Study On Prokaryotic Expression Of Fusion Gene Of VacA-CtxB In Helicobacter Pylori And On Immunologenicity Of VCTB Fusion Protein

5 Objective: To construct and express the fusion gene of H.pylori vacA and cholera toxin subunit B (ctxB), To explore the immune response with the conjugated antigen of VacA-CtxB, which would lay a foundation for prophylaxis and therapy of H.pylori infection.Methods: A recombinant strain which could express bivalent antigen of VacA and CtxB subunit was constructed.1) vacA gene was amplified by PCR and cloned into plasmid pQE30which was plasmid pQE30-vacA.2) ctxB gene was amplified by PCR and cloned into plasmid pQE30-vacA, which was plasmid pQE-vctB.3) translated into E.coli strain DH5 a to express VCTB fusion protein. Its immunogenicity was analyzed by Western blotting.4) After purification,to fedm ice by oral immunization. antibody(IgG and IgA) in the delected by ELISA assay.Results: vctB fusion gene was sequenced as 1092bp, the fusion protein encoded polypeptides of 364 amino acid residues. The molecular weight was 40kD analysiced by SDS-PAGE.The levels of soluble expression product was about 20% of total cell protein.After affinity chromatography,the purity of fusion protein was above 92%.Western blotting analysis of confirmed that fusion protein could be specicifically recognized by the serum of anti-VacA and anti-CT. The specific antibody response in mice immunized with VCTB was determined by ELISA. The results showed that the levels of specific antibody were higher than those of controls.Conclusion:The successful construction and expression fusion gene of Helicobacter pylori vacA and cholera toxin subunit B(ctxB). Experiment indicated that the oral immunization with VCTB induces effective mucosal immune responece and produced higher levels IgA. The recombinant fusion protein VCTB can be used as an effective oral vaccine for prevention infection of H.pylori.