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Construction of recombinant attenuated Salmonella typhimurium DNA vaccine expressing H pylori ureB and IL-2

World Journal of Gastroenterology 2007年第6期13卷 939-944页

作者： Can Xu Zhao-Shen Li Yi-Qi Du Yan-Fang Gong Hua Yang Bo Sun Jing Jin Department of Gastroenterology Changhai Hospital Second Military Medical University Shanghai 200433 China

AIM： To construct a recombinant live attenuated Salmonella typhimurium DNA vaccine encoding H pylori ureB gene and mouse IL-2 gene and to detect its immuno.enicity in vitro and in vivo. METHODS： Hpylori ureB and mouse IL-2 gene fragments were amplified by polymerase chain reaction （PCR） and cloned into pUCmT vector. DNA sequence of the amplified ureB and IL-2 genes was assayed, then cloned into the eukaryotic expression vector pIRES through enzyme digestion and ligation reactions resulting in pIRES-ureB and pIRES-ureB-IL-2. The recombinant plasmids were used to transform competent E. co/i DH5α, and the positive clones were screened by PCR and restriction enzyme digestion. Then, the recombinant pIRES-ureB and pIRES-ureB-IL-2 were used to transform LB5000 and the recombinant plasmids extracted from LB5000 were finally introduced into the final host SL7207. After that, recombinant strains were grown in vitro repeatedly. In order to detect the immuno.enicib/of the vaccine in vitro, pIRES-ureB and pIRES-ureB-IL-2 were transfected to COS-7 cells using LipofectamineTM2000, the immuno.enicity of expressed UreB and IL-2 proteins was assayed with SDS-PAGE and Western blot. C57BL/6 mice were orally immunized with 1 × 10^8 recombinant attenuated Salmonella typhimurium DNA vaccine. Four weeks after vaccination, mice were challenged with 1 × 10^7 CFU of live Hpylori SS1. Mice were sacrificed and the stomach was isolated for examination of H pylon 4 wk post-challenge. RESULTS： The 1700 base pair ureB gene fragment amplified from the geno.ic DNA was consistent with the sequence of H pylori ureB by sequence analysis. The amplified 510 base pair fragment was consistent with the sequence of mouse IL-2 in gene bank. It was confirmed by PCR and restriction enzyme digestion that H pylori ureB and mouse IL-2 genes were inserted into the eukaryotic expression vector pIRES. The experiments in vitro showed that stable recombinant live attenuated Salmonella typhimurium DNA vaccine carrying

关键词： Hpylori DNA vaccine ureB gene Salmonella typhimurium

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Construction of an oral recombinant DNA vaccine from H pylori neutrophil activating protein and its immuno.enicity

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World Journal of Gastroenterology 2006年第43期12卷 7042-7046页

作者： Bo Sun Zhao-Shen Li Zhen-Xing Tu Guo-Ming Xu Yi-Qi Du Department of Gastroenterology Changhai Hospital SecondMilitary Medical University Shanghai 200433 China

AIM： To construct a live attenuated Salmonella typhimurium （S. typhimurium） strain harboring the H pylori neutrophil activating protein （HP-NAP） gene as an oral recombinant DNA vaccine, and to evaluate its immuno.enicity. METHODS： By genetic engineering methods, the geno.ic DNA of Hpylori was extracted as a template. The total length of the HP-NAP gene was amplified by polymerase chain reaction （PCR） and cloned into pBT vector for sequencing and BLAST analysis, then subcloned into a eukaryotic expression vector pIRES followed by PCR identification and restriction enzyme digestion. The identified recombinant plasmid pIRES-NAP was transfected into COS-7 cells for target fusion protein expression, and its antigenicity was detected by Western blotting. Then the recombinant plasmid was transformed into a live attenuated S. typhimurium strain SL7207 as an oral vaccine strain, and its immuno.enicity was evaluated with animal experiments. RESULTS： A 435 bp product was cloned using high homology with HP-NAP gene in GenBank （more than 98%）. With identification by PCR and restriction enzyme digestion, a recomoinant eukaryotic expression plasmid pIRES-NAP containing the HP-NAP gene of H pylori was successfully constructed. The expressed target protein had a specific reaction with Hpyloril whole cell antibody and showed a single strip result detected by Western blotting. Oral immunization of mice with recombinant DNA vaccine strain SL7207 （pIRES-NAP） also induced a specific immune response. CONCLUSION： The successful construction of HP-NAP oral DNA vaccine with good immuno.enicity may help to further investigate its immuno.rotection effects and develop vaccine against Hpylori infection.

关键词： H pylori Neutrophil activating protein DNAvaccine

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Construction of a recombinant attenuated Salmonella typhimurium DNA vaccine carrying Helicobacter pylorihpaA

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World Journal of Gastroenterology 2005年第1期11卷 114-117页

作者： CanXu Zhao-ShenLi Yi-QiDu Zhen-XingTu Yan-FangGong JingJin Hong-YuWu Guo-MingXu DepartmentofGastroenterology ChanghaiHospitalSecondMilitaryMedicalUniversityShanghai200433China

AIM: To construct a recombinant attenuated Salmonella typhimurium DNA vaccine carrying Helicobacter pylori hpaA gene and to detect its immuno.enicity. METHODS: Geno.ic 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 doned into the eukaryotic expression vector pIRES through enzyme digestion and ligation reactions. The recombinant plasmid was used to transform competent Escherichia coliDH5α, 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 immuno.enicity of the vaccine in vitro, the recombinant pIRES-hpaA was transfected to COS-7 cells using Lipofectamine^(TM)2000, the immuno.enicity of expressed HpaA protein was detected with SDS-PAGE and Western blot. RESULTS: The 750-base pair hpaA gene fragment was amplified from the geno.ic 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 immuno.enicity can be constructed and it may be helpful for further investigating the immune action of DNA vaccine in vivo.

关键词： Helicobacter pylori hpaA Gene DNA vaccine

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