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...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 immunogenicity. METHODS： By genetic engineering methods, the genomic 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 immunogenicity 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 immunogenicity may help to further investigate its immunoprotection effects and develop vaccine against Hpylori infection.展开更多
基金Supported by the National Natural Science Foundation of China, No. 30170427
文摘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 immunogenicity. METHODS： By genetic engineering methods, the genomic 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 immunogenicity 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 immunogenicity may help to further investigate its immunoprotection effects and develop vaccine against Hpylori infection.
