Experimental Study On Helicobacter Pylori Polyvalent Oral Recombinant DNA Vaccine

Helicobacter pylori (H. pylori) is a major pathogen of chronic active gastritis and peptic ulcer, and plays an important role in the pathogenesis of gastric adenocarcinoma and mucosa-associated lymphoid tissue lymphoma. Although significant progresses have been made in treating H. pylori infection with current triple or quadruple therapy based on antibiotics and proton pump inhibitor, the limitations of pharmacological therapy such as side effects, poor compliance, high cost, and especially, rapid emergence of antibiotic resistance have been hindering the development of more efficient means to prevent and control H. pylori infections. Previous experiences suggest that vaccination may be an alternative. More and more attentions have been paid on the development of oral H. pylori vaccine with features such as easy inoculation, low cost and quick extensive generalization.It is important for H. pylori vaccine investigation to screen highly conservative surface antigen with good immunogenicity and immunoreactivity. The urease (Ure) necessary for H. pylbrisurvival is not only a colonization factor, but a major factor of virulence, its B subunit is a certain candidate antigen with good antigenicity. The neutrophil activating protein (HP-NAP) is a new major virulence factor of H. pylorimore recently identified and termed for its ability of inducing adhesion of neutrophils to gastric endothelial cells and production of reactive oxygen radicals. And the H. pylori adhesion A (HpaA), one of the major adhesion factor, is a flagellar sheath protein of H. pylori. The nucleotide sequences of Ure, NapA (oligomer of HP-NAP) and HpaA are certificated and highly conservatived, and they may be used for H. py/orivaccme development as effective protection antigens binding to the surface of bacterium with powerful immunogenicity.At present, investigation of H. py/orioral vaccines is mainly focused on thedevelopment of protein vaccines. However, the preparation and purification of protein antigen is laborious, and effective immune responses are induced only at the presentation of mucosal adjuvant. Most of the mucosal adjuvants are harmful to human body to different extents. It is of considerable significance to explore new generation vaccine system for the development of H. pylori oral vaccine.DNA vaccine has shown large potential in protecting and treating many diseases since it was born. It can induce complete immune responses, provide heterologous cross protection, and be easily prepared as multivalency vaccine. On the other hand, compared with traditional vaccines, live attenuated Salmonella typhimurium (S. typhimurium) used as neotype of vector delivery system for heterologous antigens does not require antigen purification, and can not only protect antigen from degradation and denaturation in stomach but also express adjuvant activity preventing induction of oral tolerance. Massive trials have proved that attenuated 5. typhimurium is safe to immunize human as vaccine bearer.The immunifaction of simple protective antigen of H. pylori is not satisfactory. And polyvalent vaccine will be tendency of H. pylori vaccine investigation. The objective of our study was to construct a fusion gene of UreB, NapA and HpaA, the main candidates of H. pylori protective antigen, then the fusion gene of ureB-napA-hpaA was subcloned into an eukaryotic expression vector of pIRES, and the recombinant plasmid of plRES-ureB-napA-hpaA was then transformed into the ending host bacteria of live attenuated 5. typhimurium strain SL7207, then an oral recombinant DNA vaccine of UreB-NapA-HpaA was constructed, and its immunogenicity was evaluated as a fundament for the development of oral H. pylori DNA vaccine.Objectives1. To construct a prokaryotic expression system of ureB, napA and hpaA gene, and to induce and purify the expression of UreB, NapA and HpaA protein. To prepare antiserum of relevant protein and to evaluate its immunogenicity, respectively.2. To construct and identify a fusion gene of ureB-napA-hpaA, then to subclone the fusion gene int...