The Study Of Oral Vaccination With Attenuated Salmonella Choleraesuis C500Expreeing Recombinant UreB And CagA Of Helicobacter Pylori

Helicobacter pylori is a gram-negative bacterium, specialized in the colonization of the stomach, and well known as the major gastro-duodenal pathogen of peptic ulcer disease, mucosa-associated lymphoid tissue lymphoma, and gastric cancer. In90%of H. pylori-infected patients treated with antibiotics and strong acid suppressor drug such as proton pump inhibitors, its eradication is successful. However, failure of H. pylori eradication treatment has recently increased due to the proliferation of antibiotic-resistant strains. Artificial passive immunization is one way to against H. pylori, and vaccination against H. pylori is therefore one of the most effective ways to control H. pylori infection and, indeed, administration of oral bacterial antigens can protect mice against H. pylori infection.To observe the effect of intra-muscular injection with H. pylori NCTC11637on the induction of immunity,14lactating cows were randomly divided into2groups:whole bacterial antigen of H. pylori (2×1010cfu) with Freund’s adjuvant and PBS with adjuvant. The immunizations were performed on days0,14and28, and milk and serum samples were taken by at intervals of7days and then centrifugalizated and stored at-20℃until use. Serum and milk samples were collected and the levels of anti-Hp IgG assayed by indirect enzyme-linked immunosorbent assay. The results of ELISA proved that specific IgG in serum and milk increased significantly(P≤0.05). But the levels of anti-Hp IgG in serum increaser quickly than that the milk. The temperature, feed and oestrus have no changing. But the whole bacterial antigen of H.pylori made more trouble, the cows had pain when intra-muscular injected, and had several times vaccination. The intra-muscular injection of whole bacterial antigen with adjuvant can induce systemic immune response, and may be a safe and effective immunization route. But the whole bacterial antigen of H.pylori made more trouble, expensive and trouble in used.So one other vaccine which been safe, effective and convenient should be constructed. Recombinant H. pylori vaccine comprising a single subunit antigen can only induce immune response with limited protection efficiency. Development of oral vaccine would be a new effective strategy for prevention of H. pylori infection. In this study, the protective effect of H. pylori multicomponent vaccine consisting of UreB and CagA subunit antigens was constructed and investigated in mice. To develop an attenuated salmonella choleraesuis vaccine strain C500expressing Helicobacter pylori CagA and UreB, and to investigate the effects of the two-valence vaccine consisting of Helicobacter pylori CagA and UreB in preventing H. pylori infection in mice and the safety of vaccine. The Hp-UreB and Hp-CagA gene were amplified by PCR respectively and were cloned into prokaryotic expression plasmid pYA3493to construct recombinant plasmid pYA3493-UreB-CagA. The pYA3493-UreB-CagA was performed successively by PCR, enzyme digestion analysis and sequencing. The homology was compared between the cloned Hp-CagA and Hp-UreB gene and related genes in GenBank. The plasmid pYA3493-UreB-CagA was purified and transformed into Salmonella choleraesuis C500strain by electroporation. After be cultivated, positive colonies were picked out and the plasmid was amplified by PCR and enzyme digestion analysis again. The expression of pYA3493-UreB-CagA protein in the C500bacteria was proved by SDS-PAGE and Western Blotting.50Kunming mice were randomly divided into5groups:inoculated orally0.2mL (1×1010CFUmL-1) of C500expressing PYA3493-UreB-CagA, C500with pYA3493, C500, PBS and injection0.2mL (2×109) of H, pylori SS1with adjuvant. Oral doses of1×1010CFUmL-1were administered via an intragastric gavage. Immunizations were performed on days0,7,14,21and28, and serum samples were taken by at intervals of7days or14days. Vaccinated mice were challenged with0.2mL (2×109) H. pylori SSI instilled into stomach one time every day for three days after vaccination in6th week.The immune response was assessed by mice immunity IgG ELISA, Immunohistochemistry and Helicobacter pylori SSl attacking. The safety of vaccine was assessed by the hematoxylin-eosin staining.The results show the equencing and homologous analysis showed that the homology between the cloned Hp-CagA and Hp-UreB gene and related genes in GenBank reached98%(472/480) and100%(1134/1134) respectively for their nucleotide and deduced amino acid sequences. The expression of UreB-CagA protein could be detected in the culture fluid of C500bacteria. Noticeable IgG response was induced in the sera of mice orally immunized with the Salmonella choleraesuis C500strain consisting of UreB and CagA subunit antigens. Mice vaccinated orally were significantly protected against gastric helicobacter infection following a challenge with Helicobacter pylori strain SSI and the liver, lung, spleen, duodenum and pylorus had no lesions. The immunohistochemical analysis showed that the anti-UreB antibody was positively expressed in both the duodenum and pylorus in immunized orally with C500that expresses the UreB-CagA protein. On the contrary, there was no positive reaction in control groups.The results showed that the attenuated salmonella choleraesuis strain C500has been established successfully, which lays the foundation for further developing oral H. pylori vaccine. The orally vaccination with expressing UreB-CagA could prevent gastric infection with helicobacter pylori and safe. But didn’t against the helicobacter pylori infection completely.Experiment in vivo showed the orally vaccination with expressing UreB-CagA could prevent gastric infection with helicobacter pylori and safe. But effective in vitro has not been reported.To investigate the effect of helicobacter pylori on apoptosis of the GES-l transfected pEGFP-N1-UreB-CagA eukaryotic expression plasmid in vitro, the Hp-UreB and Hp-CagA gene were amplified by PCR respectively and were cloned into eukaryotic expression plasmid pEGFP-NI to construct recombinant plasmid pEGFP-NI-UreB-CagA and transfected into GES-1cells and choiced by G418. GES-1cells transfected into plasmid pEGFP-NI-UreB-CagA were cocultured with H. pylori strain SSI and its apoptosis was quantified by flow cytometry using Annexin V-APC and AAD. The recombinant PEGFP-N1-UreB-CagA was constructed and stable transfected into GES-1cells. The apoptotic rates of cells transfected into pEGFP-N1-UreB-CagA plasmids were significantly lower than those of control cells (P<0.01). The GES-1cells were died when H. pylori strain SS1cocultured with cells for a long time.The results showed that the GES-1cells which transfected into pEGFP-Nl-UreB-CagA plasmids can block apoptisis of GES-1cells. But helicobacter pylori also caused cell necrosis for a long time and high dose.