Construction of attenuated Salmonella typhimurium Strain expressing Helicobacter pylori conservative region of adhesin antigen and its immunogenicity.
OBJECTIVE
To construct a non-resistant and attenuated Salmonella typhimurium (S. typhimurium) strain which expresses conservative region of adhesion AB of Helicobacter pylori (H pylori) and evaluate its immunogenicity.


METHODS
The AB gene amplified by PCR was inserted into the expression vector pYA248 containing asd gene and through two transformations introduced into the delta Cya, delta Crp, delta Asd attenuated Salmonella typhimurium strain, constructing balanced lethal attenuated Salmonella typhimurium strains X4072 (pYA248-AB). Bridged ELISA method was used to measure the expression of AB antigen in sonicate and culture supernatant. According to the method described by Meacock, stability of the recombinant was evaluated. Semi-lethal capacity test was used to evaluate the safety of recombinant. The immunogenicity of recombinant was evaluated with animal experiments.


RESULTS
The attenuated S. typhimurium X4072 (pYA248-AB) which expresses AB was successfully constructed. Furthermore, bridged ELISA assay showed that the content of AB in recombinant X4072 (pYA248- AB) culture supernatant was higher than that was in thallus lytic liquor. And after recombinant X4072 (pYA248- AB) was cultured for 100 generations without selection pressure, the entire recombinant bacteria selected randomly could grow, and the AB antigen was defected positive by ELISA. The growth curve of the recombinant bacteria showed that the growth states of X4072 (pYA248) and X4072 (pYA248-AB) were basically consistent. The survival rate of C57BL/6 was still 100%, at 30 d after mice taking X4072 (pYA248-AB) 1.0 x 10(10) cfu orally. Oral immunization of mice with X4072 (pYA248-AB) induced a specific immune response.


CONCLUSIONS
In vitro recombinant plasmid appears to be stable and experiments on animals showed that the recombinant strains were safe and immunogenic in vitro, which providing a new live oral vaccine candidate for protection and care of H pylori infection.
Bai
Yang
Y
PLA Institute for Digestive Medicine, Nanfang Hospital, the First Military Medical University, Guangzhou 510515, Guangdong Province, China. baiyang1030@hotmail.com
Zhang
Ya-Li
YL
Wang
Ji-De
JD
Zhang
Zhao-Shan
ZS
Zhou
Dian-Yuan
DY
D018829:Adhesins, Bacterial / Q000235:genetics* / Q000276:immunology; D000818:Animals; D001428:Bacterial Vaccines / Q000235:genetics / Q000276:immunology; D016481:Helicobacter Infections / Q000517:prevention & control*; D016480:Helicobacter pylori / Q000235:genetics* / Q000276:immunology; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D010957:Plasmids; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology; D012486:Salmonella typhimurium / Q000235:genetics* / Q000254:growth & development / Q000276:immunology; D013047:Specific Pathogen-Free Organisms; D014613:Vaccines, Attenuated / Q000235:genetics* / Q000276:immunology
D018829:Adhesins, Bacterial; D001428:Bacterial Vaccines; D011994:Recombinant Proteins; D014613:Vaccines, Attenuated; C073899:adhesin, helicobacter