Cloning,Expression Of Helicobacter Pylori Adhesin HpaA And Evaluation Of Its Lmmunogenicity And Protective Effecacy In H.pylori Vaccination

BackgroudlObjectlves: More than half of the population in the world is chronically infected by the gastric pathogen, Helicobacter pylon, which is responsible for most peptic ulcer disease, chronic active gastritis, and is closely associated with gastric cancers such as adenocarcinoma and MALT lymphoma. Current therapies for eradicating H. pylon depend on the use of combined antibiotics. However the high cost, low patient compliance, and increasing of resistant strains make these therapies impractical on a large scale. Therefore, there is an urgent need for an effective vaccine against H. pylon infection. Since the adhesion to human gastric mucosa is the initial step for H. pylon colonization, inhibition of adhesion is thus an effective strategy to prevent H. pylon infection. Recent studies showed that HpaA was an adhesin presenting on bacterial surface of H.pyloni and involved in the attachment to gastric epithelial cell receptors. So we choose HpaA as a target to obtain the recombinant HpaA by genetic engineering and then evaluate its immunogenicity and protective efficacy against H.pyloni infection both in vitro and in mouse model, finally to determine the feasibility of rHpaA in H.pyloni vaccination. Methods: (1)Gene hpaA, which was amplified from H.pyloni chromosomal DNA by PCR technique, was cloned into plasmid pUC 19 and sequenced. (2)The gene was subcloned into expression vectors pTrc99A and expressed in E.coli JM1O9. The recombinant prbtein(rHpaA) was purified with two steps of ammoniuni sulfate fractionation and anion exchange chromatography. Antigenicity of rHpaA was identified by Western blot. (3)The effect of rHpaA or anti-HpaA serum on H.pyloni binding to human gastric epithelial cell lines was investigated by flow cytometry and light microscopy. (4)ELISA method was used to measure HpaA-specific antibodies in sera of H.pyloni infected patients. And the proliferation of peripheral blood lymphocytes (PBL) in response to rHpaA was examined by 3H incorporation test. (5)Flow cytometry was adopted to detect if rHpaA had the selective proliferation effect on different phenotypes ofT helper cells in PBL. (6)H.pyloni infected mouse model was established and applied in oral vaccination. The bacterial colonizing density was determined by semi- quantitative urease test and histological examination with Giemsa staining. (7)HE-stained slides were observed for gastric histopathological changes in immunized mice. (8)Mice sera were tested for HpaA-specific IgG by ELISA. Results: (1)A recombinant plasmid pUC-hpa was constructed. DNA sequence analysis showed one open reading frame of 783bp, which coded for polypeptides of 261 amino acids. The HpaA contained a short sequence of amino acids(KRTIQK) which was similar to those composing the sialic acid- binding motif of E. coli SfaS, K99 and CFAII. (2)SDS-PAGE showed that the molecular weight of expressed protein rHpaA was about 29 kD. The level of soluble expression was above 20% of total cell protein. After purified with two steps, the purity of rHpaA was above 90%. Western blot showed it could be recognized by antibodies against H. pylon whole cell. (3)Both rHpaA and anti-HpaA serum could partially inhibit H.pylori binding to gastric epithelial cells. Flow cytometry showed that mean fluorescent intensity(MRI) of Kato III bound with labeled bacteria descended but did not drop to zero. Under light microscopy, the adhesion of H. pylon to MGC-803 was significantly inhibited when pret...