H. Pylori
Copyright ©The Author(s) 2003. Published by Baishideng Publishing Group Inc. All rights reserved.
World J Gastroenterol. Jul 15, 2003; 9(7): 1529-1536
Published online Jul 15, 2003. doi: 10.3748/wjg.v9.i7.1529
Construction of hpaA gene from a clinical isolate of Helicobacter pylori and identification of fusion protein
Ya-Fei Mao, Jie Yan, Li-Wei Li, Shu-Ping Li
Ya-Fei Mao, Jie Yan, Li-Wei Li, Shu-Ping Li, Department of Medical Microbiology and Parasitology, College of Medical Sciences, 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 Research Plan of Science and Technology Department of Zhejiang Province, No. 001110438
Correspondence to: Dr. Jie Yan, Department of Medical Microbiology and Parasitology, College of Medical Sciences, Zhejiang University, 353 Yan an Road, Hangzhou 310031, Zhejiang Province, China. yanchen@mail.hz.zj.cn
Telephone: +86-571-87217385 Fax: +86-571-87217044
Received: March 2, 2003
Revised: March 14, 2003
Accepted: March 28, 2003
Published online: July 15, 2003
Abstract

AIM: To clone hpaA gene from a clinical strain of Helicobacter pylori and to construct the expression vector of the gene and to identify immunity of the fusion protein.

METHODS: The hpaA 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 recombinant expression vector inserted with hpaA gene was constructed. The expression of HpaA fusion protein in E.coli BL21(DE3) induced by IPTG at different dosages was examined by SDS-PAGE. Western blot with commercial antibody against whole cell of H. pylori as well as immunodiffusion assay with self-prepared rabbit antiserum against HpaA fusion protein were applied to determine immunity of the fusion protein. ELISA was used to detect the antibody against HpaA in sera of 125 patients infected with H. pylori and to examine HpaA expression of 109 clinical isolates of H. pylori.

RESULTS: In comparison with the reported corresponding sequences, the homologies of nucleotide and putative amino acid sequences of the cloned hpaA gene were from 94.25%-97.32% and 95.38%-98.46%, respectively. The output of HpaA fusion protein in its expression system of pET32a-hpaA-BL21(DE3) was approximately 40% of the total bacterial proteins. HpaA fusion protein was able to combine with the commercial antibody against whole cell of H. pylori and to induce rabbit producing specific antiserum with 1:4 immunodiffusion titer after the animal was immunized with the fusion protein. 81.6% of the serum samples from 125 patients infected with H. pylori (102/125) were positive for HpaA antibody and all of the tested isolates of H. pylori (109/109) were detectable for HpaA.

CONCLUSION: A prokaryotic expression system with high efficiency of H. pylorihpaA gene was successfully established. The HpaA expressing fusion protein showed satisfactory immunoreactivity and antigenicity. High frequencies of HpaA expression in different H. pylori clinical strains and specific antibody production in H. pylori infected patients indicate that HpaA is an excellent and ideal antigen for developing H. pylori vaccine.

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