Construction and evaluation of a D-amino acid auxotrophic attenuated oral vaccine against Salmonella typhimurium
OBJECTIVE To study the safety and immunogenicity of D-glutamate(D-glu)and D-alanine(D-ala)aux-otrophic Salmonella typhimurium and explore the feasibility of D-amino acid auxotrophic strain as an oral live at-tenuated vaccine or carrier.METHODS Polymerase chain reaction(PCR)amplification and sequencing were per-formed for evaluation of genotypes.The growth status and morphological features of the strain were observed by growth curve experiment,scanning electron microscopy and transmission electron microscopy.The phenotype of D-amino acid auxotrophic S.typhimurium was confirmed stable after knocking out the corresponding genes.The sensitivities of the auxotrophic strains to penicillin and ampicillin acting on the cell walls were explored by drug susceptibility testing after exogenous supplement of D-amino acid.Safety evaluation was carried out by counterac-ting toxic test with various immunizing doses,pathological sections of organs,living imaging of rats,counting of strains and 16s rRNA full-length sequencing were performed to test the colonization and invasion capacity of the D-amino acid auxotrophic strains.The antibody titers of the immune rats was tested to detect the immune response induced by the vaccine,and the protective effect on the D-amino acid auxotrophic strains was determined by coun-teracting toxic test after immunization.RESULTS The growth of D-amino acid auxotrophic S.typhimurium was stable,which was effectively attenuated by oral administration and stayed for short period of time in the rats,and the trigged inflammatory response was lower.The D-glutamate auxotrophic strains,in particularly,showed the remarkable decline of drug resistance even under the exogenous supplement of D-glutamate,and they reduced the damage to the bodies of the rats and triggered the effective protection of immune response.CONCLUSION The D-glutamate auxotrophic S.typhimurium is expected to be the attenuated live vaccine or carrier for delivery of anti-gen so as to prevent pathogens infections.
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