RESUMEN
Objective:To develop a host-vector balanced lethal system of attenuated Salmonella typhinurium secreted effector K1 mutant,and an live vaccine vector which stably carries exogenous genes. Methods:We constructed SL1344ΔsseK1Δasd mutant strain by recombinant suicide plasmid( pREΔasd) ,and screened by two-step method,transformed pYA3493 plasmid containing the asd gene without resistance electric into the mutant strain of SL1344ΔsseK1Δasd, then the recombinant strain SL1344ΔsseK1Δasd (pYA3493) was constructed successfully. Results:The results of PCR and sequencing showed that SL1344ΔsseK1Δasd(pYA3493)was constructed successfully. Further studies had shown that the serotype of the recombinant strain was identical to the parent SL1344ΔsseK1 and wild SL1344 strains,and the mutant was stable with the recombinant Δasd gene in vitro. It was found that the re-combinant strain had displayed identical growth profile and biochemical characteristics compared with the parent SL1344ΔsseK1 strain and wild SL1344 strain. The oral challenge of bacteria in mice revealed that LD50 of the recombinant strain was 5. 24×108 CFU,and the toxicity reduced to about 0. 048%;the immunoprotective effect assay showed that the protection rate infected with wild strain of Salmonella typhimurium was 62. 5% on the 17th day post-immunization,which was identical to the parent SL1344ΔsseK1. Conclusion:These results show that the secreted effector K1 gene deleted mutant host-vector balanced lethal system of Salmonella typhimurium SL1344 strain has been successfully constructed,and genetic stability,significantly reduced virulence,which has laid a foundation for developing potential oral live vaccin vector to express foreign genes.
RESUMEN
Objective:To develop a host-vector balanced lethal system of attenuated Salmonella typhinurium secreted effector K1 mutant,and an live vaccine vector which stably carries exogenous genes. Methods:We constructed SL1344ΔsseK1Δasd mutant strain by recombinant suicide plasmid( pREΔasd) ,and screened by two-step method,transformed pYA3493 plasmid containing the asd gene without resistance electric into the mutant strain of SL1344ΔsseK1Δasd, then the recombinant strain SL1344ΔsseK1Δasd (pYA3493) was constructed successfully. Results:The results of PCR and sequencing showed that SL1344ΔsseK1Δasd(pYA3493)was constructed successfully. Further studies had shown that the serotype of the recombinant strain was identical to the parent SL1344ΔsseK1 and wild SL1344 strains,and the mutant was stable with the recombinant Δasd gene in vitro. It was found that the re-combinant strain had displayed identical growth profile and biochemical characteristics compared with the parent SL1344ΔsseK1 strain and wild SL1344 strain. The oral challenge of bacteria in mice revealed that LD50 of the recombinant strain was 5. 24×108 CFU,and the toxicity reduced to about 0. 048%;the immunoprotective effect assay showed that the protection rate infected with wild strain of Salmonella typhimurium was 62. 5% on the 17th day post-immunization,which was identical to the parent SL1344ΔsseK1. Conclusion:These results show that the secreted effector K1 gene deleted mutant host-vector balanced lethal system of Salmonella typhimurium SL1344 strain has been successfully constructed,and genetic stability,significantly reduced virulence,which has laid a foundation for developing potential oral live vaccin vector to express foreign genes.