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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.
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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.
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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.
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Objective To achieve arabinose-controlled expression of HtrA strain and detect the expression of HtrA protein.Methods Arabinose promoter with htrA100 was amplified from pACD-htrA vector by PCR and cloned into pGP704 vector.Then, Shigella flexneri 2a strain 301 was transferred with the recombinant plasmid pGD-htrA and an AraC-expression vector .The expressions of HtrA in whole-cell and periplasmic space were detected by Western blotting .Results The suicide plasmid-mediated homologous recombinant vector and the inducible HtrA expression strain were successfully constructed.Without arabinose,HtrA protein was hardly detected ,but in the presense of arabinose , HtrA protein could be detected in whole-cell lysate and in periplasmic space lysate by Western blot .Conclusion Homologous recombination using suicide plasmid can significantly knock down the expression of HtrA protein .After being induced with arabinose , HtrA protein can be expressed normally .
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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.
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Construction of mutant strain is an essential method in pathogenesis researches. The conventional method for Brucella unmarked deletion mutant construction is based on suicide plasmid, but the efficiency is very low. In the present study, we first optimized the electroporation parameters, and then, the cloning plasmid pEX18Gm containing sacB was successfully used to construct unmarked deletion mutant of the type IV secretion system. This indicated that by using conventional cloning plasmid as suicide plasmid in Brucella, unmarked deletion mutants can be constructed with high efficiency.
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Streptococcus pneumoniae(S.pn) is an opportunity pathogenic bacteria,environmental factors play a key role in the pathogenicity of S.pn. It is important to study virulent gene in vivo. The S.pn suicide plasmid containing gfp reporter was constructed by fusing the genes pneumolysin and gfp,in which gfp is an excellent molecule probe in vivo. The plasmid was integrated to No.22 S.pn by homologous recombination. The recombinant S.pn was gained and evaluated in aspects of fluorescence excitation, biological character and physio-activity. The results showed it is efficient and available to report the expression of virulent genes in vivo and in vitro, which will provide a new easy method for evaluating and screening the virulent genes of S.pn in vivo.