Construction and characterization of host-vector balanced lethal system of attenuated Salmonella typhimurium SL1344ΔsseK1Δasd / 中国免疫学杂志

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.

Construction and characterization of host-vector balanced lethal system of attenuated Salmonella typhimurium SL1344ΔsseK1Δasd / 中国免疫学杂志

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.

Construction and characterization of invasion protein B gene deleted mutant asd host-vector balanced lethal system / 中国免疫学杂志

Objective: To develop an oral live vaccine vector which stably carries exogenous genes.Methods:SL1344ΔsipBΔasd host-vector balanced lethal system was constructed by the method of recombinant suicide plasmid-mediated allelic exchange on the basis of attenuated Salmonella typhinurium SL1344ΔsipB.Then,the biological characteristics of SL1344ΔsipBΔasd was analyzed.Results:The results showed that the mutant was stabile with the Δasd gene in vitro;the serotype and growth rate of SL1344ΔsipBΔasd strain was almost same as the parent SL1344ΔsipB and SL1344 strain.And the mutant strains remain swim ming zones.Virulence test in mice showed that the virulence of SL1344ΔsipBΔasd which carried complementary plasmid pYA3493 by electro-transformation decreased by 1.4%compared with SL1344.Conclusion: These results showed that the SL1344ΔsipBΔasd mutant was successfully constructed.It is likely that this mutant should be used as a live vector to express foreign genes.

Construction and characterization of type III secretion system of attenuated Salmonella typhimurium / 生物工程学报

In order to develop a recombinant attenuated Salmonella typhimurium as oral live vaccine vector, we constructed recombinant plasmid pYA-sopENt100 by replacing the trc promoter with the sopE promoter and secretion signal sequence sopENt100 of Salmonella typhimurium on the basis of plasmid pYA3493. Then, the complementary plasmid pYA-sopENt100 was transformed into ΔcrpΔasdSL1344 by electroporation to generate attenuated Salmonella typhimurium type III secretion system ΔcrpΔasdSL1344 (pYA-sopENt100). We further characterized ΔcrpΔasdSL1344 (pYA-sopENt100). We also constructed a recombinant strain ΔcrpΔasdSL1344 (pYA-sopENt100-egfp) that harbored the reporter gene-enhanced green fluorescent protein (egfp) gene. Vero cells were infected with ΔcrpΔasdSL1344 (pYA-sopENt100-egfp) and the ability of delivery foreign antigens was tested via Western blotting analysis. The results of PCR, enzyme digestion and sequencing showed that the ΔcrpΔasdSL1344 (pYA-sopENt100) type III secretion system was constructed successfully. The serotype of ΔcrpΔasdSL1344 (pYA-sopENt100) was identical to ΔcrpΔasdSL1344 and SL1344. Compared with wild strain SL1344, the biochemical characteristics of ΔcrpΔasdSL1344 (pYA-sopENt100) had obvious change, but it was basically the same with ΔcrpΔasdSL1344. The growth speed was much slower than that of the wild strain SL1344. The chicken virulence test (LD₅₀) showed that the virulence of ΔcrpΔasdSL1344 (pYA-sopENt100) was 7×10⁴ times lower than SL1344. In addition, we observed the 37 kDa SopENt100-egfp protein in the cultured supernatant of ΔcrpΔasdSL1344 (pYA-sopENt100-egfp) strain by Western blotting analysis. However, both the 37 kDa SopENt100-egfp protein and 27 kDa EGFP protein were detected in ΔcrpΔasdSL1344 (pYA-sopENt100-egfp)-infected Vero cells. These results demonstrated that the recombinant Salmonella typhimurium type III secretion system ΔcrpΔasdSL1344 (pYA-sopENt100) was successfully constructed, and it should be used as a live vaccine vector for expressing foreign genes.

Construction and characterization of host-vector balanced lethal system of attenuated salmonella choleraesuisΔcrpΔcyaΔasdC78-1 (pYA3493) / 中国免疫学杂志

Objective:In order to develop an oral live vaccine vector of swine that can stably carry exogenous genes.Methods:Mutant ΔcrpΔcyaΔasdC78-1 was constructed by the method of suicide plasmid pREasd-mediated bacteria homologous recombination on the basis of attenuated Salmonella choleraesuisΔcrpΔcyaC78-1.Complementary plasmid pYA3493 with asd was electrotransformed into the mutant,and thenΔcrpΔcyaΔasdC78-1(pYA3493) host-vector balanced lethal system was constructed.Its biological characteristics were analyzed further.Results:The results of PCR and sequencing showed thatΔcrpΔcyaΔasdC78-1(pYA3493) was constructed suc-cessfully.Biological characteristics showed that the serotype of ΔcrpΔcyaΔasdC78-1(pYA3493) was identical to ΔcyaΔasdC78-1 and vaccine strain C500 and it can stably carry theΔasd gene in vitro;its growth speed was a little slower than ΔcrpΔcyaC78-1 strain,but both of their growth speeds were significantly slower than vaccine strain C500;the biochemical characteristics of ΔcrpΔcyaΔasdC78-1 ( pYA3493 ) were basically the same with ΔcrpΔcyaC78-1 strain.Oral virulence test in mice showed that the virulence ofΔcrpΔcyaΔasdC78-1 ( pYA3493 ) was similar with ΔcrpΔcyaC78-1, but its median lethal dose is 412 times of vaccine strain C500.Conclusion:These results demonstrated that attenuated Salmonella choleraesuisΔcrpΔcyaΔasdC78-1(pYA3493) strain had the potential to be used as an oral live vaccine vector for expressing foreign genes efficiently.

Construction of Salmonella Pullorum C79-13ΔcrpΔasd mutant balanced-lethal host-vector system and its biological features / 中国免疫学杂志

In order to develop a safer vaccine strain exploit Salmonella Pullorum vaccine strain as a live vaccine vector.Methods:AΔcrpΔasd mutant of S.pullorum C79-13 strain was constructed and it was developed E.coli donor strain mutant was generated through the two-step method introduced by χ7213 ( pREΔasd) was conjugated with the recipient of C 79-13Δcrp.The C79-13ΔcrpΔasd the transduction of recombinant suicide plasmid .Results:PCR and sequencing results showed that ΔsipBSL1344 was suc-cessfully constructed.The further study indicated that foreign DAP must be supplied for the ΔcrpΔasd mutant to grow,in addition,the asd gene was transmitted stably .Compared with C79-13 strain,the O antigens was identical to C79-13 strain,but the growth velocity was reduced significantly ,significantly reduced virulence .Conclusion: The ΔcrpΔasd mutant can accept asd+plasmid to construct host-vector balance lethal system , and this system can be used to express foreign gene efficiently and to develop potential oral multivalent vaccines.