Deletion of Helicobacter pylori vacuolating cytotoxin gene by introduction of directed mutagenesis

doi:10.3748/wjg.v9.i10.2251

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Oct 15, 2003 (publication date) through Nov 8, 2024

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World Journal of Gastroenterology

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1007-9327

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Baishideng Publishing Group Inc, 7041 Koll Center Parkway, Suite 160, Pleasanton, CA 94566, USA

H Pylori

World J Gastroenterol. Oct 15, 2003; 9(10): 2251-2257
Published online Oct 15, 2003. doi: 10.3748/wjg.v9.i10.2251

Deletion of Helicobacter pylori vacuolating cytotoxin gene by introduction of directed mutagenesis

Jian-Ping Yuan, Tao Li, Xiao-Dong Shi, Bao-Yu Hu, Gui-Zhen Yang, Shan-Qing Tong, Xiao-Kui Guo

Jian-Ping Yuan, Tao Li, Xiao-Dong Shi, Bao-Yu Hu, Gui-Zhen Yang, Shan-Qing Tong, Xiao -Kui Guo, Department of Microbiology and Parasitology, Shanghai Second Medical University, Shanghai 200025, China

Author contributions: All authors contributed equally to the work.

Supported by Ministry of Education Research Foundation for Returned Overseas Chinese Scholars Abroad (2001) 498

Correspondence to: Xiao-Kui Guo, Department of Microbiology and Parasitology, Shanghai Second Medical University, 280 Chongqingnan Road, Shanghai 200025, China. xkguo@shsmu.edu.cn

Telephone: +86-21-64671226 Fax: +86-21-64671226

Received: March 2, 2003
Revised: May 9, 2003
Accepted: May 16, 2003
Published online: October 15, 2003

Abstract

AIM: To construct a vacA-knockout Helicobacter pylori mutant strain, whose only difference from the wild strain is its disrupted vacA gene.

METHODS AND RESULTS: A clone containing kanamycin resistance gene used for homologous recombination was constructed in a directional cloning procedure into pBluescript II SK, and then transformed into vacA⁺H pylori by electroporation. Colonies growing on the selective media containing kanamycin were harvested for chromosomal DNA extraction, and the allelic exchange was determined by polymerase chain reactions and sequencing. Loss of vacuolating activity of the vacA-knockout strain was confirmed by examining the gastric cells co-cultured with cell-free supernatants from H pylori wild strain or the mutant.

CONCLUSION: We constructed a vacA-knockout strain of H pylori through direct mutagenesis, which creates an important precondition for the future research on virulence comparison with gene expression analysis.

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