Preliminary Studies On Preparation And Immune Protective Effectiveness Of The Oral Liposome-encapsulated Recombinant H.pylori Vaccine

Backgroud/ObjectiveHelicobacter pylori(H.pylori) infection is the major etiological factor of chronic active gastritisis and most peptic ulcer disease, and is closely associated with gastric cancer tumors such as adenocarcinoma and MALT lymphoma. A working group of the International Agency for Reseach on Cancer sponsored by the World Health Organization has categorized Hp infection as a class I carcinogen since 1994. Current therapies for eradicating Hp 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 Hp infection. Since CT and LT is essential for Hp vaccine, the Hp vaccines were bouned for human. In this study, we choose urease-B subunit (UreB) and catalase (Kat) of Hp as candidate vaccine antigens to obtain the recombinant UreB (rUreB) and the recombinant Kat (rKat) prokaryotie expression systems by genetic engineering. The recombinant proteins were purified with two or three steps by fast protein liquid chromatography (FPLC) syetem. The oral lipsome-encapsulated vaccines with phosphatidyl choline (PC) and cholesterols (Choi) were prapared using reverse evaporation method. Afterwards, their immune protective efficts and mechanism were evaluated and studied.Methods1.Gene UreB was amplified from Hp chromosmal DNA by PCR technique,cloned into plasmid pT (pGEM-T-easy vector) and sequenced.The gene was subcloned into expression vector pET-22b(+)and expressed in E.coli BL21(DE3).The recombinat protein(rUreB) was purified with Q-Sephacyl S-200 gel filtration and Q-Sepharose Fast Flow ion exchange by FPLC system. Antigenicity of rlJreB was indentified by Western-blot. Several mg rUreB protein was collected by 15L fermental container.2.Gene Kat was amplified from Hp chromosmal DNA by PCR technique,cloned into expression vectors pET-22b(+) and sequenced. The gene was expressed in BL21(DE3).The recombinat protein(rKat) was purified with Q-Sepharose Fast Flow ion Q-Octyl-Sepharose-4FF and Q-Sephacyl S-200 by FPLC system. The activity of rKat was assayed by the Beers and Sizers.3. The oral lipsome-encapsulated vaccines with/without CT used phosphatidyl choline (PC) and cholesterols (Choi) were praparated using reverse evaporation method. Its size distribution of folate liposomes was measured by electronic microscopy. BALB/c mice were divided into six groups and immunized by BPS alone, liposome alone, rUreB plus CT, liposome-encapsulated rUreB, liposome-encapsulated rUreB plus CT and liposome-encapsulated (rUreB and rKat) plus CT orally respectively once a week for four weeks. All mice were challenged by alive Hp three times in two weeks after the last immunization and sacrificed in five weeks after the last challenge. Hp was determined by the fast urease test. The bactrerial colonizing density of semi-quantitation , the inflammation severity grades and activity grades of the gastric histopathology were observed. INF- Y andIL-4 mRNA expression of spleen T lymphocyte in different groups were studied with RT-PCR.Results1. A recombinant plasmid pT-UreB was constructed. DNA sequenceanalysis showed one open reading frame of 1710bp,which coded for polypeptides of 570 amino acides.lt showed 97.0%( 1658/1710) identity comparing with nucleotide sequence of UreB gene from GeneBank.It was comfirmed that the recombinant plasmide pET-22(+)/UreB was insered into expression vector successfully by PCR and analyzed by digestion with double restriction enzymes. SDS-PAGE showed that the molecular weight of expressed protein rUreB was about 64kD.The level of soluble expression was above 18%of total cell protein.The purity of rUreB was above 95% after puified by FPLC system. Western blot showed it could be recognized by the monoclonal antibody agaist UreB.2. A recombinant plasmid pET-22b(+)/Kat was constructed. DNA sequence analysis showed one open reading frame of 1512bp,which coded for polypeptides of 5...