Genetic Engineering And Immunogenicity Of Antigen Urease B From Helicobacter Pylori

The Gram-negative bacterium Helicobacter pylori(H.pylori) colonize the human gastric epithelium and is strongly associzted with the development of chronic gastritis,peptic ulcer and gastric cancer and gastric MALT lymphoma. H.pylori has many antigens, including urease, heat shock protein and vacuolating cytotoxin, cytotoxin associated gene protein and so on,and urease is an important factor in the colonization of the gastric mucosa and suspected to cause damage to the gastric mucosa. At the same time, urease is also one of the important protective antigens. It consists of two distict subunits with apparent molecular mass of 29.5kDa(UreA) and 66.0 kDa(UreB), and urease B subunit is nontoxic,highly immunogenic,and an effective component of protective antigens.In the present study, the studies on the cloning and sequencing of H.pylori ureB gene from H.pylori Zhejiang strain,construction of expression vector,transformation to tobacco, rice, E.coli, and Lactococcus lactis, mucosal immunizations were carried out.Vaccines produced by transgenic plants would have the potential to change thetraditional means of production and inoculation of vaccines, and to reduce the cost ofvaccine production. In the present study, The nucleotide and deduced amino acidssequences of UreB of H, pylori strain ZJC02 were compared with those from other H.pylori strains reported in GenBank.It was revealed that the ureB gene from the ZJC02strain shares a high level of similarity with those of other strains. The UreB antigengene was cloned into the binary vector pBI121 which contains a CaMV35S promoterand a kanamycin resistance gene, and then transformed UreB into tobacco leaf-disc byAgrobacterium-mediated method. A total of 50 regenerated plants with kanamycinresistance were obtained in the selection media. The 35 putative transgenicindividuals were tested and verified the presence and integration of the UreB into thenuclear genome of tobacco plants by PCR, PCR-southern analyses. Expression ofUreB gene in the tobacco plants was confirmed by RT-PCR for transcription level andwestern blot analysis using polyclonal human antiserum for translation levelrespectively. To our knowledge, this is the first report that expression of Helicobacterpylori UreB antigen gene in a plant system, suggesting a major step in the productionof plant-based vaccines for Helicobacter pylori.A system of production of UreB antigen in transgenic rice plants was established.UreB gene was cloned to 5' end of GUS reporter gene betweenCaMV35S promoter and the OCS terminator in the plasmid pCAMBIA13011-UreB. The rice calli induced from mature embryos of zhonghuaW cultivar were transformed by Agrobacterium tumefaciens EHA105 with UreB gene.The recombinant pCAMBIA 13011-UreB plasmid enables selection of transformants on madia containing hygromycin and stable integration into nuclear chromosomal plant DNA.After twice selections of hygromycin cultures, a total of 12 regenerated plants with hygromycin resistance were obtained in the selection media, and about 75% of them turned out to be transgenic.PCR and PCR-Southern confirmed that the UreB gene has been integrated into the chromosomal DNA of these transgenic plants,and RT-PCR confirmed the transcription of the genes.The result of Western-blot using polyclonal human antiserum showed that UreB protein was expressed in the transgenic rice.The result obtained provide basic for the research of level of UreB recombinant protein accumulation in transgenic rice and future study of transgenic rice for delivery of edible vaccines agaist Helicobacter pylori.To elucidate the efficacy of individual urease beta subunits and it's differentfractions to act as immunogens, the genes and fraction of ureB encoding therespective urease beta subunits of Helicobacter pylori and peptide were cloned in anexpression vector (pAMJ399) and expressed in Escherichia coli cells. Therecombinant UreB proteins and E fractions of UreB had predicted molecular massesof approximately 66 and 28 kDa, respectively. Western blotting (immunoblotting)studies indicated that the proteins or peptides were strongly immunogenic and werespecifically recognized by polyclonal human anti-Helicobacter pylori sera. Therecombinant proteins and E fractions were used, in combination with a adjuvant(Freund's complete adjuvant. FCA), to inject immunize mice.Sera from Balb/c miceimmunized 37 days previously were tested by ELISA for the presence ofUreB-specific IgG.Mice vaccined with the UreB and E peptide-expressor strainresponded with UreB-specific serum antibody.The mean titers were significantlyhigher than those of the control groups.The study demonstrated that a recombinanturease subunit beta antigen and E pepite could elicited a protective immune responseagainst gastric Helicobacter pylori infection.They may be an effective protein orpeptide vaccine for prevention and treatment of the infection of Helicobacter pylori.Lactococcus lactis as E fractions of//, pylori urease subunit B delivery vehicle for mucosal immunisation was first reported. To determine whether Lactococcus lactis could effectively deliver Helicobacter pylori antigens to the immune system, arecombinant L.lactis expressing UreB and E fractions was constructed. Secretion expression of E fractions by a pAMJ399-e vector resulted in the extracellular accumulation of E fractions of soluble protein.Oral regimens were used to vaccinate Balb/c mice and the immune response measured by ELISA.After an interval 5 weeks by four successive daily doses, it was able to elicit anti-UreB serum antibody responses to UreB in the mice. The data therefore provide further evidence of the potential of recombinant lactococcal vaccines for inducing immune responses.