background: H. pylori is closely related to the occurrence and development of various digestive gastritis, peptic ulcer and mucosa-associated lymphoid tissue (MALT) lymphoma. H. pylori is also a class I carcinogen of gastric cancer. VacA is the only exocrine toxin of H. pylori, which plays a very important role in the pathogenesis of H. pylori. The production of VacA in natural circumstances is complex with heavy workload and low yield. Therefore, it is very important to obtain recombinant VacA protein which is stable and biologically active. This study therefore aims to explore the expression, purification and stable storage of VacA toxin of H. pylori in E.coli, and to provide experimental basis for further ex... More
background: H. pylori is closely related to the occurrence and development of various digestive gastritis, peptic ulcer and mucosa-associated lymphoid tissue (MALT) lymphoma. H. pylori is also a class I carcinogen of gastric cancer. VacA is the only exocrine toxin of H. pylori, which plays a very important role in the pathogenesis of H. pylori. The production of VacA in natural circumstances is complex with heavy workload and low yield. Therefore, it is very important to obtain recombinant VacA protein which is stable and biologically active. This study therefore aims to explore the expression, purification and stable storage of VacA toxin of H. pylori in E.coli, and to provide experimental basis for further exploration of the role of VacA in H. pylori -induced inflammation of cancer.
results: A 2502-bp fragment and VacA gene were identified. An 89.7-kDa VacA recombinant protein was expressed and purified from the recombinant engineering bacteria and was preserved stably in 50 mM acetic acid buffer (pH 2.9). The amount of the recombinant protein was larger in the inclusion bodies than in the supernatant. In addition, after a 24-h culture with VacA recombinant protein, GES-1 cells demonstrated evidence of apoptosis including early nuclear immobilization and clustering under inverted microscope and TEM. It was found that VacA recombinant protein induced apoptosis by TUNEL assay.
conclusions: A VacA recombinant protein that is stably and highly expressed and possesses pro-apoptotic activity is successfully constructed. The protein is stably preserved in 50 mM acetic acid buffer (pH 2.9).
