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AIM:To construct a non-resistant and attenuated Salmonellatyphimurium(S.typhimurium)strain which expressesconservative region of adhesion AB of Helicobacter pylori(Hpylon)and evaluate its immunogenicity.METHODS:The AB gene amplified by PCR was insertedinto the expression vector pYA248 containing asd geneand through two transformations introduced into the deltaCya,delta Crp,delta Asd attenuated Salmonella typhimuriumstrain,constructing balanced lethal attenuated Salmonellatyphimurium strains X4072(pYA248-AB).Bridged ELISAmethod was used to measure the expression of AB antigenin sonicate and culture supematant.According to the methoddescribed by Meacock,stability of the recombinant wasevaluated.Semi-lethal capacity test was used to evaluate thesafety of recombinant.The immunogenicity of recombinantwas evaluated with animal experiments.RESULTS:The attenuated S.typhimunum X4072(pYA248-AB)which expresses AB was successfully constructed.Furthermore,bridged ELISA assay showed that the contentof AB in recombinant X4072(pYA248- AB)culture supematantwas higher than that was in thallus lytic liquor.And afterrecombinant X4072(pYA248- AB)was cultured for 100generations without selection pressure,the entire recombinantbacteria selected randomly could grow,and the AB antigenwas defected positive by ELISA.The growth curve of therecombinant bacteria showed that the growth states ofX4072(pYA248)and X4072(pYA248-AB)were basicallyconsistent.The survival rate of C57BL/6 was still 100%,at30 d after mice taking X4072(pYA248-AB)1.0×10~(10)cfuorally.Oral immunization of mice with X4072(pYA248-AB)induced a specific immune response.CONCLUSION:In vitro recombinant plasmid appears tobe stable and experiments on animals showed that therecombinant strains were safe and immunogenic in vitro,which providing a new live oral vaccine candidate for protectionand care of Hpyloriinfection.
AIM: To construct a non-resistant and attenuated Salmonella typhimurium (S. typhimurium) strain which expressesconservative region of adhesion AB of Helicobacter pylori (Hpylon) and evaluate its immunogenicity. METHODS: The AB gene amplified by PCR was inserted in the expression vector pYA248 containing asd gene and through two transformations introduced into the deltaCya, delta Crp, delta Asd attenuated Salmonella typhimuriumstrain, establishing balanced lethal attenuated Salmonella typhimurium strain X4072 (pYA248-AB). Bridged ELISA method was used to measure the expression of AB antigenin sonicate and culture supematant. Accredited to the methoddescribed by Meacock, stability of the recombinant wasevaluated. Semi-lethal capacity test was used to evaluate the safety of recombinant. immunogenicity of recombinant as evaluated with animal experiments .RESULTS: The attenuated S. typhimunum 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 supematantwas higher than that was in thallus lytic liquor. End after recombination X4072 (pYA248-AB) was cultured for 100 generations without selection pressure, the entire recombinant bacterium selected randomly grew, and the AB antigen was defected positive by ELISA. The growth curve of therecombinant bacteria showed that the growth states of X4072 (pYA248) and X4072 (pYA248-AB) were substantiallyconsistent.The survival rate of C57BL / 6 was still 100% at30d after mice taking X4072 (pYA248- AB) 1.0 × 10 ~ (10) cfuorally. Oral immunization of mice with X4072 (pYA248-AB) induced a specific immune response. CONCLUSION: In vitro recombinant plasmid appeared to be stable and experiments on animals showed that therecomcombinant strains were safe and immunogenic in vitro, which providing a new live oral vaccine candidate for protection and care of Hpylori infection.