[Construction of hpaA gene-engineered attenuated Salmonella typhimurium vaccine strain].
To express Helicobacter pylori hpaA gene in attenuated Salmonella typhimurium vaccine vehicle, and elucidate the potential value of attenuated Salmonella typhimurium as a vector expressing Helicobacter pylori antigens, by means of molecular biology, 783 bp hpaA gene was cloned into NcoI-SalI site of a procaryotic expression plasmid pTrc99A, and the recombinant plasmid was then used to transform an attenuated Salmonella typhimurium vaccine strain SL3261, and the positive clones were screened by PCR and restriction enzyme digestion. HpaA expression was analyzed by SDS-PAGE and Western blot. Two and 10 days after recombinant strain intragastric immunization, the C57BL/6 mice was sacrificed, and the spleen and terminal ileum was cultured for recombinant strain. The results showed that a recombinant procaryotic expression plasmid pTrc99A-hpaA was constructed, and the recombinant plasmid was then introduced into an attenuated Salmonella typhimurium vaccine strain SL3261 successfully. HpaA was expressed in the recombinant strains as a 30 kD protein, and also its immunogenicity was confirmed by Western blot. Recombinant strain was found in both spleen and terminal ileum of each mouse two and ten days after intragastric immunization. We concluded that a recombinant live attenuated Salmonella typhimurium vaccine strain expressing Helicobacter pylori hpaA gene was constructed and identified, and this work will help to develop oral recombinant live vaccine strains against Helicobacter pylori infection.
Zhu
Senlin
S
Department of Gastroenterology, First Affiliated Hospital, Sun Yat-Sen University of Medical Sciences, Guangzhou 510080, China.
Chen
Minhu
M
Chen
Jie
J
Hu
Pinjin
P
Li
Guoqing
G
D000595:Amino Acid Sequence; D000818:Animals; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000235:genetics; D001428:Bacterial Vaccines; D001483:Base Sequence; D005798:Genes, Bacterial; D016480:Helicobacter pylori / Q000235:genetics* / Q000302:isolation & purification; D006388:Hemagglutinins / Q000096:biosynthesis / Q000235:genetics*; D007082:Ileum / Q000382:microbiology; D037102:Lectins; D008074:Lipoproteins / Q000096:biosynthesis / Q000235:genetics*; D051379:Mice; D008810:Mice, Inbred C57BL; D008969:Molecular Sequence Data; D010957:Plasmids; D012486:Salmonella typhimurium / Q000235:genetics* / Q000378:metabolism; D013154:Spleen / Q000382:microbiology; D014170:Transformation, Genetic; D014613:Vaccines, Attenuated
D001426:Bacterial Proteins; D001428:Bacterial Vaccines; D006388:Hemagglutinins; C096437:HpaA protein, bacteria; D037102:Lectins; D008074:Lipoproteins; D014613:Vaccines, Attenuated