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


CONCLUSIONS
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.
Sun
Bo
B
Department of Gastroenterology, Changhai Hospital, Second Military Medical University, 174 Changhai Road, Shanghai 200433, China.
Li
Zhao-Shen
ZS
Tu
Zhen-Xing
ZX
Xu
Guo-Ming
GM
Du
Yi-Qi
YQ
D000284:Administration, Oral; D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics / Q000276:immunology; D001428:Bacterial Vaccines / Q000008:administration & dosage / Q000235:genetics / Q000276:immunology; D019556:COS Cells; D002522:Chlorocebus aethiops; D004269:DNA, Bacterial / Q000235:genetics / Q000276:immunology; D016481:Helicobacter Infections / Q000235:genetics / Q000276:immunology* / Q000517:prevention & control*; D016480:Helicobacter pylori / Q000235:genetics / Q000276:immunology*; D007125:Immunogenetics; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D021141:Nucleic Acid Amplification Techniques; D012486:Salmonella typhimurium / Q000235:genetics; D019444:Vaccines, DNA / Q000008:administration & dosage / Q000235:genetics / Q000276:immunology*
D001426:Bacterial Proteins; D001428:Bacterial Vaccines; D004269:DNA, Bacterial; D019444:Vaccines, DNA; C092412:neutrophil-activating protein A, Helicobacter pylori