| Backgroud/Objectives: Helicobacter pylori(H.pylori) infection is the major etiological factor of chronic active gastritis and most peptic ulcer disease, and is closely associated with gastric cancers tumors such as adenocarcinoma and MALT lymphoma. Clinic practice proved the peptic ulcer recurrence rate was above 50% one year after cure in patients infected with Hp, Eradication of Hp cured peptic ulcer and reduced the recurrence rate of one year to below 10%. In addition, Eradication of Hp was able to cure MALT lymphoma. Current therapies for eradicating H. pylori depend on the use of combined antibiotics, although having relatively high eradication rate, the high cost, low patient compliance, side effect, and increasing of resistant strains make these therapies imperfect. In 1994, Doige et al discovered the treatment of Hp vaccination. Scince then, Immunization against Hp infection has been one of the most prospective treaments. Since the Hp adhesin conservation region(AB) is outer membrane protein and porin type component, while these two proteins are the excellent immunogen candidates of vaccination, we prepared AB as a target to obtain the recombinant AB by genetic engineering and evaluated its safety, biological activity and immunogenicity in vitro, Furthmore, we constructed the attenuated Salmonella typhimurium strain expressing AB which was a balanced lethal recombinant and investigated its efficacy and the potential mechanism of treatment in mouse model infected with Hp. Finally the feasibility of AB and the attenuated Salmonella typhimurium strain expressing AB in H. pylori vaccination was determined.Methods: (l)Gene ab which was amplified from Hp chromosomalDNA by PCR technique, was sequenced and the biological information was analysised. Then it was cloned into the expression vector pET-22b ( + ) and expressed in E. coli BL2KDE3). The recombinant protein(rAB) was purified with affinity chromatography. Antigenicity of rAB was identified by Western blot. (2) Flow cytometry was used to evaluate the increase in FasL expression on T cells under the stimulation of Hp AB,T cell apoptosis induced by AB was detected by DAP assay .The effect of anti-AB serum on Hp binding of human gastric carcinoma cell lines was determined by light microscopy. ELISA assay was used to measure AB-specific antibody in serum of Hp infected patients. The proliferation of peripheral blood lymphocytes(PBL) in response to rAB was examined by MTT test. (3) The ab gene was amplified by PCR and inserted into the expression vector pYA248 containing asd gene and was introduced into the delta Cya,delta Crp,delta Asd attenuated Salmonella typhimurium strain that is a balanced lethal recombinant by twice transformations. ELISA assay was used to measure AB expressed in sonicate and culture supernatant. According to Meacock's way and growth curve, stability of the recombinant is evaluated. Semi-lethal capacity test was used to evaluate the safty of recombinant. (4) H. pylori infected mouse model was established and applied in oral vaccination. The density of bacterial colonization was determined by the semi-quantitative bacterial culture assay. The T cell subsets of spleenocytes were assayed by flow cytometry (FCM) after immunization. IL-2 and IL-4 were detected through MTT. Mice sera and intestinal fluid were separatly tested for AB-specific IgG and IgA by ELISA.Results: (1) The recombinant plasmid pET-22b(+)/AB was succefully constructed.DNA sequencing showed one open reading frame with the length of 588 bp.lt encoded 7 conservative regions that showed good antigencity and hydrophobicity by Parker and Welling method. Furthmore, INTERNET ExPASY, NNPREDICT and ISREC predicted it was a porin like structure consisting of 3 -pleated sheets, that were embeded in the outer membrane. BLAST analysed 836767 protein sequences and found the similar sequences were all belong to Hp OMP sequences. SDS-PAGE and scan analysis showedthe molecular weight of AB was 22.5kD and recombinant protein amounted to 29% of the total bacterial protein...
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