Construction of an oral recombinant DNA vaccine from H pylori neutrophil activating protein and its immunogenicity

Abstract

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 H pylori 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 recombinant 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 H pylorii 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 H pylori infection.

Figure 1

Electrophoresis of HP-NAP PCR products. Lane 1: 100 bp DNA ladder marker; lanes 2 and 3: PCR products of HP-NAP; lane 4: Blank control.

Figure 2

Identification map of recombinant plasmid pIRES-NAP. Lane 1: 100 bp DNA ladder marker; lane 2: PCR products templated on pIRES-NAP; lane 3: pIRES-NAP digested by endonucleases Xho I and Mlu I; lane 4: Blank control.

Figure 3

Western blot analysis of HP-NAP fusion protein expression. Lane 1: Protein standard; lane 2: COS-7 cells transfected with pIRES-NAP; lane 3: COS-7 cells without transfection as control.