA gene (3864bp) was synthesized and constructed intopUC57plasmid. Then the vector plasmid pQE30and VacA gene was restrictionenzyme digested by Sal Ⅰ+BamH Ⅰ. Recycling fragment of plasmid pQE30and VacAgene was linked and transformed into JM109competent bacteria, coated on LB plates,incubated in37℃incubator upside down overnight. Well grown colonies were choosedand inoculated in LB culture medium, cultured in bed temperature incubator, at37℃and250r/min overnight, the plasmids were extracted using plasmid extraction kit and identifiedby Sal Ⅰ+BamH Ⅰ restriction endonuclease digestion. The identified VacA plasmidswere transformed into competent cells M15, coated on A+plates, incubated at37℃overnight. A single colony was inoculated into LB culture medium and activated to culturein bed temperature incubator overnight. Inducible expressed VacA recombinant protein was purified by Ni2+-NTA resin and identified by SDS-PAGE electrophoresis.3. Biological effects of VacA protein secreted H.pylori and VacA recombinant protein.The two kinds of VacA protein were incubated with AGS cells respectively, and thedifference of protein-induced biological effect such as apoptosis and vacuolar effectsbetween the natural protein and recombinant protein was identified by flow cytometry andmicroscopy. protein compare these two methods of.4. Construction, packaging of PLVX-siRNA2-Puro-hScramble lentiviral vector, andscreening of lentivirus-stably transfected human gastric cancer AGS cell lines. ThepLVX-cortactin-mCMV-ZsGreen-PGK-Puro plasmids and pLVX-siRNA2-Puro-cortactinplasmids were constructed, identified by gene sequencing. Then the plasmids werepackaged and stably transfected to human293T cells, HEK293cells, AGS gastric cancercell lines. We successfully obtained cortactin-overexpressed AGS cell lines and cortactinsiRNA-infected AGS cell lines.5. Role of cortactin in VacA-induced apoptosis. VacA protein secreted H.pylori wasincubated with AGS cells, cortactin-overexpressed AGS cells and cortactin siRNA-infectedAGS cells, respectively, Flow cytometry and Western blot were used to detect apoptosis.Results:1Successfully obtained purified VacA protein secreted by H.pylori, SDS-PAGEanalysis indicated the protein was consistent with the expected molecular weight, theprotein was identified VacA protein by Western blotting.2Successfully obtained purified the recombinant VacA protein, SDS-PAGE analysisindicated the protein was consistent with the expected molecular weight.3. VacA protein secreted by H.pylori can significantly induce AGS apoptosis andvacuoles effect, but the recombinant VacA protein did not.4. Successfully constructed cortactin-overexpressed AGS cell lines and cortactinsiRNA-infected AGS cell lines.5. VacA protein-induced apoptosis was enhanced in in cortactin-overexpressed AGScell lines, Bax protein expression was upregulated and anti-apoptotic Bcl-2protein wasdownregulated. And VacA protein-induced apoptosis in cortactin siRNA-infected AGS celllines showed the opposite effects. Conclusion:1. VacA protein secreted by H.pylori has better activity to induce cell vacuolizationand apoptosis, and the recombinant VacA protein shows no effects.on……2. Cortactin is involved in the regulation of AGS cell apoptosis induced by VacA.