Construction of prokaryotic expression system of ureB gene from a clinical Helicobacter pylori strain and identification of the recombinant protein immunity.
OBJECTIVE
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-beta-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.


CONCLUSIONS
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 H pylori isolates 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.
Mao
Ya-Fei
YF
Department of Medical Microbiology and Parasitology, College of Medical Science, Zhejiang University, 353 Yanan Road, Hangzhou 310031, Zhejiang Province, China.
Yan
Jie
J
D000595:Amino Acid Sequence; D001428:Bacterial Vaccines / Q000276:immunology*; D001483:Base Sequence; D004926:Escherichia coli / Q000201:enzymology; D016480:Helicobacter pylori / Q000201:enzymology / Q000276:immunology*; D006801:Humans; D007527:Isoenzymes / Q000235:genetics / Q000276:immunology; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011993:Recombinant Fusion Proteins / Q000276:immunology*; D017386:Sequence Homology, Amino Acid; D014510:Urease / Q000235:genetics* / Q000276:immunology; D014614:Vaccines, Synthetic / Q000276:immunology*
D001428:Bacterial Vaccines; D007527:Isoenzymes; D011993:Recombinant Fusion Proteins; D014614:Vaccines, Synthetic; D014510:Urease