Zhe Chen1, Jie Yan, Ya-Fei Mao. 1. Department of Medical Microbiology and Parasitology, College of Medical Sciences, Zhejiang University, Hangzhou 310031, China.
Abstract
OBJECTIVE: To clone Helicobacter pylori ureB gene, to construct prokaryotic expression system of the gene and to identify immunogenicity of the fusion protein. METHODS: The ureB gene from a clinical isolate Y06 of H.pylori was amplified by high fidelity PCR. The nucleotide sequence of the target DNA amplification fragment was sequenced after T-A cloning. The expression vector pET32a with inserted ureB gene was constructed. ureB fusion protein was expressed in E.coli strain BL21DE3 induced by IPTG at different dosages. Western blot using antibody against whole cell of H.pylori as well as immunodiffusion assay using antiserum of rabbit against the fusion protein was applied to determine immunogenicity of the fusion protein. RESULTS: In comparison with the reported corresponding sequences, the homology of nucleotide sequence of the cloned ureB gene was from 96.88% approximate, equals 97.82%, while the homology of its putative amino acid sequence was as high as 99.65% approximate, equals 99.82%. The expression output of UreB protein in pET32a-ureB-BL21DE3 system was approximately 40%of the total bacterial proteins. UreB protein was able to combine with antibody against whole cell of H.pylori and induce rabbit to produce high titer antibody after the animal was immunized with the protein. CONCLUSION: An expression system with high efficiency of H.pylori ureB gene has been established successfully. The expressed UreB protein with satisfactory immunogenicity and immunoreactivity can be used as antigen in H.pylori vaccine.
OBJECTIVE: To clone Helicobacter pylori ureB gene, to construct prokaryotic expression system of the gene and to identify immunogenicity of the fusion protein. METHODS: The ureB gene from a clinical isolate Y06 of H.pylori was amplified by high fidelity PCR. The nucleotide sequence of the target DNA amplification fragment was sequenced after T-A cloning. The expression vector pET32a with inserted ureB gene was constructed. ureB fusion protein was expressed in E.coli strain BL21DE3 induced by IPTG at different dosages. Western blot using antibody against whole cell of H.pylori as well as immunodiffusion assay using antiserum of rabbit against the fusion protein was applied to determine immunogenicity of the fusion protein. RESULTS: In comparison with the reported corresponding sequences, the homology of nucleotide sequence of the cloned ureB gene was from 96.88% approximate, equals 97.82%, while the homology of its putative amino acid sequence was as high as 99.65% approximate, equals 99.82%. The expression output of UreB protein in pET32a-ureB-BL21DE3 system was approximately 40%of the total bacterial proteins. UreB protein was able to combine with antibody against whole cell of H.pylori and induce rabbit to produce high titer antibody after the animal was immunized with the protein. CONCLUSION: An expression system with high efficiency of H.pylori ureB gene has been established successfully. The expressed UreB protein with satisfactory immunogenicity and immunoreactivity can be used as antigen in H.pylori vaccine.