摘要
AIM: To clone ureB gene from a clinical isolate of Helicobacter pyloriand construct a prokaryotic expression system of the gene and identify immunity of the expressed recombinant protein. METHODS: ureBgene from a clinical Hpyloristrain Y06 was amplified by the high fidelity polymerase chain reaction technique. The target DNA fragment amplified from ureB gene was sequenced after T-A cloning. Prokaryotic recombinant expression vector pET32a inserted with ureB gene (pET32a-ureB) was constructed. The expression of recombinant UreB protein (rUreB) in E. coli BL21DE3 induced by isopropylthio-β-D-galactoside (IPTG) at different concentrations was examined by SDS-PAGE. Western blot using commercial antibodies against whole cell of Hpylori and an immunodiffusion assay using a self-prepared rabbit anti-rUreB antibody were applied to determine immunity of the target recombinant protein. ELISA was used to detect the antibody against rUreB in sera of 125 Hpyloriinfected patients and to examine rUreB expression in 109 Hpylori isolates. RESULTS: In comparison with the reported corresponding sequences, the nucleotide sequence homology of the cloned ureB gene was from 96.88-97.82% while the homology of its putative amino acid sequence was as high as 99.65-99.82%. The rUreB output expressed by pET32a-ureB-BL21DE3 was approximate 30% of the total bacterial proteins, rUreB specifically combined with the commercial antibodies against whole cell of Hpylori and strongly induced rabbits to produce antibody with a 1:8 immunodiffusion titer after the animals were immunized with the recombinant protein. Serum samples from all Hpyloriinfected patients were positive for UreB antibody and UreB expression were detectable in all tested Hpyloriisolates. CONCLUSION: A prokaryotic expression system with high expression efficiency of Hpylori ureBgene was successfully established. The expressed rUreB showed qualified immunoreactivity and antigenicity. High frequencies of UreB expression in different Hpyloriisolates and specific antibody against UreBin sera of Hpyloriinfected patients indicatet hat UreB is an excellent antigen candidate for developing H pylori vaccine.
AIM:To clone ureB gene from a clinical isolate of Helicobacter pylori and construct a prokaryotic expression system of the gene and identify immunity of the expressed recombinant protein. METHODS:ureB gene from a clinical H pylori strain Y06 was amplified by the high fidelity polymerase chain reaction technique.The target DNA fragment amplified from ureB gene was sequenced after T-A cloning.Prokaryotic recombinant expression vector pET32a inserted with ureB gene (pET32a-ureB) was constructed.The expression of recombinant UreB protein (rUreB) in E.coli BL21DE3 induced by isopropylthio-β-D-galactoside (IPTG) at different concentrations was examined by SDS-PAGE.Western blot using commercial antibodies against whole cell of H pylori and an immunodiffusion assay using a self-prepared rabbit anti-rUreB antibody were applied to determine immunity of the target recombinant protein.ELISA was used to detect the antibody against rUreB in sera of 125 H pylori infected patients and to examine rUreB expression in 109 H pylori isolates. RESULTS:In comparison with the reported corresponding sequences,the nucleotide sequence homology of the cloned ureB gene was from 96.88-97.82% while the homology of its putative amino acid sequence was as high as 99.65-99.82%. The rUreB output expressed by pET32a-ureB-BL21DE3 was approximate 30% of the total bacterial proteins,rUreB specifically combined with the commercial antibodies against whole cell of H pylori and strongly induced rabbits to produce antibody with a 1:8 immunodiffusion titer after the animals were immunized with the recombinant protein. Serum samples from all H pylori infected patients were positive for UreB antibody and UreB expression were detectable in all tested H pylori isolates. CONCLUSION:A prokaryotic expression system with high expression efficiency of H pylori ureB gene was successfully established.The expressed rUreB showed qualified immunoreactivity and antigenicity.High frequencies of UreB expression in different Hpyloriisolates and specific antibody against UreB in sera of H pylori infected patients indicate that UreB is an excellent antigen candidate for developing H pylori vaccine.
基金
Supported by the Excellent Young Teacher Fund of Chinese Education Ministry and the General Science and Technology Research Plan of Zhejiang Province,No.001110438
