H Pylori
Copyright ©The Author(s) 2004. Published by Baishideng Publishing Group Inc. All rights reserved.
World J Gastroenterol. Apr 1, 2004; 10(7): 977-984
Published online Apr 1, 2004. doi: 10.3748/wjg.v10.i7.977
Construction of prokaryotic expression system of ureB gene from a clinical Helicobacter pylori strain and identification of the recombinant protein immunity
Ya-Fei Mao, Jie Yan
Ya-Fei Mao, Jie Yan, Department of Medical Microbiology and Parasitology, College of Medical Science, Zhejiang University, Hangzhou 310031, Zhejiang Province, China
Author contributions: All authors contributed equally to the work.
Supported by the Excellent Young Teacher Fund of Chinese Education Ministry and the General Science and Technology Research Plan of Zhejiang Province, No. 001110438
Correspondence to: Jie Yan, Department of Medical Microbiology and Parasitology, College of Medical Science, Zhejiang University, 353 Yanan Road, Hangzhou 310031, Zhejiang Province, China. yanchen@mail.hz.zj.cn
Telephone: +86-571-87217385 Fax: +86-571-87217044
Received: October 8, 2003
Revised: November 18, 2003
Accepted: December 6, 2003
Published online: April 1, 2004

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 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.

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