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    刘翠翠, 马思思, 王佳莹, 吴云燕, 易琳, 赵明秋, 陈金顶. 布鲁菌缺失疫苗株M5-ΔznuA和M5-Δbp26-ΔznuA的构建及毒力和免疫原性的评估[J]. 华南农业大学学报, 2015, 36(5): 18-25. DOI: 10.7671/j.issn.1001-411X.2015.05.004
    引用本文: 刘翠翠, 马思思, 王佳莹, 吴云燕, 易琳, 赵明秋, 陈金顶. 布鲁菌缺失疫苗株M5-ΔznuA和M5-Δbp26-ΔznuA的构建及毒力和免疫原性的评估[J]. 华南农业大学学报, 2015, 36(5): 18-25. DOI: 10.7671/j.issn.1001-411X.2015.05.004
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    LIU Cuicui, MA Sisi, WANG Jiaying, WU Yunyan, YI Lin, ZHAO Mingqiu, CHEN Jinding. Construction of M5-ΔznuA and M5-Δbp26-ΔznuA mutant strains of Brucella melitensis and assessment of their virulence and immunogenicity[J]. Journal of South China Agricultural University, 2015, 36(5): 18-25. DOI: 10.7671/j.issn.1001-411X.2015.05.004
    Citation: LIU Cuicui, MA Sisi, WANG Jiaying, WU Yunyan, YI Lin, ZHAO Mingqiu, CHEN Jinding. Construction of M5-ΔznuA and M5-Δbp26-ΔznuA mutant strains of Brucella melitensis and assessment of their virulence and immunogenicity[J]. Journal of South China Agricultural University, 2015, 36(5): 18-25. DOI: 10.7671/j.issn.1001-411X.2015.05.004
    shu

    布鲁菌缺失疫苗株M5-ΔznuA和M5-Δbp26-ΔznuA的构建及毒力和免疫原性的评估

    Construction of M5-ΔznuA and M5-Δbp26-ΔznuA mutant strains of Brucella melitensis and assessment of their virulence and immunogenicity

      摘要
    • 摘要:
      目的 利用分子生物学技术，通过同源重组的方法构建了新型的羊种布鲁菌Brucella melitensis减毒活疫苗株，为布鲁菌的防治提供新的选择.
      方法 以羊种布鲁菌减毒活疫苗M5株及缺失bp26基因的M5-Δbp26缺失突变株为亲本株，利用电击转化法，将含有znuA基因上下游同源臂的自杀质粒转入到亲本株中，通过同源重组敲除znuA基因.对构建的新型基因缺失株进行生物学特性及毒力的鉴定与分析.
      结果和结论 PCR及核苷酸序列测序结果表明，羊种布鲁菌减毒活疫苗znuA单基因缺失株和bp26、znuA双基因缺失株均构建成功，分别将其命名为M5-ΔznuA和M5-Δbp26-ΔznuA.与亲本株相比，2种缺失突变株的生物学特性没有显著性差异；体外连续传至20代后，菌落PCR和核苷酸测序结果显示M5-ΔznuA和M5-Δbp26-ΔznuA株具有良好的遗传稳定性.小鼠脾脏质量和脾脏菌落数表明，M5-Δbp26-ΔznuA双基因缺失株毒力最弱，单基因缺失株M5-ΔznuA和M5-Δbp26次之.血清中抗体水平的监测显示，znuA基因的缺失对布鲁菌减毒活疫苗诱导机体体液免疫的能力没有影响.获得了免疫原性保持良好而毒力减弱的羊种布鲁菌减毒活疫苗基因突变株M5-ΔznuA和M5-Δbp26-ΔznuA.

       

      Abstract:
      Objective To harvest a kind of live vaccine of Brucella melitensis with less virulence, the novel B.melitensis live vaccine was constructed through homologous recombination.
      Method M5 and M5-Δbp26 were used as the parental strains.znuA gene was knocked out through the recombined suicide plasmid.The biological characteristic of mutant strains was examined by inspecting and annlyzing the growth curve, stability of generating the virulence in Balb/c mice.
      Result and conclusion The results of PCR amplification and sequencing showed that two types of genic deleted mutant strains were constructed successfully, which were designated as M5-ΔznuA and M5-Δbp26-ΔznuA.Compared with the parental strain M5, these two mutant stains had the growing characteristics similar to M5 and possessed a stable genic generation.Based on the mass and counts of spleen of Balb/c, the results demonstrated that M5-Δbp26-ΔznuA exhibited the lowest virulence among all strains, followed by M5-ΔznuA and M5-Δbp26.The quantity of specific antibody of serum revealed that the ability to induce humoral immune reaction was not affected by znuA deletion.In this study, two gene deletion mutants of B.melitensis live vaccine have been yielded, and they might be a new candidate vaccine.

       

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