Isolation and purification of recombinant VacA and Helicobacter pylori-secreted VacA and VacA-induced cell vacuolar change and apoptosis / 军事医学

ABSTRACT

Objective To isolate and purify VacA protein secreted by Helicobacter pylori or recombinant VacA , and to investigate the effect of VacA-induced cell vacuolar change and apoptosis .Methods VacA proteins were separated and pu-rified from the culture supernatant of H.pylori ( ATCC26695 ) or from the split products of genetically engineered bacteria (pQE30-VacA-E.coli M15) expressing recombinant VacA.The VacA protein obtained was acidified and then incubated with AGS cells for 24 h at different final concentrations of 5 and 10 ng/ml before the vacuolar change and apoptosis of AGS cells were detected via microscopy and flow cytometry assay , respectively .Results H.pylori-secreted VacA and recombi-nant VacA were successfully separated and purified .The H.pylori-secreted VacA significantly induced the vacuolar change and apoptosis of AGS cells (P<0.01) while the recombinant VacA did not.Conclusion H.pylori-secreted VacA protein can effectively induce cell vacuolar change and apoptosis, but recombinant VacA can not, suggesting that the purified VacA protein secreted by H.pylori can be used to explore VacA-induced pathogenesis.