H Pylori
Copyright ©2005 Baishideng Publishing Group Inc. All rights reserved.
World J Gastroenterol. Jan 7, 2005; 11(1): 114-117
Published online Jan 7, 2005. doi: 10.3748/wjg.v11.i1.114
Construction of a recombinant attenuated Salmonella typhimurium DNA vaccine carrying Helicobacter pylori hpaA
Can Xu, Zhao-Shen Li, Yi-Qi Du, Zhen-Xing Tu, Yan-Fang Gong, Jing Jin, Hong-Yu Wu, Guo-Ming Xu
Can Xu, Zhao-Shen Li, Yi-Qi Du, Zhen-Xing Tu, Yan-Fang Gong, Jing Jin, Hong-Yu Wu, Guo-Ming Xu, Department of Gastroenterology, Changhai Hospital, Second Military Medical University, Shanghai 200433, China
Author contributions: All authors contributed equally to the work.
Supported by the National Natural Science Foundation of China, No. 30170427
Correspondence to: Dr. Can Xu, Department of Gastroenterology, Changhai Hospital, Second Military Medical University, Shanghai 200433, China. xucan9@hotmail.com
Telephone: +86-21-25070556 Fax: +86-21-25074635
Received: February 21, 2004
Revised: February 25, 2004
Accepted: March 2, 2004
Published online: January 7, 2005
Abstract

AIM: To construct a recombinant attenuated Salmonella typhimurium DNA vaccine carrying Helicobacter pylori hpaA gene and to detect its immunogenicity.

METHODS: Genomic DNA of the standard H pylori strain 17 874 was isolated as the template, hpaA gene fragment was amplified by polymerase chain reaction (PCR) and cloned into pUCmT vector. DNA sequence of the amplified hpaA gene was assayed, then cloned into the eukaryotic expression vector pIRES through enzyme digestion and ligation reactions. The recombinant plasmid was used to transform competent Escherichia coli DH5α, and the positive clones were screened by PCR and restriction enzyme digestion. Then, the recombinant pIRES-hpaA was used to transform LB5000 and the recombinant plasmid isolated from LB5000 was finally used to transform SL7207. After that, the recombinant strain was grown in vitro repeatedly. In order to identify the immunogenicity of the vaccine in vitro, the recombinant pIRES-hpaA was transfected to COS-7 cells using LipofectamineTM2000, the immunogenicity of expressed HpaA protein was detected with SDS-PAGE and Western blot.

RESULTS: The 750-base pair hpaA gene fragment was amplified from the genomic DNA and was consistent with the sequence of H pylori hpaA by sequence analysis. It was confirmed by PCR and restriction enzyme digestion that H pylori hpaA gene was inserted into the eukaryotic expression vector pIRES and a stable recombinant live attenuated Salmonella typhimurium DNA vaccine carrying H pylori hpaA gene was successfully constructed and the specific strip of HpaA expressed by pIRES-hpaA was detected through Western blot.

CONCLUSION: The recombinant attenuated Salmonella typhimurium DNA vaccine strain expressing HpaA protein with immunogenicity can be constructed and it may be helpful for further investigating the immune action of DNA vaccine in vivo.

Keywords: Helicobacter pylori; hpaA Gene; DNA vaccine